EFFECT OF HYPOCHOLESTERIMIC DRUGS FOR MEMORY … · 2016. 10. 23. · I, Srinivasa Rao Talasila declare that the thesis entitled “Effect of Hypocholesteremic Drugs for Memory Enhancement

EFFECT OF HYPOCHOLESTERIMIC DRUGS FOR MEMORY ENHANCEMENT AMONG ANIMALS

Thesis submitted in

Partial Fulfilment for the award of

Degree of Doctor of Philosophy

in Pharmacy

By

Mr. Srinivasa Rao Talasila, M.Pharm.,

Reg. No.: J86360001

VINAYAKA MISSIONS UNIVERSITY

SALEM, TAMILNADU, INDIA

October 2016

CERTIFICATE BY THE GUIDE

I, Dr. S Kavimani, M. Pharm., Ph.D, certify that the thesis entitled “Effect of

Hypocholesteremic Drugs for Memory Enhancement Among Animals”

submitted for the Degree of Doctor of Philosophy by Mr. Srinivasa Rao

Talasila is the record of research work carried out by him during the period

from July 2008 to December 2015 under my guidance and supervision and

that this work has not formed the basis for the award of any degree diploma,

associate-ship, fellowship, titles in this University or any other University or

other institution of higher learning.

Place: Signature of the Supervisor with designation

Date:

DECLARATION

I, Srinivasa Rao Talasila declare that the thesis entitled “Effect of

Hypocholesteremic Drugs for Memory Enhancement Among Animals”

submitted by me for the Degree of Doctor of Philosophy is the record of work

carried out me for the period from July 2008 to December 2015 under

guidance of Dr S. Kavimani, M. Pharm., Ph. D and has not formed the basis

for the award of any degree, diploma, associate-ship, fellowship, titles in

this or any other University other similar institutions of higher learning.

Place: Signature of the Candidate

Date:

ACKNOWLEDGEMENT

“The act of thanks giving does not exhibit ones sense of gratitude, but the true

tendency of leading a helping hand during emergency and the fact that every

work has thousands of hands behind”.

I humbly owe the completion of this dissertation work to the Almighty and

family members whose love and blessing will be with me every moment of my

life.

It is my pleasure to express deep sense of gratitude and thankfulness to my

esteemed guide Dr. S. Kavimani, M. Pharm., Ph.D., Professor and Head,

Department of Pharmacology, Mother Theresa Postgraduate and Research

Institute of Health Sciences, Puducherry for his invigorate guidance, constant

encouragement and invaluable suggestions during the course of my

dissertation work. Indeed without his guidance and optimistic support this

project couldn’t have been successful.

I express my sincere thanks to Dr. Suthakaran, M. Pharm., Ph.D., Principal,

Teegala Ram Reddy College of Pharmacy, Hyderabad, for his guidance, deep

inspiration and co-operative nature with due respect in my heart. I thank him

for his never-ending willingness to render generous help whenever needed.

I would like to extend my deep sense of gratitude to Dr. Arihara Sivakumar,

M. Pharm., Ph.D., Associate Professor, Department of Pharmacology, KMCH

i

college of Pharmacy, Coimbatore, for their untire support in offering the

facilities and instrumentation for carrying out the pharmacological studies.

I take pleasure to express my sincere thanks to Dr. Rajashekar Jaladi,

Associate Director, DMPK, Dr Reddy’s Laboratories, Hyderabad and Dr

Veeresh, Associate professor, Department of Pharmacology, Gokaraju

Rangaraju college of Pharmacy, Hyderabad for their continuous support,

guidance and motivation during writing of my thesis.

I wish to extend my thanks to my M. Pharm junior Dr. Alen JS Jinu for

introducing me to my esteemed guide and enabled my Ph.D. registration.

I wish to than my beloved parents Shri. Shyamsunder Rao and Smt.

Laxmirani for giving me life in the first place, educating me and their

encouragement to pursue my interests.

My family has always been an inspiration and the credit of this work also goes

to them, my beloved wife Mrs. Varalakshmi and my dearest daughters

Keerthisree and Sanvisree who always covered under the shade of their love

and affection, the spirit of cooperation for completion of my research work.

Srinivasa Rao T

ii

CONTENTS

Page No.

1. Introduction 1

2. Review of literature 21

3. Need for the study 58

4. Objectives 59

5. Methodology

5.1. Animals 60

5.2. Drugs 60

5.3. Study design 65

5.4. Experimental design for scopolamine induced

amnesia

66

5.5. Experimental design for high fat diet induced amnesia 67

5.6. Experimental methods

5.6.1. Elevated plus maze 68

5.6.2. Rectangular maze 69

5.6.3. Locomotor activity 71

5.7. Biochemical estimation

5.7.1. Lipid profile 72

5.7.2. Estimation of cholinesterase enzyme 78

5.8. Brain Histopathology 79

6. Results

6.1. Effect of hypocholesteremic drugs on scopolamine

induced amnesia




iii

Page No.

6.1.4. Brain acetylcholinesterase activity 90

6.1.5. Brain Histopathology 92

6.2. Effect of hypocholesteremic drugs on high fat diet

induced amnesia

6.2.1. Effect of hypocholesteremic drugs on body

weights

99




6.2.5. Brain acetylcholinesterase activity 111

6.2.6. Serum lipid profiles 113

6.2.7. Brain Histopathology 119

7. Discussion 126

8. Conclusions 131

9. Bibliography 133

10. Annexures

11. Publications

iv

List of Abbreviations

5-HT – 5-hydroxytryptamine

ABCA1 – ATP-binding cassette transporter

ACAT transferase – Acyl-CoA-cholesterol acyl

ACh – Acetylcholine

AChE – Acetylcholinesterase

AChIs – Acetylcholinesterase inhibitors

AD – Alzheimer’s disease

Akt – Protein Kinase B

AMPA – α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic

acid

ApoA1 – Apolipoprotein A1

ApoB – Apolipoprotein B

ApoE – Apolipoprotein E

APP – Amyloid precursor protein

AT – Acquisition trial

Aβ – Amyloid beta

BBB – Blood brain barrier

BCEC – Brain capillary endothelial cells

BZD – Benzodiazepines

Ca2+ – Calcium

cAMP – Cyclic adenosine monophosphate

v

CDC – Centers for Disease Control

CE – Cholesteryl esters

CNS – Central nervous sysyem

Co-A – Coenzyme A

CSF – Cerebrospinal fluid

CYP – Cytochrome P450

DAG – Diacylglycerol

dl – Decilitre

DTNB – (5,5'-dithiobis-(2-nitrobenzoic acid)

ELT – Escape latency time

ERK – Extracellular signal-regulated kinase

FDA – Food and Drug Administration

GABA – Gamma amino butyric acid

GC-MS – Gas chromatography–mass spectrometry

Gms – Grams

GSH – Glutathione stimulating hormone

HFD – High fat diet

HM – Hippocampus and Memory

HMG-CoA – 3-hydroxy-3-methylglutaryl-coenzyme A

i.p – Intraperitoneal

IAEC – Institutional animal ethics committee

ICAM – Intercellular adhesion molecules

ICV – Intracerebroventricular

vi

IL-1β – Interleukin 1β

IL6 – Interleukin-6

INSIG1 – Insulin Induced Gene 1

IP3 – Inositol trisphosphate

Kg – Kilograms

KU/l – Kilounits per liter

LDL – Low-density lipoproteins

LDLR – Low-density lipoprotein receptors

LTM – Long term memory

LXR – Liver x receptor

mAChRs – Muscarinic cholinergic receptors

MAPK – Mitogen-Activated Protein Kinase

mg – Milligram

min – Minutes

ml – Millilitre

mM – Milli moles

mmol – Milli mole

MMSE – Mini-Mental State Examination

MRI – Magnetic resonance imaging

mRNA – Messenger RNA

MWM – Morris water maze

NaCMC – Sodium carboxy methyl cellulose

NF-κB – Nuclear factor kappa-light-chain-enhancer of

vii

activated B cells

NMDA – N-methyl D-aspartate

NSAIDs – Non-steroidal anti-inflammatory drugs

OCD – Obsessive compulsive disorder

p.o. – Oral administration

PI3K – Phosphoinositide 3-kinase

PIP2 – Phosphatidylinositol 4,5-bisphosphate

PKC – Protein kinase C

PSEN1 – Presenilin 1 gene

PSEN2 – Presenilin 2 gene

rpm – Revolutions per minute

RT – Retention trial

SEM – Standard error mean

SRB1 – Scavenger receptor class B member 1

SREBP – Sterol regulatory element-binding protein

STM – Short term memory

STZ – Streptozotocin

TAG – Triacylglycerols

TBARS – Thiobarbituric acid reactive species

TBI – Traumatic brain injury

TC – Total cholesterol

TG – Triglycerides

TL – Transfer latency

viii

http://www.human-memory.net/disorders_ocd.html

https://en.wikipedia.org/wiki/Phosphoinositide_3-kinase

TLR4 – Toll-like receptor 4

TNFα – Tumor necrosis factor –alpha

Tris– HCl – Tris hydrochloride

U/l – Units per litre

VLDL – Very low-density lipoproteins

βA – β-amyloid

μl – Microliter

ix

List of Figures

Figure No. Title Page

No. 1. Human Brain 1

2. Memory processes 2

3. Classification of different Types of Memory 3

4. Cholesterol Biosynthetic pathway 24

5. Effect of hypocholesterolemic drugs on transfer latency

using EPM in Scopalamine treated animals. 83

6. Effect of hypocholesterolemic drugs on transfer latency

using Rectangular maze in Scopalamine treated animals. 86

7. Effect of hypocholesterolemic drugs on locomotor activity

using Actophotometer in Scopalamine treated animals. 89

8. Effect of hypocholesterolemic drugs on brain

Acetylcholinesterase levels in Scopalamine treated animals. 91

9. Basal ganglia of normal rat (40X) 93

10. Basal ganglia of rat after Scopolamine induced amnesia

(40X) 93

11. Basal ganglia of rat treated with Donepezil in Scopolamine

induced amnesia (40X) 93

12. Basal ganglia of rat treated with Simvastatin in Scopolamine


13. Basal ganglia of rat treated with Rosuvastatin in

Scopolamine induced amnesia (40X) 94

14. Basal ganglia of rat treated with Fenofibrate in Scopolamine


15. Basal ganglia of rat treated with Nicotinic acid in


16. Basal ganglia of rat treated with Donepezil + Simvastatin in 94

x

Scopolamine induced amnesia (40X)

17. Basal ganglia of rat treated with Donepezil + Rosuvastatin in


18. Basal ganglia of rat treated with Donepezil + Fenofibrate in


19. Basal ganglia of rat treated with Donepezil + Nicotinic acid

in Scopolamine induced amnesia (40X) 95

20. Cerebellum of normal rat (40X) 95

21. Cerebellum of rat after Scopolamine induced amnesia (40X) 96

22. Cerebellum of rat treated with Donepezil in Scopolamine


23. Cerebellum of rat treated with Simvastatin in Scopolamine


24. Cerebellum of rat treated with Rosuvastatin in Scopolamine


25. Cerebellum of rat treated with Fenofibrate in Scopolamine


26. Cerebellum of rat treated with Nicotinic acid in Scopolamine


27. Cerebellum of rat treated with Donepezil + Simvastatin in


28. Cerebellum of rat treated with Donepezil+Rosuvastatin in

scopolamine induced amnesia induced amnesia (40X) 97

29. Cerebellum of rat treated with Donepezil+Fenofibrate in

scopolamine induced amnesia (40X) 98

30. Cerebellum of rat treated with Donepezil+Nicotinic acid in

scopolamine induced amnesia (40X) 98

31. Effect of hypocholesterolemic drugs on Body Weights in

animals receiving high fat diet (HFD). 101

32. Effect of hypocholesterolemic drugs on transfer latency 104

xi

using Elevated plus-maze in animals receiving high fat diet

(HFD).

33.

Effect of hypocholesterolemic drugs on transfer latency

using Rectangular maze in animals receiving high fat diet

(HFD). 107

34.

Effect of hypocholesterolemic drugs on locomotor activity

using Actophotometer in animals receiving high fat diet

(HFD). 110

35.

Effect of hypocholesterolemic drugs on Brain

Aceylcholinesterase levels in animals receiving high fat diet

(HFD). 112

36. Effect of hypocholesterolemic drugs on serum lipid profile in

animals receiving high fat diet (HFD). 118

37. Basal ganglia of normal rat (40X) 120

38. Basal ganglia of HFD induced rat (40X) 120

39. Basal ganglia of rat treated with Donepezil in HFD induced

amnesia (40X) 120

40. Basal ganglia of rat treated with Simvastatin in HFD induced

amnesia (40X) 120

41. Basal ganglia of rat treated with Rosuvastatin in HFD


42. Basal ganglia of rat treated with Fenofibrate in HFD induced

amnesia (40X) 121

43. Basal ganglia of rat treated with Nicotinic acid in HFD


44. Basal ganglia of rat treated with Donepezil + Simvastatin in

HFD induced amnesia (40X) 121

45. Basal ganglia of rat treated with Donepezil + Rosuvastatin in


46. Basal ganglia of rat treated with Donepezil + Fenofibrate in 122

xii

HFD induced amnesia (40X)

47. Basal ganglia of rat treated with Donepezil + Nicotinic acid

in HFD induced amnesia (40X) 122

48. Cerebellum of normal rat (40X) 122

49. Cerebellum of rat after HFD induced amnesia (40X) 123

50. Cerebellum of rat treated with Donepezil in HFD induced

amnesia (40X) 123

51. Cerebellum of rat treated with Simvastatin in HFD induced

amnesia (40X) 123

52. Cerebellum of rat treated with Rosuvastatin in HFD induced

amnesia (40X) 123

53. Cerebellum of rat treated with Fenofibrate in HFD induced

amnesia (40X) 124

54. Cerebellum of rat treated with Nicotinic acid in HFD induced

amnesia (40X) 124

55. Cerebellum of rat treated with Donepezil + Simvastatin in

HFD induced Amnesia (40X) 124

56. Cerebellum of rat treated with Donepezil + Rosuvastatin in

HFD amnesia induced amnesia (40X) 124

57. Cerebellum of rat treated with Donepezil + Fenofibrate in


58. Cerebellum of rat treated with Donepezil + Nicotinic acid in


xiii

List of Tables

Table No.

Title Page No.

1. Different brain areas and their role in learning and memory 9

2. Potential neurotransmitters, neuromodulators and their

respective receptors involved in learning and memory 13

3.

Drugs used along with the dose of each drug selected for

the study and route of administration followed for the

corresponding drugs.

63

4. Groups allocation for scopalamine induced amnesia 66

5. Groups allocation for high fat diet induced amnesia 67

6. Transfer latency recordings of hypocholesteremic drugs

using elevated plus maze. 82


using rectangular maze 85

8. Activity scores of hypocholesteremic drugs using

actophotometer 88

9. Acetylcholinesterase levels of animals treated with

hypocholesteremic drugs in scopalamine induced rats. 91

10. Body weight summary of animals at different time points 100


using elevated plus maze. 103


using rectangular maze (HFD). 106

13. Activity score recordings of hypocholesteremic drugs using

Actophotometer (HFD) 108

14. Acetylcholinesterase levels of animals treated with

hypocholesteremic drugs in high fat diet induced rats. 112

15. Changes in lipid profiles upon high fat diet induction at

different time points. 114 - 117

xiv

INTRODUCTION

1. INTRODUCTION

The brain is the center of the nervous system in all vertebrates and most

invertebrates. The brain allows them to collect information (sensory system),

act on the collected information (motor system) and stores the result for future

reference (memory), thus effectively making life possible.

The human brain is the most complex of all, and

perhaps the most complex structure known in the

universe. The adult brain weighs an average about

1.5 kg (3 lbs). It continuously receives and analyses

sensory information and responds by controlling all

bodily actions and functions. The brain is the centre

of higher order thinking, learning and memory, and

gives the power to think, plan, speak, imagine,

dream, reason and experience emotions.

MEMORY

Memory can be stated as an organism’s ability to encode, store, retain and

subsequently recall information and past experiences. It is the store of things

learned and retained from the activity or experience, as evidenced by

modification of structure or behaviour, or by recall and recognition.

In physiological or neurological terms, memory is a set of encoded neural

connections in the brain. It is the re-creation or reconstruction of past

experiences by synchronous firing of neurons that were involved in the original

experience.

Fig-1: Human Brain

1

Learning and memory are the two fundamental cognitive functions that provide

us the ability to accumulate knowledge from our experiences (Kulkarni et al.,

2010)1. Memory is closely related but distinct from learning. Learning is the

acquisition of skill or knowledge of the world while memory is the expression of

what have been acquired. Another difference between the two is the speed

with which the two things happen. Acquiring the new skill or knowledge slowly

and laboriously is learning while instant acquisition is memory (Puchchakayala

Goverdhan et al., 2012)2. Memory depends on learning as it stores and

retrieves the learned information. In the process of formation and retrieval of

memory, three main stages are being suggested viz., acquisition,

consolidation and recall of the learned task (Milind Parle & Nitin Bansal,

2011)3.

Fig-02: Memory processes

Retrieval

Aquisition

Engra

Consolidation

Perception of stimuli

Long term

Storage

Recognition/Reconstructi

2

1. Acquisition: This mainly involves receiving, processing and combining of

the received information.

2. Storage or consolidation: This stage involves complex neuro-chemical

process creating the temporary/permanent record of the information.

3. Retrieval, recall or recollection: This process involves the decoding and

retrieval of the encoded stored memory (Ji Hoon Ohet al., 2009)4.

TYPES OF MEMORY

With duration as the criterion, three different types of memory can be

distinguished: sensory memory, short-term memory, and long-term memory.

Fig-3: Classification of different Types of Memory

Memory

Sensory Short term

Long term

Explicit/Conscious Implicit/Unconscious

Procedural Declarative

Episodic Semantic

3

• Sensory memory is the shortest-term element of memory. It takes the

information provided by the senses and retains it accurately but very briefly.

Sensory memory lasts such a short time (from a few hundred milliseconds

to one or two seconds) that it is often considered as a buffer for stimuli

received through the sense of sight, hearing, smell, taste and touch.

Nevertheless, it represents an essential step for storing information in

short-term memory via the process of attention.

• Short-term memory (STM) temporarily recalls the information which is

being processed at any point of time. It can be thought as the ability to

remember and process information at the same time. It records the

succession of events occurring in lives such as registering a face seen in

the street, or a telephone number overheard when someone giving out, but

this information quickly disappears forever unless a conscious effort is

made to retain it.

STM has a storage capacity for small amount of information; only about

seven items and lasts only a few dozen seconds. After entering sensory

memory, limited amount of information is transferred into STM. There are

three basic operations that occur within STM.

• Iconic memory – the ability to hold visual images

• Acoustic memory – the ability to hold sounds. This can be held longer

than the iconic memory.

• Working memory – it’s an active process to keep it until its being put to

use. For example, repeating a phone number until its being dialed. Here,

4

the goal is to merely put the information to immediate use and not move the

information from STM to LTM. Just as sensory memory is a necessary step

for short-term memory, short-term memory is a necessary step toward the

next stage of retention, long-term memory (Atish Prakash, 2007)5.

The central executive part of the prefrontal cortex at the front of the brain

appears to play a fundamental role in short-term and working memory. It

cooperates with other parts of the cortex to extract information for brief

periods. It was reported in 1935 that damage to prefrontal cortex caused

short-term memory deficits in primates.

• Long-term memory (LTM) is intended for storage of information over a

long period of time. This process relates to three main activities: storage,

deletion and retrieval. Information from STM is stored in LTM by repeated

exposure to a stimulus or by rehearsal of a piece of information. Deletion is

mainly caused by decay and interference. Information that had been

forgotten sometimes may be recognised or may be recalled with

prompting. This leads to information retrieval. In recall, information is

reproduced from memory and in recognition; the presentation of

information provides the knowledge of the information that has been seen

before. The hippocampus area of the brain not itself is used to store

information but acts as an essential kind of temporary transit point for long-

term memories. Two main forms of long-term memory can be

distinguished:

5

Declarative memory requires conscious recall. It is sometimes called

explicit memory, since it consists of information explicitly stored and

retrieved. Declarative memory can be further sub-divided into semantic

memory, which is a record of facts, meanings, concepts and knowledge

about the external world that have been acquired; and episodic memory,

represents our memory of experiences and specific events in time.

Declarative memories are encoded by the hippocampus, entorhinal

cortex and perirhinal cortex (all within the medial temporal lobe of the

brain), but are consolidated and stored in the temporal cortex and

elsewhere. Semantic memory mainly activates the frontal and temporal

cortex whereas episodic memory activity is concentrated in the

hippocampus initially. After processing in the hippocampus, it is

consolidated and stored in the neocortex. The memories of different

elements of particular event are distributed to visual, olfactory and auditory

areas of the brain; all are connected together by the hippocampus to form

an episode than remaining as a collection of separate memories.

Non-declarative memory (Implicit memory) also known procedural

memory is the unconscious memory of skills such as riding a bike, juggling

some balls or simply tying shoelaces. Procedural memory should be

considered as a subset of implicit memory as no new explicit memories are

being formed when a given task is properly performed due to repetitions but

previous experiences are being unconsciously accessed. Procedural

memories, on the other hand, do not appear to involve the hippocampus at

6

http://www.human-memory.net/processes_consolidation.html

http://www.human-memory.net/processes_storage.html

all, and are encoded and stored by the cerebellum, putamen, caudate

nucleus and the motor cortex, all of which are involved in motor control.

Autobiographical memory refers to a memory system which consists of

episodes that is recollected from an individual’s own life. It is often based

on a combination of episodic and semantic memory.

Based on the temporal direction of the memory, long-term memory can be

classified as:

• Retrospective memory refers to the recollection of past episodes, i.e. the

content to be remembered is from the past. It can be semantic, episodic or

autobiographical memory.

• Prospective memory refers to the contents to be remembered in the

future or can also be defined as ‘remembering to remember’ or

‘remembering to perform an action’.

Anatomical areas involved in learning and memory (Kamalraj, 2011)6

The human brain has three major components: the cerebrum, the cerebellum

and the brain stem. The brain stem includes the medulla, the pons and the

midbrain and connects the brain with the spinal cord and rest of the body.

The cerebellum plays an important role in balance, motor control and some

cognitive functions such as attention, language, emotional functions like

regulating fear and pleasure responses; and also in the processing of

procedural memories.

7

http://www.human-memory.net/processes_encoding.html


The cerebrum is covered by a sheet of neural tissue known as cerebral cortex

(or neocortex), which envelops other brain organs such as the thalamus,

hypothalamus and pituitary gland.

The cerebral cortex plays a key role in memory, attention, perceptual

awareness, thought, language and consciousness. It is divided into four main

lobes which covers both hemispheres: frontal, parietal, temporal and occipital

lobe. The frontal lobe is involved in conscious thought and higher mental

functions such as decision-making. The prefrontal cortex plays an important

role in processing short-term memories and retaining long-term memories that

are not task-based. Also temporal lobe plays a key role in the formation of

LTM. The medial temporal lobe is thought to be involved in declarative and

episodic memory. Deep inside medial temporal lobe is the limbic system which

consists of a number of structures, including the hippocampus, cingulate

gyrus, amygdala, the parahippocampal gyrus and parts of the thalamus.

Limbic system is a group of interconnected structures that mediate emotions,

learning and memory. Main areas involved in memory are the hippocampus,

the amygdala and the striatum. The hippocampus is a major component of

the brain. It is essential for memory function; mainly plays an important role in

the consolidation of information from short-term memory to long-term memory.

It is believed to be involved in spatial learning and declarative learning. The

hippocampus is one of the first areas affected by Alzheimer's disease. As the

disease progresses, damage extends throughout the lobes. Amygdala is

8

http://www.brightfocus.org/alzheimers/about/understanding/brain-with-alzheimers.html

http://www.brightfocus.org/alzheimers/about/understanding/brain-with-alzheimers.html

involved in many brain functions, including emotion, learning and memory. It is

part of a system that processes "reflexive" emotions like fear and anxiety.

Inside the cerebral cortex, another sub-cortical system essential in memory

function is the basal ganglia system, particularly the striatum which is

important in the formation and retrieval of procedural memory.

Table–01: Different brain areas and their role in learning and memory

Type of Learning/Memory Brain Areas Involved

Spatial learning

Hippocampus, Parahippocampus,

Subiculum, Cortex (Temporal cortex &

Posterior parietal cortex)

Emotional memory Amygdala

Recognition memory Hippocampus, Temporal lobe

Working memory Hippocampus, Prefrontal cortex

Motor skills Striatum, Cerebellum

Sensory (visual, auditory, tactile) Various cortical areas

Classical conditioning Cerebellum

Habituation Basal ganglia

Pathological changes leading to impairment of learning and memory

The average human brain has about 100 billion neurons (or nerve cells) which

is the core component of the nervous system. A neuron an electrically

excitable cell; processes and transmits information by electro-chemical

9

signalling. Within the brain, information transmission takes places during the

processes of memory encoding and retrieval.

Each axon terminal has thousands of membrane-bound sacs called vesicles

which contain thousands of neurotransmitter molecules in each.

Neurotransmitters are chemical messengers which relay, amplify and

modulate signals between neurons and other cells. The two most common

neurotransmitters in the brain are the amino acids glutamate and gamma

amino butyric acid (GABA). Other important neurotransmitters

include acetylcholine, dopamine, nor-adrenaline, serotonin, glycine

and histamine.

When stimulated by an electrical pulse, the neurotransmitter gets released

from the vesicles and they cross the cell membrane into the synaptic gap

between neurons. These then binds to the chemical receptors of the post-

synaptic neuron which causes changes in the permeability of the cell

membrane to specific ions, opening up special gates or channels through

which charged particles of ions such as calcium, sodium, potassium and

chloride flows. This passage of ions through the gates or channels affects the

potential charge of the receiving neuron which then starts up a new electrical

signal in the receiving neuron. Thus a message within the brain is converted

from an electrical signal to a chemical signal and back again; moving from one

neuron to another.

The electro-chemical signal released by a particular neurotransmitter may be

such as to encourage to the receiving cell to also fire, or to inhibit or prevent it

10

from firing. Different neurotransmitters tend to act as excitatory (e.g.

acetylcholine, glutamate, aspartate, noradrenaline, histamine) or inhibitory (e.g.

GABA, glycine, serotonin), while some neurotransmitter (e.g. dopamine) may be

either excitatory or inhibitory. Subtle variations in the mechanisms of

neurotransmission allow the brain to respond to the various demands made on

it, including the encoding, consolidation, storage and retrieval of memories.

Role of neurotransmitter

Thinking is a biologically demanding task which involves the firing of neurons

requiring plenty of neurotransmitters. Depletion of neurotransmitters results in

reduced mental performance such as slowed reasoning, decreased learning

efficiency, impaired recall, reduced coordination, lowered moods, inability to

cope and increased response time. Stress causes the neurotransmitter to

deplete faster. Maintenance of neurochemicals at optimum level has

corresponding effect on the brain’s performance. The ability to produce and

maintain the optimum levels of neurotransmitter declines as the brain ages.

The brain can therefore be provided with ample raw materials to restore the

levels of neurotransmitter and thus can maintain the cognitive functions.

Central cholinergic system is considered as the most important

neurotransmitter involved in regulation of cognitive functions. Decrease in

cholinergic neurotransmission claims to decline the cognitive functions.

Scopolamine, a nonselective cholinergic muscarinic antagonist

inhibits muscarinic receptors for ACh and produces amnesic effects. It is a

11

http://www.human-memory.net/processes_encoding.html

http://www.human-memory.net/processes_consolidation.html


http://www.human-memory.net/processes_recall.html

very potent psychoactive drug that is used as a standard/reference drug for

inducing amnesia in mammals. The subjects do not recall memories of the

time they were intoxicated and lose all sense of reality. It is highly selective for

muscarinic receptors but high doses also blocks nicotinic receptors (6).

Acetylcholinesterase inhibitors (AChIs) such as donepezil enhances the

availability of ACh in the synaptic cleft, which are able to reverse the

scopolamine induced deficit indicating the role of ACh in learning and memory

functions.

Other levels neurotransmitter such as GABA, dopamine and serotonin plays a

role in the process of learning and memory. The mesocortical dopamine

system plays a crucial role in cognitive processes since dopamine

predominantly controls the functions of prefrontal cortex. Stimulation of

serotonergic neurotransmission disrupts behavioural performance while

inhibition enhances performance in experimental animals.

12

Table–02: Potential neurotransmitters, neuromodulators and their respective

receptors involved in learning and memory.

Neurotransmitter/Neuromodulator Receptor systems

Glutamate NMDA, AMPA receptors

Acetylcholine Muscarinic, nicotinic

Dopamine D1, D2 receptors

Serotonin 5-HT3, 5-HT1A receptors

Neuropeptides G-protein coupled receptors

GABA GABAA/BZD receptor complex

Neurosteroids NMDA/GABAA receptors

Memory Disorders

Memory disorders ranges from mild to severe, all disorders results from some

kind of neurological damage to the brain, which hinders the storage, retention

and recollection of memories.

They can be progressive, like Alzheimer's or Huntington’s disease, or can be

immediate, like those resulting from traumatic head injury. Most disorders are

exacerbated by the effects of ageing, which remains as the single greatest risk

factor for neurodegenerative diseases.

Some of the memory disorders are:

• Age Associated Memory Impairment

• Alzheimer's disease

• Amnesia

• Autism

13

http://www.human-memory.net/brain.html

http://www.human-memory.net/disorders_alzheimers.html


http://www.human-memory.net/disorders_traumatic.html

http://www.human-memory.net/disorders_age.html

http://www.human-memory.net/disorders_age.html


http://www.human-memory.net/disorders_amnesia.html

http://www.human-memory.net/disorders_autism.html

• Dementia

• Huntington's disease

• Korsakoff's syndrome

• Obsessive-Compulsive Disorder (OCD)

• Parkinson's disease

• Schizophrenia

• Tourette syndrome

Amnesia

Amnesia is a condition in which the stored memory; such as facts, information

and experiences is either disturbed or lost. It occurs when the portion of the

brain vital for memory processing is somehow compromised. The limbic

system; comprising the hippocampus, the amygdala, and portions of the cortex

are mainly responsible for retrieving memory. People are amnesiac when the

memory retrieval portion of the limbic system isn’t working properly but there is

otherwise no change in language, attention span, visual/spatial functioning, or

motivation. Limbic system together with several areas of the brain is involved

in the storage of memory and it mainly depends on the type of information

being assimilated. For example, temporal lobe stores the visual and auditory

patterns whereas the parietal lobe stores language, speech, word usage, and

comprehension. People with amnesia; also called amnestic syndrome

generally does not loose self-identity but may have trouble in learning new

14

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http://www.human-memory.net/disorders_huntingtons.html

http://www.human-memory.net/disorders_korsakoffs.html

http://www.human-memory.net/disorders_ocd.html

http://www.human-memory.net/disorders_parkinsons.html

http://www.human-memory.net/disorders_schizophrenia.html

http://www.human-memory.net/disorders_tourettes.html

information and forming new memories. Amnesia can be a temporary

(transient global amnesia) or permanent.

Types of Amnesia

Two main types of amnesia are

a. Anterograde amnesia

b. Retrograde amnesia

a. Anterograde amnesia is characterized by the inability to store, retain,

or recall new knowledge following the onset of amnesia. This occurs when the

ability to memorize new things gets impaired or lost as data is not being

successfully transferred from the conscious short-term memory into

permanent long-term memory. This is the typeof amnesia is seen in dementia

and Alzheimer’s disease.

Anterograde amnesia results from damage to the hypothalamus, thalamus and

the surrounding cortical structures. The encoded memories are never being

stored as the connections between the hippocampus and cortex are being

disrupted.

b. Retrograde amnesia is the ability to recall pre-existing memories such

as past events or previously familiar information are impaired or lost. The

ability to memorize new things that occur after the onset of amnesia may not

be comprised. It usually results from damage to the brain regions such as

the temporal lobe and prefrontal cortex.

15

http://www.human-memory.net/types_short.html

http://www.human-memory.net/types_long.html

Other types of amnesia include:

c. Psychogenic amnesia known as functional amnesia or dissociative

amnesia, is a disorder characterized by abnormal memory functioning

resulting from the effects of severe stress or psychological trauma on

the brain, rather than from any physical or physiological cause.

d. Post-traumatic amnesia is a state of confusion or memory loss that

occurs immediately following a traumatic brain injury.

Causes of Amnesia (Ken-IchiNezasa et al., 2002)7

Amnesia may result either from organic or neurological causes or

from functional or psychogenic causes. Psychogenic or dissociative amnesia

may be caused due to emotional shock or trauma. Causes of neurological

amnesia include:

• Stroke

• Brain inflammation (encephalitis)

• Lack of adequate oxygen in the brain (for example, from heart

attack, respiratory distress or carbon monoxide poisoning)

• alcohol

• Tumors in areas of the brain that control memory

• Degenerative brain diseases, such as Alzheimer's disease and

other forms of dementia

• Seizures

16

http://www.human-memory.net/brain.html

• Electroconvulsive therapy

• Certain medications, such as benzodiazepines

• Head injuries.

Treatment of Amnesia

The major feature of Alzheimer’s disease (AD) is impaired cognitive

functions1. Alzheimer's disease (AD) is a progressive neurodegenerative

disorder is slow in onset characterized by memory loss leading to dementia,

unusual behaviour, personality change and ultimately death The cholinergic

neuronal system plays an important role in learning and memory (Srikanth

Jeyabalan & Muralidharan Palayan, 2009)8. Presence of ACh within the

neocortex is sufficient to ameliorate learning deficits and restore memory. The

treatment with AChE inhibitors and muscarinic receptors agonists increases

cholinergic neurotransmission causing an improvement in cognitive deficits in

AD. Few synthetic AChIs, e.g. tacrine, donepezil, and the natural product

based rivastigmine are used for treatment of cognitive dysfunction and

memory loss associated with AD. Another class of drugs includes the

nootropic agents such as the piracetam.

Recently, focus has been drawn to the role lipids, specifically cholesterol, play

in AD. This attention is overdue, given the disproportionate amount of

cholesterol that is concentrated in the central nervous system. Given the

importance of cholesterol in the brain, dysregulation of cholesterol metabolism

17

may be detrimental to normal cognitive function and may play a role in the

aetiology of AD.

Most of the cholesterol in the brain is concentrated in myelin, which makes up

the white matter. In both humans and monkeys, there is a decrease in white

matter as the brain ages, most of this being attributed to a reduction in myelin.

Cholesterol levels also seem to increase in the exofacial layer of cellular

membranes in mice, which leads to the loss of fluidity, which if occurring in the

synapse may cause a “hardening of the synapse”, therefore effecting synaptic

communication.

Lipids and lipid peroxidation products play an important role in homeostasis of

the central nervous system. Furthermore, lipid transport genes and vascular

changes associated with peripheral dyslipidemia were reported to increase

risk of AD. Brain cholesterol is an essential component of neuronal cell

membranes and is involved in several biological functions, such as membrane

trafficking, signal transduction, myelin formation and synaptogenesis. Given

these widespread activities, it is not surprising that dysfunctions in cholesterol

synthesis, storage, transport and removal can lead to human brain diseases.

Some of these diseases emerge as a consequence of genetic defects in the

enzymes involved in cholesterol biosynthesis. In other diseases, such as AD,

there is a link between cholesterol metabolism and formation and deposition

of βA. Cholesterol overabundance in the brain plasma membrane lipid-raft

domains appears to be fundamental in the generation of the more neurotoxic

forms of βA from the β-amyloid holoprotein precursor.

18

Whether high or low cholesterol serum levels are detrimental or beneficial to

brain function is still unclear. Recent epidemiological results suggest that low

levels of serum cholesterol in the elderly (>65 years of age) is actually a frailty

marker, and is indicative of increased chances of experiencing cognitive

decline. Yet, high cholesterol at midlife is associated with impaired cognitive

function several decades later. Thus, as with most biological processes, either

too little or too much is not optimal. In addition, the studies in humans also

indicate that progressive changes in cholesterol requirements with age may

alter brain cholesterol dynamics and homeostasis.

The first links between cholesterol and AD pathology were observed more

than a decade ago, when it was observed that non-demented patients with

cardiovascular disease and hypercholesterolemia, had significantly greater

numbers of senile plaques in their brains, one of the pathological hallmarks of

the disease. This suggested that peripheral cholesterol levels may be having a

direct effect on the brain. In fact, high cholesterol diets can induce the

formation of greater numbers of senile plaques in the brains of rabbits. In

humans, high peripheral levels of cholesterol have been associated with

increased risk of AD; conversely, using drugs that lower serum cholesterol

levels are thought to reduce AD risk. Similarly, in mouse models of AD,

cholesterol-lowering drugs have been shown to reduce AD-associated

pathology.

19

The goal of this dissertation has been to elucidate the role lipid metabolism

plays in AD and the effect of lipid lowering drugs on central lipid levels and in

turn AD.

20

REVIEW OF LITERATURE

2. REVIEW OF LITERATURE

Cholesterol: Structure and Origin

Cholesterol is essential for life as it provides integrity to plasma membranes

due to the rigidity inherent to its molecular structure. Perhaps surprising to

most is that dietary cholesterol is not essential in order to have adequate

amounts within the tissues of our bodies. Every cell has the capacity to make

as much cholesterol as it needs and the body has various mechanisms to deal

with levels that may not be optimal. Yet, hypercholesterolemia is still present

in a large section of the population. The CDC reports that approximately 20%

and 50% of the population have cholesterol levels greater than 240 mg/dL and

200 mg/dL, respectively (www.cdc.gov). This is attributed to either genetic

variations leading to an increased synthesis or, the excess intake of lipid-rich

dietary sources, with dietary factors playing a greater role. It is no surprise

then that prescribing drugs to treat dyslipidemia is common practice around

the world.

Cholesterol, as alluded to above, has two distinct origins, endogenous and

exogenous. The central regulatory organ for both pools is the liver. Ingested

cholesterol, as well as triacyl glycerols (TAG), are absorbed in the intestine

and are shuttled to the liver by chylomicrons containing Apolipoprotein B

(ApoB), Apolipoprotein E (ApoE) and Apolipoprotein C-II. Upon reaching the

plasma, some of the triglycerides associated with the chylomicrons are

21

converted to free fatty acids by extracellular lipases. The remaining TAG as

well as cholesterol is then delivered to the liver. Excess lipids are either stored

or disbursed about the body. Various mechanisms are in place for the

transport of both TAGs and cholesterol to extra hepatic tissues. The majority

of TAGs are shuttled from the liver to peripheral tissues by very low-density

lipoproteins (VLDL), whereas cholesterol is predominantly shuttled by low-

density lipoproteins (LDL). The cholesterol contained within LDL is both free

and in the form of cholesteryl esters (CE), formed via an action of acyl-CoA-

cholesterol acyl transferase (ACAT) that transfers a fatty acid onto the

cholesterol making it more hydrophobic, and typically less toxic to the cell.

Goldstein and Brown (2009)9 reported that LDL is also composed of ApoB,

which allow for their absorption into cells via the large family of LDL receptors

(LDLR). After binding to LDLR, the complex is endocytosed and digested by

lysosomes within cells and broken down into its components, which include

amino acids, CE and free cholesterol. Cholesterol, specifically, is able to then

participate in the regulation of cholesterol synthesis pathways in extrahepatic

tissues.

Endogenous cholesterol is synthesized in the liver and in extrahepatic tissues,

from acetyl Co-A (much like other lipids), yet the structure of cholesterol is

quite different from other lipids that typically contain long chain fatty acids.

Cholesterol consists of a nucleus of four fused rings that create a relatively

planar and, thus, rigid structure. The molecule has a polar moiety at C3, which

22

gives it its amphipathic nature. These characteristics make it perfectly suitable

to provide structural integrity to membranes.

The cholesterol synthesis pathway is quite complex and involves a great many

enzymes that produce a whole range of different metabolites that act to

regulate the pathway itself. Many of these metabolites act as precursors to a

variety of other isoprenoid molecules that play important roles in the

prenylation of many small G-proteins. Cholesterol also plays the role of

precursor molecule in that it is further processed to produce steroid hormones,

essential vitamins as well as bile acids that allow for the digestion of dietary

lipids. Figure-4 shows some of the major reaction products within the

cholesterol synthesis pathway including key enzymes involved in their

catalysis.

Cholesterol is composed of four fused rings which make up its non-polar,

hydrophobic body. A hydroxyl group is bonded to C3 and acts to create a

polar head giving the molecule its amphipathic nature. B. Cholesteryl esters

have their hydroxyl group replaced by a fatty acid making the molecule more

hydrophobic and typically less toxic to the cell.

23

Fig-04: Cholesterol Biosynthetic pathway

Cholesterol synthetic pathway: Key molecules within the cholesterol

synthesis pathway are represented along with several enzymes that produce

them. Acetyl-CoA is quickly converted to HMG-CoA by HMG-CoA synthase.

The action of the next enzyme in the pathway, HMG-CoA reductase, is

considered rate-limiting. HMG-CoA reductase inhibitors, commonly known as

statins, act to inhibit the actions of this enzyme, which leads to the subsequent

reduction of several reaction products. Besides cholesterol, several other

essential molecules are created as a result of this pathway including the

production of isoprenoids from cholesterol precursors. Finally, further

cholesterol modification leads to the production of steroid hormones, essential

vitamins and bile acids.

Acetyl CoA Acetoacetyl -CoA

Farnesyl Pyrophosphate

Geranyl Pyrophosphate

Isopentenyl Pyrophosphate

Mevalonate HMG CoA

Cholesterol Lanosterol Squalene

CoQ10

Steroids

Vit D

Prenylated

24

The General Nature of Cholesterol and the Nervous System: Dietschy

(2009)10 stated that brain comprises 25% of the body’s total cholesterol pool,

but is only 2% of the body’s total weight and essentially all the cholesterol that

is found in the brain is synthesized there. Brain cholesterol is at a

concentration of 15-20 g/kg of tissue weight, is mostly unesterified and

predominantly concentrated within the myelin that serves to insulate neuronal

axons and, thus, increase the efficiency of their electrical signaling. Vaya and

Schipper (2007)11 indicated that unlike in other tissues, brain cholesterol has a

slow turnover rate, with a flux rate of only 0.9% of that normally seen across

the entire body, which translates into a half-life of 4-6 months in the rat brain

and 6-12 months in a human. It is important to note that total flux rates of

cholesterol in humans, primates, and rodents differ considerably, with rodents

having a much higher metabolic rate and thus a faster turnover rate of

cholesterol. Dietschy and Turley (2001&2004)12,13 reported that various animal

models are used in the study of cognition it is important to consider these

differences since the effects of specific drugs that modulate or directly inhibit

metabolic processes involved in cholesterol synthesis and transport may differ

given the length and dose of treatment with various animal species. What

remains true for all of these mammals is that, considering the rate of

peripheral cholesterol catabolism, the turnover rate of cholesterol in the brain

is still disproportionately low.

25

Dietschy and Turley (2001)12 stated the cholesterol that is incorporated into

the plasma membranes of neurons and glia is produced de novo within the

brain and its level is tightly controlled. Even in models of peripheral nerve

regeneration, the cholesterol utilized for the regeneration of damaged sciatic

nerves is synthesized locally. Jurevics et al., (1998)14 identified there is a

direct correlation between the rate of cholesterol synthesis and the

accumulation of cholesterol within the regenerating nerve and Jurevics and

Morell(1994)15 reported this synthesis is independent of dietary cholesterol.

Similar to damaged sciatic nerves, exogenous cholesterol has little to no effect

on the brain. Jurevics and Morell (1995)16 detailed that isotopically labelled

exogenous cholesterol does not enter the brain during development, and the

accumulation of sterols during this time closely correlates with their synthesis.

Levels of myelin basic protein and cerebroside also correlate with cholesterol

synthesis and accumulation, suggesting a sophisticated level of coordination

of mechanisms involved in lipid and protein formation during myelination,

remyelination and axonal outgrowth.

Regulation of Brain Cholesterol Levels: Edmond et al.,(1991)17 specified

cholesterol levels in the brain are tightly regulated, and even during

hypercholesterolemic conditions when plasma cholesterol levels are high,

brain cholesterol levels are not altered, unlike peripheral tissues which often

display lipid accumulation. Cholesterol in the periphery is typically transported

via lipoproteins and is endocytosed by cells upon binding to LDLR. There is no

strong evidence to support that this type of transport occurs across the blood

26

brain barrier. Turley et al.,(1996)18 reported mice lacking LDLR show no

difference in brain cholesterol levels and studies in sheep show no detectable

uptake of LDL from the periphery to the brain. There also seem to be few

molecules that are rate-limiting and capable of significantly altering cholesterol

levels in the brain, apart from perhaps a complete inhibition of HMG-CoA

reductase. Various knockout mice lacking specific proteins that play vital roles

in cholesterol metabolism in the periphery (ABCA1, ApoE, ApoA1, LDLR, and

SRB1, Cyp46a) show little change in overall cholesterol levels within the brain.

However, the absence of certain molecules can lead to changes in cholesterol

synthesis rates. For instance, mice lacking the cytochrome p450 Cyp46a, also

known as cholesterol 24-hydroxylase, show a reduction in synthesis. Lund et

al., (2003)19 stated Cyp46a is almost exclusively found in the brain and

produces the primary metabolite of brain cholesterol, 24(S)-hydroxycholesterol

(24-OH-Chol), an oxysterol. Thus, Cyp46a is involved directly in cholesterol

turnover. Reductions in cholesterol breakdown (e.g., with inhibition of Cyp46a)

are accompanied by reductions in brain cholesterol synthesis and, therefore,

Quan et al., (2003)20 cholesterol levels are maintained. Together, these

findings suggest a series of finely regulated feedback pathways controlling

overall cholesterol levels in the CNS.

Interestingly though, mice lacking the Niemann-Pick disease, Type C1 (Npc1)

gene show significant decreases in CNS cholesterol synthesis as well as

overall concentrations within tissues. Ikonen and Hölttä-Vuori(2004)21 detailed

people with Niemann-Pick disease Type C1 have lower levels of the protein

27

leading to a decreased ability to process cholesterol as it is endocytosed,

leading to endosomal accumulation of cholesterol. NPC1 is associated with

progressive neurodegeneration ending in adolescent mortality. Histological

examination shows an accumulation of cholesterol and other lipids in late

endosomes of peripheral tissues, whereas in the brain, Takikita et al., (2004)22

informed cholesterol accumulation is absent while demyelination and neuronal

death are both observed.

Removal of Excess Brain Cholesterol: Dietschy (2009)10 detailed yet

cholesterol levels can become excessive in the brain. Free cholesterol, if

allowed to accumulate, is toxic to cells because of its amphipathic nature.

Unlike other tissues, the mechanism by which cholesterol is removed in the

brain differs somewhat because of the presence of the blood brain barrier

(BBB). As briefly discussed above, much of the excess cholesterol must first

be converted to an oxysterol by Cyp46 before it is able to exit through the

BBB. Vance et al., (2005)23 stated this enzyme is highly expressed in the brain

relative to other regions of the body with higher levels of expression in the

cerebrum and specifically in neurons. Vance et al., (2005)23 described that

much of Cyp46’s synthesis occurs early in life during the formation of axons

and their myelination. Given the high level of Cyp46 expression in the brain, it

would stand to reason that the amount of 24(S)-hydroxycholesterol (24-OH-

Chol), the oxysterol metabolite of the enzyme’s activity, would be higher in the

brain than any other tissue of the body. This is in fact the case with the

absolute concentration of this metabolite observed to be 3.8-4.8 ng/mg in the

28

cerebellum and 8.6-15.1 ng/mg in the cerebrum. Lutjohann et al., (1996)24

indicated that this accounts for 80% of the body’s total pool of 24-OH-Chol.

The 24-OH-Chol has a flux rate of 6.4 ±1.8 mg/24hrs across the brain.

Bjorkhem et al., (1998)25 mirrors the rate of uptake of this metabolite by the

liver at 7.6 mg/24 hours, again confirming that production of this particular

metabolite takes place predominantly in the brain. Vaya & Schipper (2007)26

reported cholesterol can also be removed to a much lesser extent by

apolipoproteins that are transported to the CSF. The transport and efflux of

cholesterol is discussed further below.

Meaney et al.,(2007)27 stated that 24-OH-Chol mostly moves unidirectionally

from the brain to the plasma, other oxysterols enter into the brain from

circulating plasma, perhaps acting as signaling molecules from the periphery.

27-OH-Chol is one oxysterol able to flux across the BBB from the plasma and

is later metabolized to 7α-hydroxyl-3-oxo-4 cholestenoic acid, which is then

fluxed back out of the brain. Given that 27-OH-Chol does not remain in the

brain, but is in fact metabolized for removal may suggest that the brain is able

to utilize the molecule to sense peripheral levels of oxysterols.

Wang et al., (2008)28 declared that 24-OH-Chol itself plays a vital role in the

cholesterol homeostasis of neurons in that high levels of this particular

metabolite can actually attenuate the expression of enzymes critical in the

production of cholesterol by activating the liver x receptor (LXR), for which it is

a ligand. A more detailed discussion of the role of LXR will follow, but briefly

LXR activation leads to the expression of a whole host of genes, involved in

29

reverse cholesterol efflux as well as enzymes that participate in various

aspects of lipid metabolism.

Cholesterol Transport in the CNS: Goldstein and Brown (2009)29 reported

most of what is known about cholesterol transport comes from studies in the

periphery. As briefly reviewed above, peripheral transport of cholesterol

involves the movement of cholesterol along with other lipid species within

lipoprotein particles like high-density lipoproteins (HDL), very low density

lipoproteins (VLDL) and low-density lipoproteins (LDL). Various

apolipoproteins associate with these lipoproteins that carry cholesterol from

the GI to peripheral tissues or from peripheral tissues to the liver in a process

known as reverse cholesterol transport, referring to the movement of

cholesterol from peripheral tissues to the liver. These

lipoprotein/apolipoprotein complexes associate with receptors in the large

LDLR family allowing for the endocytosis of the lipid molecules in the

respective tissue. Rebeck et al., (2006)30 detailed cholesterol transport in the

brain, though not dependent on outside sources, still occurs and utilizes

similar mechanisms to move cholesterol and other lipids throughout the CNS.

ApoE along with ApoA1 are thought to be the major apolipoproteins

expressed in the CNS and are contained within HDL-like lipoproteins. La Du et

al., (1998)31 identified that nascent lipoproteins are produced by astrocytes,

are discoidal in shape and are generally lipid poor when first released from the

cell indicating their readiness to collect cholesterol from other tissues.

30

McColl et al., (2007)32 reported ApoE is the most studied CNS apolipoprotein

due to its role in Alzheimer’s disease as well as stroke and ischemic events.

Rebeck et al., (1998)33 stated there are three different ApoE isoforms found in

the human population, differing only in presence or absence of one or two

amino acids (ApoE 2: Cys-112, 158; ApoE 3: Cys-112, Arg-158; ApoE 4: Arg-

112, Arg-158) at specific locations. Rebeck et al., (2006)34 identifies that CNS

highly expresses ApoE and several different LDLR family members are found

on the surface of both neurons and astrocytes ApoE will bind to LDLR, VLDR,

low density-related protein (LRP) and apolipoprotein receptor 2 (APOER2).

There is evidence that cholesterol may be recycled, as well, under conditions

of damage by being gathered by apolipoproteins and redistributed to cellular

membranes. Dietschy and Turley (2001)35 identified the large number of

lipoprotein receptors in the brain, most likely allow for a redundancy in the

recycling pathway in case of an interruption, and facilitates scavenging of free

cholesterol from the intercellular space.

Rebeck et al., (1998)33 reported the HDL-like lipoproteins, secreted from

astrocytes, are in a different state from those found in the CSF, suggesting

that perhaps the HDL-like particles derived from astrocytes may act to shuttle

cholesterol within the CNS. Boyles et al., (1985)36 indicated ApoE is physically

associated with astrocytes, especially those below the pia mater and along

large blood vessels and less so with neurons and microglia. Mauch et al.,

(2001)37 detailed the formation of synapses and neurite outgrowth are

promoted by ApoEassociated cholesterol from surrounding astrocytes. de

31

Chaves et al., (1997)38 conveyed the utilization of ApoE molecules may help

to explain how axons of sympathetic neurons that do not synthesize

cholesterol themselves, are able to incorporate cholesterol within their

membranes, especially during periods of elongation.

As indicated above, axonal growth and elongation are dependent on the

synthesis and the proper transport and allocation of cholesterol. Pfrieger

(2003a)39 stated in cultured neurons, blocking synthesis via an HMG-CoA

reductase inhibitor stopped axonal elongation after only 2.7 days. This growth

was rescued though with the addition of either mevalonic acid or an

exogenous source of cholesterol, namely lipidated lipoproteins. But neurons in

culture are in early stages of development relatively speaking, and mature

neurons in vivo act much differently and are more intimately associated with

surrounding glia. Pfrieger (2003b)40 suggested that neurons, in a proposed

attempt to conserve energy, outsources cholesterol synthesis to astrocytes.

Bjorkhem and Meaney (2004)41 reported the amount of cholesterol needed

later in life is not as great as during development at which time a neuron

supplies most of its own cholesterol. Pfrieger (2003a)39 detailed as the

neurons mature, the rate of synthesis drops and the axon terminus would be

more dependent on cholesterol supplied from neighboring astrocytes. The

needed cholesterol would be most likely delivered by apolipoproteins like

ApoE.

32

Disruption of Cholesterol Synthesis - CNS and Periphery: The rate-

limiting step in the production of cholesterol is the enzymatic activity of HMG-

CoA reductase. The enzyme is regulated by cholesterol itself, with high levels

triggering mechanisms that suppress synthesis and reduce cholesterol levels.

Corsini et al., (1995)42 reported that feedback is dependent on the products of

the mevalonic pathways as opposed to actual regulation by cholesterol itself.

Dietschy (2009)10 specified these products lead to actions resulting in the

binding of HMG-CoA reductase to insulin induced gene 1 (INSIG1) and

INSIG2 leading to its degradation, reduction of sterol regulatory element-

binding protein (SREBP) activity, and revving up LXR activation which in turn

increases the production of bile acids, the last step in reverse cholesterol

transport.

Cholesterol synthesis can be pharmacologically inhibited by blocking HMG-

CoA reductase activity, typically with the use of a class of drugs collectively

called statins. Endo et al., (1976)43 reported that statins were originally derived

from fungal metabolites that competitively inhibit HMG-CoA reductase. Corsini

et al., (1995)42 specified the earliest statins, compactin (mevastatin) and

mevinolin (lovastatin) have Ki of 10-9M, indicating an affinity to the enzyme in

the nanomolar range, which is 104-105 times greater than the affinity of the

endogenous substrate (HMG-CoA) which binds at the 30 uM range. Schachter

(2005)44 informed the statin inhibits the enzyme by mimicking HMG-CoA via

its lactone segment and blocking enzyme activity by directly binding within the

substrate binding pocket.

33

Endo (1988)45 described these early statins were able to lower serum

cholesterol levels in a number of mammals. Surprisingly, the statins are

unable to reduce serum cholesterol levels in rats which should be noted given

the data that will be presented below. Goldstein and Brown (1990)46 stated to

compensate for the potent inhibition of HMG-CoA reductase, there is a 200

fold increase in reductase protein within a few hours of exposure. Long-term

effects of statins in rodents have not been thoroughly elucidated. Statin use

increases the expression of LDLR on hepatocytes leading to a reduction in

circulating LDL levels. They also increase HDL and decrease triglycerides.

The original targets for statins were peripheral, in that they were intended to

block cholesterol synthesis in the liver, which then increases the clearance of

LDL from the plasma and thus act to help prevent atherosclerosis.

Given the importance of cholesterol in the brain and the role that statins play

in reducing synthesis and quantities in the periphery, it is no wonder that there

were questions to whether statins could in fact get across the BBB and

whether or not the presence of the compound would confer an effect on the

CNS. The effects of specific statins on the CNS are potentially dependent on

their unique chemical structures which in turn determine their relative

lipophilicity. Two commonly prescribed statins that are taken in two distinct

forms are simvastatin and atorvastatin. The maximal dose for both in humans

for LDL reduction is 80 mg/day. Wood (1999)47 stated that simvastatin is

thought to be brain penetrant whereas atorvastatin is not. The difference in

lipophilicity is due in part to the chemical attributes of different functional

34

groups of the respective drugs. Schachter (2005)44 told simvastatin, a

modified version of lovastatin, which is a direct fungal derivative, is delivered

in its lactone form, whereas atorvastatin, a completely synthetic statin, is

delivered in its acid form. Both are metabolized by CYP 3A4 and have a rather

low bioavailability at 5 and 12%, respectively, when taken orally. Tubic-

Grozdanis et al., (2008)48 detailed that lactone form of simvastatin must first

be hydrolyzed into its acid form, before it can actively inhibit HMG-CoA

reductase, by a non-specific carboxyesterase in a reversible reaction.

Schachter (2005)44 stated the lactone form of simvastatin has been found in

circulating plasma and is three times more lipophilic than its acid form.

Lennernas (2003)49 indicated atorvastatin’s bioavailability, already rather low,

can actually be reduced further when taken with food. Corsini et al., (1999)50

stated the Cmax (the maximum concentration of the drug in plasma after its

administration) for Simvastatin and Atorvastatin are 10-34 ng/ml and 27-66

ng/ml respectively.

Studies using BBB models utilizing bovine brain capillary endothelial cells

(BCEC) found that the lactone form of simvastatin and lovastatin are much

more permeable than the their acid forms. Simvastatin had a permeability

coefficient of 4.76 in the lactone form, whereas it only showed a coefficient of

0.193 in the acid form. Saheki et al.,(1994)51 reported the acid form is able to

cross the BCEC model though, and appeared to do so in a passive from, most

likely via a monocarboxylic acid carrier that requires no energy. Given that

simvastatin is taken in the lactone form, and the lactone form is found within

35

the plasma, it would stand to reason that simvastatin would be more likely to

enter the CNS than would a statin that is delivered in an acid form and is

generally less lipophilic.

There is evidence that statins are able to reduce cholesterol synthesis in the

brain, but very little to indicate that such a reduction does anything to overall

cholesterol levels in the CNS. Selley (2005)52 stated that in a rather short-term

study, 10 days of treatment in mice with simvastatin was unable to reduce

cholesterol levels in the striatum as measured with GC-MS. Yet, longer

studies also fail to show any real effect on cholesterol levels. After four weeks

of high-dose treatment with both pravastatin and simvastatin, two distinctly

different statins in regards to their lipophilicity, cholesterol synthesis was be

reduced in the brains of guinea pigs, but there was not a significant effect on

cholesterol levels. Lutjohann et al., (2004)53 affirmed that pravastatin is

considered hydrophilic and is thought to be unable to pass through the BBB,

yet it still had an effect on cholesterol synthesis despite a perceived inability to

pass through the BBB. Mok et al.,(2006)54 reported effect of statins on

cholesterol synthesis in the brain was corroborated in a later study in which

very high doses of simvastatin (100 mg/kg) given to mice for approximately

one month again showed no effect on cholesterol levels or the major

cholesterol metabolite 24S-OH-CHOL in whole brain homogenates, but

again, decreased cholesterol synthesis.

36

Though reports in the current literature of pharmacologically reducing

cholesterol levels in the CNS are rare, benefits of using drugs that target

cholesterol synthesis within the brain may have benefits independent of

grossly reducing overall levels, and there is ample evidence that statins are

able to confer positive effects in the CNS. In several studies involving

traumatic brain injury (TBI), statins were found to be beneficial in reducing

TBI-associated deficits. Wang et al., (2007)55 declared both simvastatin and

atorvastatin are able to improve performance on the rota rod paradigm after

TBI as well as reduce hippocampal degeneration and improve cerebral blood

flow. Lu et al.,(2007)56 affirmed atorvastatin and simvastatin are also able to

improve performance on the Morris water maze in rodents. In another study

with a shorter term of treatment, simvastatin improved rota rod performance in

TBI animals as well as lowered mRNA levels of toll-like receptor 4 (TLR4),

nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB),

interleukin 1β (IL-1β), tumor necrosis factor –alpha (TNFα), interleukin-6 (IL6)

and ICAM around the area of injury, concurrent with an overall reduction in the

NF-κB activity. Li et al., (2009)57 reported that statins are able to ameliorate

potentially harmful immune reactions, and the role of statins as

immunomodulators is well documented. Lu et al., (2007)56 stated benefits go

beyond reducing the deleterious effects of inflammation in TBI, statins may

also be able to increase neurogenesis and reduce neuronal death following

TBI.

37

Not all observed statin-induced effects in the brain are positive or consistent.

There are several studies that indicate that statins may actually be deleterious

to the CNS. Klopfleisch and colleagues (2008)58 found that inhibition of

cholesterol synthesis in mature oligodendrocytes in vitro can actually cause

the retraction of processes due to the disruption of Ras and Rho signaling that

is dependent on the isoprenoids produced upstream of cholesterol in the

mevalonic pathway. These metabolites are essential for the proper targeting

of Ras and Rho to the membrane, which are integral in process elongation.

Klopfleisch et al.,(2008)58 reported this disruption in normal oligodendrocyte

functioning also delays remyelination after cuprizone treatment in vivo. The

effect seen on oligodendrocyte process retraction in this study depended on

the lipophilicity of the statin, in that lovastatin induced process retraction

whereas pravastatin, a much less lipophilic statin was unable to do so. de

Chaves et al., (1997)38 contradicts this when it was reported that the

hydrophilic pravastatin retarded neurite growth of axons in neuronal culture.

Miron et al.,(2007)59 confirmed in both instances though the retraction could

be rescued with co-treatment of mevalonic acid or with the isoprenoid

analogues farnesol and geranyl-geraniol, but not with cholesterol, confirming

that the effect in both was most likely due to a lack of prenylation capability as

opposed to an overall change in lipid dynamics of the membrane. Liao and

Laufs (2005)60 detailed isoprenoids like farnesyl pyrophosphate and geranyl

pyrophosphate are needed for the prenylation for a whole host of small

GTPases. These include Rho and Ras, which without proper prenylation

38

accumulate in the cytoplasm. Cijiang He et al., (2006)61 stated these GTPases

play major roles in neuron growth and elongation. Therefore, it is not clear

how disruption of cholesterol synthesis would affect myelination. Firstly, the

retraction of oligo dendrocytic processes via inhibition of the synthesis of

isoprenoids can inhibit proper myelination of axons. Secondly, the inhibition of

neural outgrowth via the same mechanism would reduce the number of axons

available to myelinate. Thirdly, reduction of cholesterol itself would reduce

materials needed to actually myelinate axons.

The effects of potentially induced cholesterol depletion in the brain should not

be completely dismissed. Changes in cholesterol dynamics of the plasma

membrane can modulate the activity of signaling molecules found within the

lipid bilayer, specifically those associated with cholesterol rich lipid rafts. For

instance, simvastatin protected against NMDA-induced neuronal excitotoxicity

by reducing the number of NMDA receptors that are actually associated with

lipid rafts without actually modulating the number of total receptors in a

membrane fraction. Ponce et al., (2008)62 stated isoprenoids do not seem to

be involved, and the change in the fluid dynamics of the lipid rafts may affect

the efficiency of NMDA receptor activity. Another study Wang et al., (2009)63

suggested, but failed to provide strong mechanistic support, that chronic

simvastatin treatment could actually act like an NMDA receptor antagonist as

well.

39

Cholesterol and the Aging Brain:

Wood et al., (2002)64 stated cholesterol in the CNS is not static, and is subject

to regular turnover; therefore dysregulation of cholesterol metabolism could

potentially confer detrimental effects on normal neuronal function and

participate in cognitive decline. The lipid bilayer is normally asymmetrical in its

distribution of various phospholipids and cholesterol, which contributes to the

fluidity of exofacial and cytofacial leaflets of the membrane. Igbavboa et al.,

(1996)65 reported in mice, the exofacial leaflet gains cholesterol over its

lifetime and the leaflets become less asymmetrical, which leads to a loss in

fluidity. The decrease in fluidity and the increase in cholesterol in the outer

leaflet of the cell membrane could be due to a decreased capacity to efflux

cholesterol. Igbavboa et al., (1997)66 confirmed in ApoE knockout mice, the

exofacial leaflet is much more concentrated in cholesterol than age-matched

animals and as mice age, Masliah et al., (1996)67 affirmed brain ApoE

expression actually increases, suggesting that perhaps the brain is

compensating for the disproportionate amount of cholesterol in the exofacial

leaflet.

Lutjohann et al., (1996)68 declared an age-dependent effect on the level of 24-

OH-Chol exists in blood plasma. The levels appear to decrease over the

lifetime of an individual, comparing the first decade of life to the sixth. This is

most likely attributed to the fact that synthesis at earlier stages of development

is much greater due to the need for cholesterol during axonal growth. When

myelin production decreases as neural networks are established, the need for

40

cholesterol is not as great, and therefore synthesis, and the process by which

cholesterol is metabolized can be reduced. Thelen et al., (2006)69 reported

that in fact, 24-OH-Chol levels have been reported to decrease in the

hippocampal regions of elderly subjects, leading the researchers to conclude

that cholesterol synthesis is decreasing, while overall cholesterol levels in the

brain remain stable. This suggests that cholesterol turnover is reduced in

parallel with the reduction in synthesis, very similar to the phenomenon seen

in Cyp46a KO mice which show a reduction in cholesterol synthesis as a way

to compensate for the absence of the Cyp46a, which is mainly responsible for

the metabolism of cholesterol to 24-OH-Chol.

Evidence suggests that a metabolic shift in cholesterol metabolism takes

place at mid-life. Studies carried out in rats have shown that a whole host of

genes begin to be expressed differently as a part of normal aging starting at

mid-age, marking a shift in expression patterns. Kadish et al., (2009)70

reported a significantly disproportionate number of which are involved in

cholesterol metabolism. Kadish et al., (2009)70 stated several of these genes

are positively correlated with cognitive impairment including ApoE and the

sterol regulatory element-binding transcription factor (Srebf1).

Bartzokis et al., (2004)71 reported that cholesterol is involved in determining

the permeability characteristics of cellular membranes, and this point is vitally

important when considering the role membrane potentials play in neuronal

signaling. Myelin has a greater amount of cholesterol than most of the plasma

membrane and it plays a key role in lowering the capacitance of axons,

41

allowing for rapid salutatory conduction. Pakkenberg et al., (2003)72 affirmed

as the brain ages, the extent of myelination decreases with respect to the total

length of myelinated fiber in the neocortex, which is reduced by 40-50 %.

Thus, dysregulation in cholesterol metabolism over the lifespan could impact

myelin, compromising its integrity, and promoting degeneration and related

pathologies. Bartzokis et al., (2004)71 accounted in fact, those axons

myelinated later in life are thinner and less thickly sheathed in myelin, and

these regions of the brain are those first affected by AD pathologies. Bartzokis

et al., (2001)73 stated the demyelination and abnormalities in white matter

have been implicated in both brain aging and Alzheimer’s disease as well.

One must also not exclude the role vascular events may play in cognitive

decline, even though these may be beyond the scope for this work,

cholesterol levels are major risk factors in the development of atherosclerosis,

and subsequently the possibility of having a heart attack or stroke. Allen and

Bayraktutan (2008)74 stated the risk for developing atherosclerosis and

suffering from a stroke increases with age, as vasculature accumulates lipid-

laden plaques. The brain itself is extremely vascularized, and thus cholesterol

dysregulation seen in aging vasculature is potentially mirrored in the brain.

Thomas et al., (2002)75 reported occlusion of small capillary beds throughout

the brain would result in small areas of hypoxia, which can lead to cell death in

areas deprived of normal blood flow. These “mini-strokes” over time can be

visualized as hyperintensities on MRI brain scans, and over time the

recurrence of these events may lead to an underappreciated level of damage

42

with serious cognitive effects. Many see these hyperintensities as part of

normal aging, but the effects of these are not well studied.

The Hippocampus and Memory:

Scoville and Milner (1957)76 stated that one of the most studied individuals in

science was a man known by the initials HM. In order to attenuate debilitating

intractable seizures in the 1950s, HM underwent surgery to remove a

posterior portion of the temporal lobe, which in turn destroyed approximately

two-thirds of his hippocampus after which he was unable to form any new

memories for the remainder of his life. deToledo-Morrell et al., (1988)77

reported without the hippocampus, we humans along with the majority of our

mammalian relatives are unable to acquire new memories or engage in spatial

memory. As in the case of HM, older memories acquired before a lesion are

undisturbed, negating the notion that the hippocampus is where memories are

archived, but rather where memories are reinforced and acquired, as will be

discussed below, most likely through the strengthening of synaptic

connections. As we age or develop certain neurodegenerative diseases, our

ability to acquire new memories becomes impaired. DeToledo-Morrell et al.,

(1988)77 affirmed that it is no surprise then that loss of synaptic plasticity is the

hippocampus during aging is highly associated with this impairment and is

very similar to deficits seen with hippocampal ablation. Animal models for both

aging and Alzheimer’s disease that attempt to replicate this phenomenon of

age-related cognitive decline are discussed in more detail below.

43

Alzheimer’s Disease- Progression and Treatment:

Sanders and Morano (2008)78 confirmed that Alzheimer’s disease is the most

common form of dementia and was first described by Dr. Alois Alzheimer in

the early part of the 20th century. It is a progressive neurodegenerative

disease considered to be fatal. The most prominent symptom early in the

disease is the loss of episodic or declarative memory with latter symptoms

including problems speaking, loss of executive functions, and changes in

personality and behaviour. Because of the rate at which the population is

aging along with the duration of the disease, AD is becoming a major global

health concern. AD exerts a considerable burden on the healthcare industry

as well as on the financial stability of the nations of the world with the cost of

treating and caring for AD patients costing over 100 billion dollars a year.

Most cases of AD are sporadic with the majority of cases occurring after the

age of sixty-five. Age is the greatest risk factor for acquiring AD, with further

increased risk in older individuals that carry the ApoE4 allele. Shepherd et al.,

(2009)79 reported that familial forms of early-onset AD do exist and are

typically caused by the presence of an autosomal dominant mutations in the

amyloid precursor protein (APP), presenilin 1 (PSEN1) or presenilin 2

(PSEN2) genes all of which increase the production of amyloid beta (Aβ).

AD is associated with two specific pathological hallmarks, often referred to as

plaques and tangles, both of which must be present for proper post-mortem

diagnosis. Davis et al.,(1999)80 reported the symptoms of the disease tend to

44

follow the pathological changes that occur in the brain as the disease

progresses, which include the formation of these senile plaques and

neurofibrillary tangles, leading to cell death throughout various brain regions.

Dickson (1997)81 declared that these senile plaques are composed of deposits

of amyloid beta peptides, activated glia associated with increased

inflammatory responses and degenerating neurons. Shepherd et al., (2009)79

avowed neurofibrillary tangles are composed of hyper phosphorylated tau

proteins (tau being the protein that makes up the microtubules of the

cytoskeleton) which form paired helical filaments. Nelson et al., (2009)82

detailed human pathology is correlated with the progression of observed

cognitive deficits, as seen when braak staging is compared with scores on the

Mini-Mental State Examination. Davis et al., (1999)80 stated it is important to

note that there is evidence of gross pathology in the absence of cognitive

deficits.

Some of the first noticeable symptoms of AD are the effects seen in short-term

memory or the acquisition of new memories. This is often referred to as

forgetfulness or general confusion about the placement of objects, the recall of

words, or recollection of events occurring only minutes to hours before. Smith

(2002)83 reported one of the first areas of the brain affected by AD is the

hippocampus with symptoms mirroring the functions of those regions

sequentially affected by AD’s pathological progression. As the disease

progresses and more regions begin to atrophy, the severity of the symptoms

increase until one is completely demented and is only a mere shell of who

45

they used to be. Burns and Iliffe (2009)84 stated the progression from mild-

memory loss at early stages of the disease, often referred to as mild-cognitive

impairment, to the final stages of the disease can span nearly a decade.

Scarpini et al., (2003)85 affirmed there are very few FDA-approved treatments,

the most prominent of which attempt to increase the availability of the

neurotransmitter acetylcholine or reduce excite toxicity by antagonizing NMDA

receptors. Such drugs do not target the cause of the disease, and therefore

will always lack in their long-term efficacy. The development of therapeutics

for AD has proven challenging because the actual cause of the disease

remains elusive and with every hypothesized cause there is a corresponding

targeted therapeutic. Given that the most quoted hypothesis for the cause of

AD is the amyloid cascade hypothesis, it is no wonder that a great many drugs

under development target either directly or indirectly by targeting those

proteins involved in its processing. Hull et al., (2006)86 reported there are other

therapeutics under investigation including NSAIDs, fish oil, antioxidants and

various currently used drugs that may confer pleiotropic effects that may

prevent the onset or at least attenuate the progression of AD.

AD and Role of Cholesterol:

One of the major risk factors for Alzheimer’s disease, besides age, is carrying

the ApoE epsilon 4 allele (ApoE 4). Wolozin (2004)87 reported that ApoE is a

vital component in the movement of cholesterol within the brain. Transgenic

46

mice (PDAPP) that express the human ApoE 4 gene have increased levels of

A 42 at an earlier age and greater deposition of ApoA later in life. At the same

time, these animals show reduced levels of ApoE in the CSF. Bales et al.,

(2009)88 suggested the possibility that ApoE4 carriers have reduced ApoE

levels in the CSF and that increasing levels of ApoE may help reduce which in

a lipidated state aids in its degradation. Refolo et al., (2000)89 also

hypothesized that ApoE4 contributes to poor transport of cholesterol in the

brain and, thus, reduced capability of lowering levels.

Lesser et al., (2009)90 described individuals with AD pathology have higher

serum levels of total cholesterol as well as LDL. Epidemiological data has

shown that statins as well as other lipid lowering drugs may reduce the risk of

developing dementia [Jick et al., 200091; Wolozin et al., 200092; Rockwood et

al., 200293; Rodriguez et al., 200294; Sparks et al., 200695; Wolozin et al.,

200796; Sparks et al., 200897; Haag et al., 200998], however there are

instances where statins were reported to have no effect [Rea et al., 200599;

Arvanitakis et al., 2008100]. Papassotiropoulos et al., (2000)101 found that AD

patients have a significantly higher level of 24-OH-Chol in their plasma than

healthy volunteers suggestive of a dysregulation in cholesterol metabolism in

the AD brain. Abildayeva et al., (2006)102 conveyed the elevated 24-OH-Chol

may provide a biomarker of early stage AD only, given that the levels of 24-

OH-Chol are reduced in late-stage AD patients because fewer neurons are

capable of catabolizing cholesterol.

47

Interestingly, it would not appear that it is as simple as “high cholesterol is

bad, low cholesterol is good” in the context of predicting cognitive functioning

later in life. Van den Kommer et al., (2009)103 detailed in the elderly (defined

as patients over 65), low serum cholesterol levels are actually negatively

associated with cognition, and when coupled with carrying ApoE 4, the rate of

cognitive decline is actually increased. Solomon et al., (2009)104 informed, at

midlife, high serum cholesterol actually increases the risk of AD and vascular

dementia later in life, which creates a bit of a conundrum as far as trying to

determine what therapy to provide as well as how long to provide.

Amyloid precursor protein (APP) is the major protein implicated in AD

pathology. Its aberrant processing, folding, and aggregation is what some

believe is the culprit behind the development of the disease. Cholesterol plays

a part in the processing of this particular protein. Pappolla et al., (2003)105

identified animals that are fed a diet containing high levels of cholesterol tend

to develop more AD pathology. Refolo et al., (2000)89 informed

hypercholesterolemia may actually lead to cholesterol enrichment of micro

domains within the cell membrane, favouring APP cleavage, and subsequent

increased APP production via γ-secretases located within these domains. A

popular hypothesis explaining the effects of cholesterol and APP processing

considers the important role cholesterol plays in membrane fluidity. APP can

be potentially processed by three different secretases, α, β, and γ. The

sequence in which these proteases act on the APP determines whether or not

the toxic form of Aβ is formed. Alpha secretase cleavage forms the APPsα

48

fragment and precludes Aβ formation. Wolozin (2001)106 thought that high

levels of cholesterol would favour the cleavage of the APP by the beta and

gamma secretases, which would produce the Aβ40 or Aβ42 peptides, which

are thought to be responsible for AD pathology.

One mustn’t forget that the most common risk factor for acquiring AD is age.

As mice age, the lipid dynamics of neuronal membranes change and the

fluidity of the exofacial layer of the membrane decreases. Rojo et al., (2006)107

detailed this is most likely attributable to the twofold increase in cholesterol

levels within the exofacial layer of the membrane. Refolo et al., (2000)89

described high cholesterol can accelerate AD pathology in a transgenic

mouse model and its depletion can inhibit Aβ production. Papassotiropoulos et

al., (2003)108 testified polymorphisms in the Cyp46 gene, which encodes the

enzyme responsible for the majority of cholesterol turnover in the brain are

also associated with greater levels of Aβ accumulation in human brain tissue

and an increase risk of developing AD. Schneider et al., (2006)109 suggested

that by lowering cholesterol or impeding its synthesis one could reduce Aβ

levels and reduce the risk of AD. Both of these have been shown.

AD &Treatment Strategies:

It is currently possible to decrease the symptoms of AD, and improve the pa-

tients’ quality of life, within a few weeks. However, the majority of treatments is

given first when the disease has progressed to the point of diagnostic

49

certainty, and is purely symptomatic anti-dementia drugs without profound

disease-modifying effects. Treatments with cholinesterase inhibitors may

delay the breakdown of the neurotransmitter acetylcholine in the cortex and,

thus, aid the preservation of memory function, decrease the degeneration of

cognitive symptoms and reduce behavioural problems, whereas an alternative

treatment for moderate to severe AD use a NMDA (N-methylD-aspartate)

antagonist acting on glutamate, to reduce neuronal death.

Recent research on AD has focused on the possibilities to affect the pro-

gression of the disease using prevention strategies and modulations of

specific aspects of the AD pathology, such as; APP processing and secretase

inhibition; Aβ-aggregation inhibition; vaccination studies; and immunotherapy

[Ohno, 2006110; Na et al., 2007111;Chauhan and Siegel, 2007112; Nikolic et al.,

2007113]. The association between AD and inflammation has been examined

using NSAIDs. The Rotterdam study suggested that NSAIDs can delay, or

slow, the progression of AD with a protective effect up to 80%, but reports

from other trials have yielded inconclusive results [Akiyama et al., 2000114;

Aisen et al., 2003115; McGeer and McGeer, 2007116].

Solomon et al., (2007)117 reported that the connection between AD, diet,

cholesterol and lifestyle factors has been examined in a number of

investigations. The results have been unanimous in support of a strong

association, which implies that relatively simple modifications could be an

effective treatment. Sparks et al., (2006)118 specified cholesterol-lowering

therapies, with a focus on statins have, in particular, attracted rigorous atten-

50

tion. The outcome has been debated, but contradictory results may in part be

due to the different properties of the statins working in conjunction with other

factors, such as the severity of the disease and the ApoE phenotype.

The majority of all AD patients are diagnosed very late in life, and it could be

an acceptable solution for an increasingly aging population, if possible, to

simply delay the symptoms. Pasinetti et al., (2007)119 hypothesized that if the

onset of the disease could be delayed by just 5 years, the incidence would be

cut by 50%.

Cholesterol-lowering therapy:

Sparks et al., (2006)118 described the association between cholesterol-

lowering therapy and dementia has been supported by numerous studies,

including the observation that animals undergoing accelerated aging have

improved memory when they are fed a diet that lowers their cholesterol.

These findings suggest that memory and cognitive functions during aging

could benefit from a cholesterol-lowering therapy.

Statins:

Endo et al., (1976)120 detailed that statins were first introduced into clinical

practice in the late 1980s after the accidental discovery of their lipid-lowering

effects in 1976. Brown and Goldstein (1981)121 informed that the effect is

51

exerted by an up-regulated expression of the LDL-receptor in the liver, which

clear LDL-cholesterol and its precursors from the circulation. This effect is in

turn caused by the competitive inhibition of the HMG-CoA reductase, the rate-

limiting step early in the synthesis of cholesterol and many other non-steroidal

isoprenoid compounds.

The first statins were produced from fungal metabolites, but the majority is

now produced synthetically. Accordingly, the traditional method differentiates

the two types and classifies them as either natural (fungal metabolites) or

synthetic. Another classification defines the statins by their solubility: i.e.

hydrophilic (rosuvastatin, pravastatin, fluvastatin to some extent) or lipophilic

(atorvastatin, simvastatin, lovastatin, cervistatin). Simonson (2004)122 detailed

that the lipid bi-layers of the cell membranes are easily penetrated by lipophilic

compounds, whereas the uptake of hydrophilic compounds needs to be

facilitated by organic anion transporters. Kubota et al., (2004)123 indicated the

lipophilic statins may cross the BBB and have been demonstrated to cause

apoptosis in a variety of cells, whereas the hydrophilic statins do not. With this

in mind, the question have been raised if not hydrophilic statins should to be

recommended for treatment purposes, particularly as it has been demon-

strated that hydrophilic statins exert effects on the CNS despite their inability

to cross the BBB.

Clinical studies on statins have produced some promising but contradictory

results. Winblad et al., (2007)124 conveyed statins failed to prove a significant

association with maintenance of cognitive function in patients with AD,

52

whereas, on the other hand, treatment with statins has been significantly

associated with a decreased prevalence of AD. Xiu et al., (2006)125 specified

statins have also been found to significantly increase the α-secretase activity,

and decrease the levels of both Aβ1-42and Aβ1-40. Simons et al., (2002)126

reported that interest is the notion that decreased levels of Aβ1-40 in the CSF

were restricted to patients with mild AD, as no effect was observed in patients

with more severe forms of AD.

Hoglund and Blennow (2007)127 stated that majority of clinical and

experimental evidence suggests that statins are beneficial in Alzheimer’s

disease, there is a discrepancy between the effect of statins on AD

pathophysiology in experimental and clinical trials. The best known

pathophysiological features of AD are the extracellular deposition of amyloid-b

peptide and the intracellular accumulation and deposition of hyper

phosphorylated tau-protein. The formation of Ab is increased by high levels of

cholesterol, and the e4 allele of the gene that produces cholesterol transporter

Apolipoprotein E is a known risk factor for AD. Hence it was proposed that

statins are beneficial in AD by inhibiting Ab deposition. Hoglund and Blennow

(2007)127stated in vitro and in vivo tests have confirmed that statin treatment

reduces Aβ production, but the underlying mechanism remains unclear.

Ehehalt et al., (2003)128 suggested that Ab is produced at the lipid rafts, and

that depletion of lipid rafts result in less secretion. On the other hand, many in

vitro studies have reported that depletion of isoprenoids also reduces Ab-

production.

53

However, most clinical studies have not been able to confirm the experimental

effect of statins on Aβ levels in the brain. Hoglund and Blennow (2007)127

reviewed the results from cerebrospinal fluid analysis and concluded that

there is no clear effect on Aβ levels. A preliminary study of Wolozin et al.,

(2006)129 into Alzheimer subjects found no difference either, although the case

numbers were too small to yield a significant conclusion. Li et al., (2007)130 did

note a significantly reduced level of tau-deposits in statin-users. Meske et al.,

(2003)131 concluded that statins actually induce tau-phosphorylation, albeit at

toxic statin doses.

In contrast, a single study into the effect of simvastatin on an Alzheimer

mouse model reported functional improvement without changes in amyloid-b

level. This indicates that statins can improve the outcome of Alzheimer’s

disease without affecting the pathological markers. The majority of the

epidemiological evidence points to beneficial effect of statins on AD, although

more recent investigations have not always been able to replicate this

observation. Hoglund and Blennow (2007)127 suggested that statins do not

prevent AD entirely, but can slow its progression. Due to the negative clinical

data on the Aβ hypothesis, reviewers have suggested that statins’ beneficial

effect in AD is due to its effects on the vascular and immune systems.

Milind Parle and Nirmal Singh (2008)132 studied the beneficial effects of statins

(Atorvastatin, Simvastatin) in cognitive dysfunction of rats. In this study

Alprazolam, Scopolamine and high fat diet (HFD) served as interoceptive

models, whereas Water-maze and Elevated plus-maze served as

54

exteroceptive behavioural models. Rats were subjected to cholesterol rich

HFD fed for 90days to induce amnesia. Escape latency time (ELT) and

transfer latency (TL) are the parameters evaluated using Water-maze and

Elevated plus-maze respectively. It was observed that Atorvastatin and

Simvastatin treatment for 15 days produced significant reduction in (p<0.05)

total serum cholesterol levels of HFD rats. On the other side Atorvastatin and

Simvastatin failed to produce any significant decrease in total serum

cholesterol levels of control, Alprazolam and Scopolamine treated rats. It was

reported that both the test drugs were potentially reversed the amnesic affects

produced by Alprazolam, Scopolamine and HFD, however test drugs per se

did not produce any significant effect on ELT and TL. It is concluded that

Atorvastatin and Simvastatin successfully reversed the memory deficits

induced by HFD/Alprazolam/Scopolamine probably through cholesterol

dependent as well as cholesterol independent effects. However the study did

not covered the effects of test drugs on lipid profile (except total cholesterol)

brain cholinesterase levels.

Yogita Dalla et al., (2010)133 investigated the effects of Pitavastatin,

Simvastatin (lipophilic statins) and Fluvastatin (hydrophilic statin) on memory

deficits associated with Alzheimer’s type dementia in mice. Dementia was

induced with chronic administration of a high fat diet (HFD) or

intracerebroventricular streptozotocin (ICV STZ, two doses of 3 mg/kg) in

separate groups of animals. Memory of the animals was assessed by the

Morris water maze (MWM) test. Brain thiobarbituric acid reactive species

55

(TBARS) and reduced GSH levels were measured to assess total oxidative

stress. Brain acetylcholinesterase (AChE) activity and total serum cholesterol

levels were also measured. ICV STZ or HFD produced a significant

impairment of learning and memory. Higher levels of brain AChE activity and

TBARS and lower levels of GSH were observed in ICV STZ- as well as HFD-

treated animals. HFD-treated mice also showed a significant increase in total

serum cholesterol levels. Pitavastatin and simvastatin each significantly

attenuated STZ-induced memory deficits and biochemical changes. However,

Fluvastatin produced no significant effect on ICV STZ-induced dementia or

biochemical levels. Administration of any one of the three statins not only

lowered HFD-induced rise in total serum cholesterol level but also attenuated

HFD-induced memory deficits. Further Pitavastatin and simvastatin

administration also reversed HFD-induced changes in biochemical level, while

Fluvastatin failed to produce any significant effect. This study demonstrates

the potential of statins in memory dysfunctions associated with experimental

dementia and provides evidence of their cholesterol dependent and

independent actions.

Gil Atzmon et al., (2010)134 conducted a clinical study to correlate the plasma

HDL levels with cognitive function in longevity. Total plasma cholesterol, low-

density lipoprotein (LDL) cholesterol, HDL, triglycerides, and apolipoprotein

levels were measured and were correlated with their cognitive function

measured by Mini-Mental State Examination [MMSE]. It is observed that HDL

levels correlated significantly with MMSE. Each decrease in plasma HDL

56

tertile was associated with a significant decrease in MMSE. Increased plasma

apolipoprotein A-I and decreased plasma triglyceride levels were also

correlated with a significantly superior cognitive function. The study concluded

that cognitive dysfunction is associated with a progressive decline in plasma

HDL concentrations and plasma HDL and its role in maintaining superior

cognition in longevity.

57

NEED FOR STUDY

3. NEED FOR THE STUDY

From extensive literature search is evident that the beneficial effects of

different statins on learning and memory, ameliorative effects in various types

of amnesia. However differential effects of hypocholesteremic drugs from

various classes were not covered for management of various types of

dementias. Also it is observed that most of the studies were not covered the

changes in AChE levels and lipid profiles (except total cholesterol) though it is

reported that high HDL levels has beneficial effects in cognition, were as high

TG levels lead to dementia. Also the studies not presented for any histological

changes in brain upon chronic administration of hypocholesteremic drugs.

Hence the present study was undertaken to study the above uncovered areas

in a single study and report the beneficial effects of hypocholesteremic drugs

from different classes for managing various types of dementia.

Based on extensive literature review, rat was found to be a suitable animal

model to evaluate hypocholesteremic drugs for memory enhancing activity

and behavioural assessment.

58

OBJECTIVES

4. OBJECTIVES

• To evaluate the memory enhancement effect of hypocholesteremic drugs

in amnesic animals.

• To compare the memory enhancement property of hypocholesteremic

drugs with that of established drugs for memory enhancement in amnesic

animals.

• To study the effect of hypocholesteremic drugs on brain cholinesterase

levels.

• To study the effect of hypocholesteremic drugs on histology of brain.

59

METHODOLOGY

5. METHODOLOGY

5.1. Animals:

Albino wistar rats of either sex were procured from Sri Venkateshwara

Enterprises Pvt. Ltd., Bengaluru and employed in the present study. They

were acclimatized to the laboratory conditions for at least 5 days prior to the

behavioural test. Care of the animals was taken as per guidelines of

committee for the purpose and control supervision of experiments on animals

(IAEC No. KMCRET/Ph.D/17/2013-14).

The animals were housed in poly acrylic cages with not more than six animals

per cage, with 12hr-light/12hr-dark cycle. They were maintained at a

temperature of 22±3 °C and relative humidity of 60±5. Rats had free access

to standard diet and drinking water ad libitum.

5.2. Drugs:

The test and standard drugs proposed for evaluating in the present research

were procured from different pharmaceutical companies as a gift samples and

Scopolamine is purchased from Sigma chemicals are as follows.

1. Simvastatin (Artemis Biotech, Hyderabad)

2. Rosuvastatin (MSN Laboratories Ltd., Patancheru)

3. Fenofibrate (Alembic Limited, Vadodara)

4. Nicotinic acid (Dr. Reddy’s, Hyderabad)

5. Donepezil (Dr. Reddy’s, Hyderabad)

60

Simvastatin, Rosuvastatin, Fenofibrate, Nicotinic acid and Donepezil were

suspended in 1% w/v sodium carboxy methyl cellulose (NaCMC) for oral

administration. Scopolamine was dissolved in distilled water and administered

intraperitoneally.

Preparation of drug solution for dosing:

i. Scopolamine: 100 mg of scopolamine weighed and transferred in to a 100

ml volumetric flask and dissolved in distilled water and the volume is made

up to 100 ml. The procedure was repeated during each preparation. The

required quantity of solution is drawn into a 1 ml syringe according to the

body weight of individual animal, 3 mg/kg solution137 was administered

intraperitoneally.

ii. Donepezil: 1 gm of NaCMC is weighed and transferred into a 200 ml

beaker and 100 ml of distilled water added slowly under magnetic stirring.

The stirring is continued until achieving homogenous suspension. 0.5 gm of

donepezil was weighed and added slowly into the suspension under

continuous stirring and the stirring is continued for 10min to achieve uniform

suspension. The required quantity of suspension is drawn into a 1 ml

syringe according to the body weight of individual animal; 5 mg/kg

suspension139 was administered orally using oral gavage needle.

iii. Simvastatin: 1 gm of NaCMC is weighed and transferred into a 200 ml


61


simvastatin was weighed and added slowly into the suspension under





iv. Rosuvastatin: 1 gm of NaCMC is weighed and transferred into a 200 ml



rosuvastatin was weighed and added slowly into the suspension under





v. Fenofibrate: 1 gm of NaCMC is weighed and transferred into a 200 ml



fenofibrate was weighed and added slowly into the suspension under





62

vi. Nicotinic acid: 1 gm of NaCMC is weighed and transferred into a 200 ml



nicotinic acid was weighed and added slowly into the suspension under

continuous stirring and the stirring is continued for 10 min to achieve

uniform suspension. The required quantity of suspension is drawn into a 1

ml syringe according to the body weight of individual animal; 85 mg/kg


Doses:

Following test and standard drugs were chosen for the research work and the

doses for individual drugs were selected based on available literature.

Table-03: Drugs used along with the dose of each drug selected for the

study and route of administration followed for the corresponding

drugs.

S. No Drug Dose

(mg/kg) Route of

administration Formulation

strength (mg/ml)

1 Scopolamine 3 i.p 1

2 Donepezil 5 p.o 5

3 Simvastatin 5 p.o 5

4 Rosuvastatin 5 p.o 5

5 Fenofibrate 65 p.o 65

6 Nicotinic acid 85 p.o 85

Note: Oral suspension was administered at 1 ml/kg.

63

Composition and preparation of high fat diet:

The high fat diet used in the present study contains 2% of cholesterol, 1% of

cholic acid, 20% of dalda, 6% of coconut oil and 71% of rat feed. Rat feed was

finely powdered mixed thoroughly with weighed quantities of other ingredients.

The mixture was spread on to a tray to form a uniform cake. The trays are

loaded into oven and dried at 100ᵒC for 1 hr. Followed by the trays are

unloaded from the oven and the feed was then cut into small pieces and

provided to animals (Lowry et al., 1951)135.

Induction of hyperlipidaemia: Animals are weighed and initial body weights

were noted and the individual rats were bleed and samples were collected to

estimate the baseline lipid profile values. The rats were fed with prepared high

fat diet of approximately 100 gm/kg of body weight for 12 weeks. Initial lipid

profile of animal was compared with lipid profile measured after 12 weeks of

induction of hyperlipidaemia. Rats that showed elevated lipid profile were

selected for the study (Miida et al., 2007)136.

64

5.3. Study Design

5.3.1. Interoceptive Behavioral Models

Scopolamine induced amnesia

High fat diet induced amnesia

5.3.2. Exteroceptive Behavioral Models

Elevated plus maze

Rectangular maze

Locomotor activity

65

5.4. Experimental Design For Scopolamine Induced Amnesia:

The animals were divided into 11 groups. Each group comprised of 6

animals.

Table-04: Groups allocation for scopolamine induced amnesia

Group Treatment Dose (mg/kg)

1 Control (only vehicle) -

2 Scopolamine 3

3 Scopolamine + Donepezil 3+5

4 Scopolamine + Simvastatin 3+5

5 Scopolamine + Rosuvastatin 3+5

6 Scopolamine + Fenofibrate 3+65

7 Scopolamine + Nicotinic acid 3+85

8 Scopolamine + Donepezil + Simvastatin 3+5+5

9 Scopolamine + Donepezil + Rosuvastatin 3+5+5

10 Scopolamine + Donepezil + Fenofibrate 3+5+65

11 Scopolamine + Donepezil + Nicotinic acid 3+5+85

66

5.5. Experimental Design For High Fat Diet Induced Amnesia:

The animals were divided into 11 groups. Each group comprised of 6

animals.

Table-05: Groups allocation for high fat diet induced amnesia

Group Treatment Dose (mg/kg)


2 Only High Fat Diet -

3 Donepezil 5

4 Simvastatin 5

5 Rosuvastatin 5

6 Fenofibrate 65

7 Nicotinic acid 85

8 Donepezil + Simvastatin 5+5

9 Donepezil + Rosuvastatin 5+5

10 Donepezil + Fenofibrate 5+65

11 Donepezil + Nicotinic acid 5+85

67

5.6. Experimental Methods:

5.6.1. Elevated plus-maze apparatus:

The EPM served as an exteroceptive behavioural model to evaluate learning

and memory in rats. Transfer latency (TL) is a parameter of memory which

was defined as the time in seconds taken by the animal to move into one of

the closed arms with all its four legs. The training trials were carried out for

three days before initiation of behavioural study and the average was taken as

basal score. During behavioural study the animal was placed at the end of an

open arm facing away from the central platform, the time taken to place all the

four paws in the closed arm is noted as TL time. A maximum of 120 seconds

was given for the animal to explore closed arm, the animal which failed to

explore the closed arm within 120 seconds was given a score of 120. The

animal was allowed to explore the maze for 15s and then returned to its home

cage (Moreira, 1984)137.

Behavioural study for scopolamine treated groups was carried for 14 days,

where the animals were administered with vehicle or test drugs (doses in

mg/kg, p.o.) on daily basis for 14 days, scopolamine (3 mg/kg, i.p.) was

administered 30 min after administration of test drugs on days of acquisition.

Days 1, 3, 5, 7, 9, 11, 13 served the acquisition trial (AT) and days 2, 4, 6, 8,

10, 12, 14 served as retention trial (RT). RT was performed 24hrs after

scopolamine administration. TL was recorded for all animals one hour after

68

the administration of test compounds, animals were allowed to explore the

maze.

For the high fat diet treated groups behavioural study was carried on day 1, 7

and 14, where the animals were administered with vehicle or test drugs

(doses in mg/kg, p.o.) on daily basis for 14 days. TL was recorded for all

animals one hour after the administration of test compounds, animals were

allowed to explore the maze.

5.6.2. Rectangular maze:

The maze consists of completely enclosed rectangular box with an entry (A)

and reward chamber (B) appended at opposite ends. The box is partitioned

with wooden slats into blind passages leaving just twisting corridor (C) leading

from the entry (A) to the reward chamber (B). The learning assessment for

control and treated rats was conducted at end of treatment. On the first day,

all the rats were familiarized with the rectangular maze for a period of ten

minutes followed by the training trials were carried out for three days before

initiation of behavioural study. In each trial the rats were placed in the entry

chamber and the timer was activated as soon as the rat leave the chamber,

time taken by the rat to reach the reward chamber (transfer latency (TL)) was

taken as the learning score of the trial. The average of three trials was taken

as the learning score. Lower scores of assessment indicate efficient learning

while higher scores indicate poor learning in animals. During learning

assessment the animals were exposed to food and water ad libitum only for 1

69

hour after the maze exposure for the day was completed to ensure motivation

towards reward area (B) (Muldoon et al., 2000)138.

During behavioural study the animal was placed at an entry chamber (A), the

time taken by the animal to reach the reward chamber (B) (TL) was recorded.

A maximum of 10 min was given for the animal to explore the reward chamber

(B), the animal who failed to explore the B within 10 min was given a score of

600 and the animals were returned to its home cage.

Behavioural study for scopolamine groups was carried for 14 days, where the

animals were administered with vehicle or test drugs (doses in mg/kg, p.o.) on

daily basis for 14 days, scopolamine (3 mg/kg, i.p.) was administered 30 min

after administration of test drugs on days of acquisition. Days 1, 3, 5, 7, 9, 11,

13 served the acquisition trial (AT) and days 2, 4, 6, 8, 10, 12, 14 served as

retention trial (RT). RT was performed 24 hrs after scopolamine

administration. TL was recorded for all animals one hour after the

administration of test compounds, animal were allowed to explore the maze.





allowed to explore the maze.

70

5.6.3. Locomotor activity:

This method aims to evaluate the locomotor activity of the control and treated

animals. The locomotor activity will be measured using actophotometer. Each

animal will be placed individually in the actophotometer for 3 min and the

basal activity score was obtained. The training trials were carried out for three

days before initiation of behavioural study; the average was taken as basal

activity score. It was followed by behavioural study and activity scores of

individual animals were recorded (Notkola et al., 1998)139.

Behavioural study for scopolamine groups was carried for 14 days, where the

animals were administered with vehicle or test drugs (doses in mg/kg, p.o.) on

daily basis for 14 days, scopolamine (3 mg/kg, i.p.) was administered 30 min

after administration of test drugs on days of acquisition. Days 1, 3, 5, 7, 9, 11,

13 served the acquisition trial (AT) and days 2, 4, 6, 8, 10, 12, 14 served as

retention trial (RT). RT was performed 24 hrs after scopolamine

administration. Basal activity score was recorded for all animals one hour after

the administration of test compounds, animal were placed in the

actophotometer for 3 min.





allowed to explore the maze for 5 min.

71

5.7. Biochemical Estimation:

5.7.1. Lipid Profile:

• Total cholesterol

• Triglycerides

• High density lipoproteins (HDL)

• Low density lipoproteins (LDL)

• Very low density lipoproteins (VLDL)

Total cholesterol, Triglycerides and HDL were estimated using serum

samples. All the above biochemical parameters were estimated using semi-

auto analyzer (Photometer 5010 V5+, Germany) with enzymatic kits procured

from Primal Healthcare limited, Lab Diagnostic Division, Mumbai, India.

72

TOTAL CHOLESTEROL (TC)

Principle

Determination of cholesterol is done after enzymatic hydrolysis and oxidation.

The colorimetric indicator is quinoneimine, which is generated from 4-

aminoantipyrine and phenol by hydrogen peroxide under the catalytic action of

peroxidase (Trinder’s reaction) (Ireland, 1941)140.

Cholesterol ester + H2O CHE Cholesterol + Fatty acid

Cholesterol + O2 CHO Cholesterol-3-one +H2O2

2H2O2 + 4- Amino antipyrine + Phenol POD Quinonelimine + 4 H2O2

Method

CHOD-PAP: Enzymatic photometric test

Reagents

Goods buffer (pH 6.7)- 50 mmol/ l

Phenol -5 mmol/l

4-aminoantipyrine- 0.3 mmol/l

Cholesterol estrase - > 200 U/l

Cholesterol oxidase - > 100 U/l

73

Peroxidase - 3 KU/l

Standard - (5.2 mmol/l)

Assay procedure

a. 1 ml (1000 μl) of reagent-1 is taken in a 5 ml test tube.

b. Added 0.01 ml (10 μl) of serum.

c. Mixed well and incubated at 370C for 5 min.

d. Read the test sample.

TRIGLYCERIDES

Principle

Determination of triglycerides (TG) alters enzymatic splitting with lipoprotein

lipase. Indicator is quinoneimine which is generated from 4-aminoantipyrine

and 4- chlorophenol by hydrogen peroxidase under the catalytic action of

peroxidase (Gloria Buckley et al., 1966)141.

Triglycerides LPL Glycerol + fatty acid

Glycerol + ATP GK Glycerol-3-phosphate+ ADP

Glycerol-3-phosphate +O2 GPO Dihydroxyaceton phosphate + H2O2

2H2O2 + 4- Amino antipyrine + 4- chlorophenol POD Quinonelimine + HCl +

4H2O2

74

Method

Colorimetric enzymatic test using glycerol-3-phosphate-oxidase (GPO).

Reagents

Components and concentrations in the test Goods buffer pH 7.2, 50 mmol/ l

4-chloro Phenol - 4 mmol/l

ATP - 2 mmol/l

Mg2+ - 15 mmol/l

Glycerokinase - > 0.4 Kμ/l

Peroxidase -> 2 Kμ/l

Lipoprotein lipase -> 4 Kμ/l

4-aminoantipyrine - 0.5 mmol/l

Glcerol-3-phosphate- oxidase - > 1.5Kμ/l

Standard - (2.3 mmol/l)

Assay procedure

a. 1 ml (1000 μl) of reagent-1 is taken in a 5 ml test tube.

b. Added 0.01 ml (10 μl) of serum.

75

c. Mixed well and incubated at 37oC for 15 min.


HDL CHOLESTEROL

Principle

Chylomicrons, VLDL and LDL are precipitated by adding phosphotungstic acid

and magnesium ions to the sample. Centrifugation leaves only the HDL in the

supernatant. The cholesterol content in it is determined enzymatically (Jensen

et al., 2002)142.

Method

Phosphotungstic acid precipitation method.

Reagents

Phosphotungstic acid - 0.55 mmol/l

Magnesium chloride - 25 mmol/l

Assay procedure

A. Preparation of supernatant for the HDL-CHL estimation

76

Added 200 μl of serum to the 500 μl of HDL-Cholesterol precipitating reagent

(from HDL kit)in 1.5 ml centrifuge tube and mixed well. Centrifuged the above

solution at 4000 rpm for 10 min.

B. Preparation of test sample for the estimation of HDL-Cholesterol

a. Taken 1000 μl of reagent-1 (from cholesterol kit) in a 5 ml test tube.

b. Added, 100 μl of supernatant from above centrifuged solution

c. Mixed well and incubated at 370C for 15 min.


LDL & VLDL Cholesterol

LDL and VLDL cholesterol are calculated using following Friede wald

formulas.

LDL = Total Cholesterol – (HDL+VLDL)

VLDL = Triglycerides/5

77

5.7.2. Estimation of cholinesterase enzyme:

Reagents:

15 mM ATCI, 3 mM DTNB, 50 mM Tris-HCl of pH 8.0 containing 0.1 % BSA

Preparation of homogenate:

Brain was weighed, sliced into small pieces and transferred to homogenisation

container.0.5 g of tissue was homogenized in 10 mL of phosphate buffer of pH

7.4 at 4000 rpm. Homogenate was transferred to 2 L beaker on ice. It was

stirred on magnetic stirrer. Homogenisation was repeated to obtain 10 % (w/v)

homogenate.

Procedure:

A sample was prepared by adding 25 µL of 15 mM ATCI, 75 µL of 3 mM

DTNB and 75 µL of 50 mM Tris-HCl of pH 8.0 containing 0.1 % BSA.A stock

solution was prepared by adding 250μl of 15 mM ATCI, 750 μl of 3 mM DTNB

and 750 μl of 50 mM Tris–HCl, pH 8.0, containing 0.1% BSA. 25 µL of sample

was added to each well of 96 well micro titer plate and incubated.175 µL of

stock was added to each well containing sample after 5 min of incubation.2

out of 96 wells were added only stock without sample. The microtiter plate

was again incubated and OD was measured at 412 nm (Miroslav Pohanka et

al., 2011)143.Acetylcholinesterase levels were calculated by the following

formula:

78

Concentration AChE = 5.74×10-4 × ∆A/Co

Where ∆A= change in absorbance

Co= original concentration of tissue (mg/ml)

5.8. Brain Histopathology:

Histopathology is the microscopic study of tissues for pathological alterations.

This involves collection of morbid tissues from biopsy or necropsy, fixation,

preparation of sections, staining and microscopic examination.

Collection of materials:

Thin pieces of 3 to 5 mm thickness were collected from tissues showing gross

morbid changes along with normal tissue.

Fixation:

The tissue was kept in 10% formalin for 24-48 hours at room temperature. It

becomes hard and dry, becomes suitable for further histological examination.

Haematoxylin and eosin method of staining:

The section was deparaffinised by xylol for 5 to 10 minutes and xylol removed

by absolute alcohol. It was then washed in tap water. The sections were

stained with haematoxylin for 3-4 minutes and washed in tap water, allowed

79

the sections under tap water for 5-10 min. Counterstained with 0.5% eosin

until section appears light pink (15 to 30seconds), then washed in tap water.

Blot and dehydrated in alcohol, clear with xylol (15 to 30 seconds) and

mounted in DPX Moutant. Keep slide dry and remove air bubbles (Okhawa et

al., 1979)144.

5.9. Statistical Analysis:

All the results were expressed as mean ± SEM. The data were analyzed using

two way analysis of variance (ANOVA) followed by Dunnett’s ‘t’ test.

80

RESULTS

6. RESULTS

6.1. Effect of Hypocholesteremic Drugs on Scopolamine Induced

Amnesia

6.1.1. Elevated plus maze:

All animals treated with either vehicle or scopolamine or the test drugs treated

groups showed average transfer latency of less than 120 sec. Administration

of scopolamine (Group 2) prominently reduced the memory potential indicated

by higher transfer latency (TL) time compared to normal control group (Table-

06). It was observed that standard donepezil (Group 3) produced significant

(p<0.01) reduction in TL on tested days compared to Scopolamine treated

group confirming its anti-amnesic activity. Administration of test drugs (group

4 to 7) showed reduction in TL in both acquisition and retention trial

compared to that of negative control group in the order of nicotinic acid

(p<0.001) > rosuvastatin (p<0.001) >Simvastatin (p<0.001) > fenofibrate

(p<0.001) either alone or in combination (group 8 to 11) with standard drug

Donepezil (Table-06). Overall the combination treatment appears to be better

efficacious than that of individual treatments during the course of the study.

81

Table-06: Transfer latency recordings of hypocholesteremic drugs using elevated plus maze

Each group (n=6), each value represents Mean±SEM. a) Denotes *P<0.05, **P<0.01, and ***P<0.001 compared to control(Day-7) &AP<0.05, AAP<0.01, and AAAP<0.001 compared to scopolamine treated group (Day-7). b) Denotes ×××p<0.001 compared with control and ᴛ p<0.05 compared with scopolamine treated group (Day-8) c) Denotes +++P<0.001 compared to control group (Day-13) d) Denotes ˄˄˄P<0.001 compared to control group (Day-14).ANOVA followed by dunnett’s test.

Group No. Treatment & Dose Transfer Latency (Sec.)

Initial Day-1 Day-2 Day-7 Day-8 Day-13 Day-14

1 Control (1% Na CMC) 39.89±7.76 23.5±6.48 14±1.51 9.17±2.33 18.33±5.43 16.83±2.64 15.33±3.78

2 Scopolamine 3mg/kg 32.44±5.66 94.83±8.21 81.17±9.54 119.5±0.50 108.67±8.48 120.00±0.00 119.83±0.17

3 Donepezil 5 mg/kg 26.33±2.01 39.33±16.85 15.67±5.40 35.17±5.61*** 15.67±3.03××× 12.83±1.78+++ 7.67±1.26˄˄˄

4 Simvastatin 5 mg/kg 42.22±6.51 81.33±14.00 60.17±13.15 83.5±7.6*,AAA 28.67±7.95××× 25.5±4.30+++ 13.5±2.93˄˄˄

5 Rosuvastatin 5 mg/kg 56.94±10.08 74.5±12.02 34.17±4.40 71.17±9.05***,A 25.5±3.69××× 25.33±1.41+++ 12.5±1.95˄˄˄

6 Fenofibrate 65 mg/kg 23.83±3.64 79.5±13.34 45.83±8.06 78±5.05**,AA 40.5±6.98×××,ᴛ 28.83±3.59+++ 10.83±1.35˄˄˄

7 Nicotinic Acid 85 mg/kg 28.72±4.50 64.33±6.86 20.83±4.76 37±11.79*** 16.5±3.51××× 18±3.83+++ 9± 2.34˄˄˄

8 Donepezil 5 mg/kg + Simvastatin 5 mg/kg 27.72±3.54 59.33±10.37 32.67±7.68 75.33±5.65**,AA 24.17±5.74××× 24.67±6.09+++ 17.83±7.11˄˄˄

9 Donepezil 5 mg/kg + Rosuvastatin 5 mg/kg

30.56±5.39 59.33±12.36 21.33±10.43 32.83±7.91*** 23.83±3.89××× 17.33±3.37+++ 6.67±1.17˄˄˄

10 Donepezil 5 mg/kg + Fenofibrate 65 mg/kg

31.56±8.60 57.17±8.45 20.33±4.86 37.5±11.43*** 15.17±4.22××× 18.33±3.48+++ 9.17±1.85˄˄˄

11 Donepezil 5 mg/kg + Nicotinic Acid 85 mg/Kg

19.72±4.76 55.83±6.77 31.33±8.07 33.17±9.56*** 16±4.37××× 16±3.09+++ 8.5±1.52˄˄˄

82

Figure-05: Effect of hypocholesteremic drugs on transfer latency using EPM in Scopolamine treated animals

Initial

AT (day 1

)

RT � (day 2

)

AT (day 7

)

RT (day 8

)

AT� (day 1

3)

RT� (day 1

4)0

50

100

150

Group-1Group-2Group-3Group-4


Group-9Group-10Group-11

Tran

sfer L

atenc

y (Se

c)

Each group (n=6), each value represents Mean±SEM

83

6.1.2. Rectangular Maze:

Scopolamine, an anti-muscarinic drug produced dementia indicated by

enhanced TL compared to control group. Donepezil (standard) (Group-3) is

indicative of a greater (P<0.001) memory enhancing potential (Table-07).

Administration of test drugs (group 4 to 7) alone showed reduction in TL

compared to that of control group in the order of nicotinic acid (p<0.001)

>rosuvastatin (p<0.001)>Simvastatin (p<0.001)> fenofibrate (p<0.001) from

day 7 to day 14 in both acquisition and retention trials (Table-07). When the

test drugs were given in combination with donepezil (group 8 to 11)showed

reduction in TL compared to that of control group as well as individual

treatment groups in the order of donepezil with nicotinic acid (p<0.001) >

rosuvastatin (p<0.001)> Simvastatin (p<0.001)=fenofibrate (p<0.001) (Table-

07) .

84

Table-07: Transfer latency recordings of hypocholesteremic drugs using rectangular maze

Group No. Treatment & Dose

Transfer Latency (Sec.)

Initial Day-1 Day-2 Day-7 Day-8 Day-13 Day-14

1 Control (1% Na CMC p.o) 53.56±22.79 117±52.39 61.17±30.19 145.67±21.84 117.33±28.46 82.17±15.86 63.33±11.95

2 Scopolamine 3mg/kg 63.28±20.27 336.17±57.54 295.67±51.09 558.67±25.21 420.67±67.04 600±0 600±0

3 Donepezil 5 mg/kg 55.67±9.49 90.83±20.23 43.5±4.61 115.17±16.79*** 67±12.46××× 39.33±7.51+++ 21.67±3.56˄˄˄

4 Simvastatin 5 mg/kg 91.67±13.11 272±44.17 174.83±10.41 154.5±24.46*** 145±24.8×××,ᴛ 95.5±4.16+++ 42.17±10.32˄˄˄

5 Rosuvastatin 5 mg/kg 91.33±10.05 262.33±79.76 168.17±71.55 161.67±21.96*** 99±20.91××× 94.5±13.53+++ 47.17±8.98˄˄˄

6 Fenofibrate 65 mg/kg 71.06±30.48 235.33±41.6 164±25.21 142±16.04*** 103.17±8.51××× 104.17±7.27+++ 59±11.4˄˄˄

7 Nicotinic Acid 85 mg/kg 89.83±16.89 291.5±46.32 155.83±12.05 131±9.73*** 86.83±10.59××× 81±4.12+++ 50.33±11.43˄˄˄

8 Donepezil 5 mg/kg + Simvastatin 5 mg/Kg

72.5±8.35 276.5±28.8 168.33±16.46 161.5±20.11*** 94.5±18.53××× 96.5±4.89+++ 46.5±6.08˄˄˄

9 Donepezil 5 mg/kg + Rosuvastatin 5 mg/Kg

100.89±13.04 208.83±7.19 164.67±12.06 111.5±8.85*** 67.17±6.71××× 62.67±10.57+++ 29.67±12.10˄˄˄

10 Donepezil 5 mg/kg + Fenofibrate 65 mg/Kg

58.22±21.89 249.33±33.79 179±26.06 127.17±15.57*** 99.67±5.66××× 85.33±10.53+++ 59.33±14.36˄˄˄

11 Donepezil 5 mg/kg + Nicotinic Acid 85 mg/Kg

46.67±4.07 224±36.07 129.33±9.13 109.17±4.38*** 47.83±3.5××× 43±1.83+++ 33±4.73˄˄˄

Each group (n=6), each value represents Mean±SEM. a) Denotes *P<0.05, **P<0.01, and ***P<0.001 compared control group (Day-7). b) Denotes ×××p<0.001 compared with control group and ᴛ p<0.05 compared with Donepezil 5 mg/kg (Day-8). c) Denotes +++P<0.001 compared to control (Day-13) d) Denotes ˄˄˄P<0.001 compared to control (Day-14).ANOVA followed by dunnett’s test.

85

Figure-06: Effect of hypocholesteremic drugs on transfer latency using Rectangular maze in Scopolamine treated animals

Initial

AT (day 1

)

RT � (day 2

)

AT (day 7

)

RT (day 8

)

AT� (day 1

3)

RT� (day 1

4)0

200

400

600

800




Tran

sfer L

atenc

y (Se

c)


86

6.1.3. Locomotor Activity:

Rats subjected to scopolamine showed a significant (P<0.05) reduction in

basal activity score compared to normal rats. Donepezil, an anticholinesterase

agent produced higher (p<0.01) score indicating its positive effect on cognitive

related activities. Test compounds either alone or in combination improved the

locomotor activity significantly compared to scopolamine treated groups. Test

compounds alone showed similar effect compared to donepezil and the order

of increase in locomotor activity is Nicotinic acid (p<0.001) > Simvastatin

(p<0.001)>fenofibrate (p<0.001) > rosuvastatin (p<0.001) (Table-08). When

the test drugs were given in combination with donepezil the order of efficacy is

in the order of Nicotinic acid (p<0.001) =rosuvastatin (p<0.001)> fenofibrate

(p<0.001) > Simvastatin (p<0.001) (Table-08).

87

Table-08: Activity scores of hypocholesteremic drugs using actophotometer


Activity Score (Nos.)

Initial Day-1 Day-2 Day-7 Day-8 Day-13 Day-14(Nos.)

1 Control (1% Na CMC p.o) 303.22±24.4 287.33±38.24 310.5±43.74 342±20 374.5±10.27 333.33±15.40 377.17±9.46

2 Scopolamine 3mg/kg 291.56±16.31 225.67±21.8 260.17±22.59 174.67±17.77 218.17±19.33 106.50±15.50 146±19.92

3 Donepezil 5 mg/kg 334.28±16.59 326.5±19.33 362±16.59 278.33±15.07** 330.83±13.2×× 213.00±15.98++ 261.5±17.44˄˄

4 Simvastatin 5 mg/kg 304.17±27.34 278.33±12.88 313±9.83 202.67±18.21 274.67±38.79 159.83±21.53 180.5±15.49

5 Rosuvastatin 5 mg/kg 234.28±25.81 277±15.24 300.33±15.35 217.17±15.93 258.67±19.1 151.50±20.98 182.67±23.05

6 Fenofibrate 65 mg/kg 332.56±20.38 262.5±37.56 249.5±12.5 202.17±23.46 270.33±12.47 153.50±3.910 207.33±5.43

7 Nicotinic Acid 85 mg/kg 288.33±17.5 292±14.03 311±17.08 239.33±13.12 271.17±39.82 161.83±21.14 231.17±25.84

8 Donepezil 5 mg/kg + Simvastatin 5 mg/Kg 285.94±29.64 272.33±19.38 309.83±25.16 187.67±25.86A 249.67±41.14ᴛ 150.33±15.42 178.17±11.6

9 Donepezil 5 mg/kg + Rosuvastatin 5 mg/Kg 290.39±4.7 320.83±38.3 349.67±41.67 223.17±6.56 274.83±31.02 179.00±11.56 254.83±15.64

10 Donepezil 5 mg/kg + Fenofibrate 65 mg/Kg 226.56±11.52 241±26.32 288.33±28.46 191.83±17.51 276.17±25.14 127.33±22.39 199.17±21.73

11 Donepezil 5 mg/kg + Nicotinic Acid 85 mg/Kg 259.39±13.99 268.5±25.52 304.17±26.21 219.67±35.24 272.5±22.96 188.83±25.95 236.17±13.67

Each group (n=6), each value represents Mean±SEM. a) Denotes **P<0.01 compared to control group (Day-7) &AP<0.05 compared to Donepezil 5 mg/kg initial to(Day-7). b) Denotes ××p<0.01 compared with control group and ᴛp<0.05 compared with Donepezil 5 mg/kg at (Day-8) c) Denotes ++P<0.01 compared to control group (Day-13) d) Denotes ˄˄P<0.01 compared to control group (Day-14). ANOVA followed by dunnett’s test.

88

Figure-07: Effect of hypocholesteremic drugs on locomotor activity using Actophotometer in scopolamine treated animals

Initial

AT (day 1

)

RT (day 2

)

AT (day 7

)

RT (day 8

)

AT (day 1

3)

RT (day 1

4)0

100

200

300

400

500




Activ

ity S

core

(Nos

)


89

6.1.4. Acetylcholinesterase Activity:

Scopolamine treated animals showed elevated levels of acetylcholinesterase

indicating its memory reducing potential. Rats treated with standard drug

donepezil produced reduction of acetylcholinesterase enzyme activity in

comparison with negative control. Administration of test drugs (group 4 to 7)

showed improvement in enzyme levels compared to that of negative control

group in the order of nicotinic acid > rosuvastatin> fenofibrate > Simvastatin,

and similar trend was observed when administered in combination with

standard drug Donepezil (Table-09). Overall the test compounds decreased

acetyl cholinesterase levels either alone or in combination when compared to

scopolamine treated group. This supports as an evidence for the observed

improvement with the test drugs in locomotor activity as well as behavioural

activities like acquisition and retention.

90

Table-09: Acetylcholinesterase levels of animals treated with

hypocholesteremic drugs in scopolamine induced rats. Group

No. Treatment & Dose AChE (moles/min/gm)

1 Control (1% Na CMC p.o) 0.028±0.00065

2 Scopolamine 3mg/kg 0.032±0.00107

3 Donepezil 5 mg/kg 0.016±0.00095

4 Simvastatin 5 mg/kg 0.027±0.0008

5 Rosuvastatin 5 mg/kg 0.018±0.00055

6 Fenofibrate 65 mg/kg 0.024±0.00132

7 Nicotinic Acid 85 mg/kg 0.016±0.00094

8 Donepezil 5 mg/kg + Simvastatin 5 mg/Kg 0.019±0.00069

9 Donepezil 5 mg/kg + Rosuvastatin 5 mg/Kg 0.015±0.00054A

10 Donepezil 5 mg/kg + Fenofibrate 65 mg/Kg 0.016±0.00037

11 Donepezil 5 mg/kg + Nicotinic Acid 85 mg/Kg 0.012±0.00047**,AAA

Each group (n=2), each value represents Mean±SEM. a) Denotes **P<0.01compared to control group b) Denotes Ap<0.05, AAP<0.01 and AAAP<0.001 compared with Donepezil 5 mg/kg ANOVA followed by dunnett’s test.

Figure-08: Effect of hypocholesteremic drugs on brain Acetylcholinesterase levels in scopolamine treated animals.

Group-1

Group-2

Group-3

Group-4

Group-5

Group-6

Group-7

Group-8

Group-9

Group-10

Group-110.00

0.01

0.02

0.03

0.04

AChE

(mol/

min/

gm)


91

6.1.5. Brain histopathology:

Micrograph of brain section of amnesia-induced group showed severe

neuronal edema with marked gliosis and neuronal degeneration. Micrograph

of brain section of amnesia-induced rats treated with donepezil showed very

mild histopathological alteration in the basal ganglion indicate its prominent

neuroprotective effect. Brain sections of amnesia-induced rats treated with

simvastatin and rosuvastatin showed mild gliosis of the basal ganglia. While

amnesia-induced rats treated with fenofibrate and nicotinic acid showed

moderate edema, gliosis and degeneration in basal ganglion.

However the brain sections of rats exposed to combination treatment illustrate

mild to negligible damage of basal ganglionic cells indicating their synergistic

effects.

92

Brain Histopathology pictures of Scopolamine induced amnesia

Fig-09: Basal ganglia of normal rat (40X) Fig-10: Basal ganglia of rat after Scopolamine

induced amnesia (40X)

Figure-11: Basal ganglia of rat treated with

Donepezil in Scopolamine induced amnesia (40X)

Figure-12: Basal ganglia of rat treated with Simvastatin in Scopolamine induced amnesia (40X)

93


Rosuvastatin in Scopolamine induced amnesia (40X)

Figure-14: Basal ganglia of rat treated with Fenofibrate in Scopolamine induced amnesia (40X)

Figure-15: Basal ganglia of rat treated with Nicotinic acid in Scopolamine induced amnesia (40X)

Figure-16: Basal ganglia of rat treated with Donepezil + Simvastatin in Scopolamine induced amnesia (40X)

94


Donepezil + Rosuvastatin in Scopolamine induced amnesia (40X)

Figure-18: Basal ganglia of rat treated with Donepezil + Fenofibrate in Scopolamine induced amnesia (40X)


Donepezil + Nicotinic acid in Scopolamine induced amnesia (40X)

Fig-20: Cerebellum of normal (40X)

95

Fig-21: Cerebellum of rat after Scopolamine

induced amnesia (40X) Fig-22: Cerebellum of rat treated with Donepezil

in Scopolamine induced amnesia (40X)

Fig-23: Cerebellum of rat treated with

Simvastatin in Scopolamine induced amnesia (40X)

Fig-24: Cerebellum of rat treated with Rosuvastatin in Scopolamine induced amnesia (40X)

96

Fig-25: Cerebellum of rat treated with Fenofibrate in Scopolamine induced amnesia (40X)

Fig-26: Cerebellum of rat treated with Nicotinic acid in Scopolamine induced amnesia

(40X)

Fig-27: Cerebellum of rat treated with Donepezil

+ Simvastatin in Scopolamine induced amnesia (40X)

Fig-28: Cerebellum of rat treated with Donepezil + Rosuvastatin in scopolamine induced amnesia (40X)

97

Fig-29: Cerebellum of rat treated with Donepezil + Fenofibrate in scopolamine induced amnesia (40X)

Fig-30: Cerebellum of rat treated with Donepezil + Nicotinic acid in scopolamine Induced amnesia (40X)

98

6.2. Effect of Hypocholesteremic Drugs on High Fat Diet Induced

Amnesia

6.2.1. Effect of hypocholesteremic drugs on Body Weights:

Significant (p<0.01) increase in the body weight of animals over the period of

14 weeks was observed in rats receiving high fat diet. Treatment with

donepezil, test drugs alone or in combination did not show any significant

reduction on body weight compared to HFD group (Table-10).

99

Table-10: Body weight summary of animals at different time points



Initial Day-1 Day-7 Day-14

1 Control (1% Na CMC p.o) 39.89± 7.76 23.5± 6.48 9.17± 2.33 15.33± 3.78

2 Only High Fat Diet (HFD) 35.28±6.03 46.33±5.1 66.17±5.28 105.00±2.77

3 Donepezil 5 mg/kg 46.06±8.33 35.50±4.13 55.00±6.63 43.00±5.44

4 Simvastatin 5 mg/kg 22.17±2.11 44.50±5.37 82.00±2.58AAA 70.67±2.09+++,BBB

5 Rosuvastatin 5 mg/kg 16.72±2.29 34.33±4.37 71.17±6.52 72.33±3.16+++,BBB

6 Fenofibrate 65 mg/kg 21.72±5.62 69.67±7.86 80.83±5.69AAA 84.5±4.59+,BBB

7 Nicotinic Acid 85 mg/kg 37.22±5.26 54.50±7.22 79.17±5.67AA 104.83±3.40BBB

8 Donepezil 5 mg/kg + Simvastatin 5 mg/kg 23.61±2.42 26.00±3.96 39.50±4.28*** 33.00±3.67+++

9 Donepezil 5 mg/kg + Rosuvastatin 5 mg/kg 32.67±5.66 39.83±3.08 78.83±3.12AA 68.83±2.6+++,BBB

10 Donepezil 5 mg/kg + Fenofibrate 65 mg/kg 32.67±7.75 59.17±5.02 84.33±6.94*, AAA 97.00±4.59.00BBB

11 Donepezil 5 mg/kg + Nicotinic Acid 85 mg/kg 28.72±2.51 47.67±6.21 77.67±3.57 AA 74.50±7.50+++,BBB

Each group (n=6), each value represents Mean±SEM. a) Denotes *P<0.05, **P<0.01, and ***P<0.001 compared to initial body weight. b) Denotes Ap<0.05 compared with HFD-group and Bp<0.05 and BBP<0.01 compared with Donepezil 5 mg/kg at week-14, ANOVA followed by dunnett’s test. NA - Not determined.

100

Figure-31: Effect of hypocholesteremic drugs on Body Weights in animals receiving high fat diet (HFD)

Group-1

Group-2

Group-3

Group-4

Group-5

Group-6

Group-7

Group-8

Group-9

Group-10

Group-110

100

200

300

400InitialWeek 12Week 14

Body

Weig

ht (g

ms)

Each group (n=6), each value represents Mean±SEM.

101

6.2.2. Effect of hypocholesteremic drugs on transfer latency using

Elevated plus-maze:

Rats treated with high fat diet showed increase in TL indicate its amnesic

effect. Treatment with donepezil prominently reduced TL compared with

negative control. Rats treated with test drugs (group 4 to 7) showed reduction

in TL compared to that of negative control group in the order of rosuvastatin

(p<0.001) >Simvastatin (p<0.001) > nicotinic acid (p<0.05)> fenofibrate

(p<0.05). Overall statins showed significant effect compare to fenofibrate and

nicotinic acid when administered alone or in combination with standard drug

Donepezil (Table-11).

102

Table-11: Transfer latency recordings of hypocholesteremic drugs using elevated plus maze (HFD)

Each group (n=6), each value represents Mean±SEM. a) Denotes *P<0.05, **P<0.01, and ***P<0.001 (Day-7) &+P<0.05, ++P<0.01, and +++P<0.001 compared to initial total latency (Day-14). b) Denotes Ap<0.05 compared with HFD-group and Bp<0.05 compared with Donepezil 5 mg/kg at week-14, ANOVA followed by dunnett’s test.




1 Control (1% Na CMC p.o) 39.89± 7.76 23.5± 6.48 9.17± 2.33 15.33± 3.78


3 Donepezil 5 mg/kg 46.06±8.33 35.50±4.13 55.00±6.63 43.00±5.44








11 Donepezil 5 mg/kg + Nicotinic Acid 85 mg/kg 28.72±2.51 47.67±6.21 77.67±3.57 AA 74.50±7.50+++,BBB

103

Figure-32: Effect of hypocholesteremic drugs on transfer latency using Elevated plus-maze in animals receiving high fat diet (HFD)

Group-1

Group-2

Group-3

Group-4

Group-5

Group-6

Group-7

Group-8

Group-9

Group-10

Group-110

50

100

150 InitialDay-1

Day-7Day-14

Tran

sfer L

aten

cy (S

ec)


104

6.2.3. Effect of hypocholesteremic drugs on transfer latency using

Rectangular maze:

High fat diet produced dementia indicated by enhanced TL compared to

control group. Donepezil (standard) significantly (P<0.001) decreased TL

indicative of a greater memory enhancing potential. Administration of test

drugs (group 4 to 7) showed reduction in TL compared to that of negative

control group in the order of Simvastatin (p<0.001) >rosuvastatin (p<0.001)

>fenofibrate (p<0.001) >nicotinic acid (p<0.001). Similarly statins showed

better effect compare to fenofibrate and Nicotinic acid when administered in

combination with standard drug Donepezil (Table-12).

105

Table-12: Transfer latency recordings of hypocholesteremic drugs using rectangular maze (HFD)

Each group (n=6), each value represents Mean±SEM a) Denotes *P<0.05, **P<0.01, and ***P<0.001 (Day-7) &+P<0.05, ++P<0.01, and +++P<0.001 compared to initial total latency (Day-14). b) Denotes Ap<0.05 compared with HFD-group and Bp<0.05 compared with Donepezil 5 mg/kg at week-14, ANOVA followed by dunnett’s test.

Group No. Treatment & Dose Transfer Latency (Sec.)


1 Control (1% Na CMC p.o) 53.56±22.79 117.00±52.39 145.67±21.84 63.33±11.95


3 Donepezil 5 mg/kg 69.11±10.07 127.67±16.73 204.33±14.57*** 176.17±13.93+++

4 Simvastatin 5 mg/kg 60.11±8.53 210.00±13.80 253.67±16.35*** 257.17±16.66+++, BB

5 Rosuvastatin 5 mg/kg 63.39±16.98 316.67±21.74 288.67±15.26***, AA 255.33±13.29+++, BB

6 Fenofibrate 65 mg/kg 36.11±6.82 252.83±8.08 373.50±15.68**, AAA 444.83±25.32+++, BBB

7 Nicotinic Acid 85 mg/kg 44.56±6.41 280.00±7.94 405.67±21.04AAA 487.83±15.5 BBB

8 Donepezil 5 mg/kg + Simvastatin 5 mg/kg 35.17±10.23 106.17±15.01 108.67±17.45***, AAA 111.33±15.12+++, B

9 Donepezil 5 mg/kg + Rosuvastatin 5 mg/kg 75.39±12.45 173.67±14.36 154.00±14.44*** 160.00±10.66+++

10 Donepezil 5 mg/kg + Fenofibrate 65 mg/kg 48.33±10.48 252.33±13.55 237.83±18.24*** 213.67±10.92+++

11 Donepezil 5 mg/kg + Nicotinic Acid 85 mg/kg 85.50±21.88 295.33±34.52 311.17±26.55***, AAA 436.5±22.22+++, BBB

106

Figure-33: Effect of hypocholesteremic drugs on transfer latency using Rectangular maze in animals receiving high fat diet (HFD)

Group-1

Group-2

Group-3

Group-4

Group-5

Group-6

Group-7

Group-8

Group-9

Group-1

0

Group-1

10

100

200

300

400

500

InitialDay-1

Day-7Day-14

Activ

ity S

core

(Nos

)


107

6.2.4. Effect of hypocholesteremic drugs on locomotor activity using

Actophotometer:

Rats subjected to high fat diet showed reduction in basal activity score

compared to normal rats. Donepezil, an anticholinesterase produced higher

(p<0.001) score indicating its positive effect on cognitive related activities. All

test drugs showed protection against HFD induced reduction in locomotor

activity in the similar manner. When given in combination with Donepezil the

order of potency was nicotinic acid (p<0.001)>rosuvastatin

(p<0.001)>Simvastatin (p<0.001)> fenofibrate (p<0.001) (Table-13). Overall

the combination treatment showed superior effect than that of individual

treatments because of the similarity in their net effects.

108

Table-13: Activity score recordings of hypocholesteremic drugs using Actophotometer (HFD)

Group No. Treatment & Dose Activity Score (Nos.)


1 Control (1% Na CMC p.o) 303.22±24.40 287.33±38.24 342.00±20.00 377.17±9.46


3 Donepezil 5 mg/kg 325.67±28.03 298.00±15.07 223.00±20.07*** 429.00±27.71+++

4 Simvastatin 5 mg/kg 286.67±14.52 284.00±24.91 270.00±26.62*** 328.00±22.89+++

5 Rosuvastatin 5 mg/kg 421.00±28.69 380.00±34.87 380.00±25.09*** 320.00±27.23+++, B

6 Fenofibrate 65 mg/kg 330.67±12.24 260.00±18.34 261.00±13.28** 320.00±28.24+++, B

7 Nicotinic Acid 85 mg/kg 314.67±9.96 242.00±6.44 232.00±9.93 218.00±7.51+++, BBB

8 Donepezil 5 mg/kg + Simvastatin 5 mg/kg 357.33±18.92 298.00±19.55 218.00±41.58***, A 310.00±28.80+++



11 Donepezil 5 mg/kg + Nicotinic Acid 85 mg/kg 350.33±11.72 291.00±14.31 320.00±22.63*** 487.00±26.03+++

Each group (n=6), each value represents Mean±SEM a) Denotes *P<0.05, **P<0.01, and ***P<0.001 (Day-7) &+P<0.05, ++P<0.01, and +++P<0.001 compared to initial basal activity (Day-14). b) Denotes Ap<0.05 compared with HFD-group and Bp<0.05 compared with Donepezil 5 mg/kg at week-14, ANOVA followed by dunnett’s test.

109

Figure-34: Effect of hypocholesteremic drugs on locomotor activity using Actophotometer in animals receiving

high fat diet (HFD) .

Group-1

Group-2

Group-3

Group-4

Group-5

Group-6

Group-7

Group-8

Group-9

Group-10

Group-110

100

200

300

400

500InitialDay-1

Day-7Day-14

Activ

ity Sc

ore (N

os)


110

6.2.5. Effect of hypocholesteremic drugs on Brain

Aceylcholinesterase levels:

High fat diet treated animals showed elevated levels of acetylcholinesterase

indicating its memory reducing potential. Rats treated with standard drug

donepezil produced significant (p<0.01) reduction of acetylcholinesterase

enzyme activity in comparison with negative control. Administration of test

drugs (group 4 to 7) showed reduction in enzyme levels compared to that of

negative control group in the order of Simvastatin > rosuvastatin > nicotinic

acid > fenofibrate, however simvastatin alone showed significant (p<0.05)

reduction in enzyme levels than other drugs when administered in

combination with standard drug Donepezil (Table-14).

Overall the combination treatment showed superior effect than that of

individual treatments may be due to their synergistic actions.

111

Table-14: Acetylcholinesterase levels of animals treated with

hypocholesteremic drugs in high fat diet induced rats. Group

No. Treatment & Dose AChE (moles/min/gm)

1 Control (1% Na CMC) 0.028±0.00065

2 Only High Fat Diet (HFD) 0.032±0.0004

3 Donepezil 5 mg/kg 0.014±0.0009**,AAA





8 Donepezil 5 mg/kg + Simvastatin 5 mg/kg 0.017±0.0004*,AA

9 Donepezil 5 mg/kg + Rosuvastatin 5 mg/kg 0.019±0.0005

10 Donepezil 5 mg/kg + Fenofibrate 65 mg/kg 0.022±0.0008

11 Donepezil 5 mg/kg + Nicotinic Acid 85 mg/kg 0.020±0.0008

Each group (n=2), each value represents Mean±SEM. a) Denotes *P<0.05, **P<0.01, and ***P<0.001 (Day-7) &+P<0.05, ++P<0.01, and +++P<0.001 compared to initial basal activity (Day-14). b) Denotes Ap<0.05 compared with HFD-group and Bp<0.05 compared with Donepezil 5 mg/kg at week-14, ANOVA followed by dunnett’s test.

Figure-35: Effect of hypocholesteremic drugs on Brain Acetylcholinesterase levels in animals receiving high fat diet

Group-1Group-2

Group-3Group-4

Group-5Group-6

Group-7Group-8

Group-9

Group-10

Group-110.00

0.01

0.02

0.03

0.04

AChE

(mol/m

in/gm)


112

6.2.6. Lipid Profile:

Rats subjected to high fat diet for 14 weeks showed a significant (P<0.001)

increase in their total cholesterol, triglycerides, LDL, VLDL and decreased

HDL, when compared to normal rats. Treatment with donepezil did not show

marked alteration in lipid profile compared to high fat diet treated rats.

Simvastatin, rosuvastatin, fenofibrate and nicotinic acid treatment produced a

significant (P<0.001) reduction in lipid profile (Table-15).

The combination treatment also produced significant (p<0.001) reduction in

lipid profile but there was no much difference in the activity when compared to

individual treatment (Table-15).

113

Table-15: Changes in lipid profiles upon high fat diet induction at different time points

Group No. Treatment & dose Test Initial Post induction1 Final

1 Control

(1% Na CMC)

HDL mg dl 98.60±2.24 - 103.15±0.99

LDL mg/dl 73.37±7.45 - 131.33±7.82

TC mg/dl 47.63±2.11 - 56.55±0.52

TG mg/dl 36.29±0.8 - 20.33±1.33

VLDL mg/dl 14.67±1.49 - 26.27±1.56

2

HDL mg/dl 106.10±3.41 214.90±6.90*** 217.5±7.85

Only High Fat Diet

(HFD)

LDL mg/dl 91.92±10.71 197.98±22.55*** 190.98±20.33

TC mg/dl 56.88±3.88 27.55±1.33*** 31.48±1.36

TG mg/dl 30.83±2.84 147.75±7.03*** 147.82±9.6

VLDL mg/dl 18.38±2.14 39.60±4.51*** 38.20±4.07

3 Donepezil 5 mg/kg

HDL mg/dl 103.82±4.25 172.92±10.7*** 176.9±9.51AAA

LDL mg/dl 70.90±4.87 119.77±4.49*** 99.95±2.66AAA

TC mg/dl 55.23±2.78 41.28±2.70*** 46.42±2.98AAA

TG mg/dl 34.40±4.47 107.68±11.73*** 110.49±10.60AAA,BBB

VLDL mg/dl 14.18±0.97 23.95±0.90*** 19.99±0.53AAA

114


4 Simvastatin 5 mg/kg

HDL mg/dl 99.30±2.52 204.97±2.29 128.73±2.54A,BB

LDL mg/dl 89.25±2.71 176.60±6.77 129.07±6.88 AAA,BBB

TC mg/dl 46.23±1.16 30.43±1.04*** 38.05±1.39AAA, BBB

TG mg/dl 35.22±2.70 139.21±1.77 64.87±2.17AAA,BBB

VLDL mg/dl 17.85±0.54 35.32±1.35 25.81±1.38AAA,BBB

5 Rosuvastatin 5 mg/kg

HDL mg/dl 102.87±0.75 207.08±1.03 129.88±1.95AAA,BB

LDL mg/dl 75.87±0.77 155.15±2.08 103.10±1.63AAA,BBB

TC mg/dl 57.68±1.11 42.77±0.9*** 54.13±1.41AAA, BBB

TG mg/dl 30.01±1.2 133.29±1.5*** 55.13±2.72AAA

VLDL mg/dl 15.17±0.15 31.03±0.42 20.62±0.33AAA

6 Fenofibrate 65 mg/kg

HDL mg/dl 103.92±1.28 210.63±0.91 131.23±1.75AAA,B

LDL mg/dl 73.75±1.22 144.52±1.47 95.42±2.21AAA,BBB

TC mg/dl 55.67±1.24 39.33±0.91*** 52.48±1.20AAA, BBB

TG mg/dl 33.50±1.99 142.4±1.66*** 59.67±2.65AAA

VLDL mg/dl 14.75±0.24 28.90±0.29 19.08±0.44AAA

115


7 Nicotinic Acid 85

mg/kg

HDL mg/dl 104.17±1.5 217.53±2.21 196.47±2.55AAA

LDL mg/dl 83.62±1.86 169.43±4.51 144.85±6.17AAA

TC mg/dl 55.08±1.89 41.37±2.83*** 50.92±3.76AA, BBB

TG mg/dl 32.36±2.98 142.28±3.97*** 116.58±3.89 AAA,BBB

VLDL mg/dl 16.72±0.37 33.89±0.90 28.97±1.23AAA,BBB

8 Donepezil 5 mg/kg +

Simvastatin 5 mg/kg

HDL mg/dl 101.47±1.06 204.02±4.31 158.77±2.9AAA

LDL mg/dl 63.72±4.03 125.68±7.16 80.43±1.66AAA,B

TC mg/dl 59.95±1.37 37.13±1.44*** 47.00±1.36AAA, BB

TG mg/dl 28.77±1.32 141.75±5.97 95.68±3.97AAA,B

VLDL mg/dl 12.74±0.81 25.14±1.43 16.09±0.33AAA,B


Rosuvastatin 5 mg/kg

HDL mg/dl 104.73±1.76 204.23±3.31 147.78±3.74AAA

LDL mg/dl 69.75±3.57 138.25±5.25 104.00±2.53AAA,BBB

TC mg/dl 56.78±1.34 41.4±1.28*** 50.78±1.57AAA, BBB

TG mg/dl 34.00±1.22 135.18±2.98 76.20±3.51AAA

VLDL mg/dl 13.95±0.71 27.65±1.05 20.80±0.51AAA

116



Fenofibrate 65 mg/kg

HDL mg/dl 102.05±0.97 211.18±1.07 143.15±2.76AAA

LDL mg/dl 92.90±7.44 148.15±3.23 110.15±3.21AAA,BBB

TC mg/dl 54.73±0.71 39.38±0.97*** 47.08±0.90AAA, BBB

TG mg/dl 28.74±1.68 142.17±0.77 74.04±2.05AAA

VLDL mg/dl 18.58±1.49 29.63±0.65 22.03±0.64AAA

11

Donepezil 5 mg/kg +

Nicotinic Acid 85

mg/kg

HDL mg/dl 106.42±2.25 218.63±3.28 150.28±4.4 AAA

LDL mg/dl 72.44±9.03 141.43±4.60 94.57±3.92AAA,BBB

TC mg/dl 55.83±1.54 40.92±1.53*** 47.22±1.21AAA, BBB

TG mg/dl 36.09±2.77 149.43±4.21 84.15±4.54AAA

VLDL mg/dl 14.49±1.81 28.29±0.92 18.91±0.78AAA

Each group (n=6), each value represents Mean±SEM. a) Denotes *P<0.05, **P<0.01, and ***P<0.001 compared to initial lipid profile. b) Denotes Ap<0.05 compared with HFD-group and Bp<0.05 and BBP<0.01 compared with Donepezil 5 mg/kg at week-14, ANOVA followed by Dunnett’s ‘t’ test.

117

Figure-36: Effect of hypocholesteremic drugs on serum lipid profile in animals receiving high fat diet (HFD)

Initia

l TC

Initia

l TG

Initia

l HDL

Initia

l LDL

Initia

l VLD

L

Post In

ducti

on TC

Post In

ducti

on TG

Post In

ducti

on H

DL

Post In

ducti

on LD

L

Post In

ducti

on VLD

L

Final T

C

Final T

G

Final H

DL

Final L

DL

Final V

LDL

0

50

100

150

200

250




Group-10Group-11

mg/

dl


118

6.2.7. Brain histopathology:

Micrographs of brain section of amnesia-induced (HFD) group showed

neuronal edema with marked gliosis and neuronal degeneration. Micrograph

of brain section of amnesia-induced rats treated with donepezil showed mild

histopathological alteration in the basal ganglion. Brain sections of amnesia-

induced rats treated with statins (Group-4 &5) showed mild gliosis of the basal

ganglia. While amnesia-induced rats treated with fenofibrate and nicotinic acid

(Group-6&7) showed moderate edema, gliosis and degeneration in basal

ganglion. The combination treatment also demonstrates superior protective

action of statins than other test drugs.

Results of histopathological studies suggest better neuroprotective action of

statins than that of fenofibrate and nicotinic acid.

119

Brain Histopathology pictures of high fat diet (HFD) induced amnesia

Fig-37: Basal ganglia of normal rat (40X) Fig-38: Basal ganglia of HFD induced rat (40X)


Donepezil in HFD induced amnesia (40X)

Figure-40: Basal ganglia of rat treated with Simvastatin in HFD induced amnesia (40X)

120

Figure-41: Basal ganglia of rat treated with Rosuvastatin in HFD induced amnesia (40X)

Figure-42: Basal ganglia of rat treated with Fenofibrate in HFD induced amnesia (40X)

Figure-43: Basal ganglia of rat treated with Nicotinic acid in HFD induced amnesia (40X)

Figure-44: Basal ganglia of rat treated with Donepezil + Simvastatin in HFD induced amnesia (40X)

121


Donepezil + Rosuvastatin in HFD induced amnesia(40X)

Figure-46: Basal ganglia of rat treated with Donepezil + Fenofibrate in HFD induced amnesia (40X)


Donepezil + Nicotinic acid in HFD induced amnesia(40X)

Fig-48: Cerebellum of normal rat (40X)

122

Fig-49: Cerebellum of rat after HFD induced amnesia (40X)

Fig-50: Cerebellum of rat treated with Donepezil in HFD induced amnesia (40X)


Simvastatin in HFD induced amnesia (40X)

Fig-52: Cerebellum of rat treated with Rosuvastatin in HFD induced amnesia (40X)

123


Fenofibrate in HFD induced amnesia (40X)

Fig-54: Cerebellum of rat treated with Nicotinic acid in HFD induced amnesia (40X)

Fig-55: Cerebellum of rat treated with Donepezil + Simvastatin in HFD induced amnesia (40X)

Fig-56: Cerebellum of rat treated with Donepezil + Rosuvastatin in HFD Induced amnesia induced amnesia (40X)

124


Donepezil + Fenofibrate in HFD induced amnesia (40X)

Fig-58: Cerebellum of rat treated with Donepezil + Nicotinic acid in HFD induced amnesia (40X)

125

DISCUSSION

7. DISCUSSION:

Central cholinergic system plays a crucial role in the process of learning and

memory. Cholinomimetic drugs have been shown to enhance memory,

whereas centrally acting cholinergic antagonists like scopolamine are reported

to impair memory and therefore have been widely used to study the

antiamnesic potential of new drugs (Bartus et al., 1982)145. In the present

study, scopolamine had prominently reduced both acquisition and retention

memory potential compared to normal control rats. The memory potential has

been significantly enhanced by standard drug donepezil in all the tested

models which was evidenced by changes in transfer latency or activity scores

during the treatment period compared to negative control. The test drugs also

produced prominent anti-amnesic effect by altering of scores at both

acquisition and retention trials compared to negative control (scopolamine

treated/HFD) and equivalent or similar effect to that of standard drug

donepezil when administered alone or in combination. The above observed

effects were supported by changes in AChE levels in treatment groups

compare with the negative control group. The current study results upon

repeated administration were in agreement with published reports. Earlier

studies have indicated that activation of muscarinic cholinergic receptors

(mAChRs) led to the activation of protein kinase A with subsequent activation

of downstream protein kinases like MAPK/ERK and PKC which are involved in

acquisition and retrieval of memory (Abel & Lattal, 2001)146. In the present

study, pretreatment with simvastatin, rosuvastatin, fenofibrate and nicotinic

126

acid have reversed scopolamine induced amnesia. Statins are known to

activatePI3K/Akt pathway (Schulz, 2005)147 which ultimately activate PLC to

hydrolyze PIP2 to produce IP3 and DAG (Reddy et al., 2002)148. DAG so

formed activates PKC and IP3 increasing free intracellular Ca2+. The

increased free intracellular Ca2+ is known to increase the release of

acetylcholine from nerve terminals. Moreover increased intracellular Ca2+

increases cAMP levels, which subsequently activates PKA. Therefore,

reversal of scopolamine induced amnesia by simvastatin and rosuvastatin

may be due to the activation of PI3K/Akt pathway. Fibrates like fenofibrate

induces β-oxidation pathway with a concomitant decrease in fatty acid and

triglyceride. Nicotinic acid inhibits triglyceride synthesis by inhibiting

hepatocyte diacylglycerol acyltransferase-2. In this way fenofibrate and

nicotinic acid reduces serum cholesterol levels with subsequent reduction of

cholesterol levels and β-amyloid deposition in the CNS. By this mechanism

fenofibrate and nicotinic acid reversed the amnesic potential of scopolamine.

In combination therapy test drugs with donepezil improved or shown similar

memory potential that of individual treatments. Bhupesh Sharma et al.,

(2008)149 reported antioxidant and anti-beta amyloid aggregatory properties of

pitavastatin enhanced antiamnesic effect of donepezil by facilitating its

anticholinesterase and neuroprotective actions. Hence the superior or similar

action of combination therapy in the present study might be due to the actions

of test drugs and donepezil reinforcing the observations of Bhupesh Sharma.

127

One more important possibility for effect of hypocholesteremic drugs on

memory could be their action on AChE activity. Lane and Farlow (2005)150

stated treatment with simvastatin for seven days had shown significant

decrease in brain AChE activity in male rats. In the present study memory

enhancing action of investigated drugs might also be due to their antiacetyl

cholinesterase activity corroborating the earlier observation.

In the present study, chronic administration (14 weeks) of high fat diet (HFD)

not only produced significant increase in the total serum cholesterol levels, but

also impaired memory. Clinical studies suggested that the net brain

cholesterol concentration is regulated by serum cholesterol level and there is

a cross talk between the CNS and peripheral cholesterol pools (Haley &

Dietschy 2000)151. Therefore, it is plausible that peripheral cholesterol levels

modulate CNS cholesterol levels and vice versa. Cholesterol turnover appears

to play a crucial role in the deposition and clearance of amyloid peptide in

brain. Furthermore, serum cholesterol, atherosclerosis, apolipoprotein-E and

AD all appear to be interconnected (Roher et al., 1999)152. ApoEis a

cholesterol transporting protein that is associated with amyloid deposits

(Hofman et al., 1997)153. Elevated serum cholesterol levels not only lead to

atherosclerosis but also carry a high risk of developing AD. Epidemiological

studies revealed that individuals with high peripheral cholesterol levels show

more susceptibility to Alzheimer’s disease, and the incidence of AD is higher

in countries with high-fat and high-calorie diets (Spark et al., 1994)154. In a

double-blind study the administration of simvastatin for 6 month period to AD

128

patients was found to decrease CNS beta-amyloid levels (Simon et al.,

2002)155. It has been reported that rats fed with a special diet having higher

amount of fats showed memory deficits (Greenwood and Winocur, 2001)156.

Therefore, HFD-induced memory deficits noted in the present study closely

mimic the clinical manifestations of AD, i.e. cognitive decline, and highlight the

importance of cholesterol in the pathophysiology of AD. A significant rise in

body weight of rats observed after 14 weeks of HFD administration. In the

present study, treatment of test drugs did not significantly altered the body

weights. Statins showed significant effect in reversing the HFD induced

amnesia than fenofibrate and nicotinic acid in all the tested models. This may

be though modifying the lipid profiles upon chronic administration for two

weeks which was indicated by improvements noted with transfer latency and

activity scores compared to HFD group or standard drug donepezil when

administered alone or in combination. Also it could be correlated with

alteration in lipid profiles contributed by hypocholesteremic drugs to that

compared with standard drug which has not altered the lipid profile as

expected. Fenofibrate and nicotinic acid might have shown effect by

preventing synthesis of peripheral cholesterol and consequent reduction of

CNS cholesterol levels and β-amyloid deposition.

However, anti-inflammatory and anti-oxidant actions of statins to enhance

memory cannot totally be ignored at this point and might have contributed to

the beneficial effect on memory. Simvastatin, rosuvastatin, fenofibrate and

nicotinic acid successfully reversed memory deficits induced by scopolamine

129

and high fat diet through their cholesterol dependent as well as cholesterol

independent effects.

Histopathological studies revealed the neuroprotective action of selected

hypocholesteremic drugs indicated by less or absence of gliosis and

degeneration of basal ganglionic cells and cerebellum. These studies further

corroborated the results obtained from in vivo studies performed.

130

CONCLUSIONS

8. CONCLUSIONS

Scopolamine and HFD significantly reduced memory marked by increased

transfer latency and activity scores. The extent of change observed in these

parameters in two different models indicated their different mechanism of

action. The selected hypocholesteremic drugs evaluated in the current study

successfully restored TL indicating their ameliorative role in cognitive

dysfunctions. The study indicated that the tested drugs have potential

therapeutic intervention for cognitive disorders like AD.

Donepezil (standard) produced significant anti-amnesic effect by inhibiting the

enzyme acetylcholinesterase (AChE) with subsequent increase in the levels of

acetylcholine. The activity produced by the test drugs was comparable to that

of standard donepezil. Hence they were found to be promising in enhancing

the learning and memory compared with that of standard drug.

The treatment with hypocholesteremic drugs found to diminish cholinesterase

activity. This activity was found to be significant compared to donepezil, a

well-known anticholinesterase drug. The data from the current study supports

cholesterol independent effect of the test drugs in rats and further studies

recommended in higher species to prove it further.

Histopathological studies supported protective effects of hypocholesteremic

drugs in brain especially the areas regulating cognitive functions. It

demonstrates potential neuroprotective effects which might be due to their

antioxidant potential.

131

The selected drugs may represent new therapeutic approaches for

intervention of the progressive neurological damage associated with

Alzheimer’s disease.

In conclusion, the findings of this thesis have helped in identifying potential

modulators of AD pathogenesis and will provide concurrent insights towards

conceptualizing effective therapeutic outcomes from the strategy for effective

prophylaxis of this devastating disorder.

132

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155

ANNEXURES

PUBLICATIONS

1001 Srinivasa Rao T. et al. / International Journal of Biological & Pharmaceutical Research. 2015; 6(12): 1001-1010.

e- ISSN 0976-3651

Print ISSN 2229 - 7480

International Journal of Biological

&

Pharmaceutical Research Journal homepage: www.ijbpr.com

NOOTROPIC EFFECT OF HYPOCHOLESTEROLEMIC DRUGS IN

SCOPOLAMINE INDUCED AMNESIC RATS

T. Srinivasa Rao1*

, S. Kavimani2, S. Sudhakaran

3, P. Veeresh Babu

4

1Research Scholar, Vinayaka Missions University, Salem, Tamil Nadu, India.

2Mother Theresa Post Graduate and Research Institute of Health Sciences, Puducherry, India.

3Teegala Ram Reddy College of Pharmacy, Hyderabad, Telangana, India.

4Gokaraju Rangaraju College of Pharmacy, Bachupally, Hyderabad, Telangana, India.

ABSTRACT

Age induced dementia as a result of progressive neurodegenerative disorders like Alzheimer’s disease (AD) has

several causes and increased cholesterol turnover, amyloid deposition and oxygen free radical mediated cell injuries are mainly

implicated ones. Hypocholesterolemic drugs along with cholesterol lowering property show many pleiotropic effects and may

help in this condition. The current study was conducted to evaluate the effect of simvastatin, rosuvastatin, fenofibrate and

nicotinic acid on learning and memory in amnesic rats. Scopolamine induced amnesia served as interoceptive memory model

whereas, elevated plus-maze, rectangular maze and locomotor activity served as exteroceptive models. The tested

hypocholesterolemic drugs significantly attenuated scopolamine induced amnesia in the tested models, reduced

acetylcholinesterase levels and the neurodegenerative changes observed at basal ganglia. The improvement in learning and

memory may be attributed to their cholesterol dependent and independent actions.

Key Words: Amnesia, Hypocholesterolemic drugs, Memory, Elevated plus-maze, Rectangular maze, Actophotometer.

INTRODUCTION

Dementia is a syndrome of progressive nature

marked by gross behavioral and personality disturbances.

This syndrome occurs in Alzheimer’s disease (AD),

cerebrovascular disease (i.e., multi-infarct dementia), and

other conditions primarily or secondarily affecting the

brain. Accumulation of peptide β-amyloid, in brains of AD

patients leads to neurotoxicity and neurodegeneration

(Corder EH et al., 1993; Bales KR et al., 1997).

Observational studies have revealed elevated serum

cholesterol level as an important risk factor for

Alzheimer’s disease (Jarvic GP et al., 1995), suggesting a

pathophysiologic relation between β-amyloid and

cholesterol levels. Cell culture and in vivo animal studies

Corresponding Author

T. Srinivasa Rao Email: [email protected]

have shown that reducing cholesterol can inhibit β-amyloid

synthesis (Sparks DL et al., 2002).

Statins are widely prescribed as cholesterol

lowering drugs, which act by inhibiting hydroxy-methyl-

glutaryl co-enzyme A (HMG Co-A) reductase, (the rate

limiting enzyme in cholesterol biosynthesis) for the

treatment of dyslipidemias. Statin therapy is associated

with distinct advantages as well as disadvantages in human

beings. The major advantages observed with statin therapy

include anti-inflammatory action, antithrombotic effect,

antioxidant activity, improvement of endothelial

dysfunction (Tandon V et al., 2005; Liao James K & Laufs

U, 2005; Spark DL et al., 2004) etc. In addition they have

also been shown to possess some benefits in cognitive

impairment. Epidemiological studies have suggested that

individuals above 50 years of age, who were receiving

statins had a substantially lowered risk of developing

dementia, independent of the presence or absence of

IJBPR


untreated hyperlipidemia, or exposure to non-statin lipid-

lowering drugs (Vaughan CJ, 2003;Jick H et al., 2000;

Austen B et al., 2002; Jakob A et al., 2006). On the other

hand, the major disadvantages associated with statin

therapy include rhabdomyolysis (Parle M et al., 2006),

immuno-suppression (Tandon V et al., 2005; Liao James K

& Laufs U, 2005)as well as cholesterol inhibition

(Wagstaff LR et al., 2003).There are conflicting

observations regarding the effect of statins on cognitive

functions. Although, there are a few studies showing

cognitive decline (Wagstaff LR et al., 2003), some studies

showing no effect on memory (Bayten SH et al., 2006;

Muldoon MF et al., 2000), but several studies suggest

improvement of cognitive functions with statin therapy. In

previous studies, ameliorative effects of simvastatin,

atorvastatin and pitavastatin in experimental amnesia was

reported (Sharma M et al., 2008). However, differential

effects of various hypocholesterolemic drugs on

modulation of experimental amnesia remain to be

elucidated. Therefore, the present study was designed to

investigate the differential effects of simvastatin,

rosuvastatin, fenofibrate and nicotinic acid on scopalamine

induced memory deficits in rats.

MATERIALS AND METHODS

Animals: Wistar rats of either sex, weighing around 200-

250 gm were employed in the present study. The animals

were exposed to alternate light and dark cycle of 12 h and

had free access to food and water. The animals were

acclimatized to the laboratory conditions for at least seven

days prior to the behavioral test. Experimental protocol

was approved by the institutional animal ethics committee

(IAEC). Care of the animals was taken as per guidelines of

committee for the purpose and control supervision of

experiments on animals (CPCSEA), Ministry of forests and

environment, government of India.

Drugs: Simvastatin (Artemis Biotech, Hyderabad),

Rosuvastatin (MSN Laboratories Ltd., Patancheru),

Fenofibrate (Alembic Limited, Vadodara), Nicotinic acid

(Dr. Reddy’s, Hyderabad), Donepezil (Dr. Reddy’s,

Hyderabad) and Scopolamine (Sigma Chemicals) were

procured. Simvastatin, Rosuvastatin, Fenofibrate, Nicotinic

acid and Donepezil were suspended in 1% w/v sodium

carboxy methyl cellulose (NaCMC) for oral

administration. Scopolamine was dissolved in distilled

water for intraperitoneal administration

Experimental Design

The animals were divided into 11 groups. Each

group comprised of 6 animals.

Group Treatment Dose (mg/Kg)


2 Scopolamine 3

3 Scopolamine + Donepezil 3+5

4 Scopolamine + Simvastatin 3+5

5 Scopolamine + Rosuvastatin 3+5

6 Scopolamine + Fenofibrate 3+65

7 Scopolamine + Nicotinic

acid

3+85

8 Scopolamine + Donepezil +

Simvastatin

3+5+5


Rosuvastatin

3+5+5


Fenofibrate

3+5+65


Nicotinic acid

3+5+85

LABORATORY MODELS Exteroceptive Behavioral Models: a) Elevated plus-maze,

b) Rectangular maze and c) Actophotometer.

Elevated Plus-maze (EPM): The EPM served as an

exteroceptive behavioural model to evaluate learning and

memory in rats (Itoh J et al., 1991; Reddy DS &Kulkarni

SK, 1998). Transfer latency (TL) is a parameter of memory

which was defined as the time in seconds taken by the

animal to move intoone of the closed arms with all its four

legs(Itoh J et al., 1991; Dhingra D et al., 2003). The

training trials were carried out for three days before

initiation of behavioral study and the average was taken as

basal score. During behavioral study the animal was placed

at the end of an open arm facing away from the central

platform, the time taken to place all the four paws in the

closed arm as noted as TL. A maximum of 120 seconds

was given for the animal to explore closed arm, the animal

who failed to explore the closed arm within 120 seconds

was given a score of 120. The animal was allowed to

explore the maze for 15s and then returned to its home

cage. Behavioral study was carried for 14 days, where the

animals were administered with vehicle or test drugs

(doses in mg/kg, p.o.) on daily basis for 14 days,

scopalamine (3 mg/kg, i.p.) (Ebert U &Kirch W, 1998;

Klinkenberg I & Blokland A, 2010) was administered 30

min after administration of test drugs on days of

acquisition. Days 1, 3, 5, 7, 9, 11, 13 served the

acquisition trial (AT) and days 2, 4, 6, 8, 10, 12, 14 served

as retention trial (RT). RT was performed 24 hrs after

scopalamine administration. TL was recorded for all

animals one hour after the administration of test

compounds, animals were allowed to explore the maze.

Rectangular maze: The maze consists of completely

enclosed rectangular box with an entry (A) and reward

chamber (B) appended at opposite ends. The box is

partitioned with wooden slats into blind passages leaving

just twisting corridor (C) leading from the entry (A) to the

reward chamber (B) (Agarwal A et al., 2002). The learning

assessment for control and treated rats was conducted at

end of treatment. On the first day, all the rats were

familiarized with the rectangular maze for a period of ten


minutes followed by the training trials were carried out for

three days before initiation of behavioral study. In each

trial the rats were placed in the entry chamber and the

timer was activated as soon as the rat leave the chamber

(Saxena Vasundhara et al., 2013), time taken by the rat to

reach the reward chamber (transfer latency (TL)) was taken

as the learning score of the trial. The average of three trials

was taken as the learning score. Lower scores of

assessment indicate efficient learning while higher scores

indicate poor learning in animals. During learning

assessment the animals were exposed to food and water ad

libitum only for 1 hour after the maze exposure for the day

was completed to ensure motivation towards reward area

(B) (Dhingra D et al., 2004; Joshi H & Parle M, 2006) .

During behavioral study the animal was placed at

an entry chamber (A), the time taken by the animal to

reach the reward chamber (B) (TL) was recorded. A

maximum of 10min was given for the animal to explore the

reward chamber (B), the animal who failed to explore the

B within 10 min was given a score of 600 and the animals

were returned to its home cage. Behavioral study was

carried for 14 days, where the animals were administered

with vehicle or test drugs (doses in mg/kg, p.o.) on daily

basis for 14 days, scopolamine (3 mg/kg, i.p.) (Ebert U

&Kirch W, 1998; Klinkenberg I & Blokland A, 2010) was

administered 30 min after administration of test drugs on

days of acquisition. Days 1, 3, 5, 7, 9, 11, 13 served the

acquisition trial (AT) and days 2, 4, 6, 8, 10, 12, 14 served

as retention trial (RT). RT was performed 24 hrs after

scopolamine administration. TL was recorded for all



Actophotometer: This method aims to evaluate the

locomotor activity of the control and treated animals. The

locomotor activity will be measured using actophotometer

(Pragati Khare et al., 2014).Each animal will be placed

individually in the actophotometer for 3 min and the basal

activity score will be obtained. Subsequently, the animals

were divided into 11 groups, each group consisting of six

animals. The training trials were carried out for three days

before initiation of behavioral study, the average was taken

as basal activity score. It was followed by behavioral study

which was conducted for 14 days and recording of activity

score was noted as described earlier (Kirti Kulkarni S et

al., 2010). The animals were administered with vehicle or

test drugs (doses in mg/kg, p.o.) on daily basis for 14 days,

scopolamine (3 mg/kg, i.p.) (Ebert U &Kirch W, 1998;

Klinkenberg I &Blokland A, 2010) was administered 30

min after administration of test drugs on days of

acquisition. Days 1, 3, 5, 7, 9, 11, 13 served the acquisition

trial (AT) and days 2, 4, 6, 8, 10, 12, 14 served as retention

trial (RT). RT was performed 24hrs after scopolamine

administration. Basal activity score was recorded for all



Estimation of cholinesterase enzyme: The cholinergic

marker, acetyl cholinesterase was estimated in the whole

brain. Briefly, the brains of the rats were removed over ice

and the brains were separated using fine forceps. The tissue

was then homogenized in 0.03 M sodium phosphate buffer,

pH 7.4. 25 µl of this homogenate will be incubated for 5

min with 75 µl of Tris HCl and 75 µl of DTNB (Ellman

GL et al., 1961; Pramodinee Kulkarni D et al., 2011).

Then, 0.1ml of freshly prepared acetyl thiocholine iodide,

pH 8 will be added and the absorbance will be read at 412

nm.

Brain histopathology: After the treatment and behavioral

studies, two animals in each group were sacrificed by

excessive ether anesthesia and the brains were isolated and

were kept in 10% formaldehyde solution. The brain was

stained with cresyl violet, cerebellum and basal ganglia

were studied under light microscope (Ashutosh Agarwal et

al., 2002).

Statistical Analysis: All the results were expressed as

mean ± SEM. The data were analyzed using two way

analysis of variance (ANOVA) followed by

Dunnet’sDunnett’s test. Significance was observed at

*P<0.05, **P<0.01 & ***P<0.001.

RESULTS

Effect of hypocholesterolemic drugs on scopolamine

induced amnesia in rats using Elevated plus-maze:

Donepezil significantly (P<0.001) reduced total latency

compared to control (Group-1).Simvastatin (Group-4),

rosuvastatin (Group-5), fenofibrate (Group-6) and nicotinic

acid (Group-7) produced significant effect (P<0.001) on

transfer latency (TL) compared to standard donepezil.

Simvastatin (Group 8), rosuvastatin (Group 9), fenofibrate

(Group 10) and nicotinic acid (Group 11) administered

with donepezil after amnesic agent, significantly (p<0.001)

attenuated scopolamine induced increase in TL measured.

The reduction in TL observed with the combination of

above compounds with donepezil was 23% better than that

of compounds alone. These observations suggested that

simvastatin, rosuvastatin, fenofibrate and nicotinic acid had

reversed scopolamine induced amnesia.


induced amnesia in rats using Rectangular maze: The

learning scores (Transfer latency) obtained by each group

were suggestive of the fact that rats took lesser time on day

8 than day 7 and also on day 14 than day 13. The transfer

latency score obtained by Group 1 (Positive control) was

higher than those afforded by Groups 4-7 indicating better

and efficient learning in them, as compared to the positive

control. Group 2 (Negative control group) (P<0.001) and

owed an increase in transfer latency score due to the

memory deficit induced by scopolamine. The transfer

latency scores observed for the standard donepezil (Group


3) are indicative of a significant (P<0.001) memory

enhancing potential compared to scopolamine treated

group (Group 2). Similarly simvastatin (Group 4),

rosuvastatin (Group 5), fenofibrate (Group 6) and nicotinic

acid (Group 7) afforded a significant reduction in transfer

latency (P<0.001) compared with the standard donepezil.

Administration of these hypocholesterolemic drugs along

with donepezil afforded 35.77% better learning scores than

that of the above drugs alone.


induced amnesia in rats using Actophotometer: Rats

subjected to scopolamine (Group 2) showed a marked

reduction in basal activity score when compared to normal

(Group 1) rats. Standard donepezil treated rats showed

significant (P<0.01) improvement in basal activity score

compared to that of scopolamine treated rats (Group 2).

Hypocholesterolemic drugs, when administered after

scopolamine, simvastatin (Group 4), rosuvastatin (Group

5), fenofibrate (Group 6) and nicotinic acid (Group 7)

slightly augmented basal activity score compared to

scopolamine. The enhancement in locomotor activity

produced by simvastatin and rosuvastatin was 18% better

than that of fenofibrate and nicotinic acid. The

enhancement of basal activity score when

hypocholesterolemic drugs administered along with

donepezil was 8% better than that of above drugs when

administered alone.

Effect of hypocholesterolemic drugs on

Acetylcholinesterase activity: Scopolamine treated animals

(Group 2) showed elevated levels of acetylcholinesterase

compared to control (Group 1) indicating its memory

reducing potential. The animals treated with standard drug

donepezil (Group 3) produced significant (P<0.001)

reduction of acetylcholinesterase enzyme activity in

comparison with scopolamine treated animals (Group 2).

In the treatment groups, the animals exposed to

scopolamine and treated with simvastatin (Group 4),

rosuvastatin (Group 5), fenofibrate (Group 6) and nicotinic

acid (Group 7) decreased the acetylcholinesterase activity

indicating their memory enhancing effects. The activity of

rosuvastatin and nicotinic acid was very close to that of

standard donepezil and 33% better than that of simvastatin

and fenofibrate. There was reduction in

acetylcholinesterase activity when hypocholesterolemic

drugs were administered along with donepezil and it was

27 % better than that of above drugs when administered

alone.

Brain Histopathology: Microscopic examination of brain

sections of negative control (Group-1) showed normal

cerebellum. The molecular layer and purkinjiec cells were

unremarkable. The basal ganglion showed normal

morphology. There was no neuronal edema or

degeneration or gliosis. Micrographs of brain section of

amnesia-induced group showed severe neuronal edema

with marked gliosis and neuronal degeneration (Group

2).The brain section of amnesia-induced rats treated with

donepezil (Group 3) showed very mild histopathological

alteration in the basal ganglion. Micrograph of brain

sections of amnesia-induced rats treated with simvastatin

(Group 4) and rosuvastatin (Group 5) showed mild gliosis

of the basal ganglia. While amnesia-induced rats treated

with fenofibrate (Group 6) and nicotinic acid (Group 7)

showed moderate edema, gliosis and degeneration in basal

ganglion. Micrograph of brain sections of amnesia-induced

rats treated with simvastatin (Group 8) and rosuvastatin

(Group 9) along with donepezil showed mild gliosis

otherwise normal in basal ganglia. Amnesia-induced rats

treated with fenofibrate (Group 10) and nicotinic acid

(Group 11) showed moderate neuronal edema and gliosis

in basal ganglia.

Table 1. Effect of hypocholesterolemic drugs on transfer latency using Elevated plus-maze Group

No.

Treatment & Dose Initial (Sec.) Day-1 (Sec.) Day-2 (Sec.) Day-7 (Sec.) Day-8 (Sec.) Day-13 (Sec.) Day-14 (Sec.)

1 Control (1% Na CMC) 39.89±7.76 23.5±6.48 14±1.51 9.17±2.33 18.33±5.43 16.83±2.64 15.33±3.78

2 Only High Fat Diet

(HFD) 32.44±5.66 94.83±8.21 81.17±9.54 119.5±0.50 108.67±8.48 120.00±0.00 119.83±0.17

3 Donepezil 5 mg/kg 26.33±2.01 39.33±16.85 15.67±5.40 35.17±5.61*** 15.67±3.03××× 12.83±1.78+++ 7.67±1.26˄˄˄

4 Simvastatin 5 mg/kg 42.22±6.51 81.33±14.00 60.17±13.15 83.5±7.6*,AAA 28.67±7.95××× 25.5±4.30+++ 13.5±2.93˄˄˄

5 Rosuvastatin 5 mg/kg 56.94±10.08 74.5±12.02 34.17±4.40 71.17±9.05***,A 25.5±3.69××× 25.33±1.41+++ 12.5±1.95˄˄˄

6 Fenofibrate 65 mg/kg 23.83±3.64 79.5±13.34 45.83±8.06 78±5.05**,AA 40.5±6.98×××,ᴛ 28.83±3.59+++ 10.83±1.35˄˄˄

7 Nicotinic Acid 85 mg/kg 28.72±4.50 64.33±6.86 20.83±4.76 37±11.79*** 16.5±3.51××× 18±3.83+++ 9± 2.34˄˄˄


Simvastatin 5 mg/kg 27.72±3.54 59.33±10.37 32.67±7.68 75.33±5.65**,AA 24.17±5.74××× 24.67±6.09+++ 17.83±7.11˄˄˄


Rosuvastatin 5 mg/kg 30.56±5.39 59.33±12.36 21.33±10.43 32.83±7.91*** 23.83±3.89××× 17.33±3.37+++ 6.67±1.17˄˄˄


Fenofibrate 65 mg/kg 31.56±8.60 57.17±8.45 20.33±4.86 37.5±11.43*** 15.17±4.22××× 18.33±3.48+++ 9.17±1.85˄˄˄


Nicotinic Acid 85 mg/Kg 19.72±4.76 55.83±6.77 31.33±8.07 33.17±9.56*** 16±4.37××× 16±3.09+++ 8.5±1.52˄˄˄



a) Denotes *P<0.05, **P<0.01, and ***P<0.001 compared to control(Day-7) &AP<0.05, AAP<0.01, and AAAP<0.001 compared to scopolamine treated group

(Day-7). b) Denotes ×××p<0.001 compared with control and ᴛ p<0.05 compared with scopolamine treated group (Day-8)

c) Denotes +++P<0.001 compared to control group (Day-13)

d) Denotes ˄˄˄P<0.001 compared to control group (Day-14).ANOVA followed by dunnett’s test.

Table 2.Effect of hypocholesterolemic drugs on transfer latency using Rectangular maze Group

No. Treatment & Dose

Initial

(Sec.) Day-1 (Sec.) Day-2 (Sec.) Day-7 (Sec.) Day-8 (Sec.) Day-13 (Sec.) Day-14 (Sec.)

1 Control (1% Na CMC p.o) 53.56±22.79 117±52.39 61.17±30.19 145.67±21.84 117.33±28.46 82.17±15.86 63.33±11.95

2 Only High Fat Diet (HFD) 63.28±20.27 336.17±57.54 295.67±51.09 558.67±25.21 420.67±67.04 600±0 600±0

3 Donepezil 5 mg/kg 55.67±9.49 90.83±20.23 43.5±4.61 115.17±16.79*** 67±12.46××× 39.33±7.51+++ 21.67±3.56˄˄˄

4 Simvastatin 5 mg/kg 91.67±13.11 272±44.17 174.83±10.41 154.5±24.46*** 145±24.8×××,ᴛ 95.5±4.16+++ 42.17±10.32˄˄˄

5 Rosuvastatin 5 mg/kg 91.33±10.05 262.33±79.76 168.17±71.55 161.67±21.96*** 99±20.91××× 94.5±13.53+++ 47.17±8.98˄˄˄

6 Fenofibrate 65 mg/kg 71.06±30.48 235.33±41.6 164±25.21 142±16.04*** 103.17±8.51××× 104.17±7.27+++ 59±11.4˄˄˄

7 Nicotinic Acid 85 mg/kg 89.83±16.89 291.5±46.32 155.83±12.05 131±9.73*** 86.83±10.59××× 81±4.12+++ 50.33±11.43˄˄˄

8 Donepezil 5 mg/kg + Simvastatin 5 mg/Kg

72.5±8.35 276.5±28.8 168.33±16.46 161.5±20.11*** 94.5±18.53××× 96.5±4.89+++ 46.5±6.08˄˄˄


Rosuvastatin 5 mg/Kg 100.89±13.0

4 208.83±7.19 164.67±12.06 111.5±8.85*** 67.17±6.71××× 62.67±10.57+++ 29.67±12.10˄˄˄

10 Donepezil 5 mg/kg + Fenofibrate 65 mg/Kg

58.22±21.89 249.33±33.79 179±26.06 127.17±15.57*** 99.67±5.66××× 85.33±10.53+++ 59.33±14.36˄˄˄


Nicotinic Acid 85 mg/Kg 46.67±4.07 224±36.07 129.33±9.13 109.17±4.38*** 47.83±3.5××× 43±1.83+++ 33±4.73˄˄˄


a) Denotes *P<0.05, **P<0.01, and ***P<0.001 compared control group (Day-7).

b) Denotes ×××p<0.001 compared with control group and ᴛ p<0.05 compared with Donepezil 5 mg/kg (Day-8). c) Denotes +++P<0.001 compared to control (Day-13)

d) Denotes ˄˄˄P<0.001 compared to control (Day-14).ANOVA followed by dunnett’s test.

Table 3. Effect of hypocholesterolemic drugs on locomotor activity using Actophotometer

Group

No. Treatment & Dose

Initial(Nos.) Day-1(Nos.) Day-2(Nos.) Day-7(Nos.) Day-8(Nos.) Day-13(Nos.) Day-14(Nos.)

1 Control (1% Na CMC

p.o) 303.22±24.4 287.33±38.24 310.5±43.74 342±20 374.5±10.27 333.33±15.40 377.17±9.46

2 Only High Fat Diet (HFD)

291.56±16.31 225.67±21.8 260.17±22.59 174.67±17.77 218.17±19.33 106.50±15.50 146±19.92

3 Donepezil 5 mg/kg 334.28±16.59 326.5±19.33 362±16.59 278.33±15.07** 330.83±13.2×× 213.00±15.98++ 261.5±17.44˄˄

4 Simvastatin 5 mg/kg 304.17±27.34 278.33±12.88 313±9.83 202.67±18.21 274.67±38.79 159.83±21.53 180.5±15.49

5 Rosuvastatin 5 mg/kg 234.28±25.81 277±15.24 300.33±15.35 217.17±15.93 258.67±19.1 151.50±20.98 182.67±23.05

6 Fenofibrate 65 mg/kg 332.56±20.38 262.5±37.56 249.5±12.5 202.17±23.46 270.33±12.47 153.50±3.910 207.33±5.43

7 Nicotinic Acid 85 mg/kg 288.33±17.5 292±14.03 311±17.08 239.33±13.12 271.17±39.82 161.83±21.14 231.17±25.84


Simvastatin 5 mg/Kg 285.94±29.64 272.33±19.38 309.83±25.16 187.67±25.86A 249.67±41.14ᴛ 150.33±15.42 178.17±11.6

9 Donepezil 5 mg/kg + Rosuvastatin 5 mg/Kg

290.39±4.7 320.83±38.3 349.67±41.67 223.17±6.56 274.83±31.02 179.00±11.56 254.83±15.64


Fenofibrate 65 mg/Kg 226.56±11.52 241±26.32 288.33±28.46 191.83±17.51 276.17±25.14 127.33±22.39 199.17±21.73


Nicotinic Acid 85 mg/Kg 259.39±13.99 268.5±25.52 304.17±26.21 219.67±35.24 272.5±22.96 188.83±25.95 236.17±13.67


a) Denotes **P<0.01 compared to control group (Day-7) &AP<0.05 compared to Donepezil 5 mg/kg initial total latency (Day-7). b) Denotes ××p<0.01 compared with control group and ᴛp<0.05 compared with Donepezil 5 mg/kg at (Day-8)

c) Denotes ++P<0.01 compared to control group (Day-13)

d) Denotes ˄˄P<0.01 compared to control group (Day-14). ANOVA followed by dunnett’s test.

Table 4. Effect of hypocholesterolemic drugs on brain Acetylcholinesterase levels

Group No. Treatment & Dose AChE (moles/min/gm)

1 Control (1% Na CMC p.o) 0.028±0.00065


3 Donepezil 5 mg/kg 0.016±0.00095







8 Donepezil 5 mg/kg + Simvastatin 5 mg/Kg 0.019±0.00069

9 Donepezil 5 mg/kg + Rosuvastatin 5 mg/Kg 0.015±0.00054A

10 Donepezil 5 mg/kg + Fenofibrate 65 mg/Kg 0.016±0.00037

11 Donepezil 5 mg/kg + Nicotinic Acid 85 mg/Kg 0.012±0.00047**,AAA Each group (n=2), each value represents Mean±SEM.

a) Denotes **P<0.01compared to control group

b) Denotes Ap<0.05, AAP<0.01 and AAAP<0.001 compared with Donepezil 5 mg/kg ANOVA followed by dunnett’s test.

Figure 1. Effect of hypocholesterolemic drugs on transfer latency using Elevated plus-maze in Scopolamine treated animals.


Figure 2. Effect of hypocholesterolemic drugs on transfer latency using Rectangular maze in Scopolamine treated animals.


Figure 3. Effect of hypocholesterolemic drugs on locomotor activity using Actophotometer in Scopolamine treated animals.



Figure 4. Effect of hypocholesterolemic drugs on brain Acetylcholinesterase levels in Scopolamine treated animals.


Brain Histopathology pictures of Scopolamine induced amnesia in Rats

Figure 5. Basal ganglia of normal rat

Figure 6. Basal ganglia of rat after Scopolamine induced amnesia

Figure 7. Basal ganglia of rat treated with Donepezil in


Figure 8. Basal ganglia of rat treated with Simvastatin in


Figure 9. Basal ganglia of rat treated with Rosuvastatin in


Figure 10. Basal ganglia of rat treated with Fenofibrate in



Figure 11. Basal ganglia of rat treated with Nicotinic acid in


Figure 12. Basal ganglia of rat treated with

Donepezil+Simvastatin in Scopolamine induced amnesia


Donepezil+Rosuvastatin in Scopolamine induced amnesia


Donepezil+Fenofibrate in Scopolamine induced amnesia

Figure 15. Basal ganglia of rat treated with Donepezil+Nicotinic acid in Scopolamine induced amnesia

DISCUSSION

Central cholinergic system plays a crucial role in

the process of learning and memory. Cholinomimetic drugs

have been shown to enhance memory, whereas centrally

acting cholinergic antagonists like scopolamine are

reported to impair memory and therefore have been widely

used to study the antiamnestic potential of new drugs. In

the present study, scopolamine has produced impairment of

both, learning ability and retention capacity (memory),

which is in agreement with previous reports.

Simvastatin, rosuvastatin, fenofibrate and

nicotinic acid have reversed scopolamine induced

augmentation of transfer latency and reduction in basal

activity score. This activity of statins might be due to

activation of PI3K/Akt pathway (Schulz R, 2005) which

ultimately activate PLC to hydrolyze PIP2 to produce IP3

and DAG (Reddy PL et al., 2002). DAG so formed

activates PKC and IP3 increasing free intracellular Ca2+

(Orban PC et al., 1999). The increased free intracellular

Ca2+

is known to increase the release of acetylcholine from

nerve terminals (Chen YQ et al., 1998). Therefore, reversal

of scopolamine induced amnesia by simvastatin and

rosuvastatin may be due to the activation of PI3K/Akt

pathway. Fibrates like fenofibrate induces β-oxidation

pathway with a concomitant decrease in fatty acid and

triglyceride synthesis (Bart Staels et al., 1998). Nicotinic


acid inhibits triglyceride synthesis by inhibiting hepatocyte

diacylglycerol acyltransferase-2 (Vaijinath Kamanna S &

Moti Kashyap L, 2008). In this way fenofibrate and

nicotinic acid reduces serum cholesterol levels with

subsequent reduction of cholesterol levels and β-amyloid

deposition in the CNS. By this mechanism fenofibrate and

nicotinic acid might have reversed the amnesic potential of

scopolamine. The tested drugs were also found to decrease

the acetylcholinesterase levels. This activity of the above

compounds might also contribute to their memory

enhancing effect by decreasing the metabolism of

acetylcholine.

Histopathological study further corroborated the

result. The photomicrographs of brain sections treated with

hypocholesterolemic drugs showed mild gliosis and lack of

neurodegenaration of basal ganglia. It indicated

neuroprotective role of hypocholesterolemic drugs.

Hypocholesterolemic drugs successfully reversed

memory deficits induced by scopolamine through their

cholesterol dependent and independent effects.

CONCLUSIONS

The current study concluded that

hypocholesterolemic drugs significantly ameliorate the

cholinergic dysfunction and inflammation-induced

neurodegeneration in scopolamine induced amnesia

characterizing Alzheimer’s disease. Noteworthy,

hypocholesterolemic drugs revealed more pronounced

modulatory effect on most of the measured physical and

biochemical parameters as well as histological feature of

the brain. The successful reversal of memory deficits

induced by scopolamine might be through their cholesterol

dependent and independent effects. However, anti-

inflammatory and antioxidant actions of

hypocholesterolemic drugs can not totally be ignored at

this point and might have contributed to the beneficial

effect on memory. The selected drugs may represent new

therapeutic approaches for intervention of the progressive

neurological damage associated with Alzheimer’s disease.

ACKNOWLEDGEMENT: None

CONFLICT OF INTEREST:

The authors declare that they have no conflict of interest.

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e- ISSN 0975 – 9328

Print ISSN 2229 – 7472 International Journal of Phytopharmacology

Journal homepage: www.onlineijp.com

AMELIORATIVE ROLE OF HYPOCHOLESTEROLEMIC DRUGS IN

HIGH FAT DIET INDUCED AMNESIA IN RATS

T. Srinivasa Rao1*

, S. Kavimani2, S. Sudhakaran

3, P. Veeresh Babu

4

1Research Scholar, Vinayaka Missions University, Salem, Tamil Nadu, India.

2Mother Theresa Post Graduate and Research Institute of Health Sciences, Puducherry, India.

3Teegala Ram Reddy College of Pharmacy, Hyderabad, Telangana, India.

4Gokaraju Rangaraju College of Pharmacy, Bachupally, Hyderabad, Telangana, India.

ABSTRACT

Investigating the effects of various classes of hypocholesterolemic drugs on memory deficits associated with

Alzheimer’s type dementia in rats can give important clues in development of drugs in this therapeutic area. In this work

learning and memory potential of dementia induced animals was assessed using exteroceptive and interoceptive models,

brain acetylcholinesterase (AChE) activity; lipid profile and histopathological studies. High fat diet (HFD) produced a

significant impairment of learning and memory indicated by increased transfer latency, decreased locomotor activity, higher

levels of AChE activity and lipid profile. The tested hypocholesterolemic drugs significantly attenuated HFD-induced

memory deficits and biochemical changes. Activity of simvastatin and rosuvastatin was found to be better than that of

fenofibrate and nicotinic acid. Histopathology study further corroborated the results. This study demonstrates the potential of

hypocholesterolemic drugs in memory dysfunctions associated with dementia and provides evidence of their cholesterol-

dependent actions.

Key words: Simvastatin, Rosuvastatin, Fenofibrate, Nicotinic acid, High fat diet, Dementia.

INTRODUCTION

Recently, the type of dementia associated with

Alzheimer’s disease (AD) has gained much concern. The

key pathological features in the AD brain are deposition

of insoluble β-amyloid peptides (βA), formation of

neurofibrillary tangles and neuroinflammation that

ultimately leads to neuronal cell death (Bales KR et al.,

1997). Brain cholesterol is an essential component of

neuronal cell membranes and is involved in several

biological functions, such as membrane trafficking, signal

transduction, myelin formation and synaptogenesis

(Valenza M &Cattaneo E, 2006). Given these widespread

activities, it is not surprising that dysfunctions in

cholesterol synthesis, storage, transport and removal can

lead to human brain diseases. In AD, there is a link

between cholesterol metabolism and formation and

Corresponding Author

T. Srinivasa Rao Email: [email protected]

deposition of βA (Korade Z &Kenworthy AK, 2008).

Dementia places an enormous burden on

individuals, families and society. Consequently, a

tremendous effort is being devoted to development of

drugs that prevent or delay neurodegeneration. Clinical

options to contain dementia are limited, and, so far,

acetylcholinesterase (AChE) inhibitors, such as donepezil,

rivastigmine and galantamine, are considered the gold

standard therapy for AD (Yoshida M, 2003).

Very recently, focus has been directed towards

3-hydroxy-3-methylglutaryl coenzyme A [HMG-CoA]

reductase inhibitors, which are better known as statins.

Statins not only lower cholesterol level but also possess

actions that are independent of their cholesterol lowering

property. For example, statins have been demonstrated to

exert potential anti-inflammatory, antioxidant and

neuroprotective actions (Suribhatla S et al, 2005).

Although few studies have reported negative effects of

statins on cognitive functions (Bayten SH et al., 2006), a

IJP


number of recent experimental and clinical reports have

documented positive effects of statins in memory

dysfunctions associated with dementias (Miida T et al.,

2007). In previous studies, ameliorative effects of

simvastatin, atorvastatin and pitavastatin in experimental

amnesia was reported (Sharma B et al., 2008). However,

differential effects of various hypocholesterolemic drugs

on modulation of experimental amnesia remain to be

elucidated. Therefore, the present study was designed to

investigate the differential effects of various classes of

hypocholesterolemic drugs (simvastatin, rosuvastatin,

fenofibrate, and nicotinic acid) on high fat diet induced

memory deficits in rats.

MATERIALS AND METHODS

Animals: Male wistar rats aged about 6 weeks were

employed in the present study. The animals were exposed

to alternate light and dark cycle of 12 h and had free

access to food and water. The animals were acclimatized

to the laboratory conditions for at least seven days prior to

the behavioral test. Experimental protocol was approved

by the Institutional Animal Ethics Committee (IAEC).

Care of the animals was taken as per guidelines of

committee for the purpose and control supervision of

experiments on animals (CPCSEA), Ministry of forests

and environment, government of India.

Drugs: Simvastatin (Artemis Biotech, Hyderabad),

Rosuvastatin (MSN Laboratories Ltd., Patanceru),

Fenofibrate (Alembic Limited, Vadodara), Nicotinic acid

(Dr. Reddy’s, Hyderabad) and Donepezil (Dr. Reddy’s,

Hyderabad) were procured. Simvastatin, Rosuvastatin,

Fenofibrate, Nicotinic acid and Donepezil were

suspended in 1% w/v sodium carboxy methyl cellulose

(NaCMC) for oral administration.

Composition and preparation of high fat diet: High fat

diet contains cholesterol (2%), cholic acid (1%), dalda

(20%) and coconut oil (6%) as the major constituents. Rat

feed was finely powdered and mixed properly with

weighed ingredients. The mixture was spread on to a tray

and baked in oven at 100ᵒC for 1 hour. It was then cut into

small pieces and provided to animals.

Induction of hyperlipidemia: Animals were fed with

prepared high fat diet 100 gm/kg of body weight for 12

weeks. Initial lipid profile of animals was compared with

lipid profile measured after 12 weeks of induction of

hyperlipidemia. Animals that showed elevated lipid

profile were selected for the study.

EXPERIMENTAL DESIGN

The animals were divided into 11 groups. Each

group comprised of 6 animals.

Group Treatment Dose

(mg/kg)


2 Only High Fat Diet -

3 Donepezil 5

4 Simvastatin 5

5 Rosuvastatin 5

6 Fenofibrate 65

7 Nicotinic acid 85

8 Donepezil + Simvastatin 5+5

9 Donepezil + Rosuvastatin 5+5

10 Donepezil + Fenofibrate 5+65

11 Donepezil + Nicotinic acid 5+85

LABORATORY MODELS:

Exteroceptive Behavioral Models: a) Elevated plus-

maze, b) Rectangular maze and c) Actophotometer

Elevated Plus-maze (EPM): The EPM serves as an

exteroceptive behavioural model to evaluate learning and

memory in rats (Itoh J et al., 1991; Reddy DS &Kulkarni

SK, 1998). Transfer latency (TL) is a parameter of

memory which was defined as the time in seconds taken

by the animal to move into one of the closed arms with all

its four legs (Dhingra D et al., 2003; Parle M &Singh N,

2007) .The training trials were carried out for three days

before initiation of behavioural study and treatment with

test drugs, the average was taken as basal score. During

behavioural study the animal was placed at the end of an

open arm facing away from the central platform, the time

taken to place all the four paws in the closed arm has

noted as TL. A maximum of 120 seconds was given to the

animal to explore closed arm, the animal which failed to

explore the closed arm within 120 seconds was given a

score of 120. The animal was allowed to explore the maze

for 15s and then returned to its home cage. Behavioural

study was carried for 14 days, where the animals were

administered with vehicle or test drugs (doses in mg/kg,

p.o.). TL was recorded for all animals one hour after the

administration of test compounds, animals were allowed

to explore the maze.

Rectangular maze: The maze consists of completely

enclosed rectangular box with an entry (A) and reward

chamber (B) appended at opposite ends. The box is

partitioned with wooden slats into blind passages leaving

just twisting corridor (C) leading from the entry (A) to the

reward chamber (B) (Prakash A et al., 2007). The

learning assessment for control and treated rats was

conducted at end of treatment. On the first day, all the rats

were familiarized with the rectangular maze for a period

of ten minutes followed by the training trials were carried

out for three days before initiation of behavioural study.

In each trial animal was placed in the entry chamber and

the timer was activated as soon as the rat leave the

chamber (Agarwal A et al., 2002), time taken by the rat to


reach the reward chamber (transfer latency (TL)) was

taken as the learning score of the trial. The average of

three trials was taken as the learning score. Lower scores

of assessment indicate efficient learning while higher

scores indicate poor learning in animals. During learning

assessment the animals were exposed to food and water

ad libitum only for 1 hour after the maze exposure for the

day was completed to ensure motivation towards reward

area (B) (SaxenaVasundhara et al., 2013; Dhingra D et

al., 2004).

During behavioural study the animal was placed

at an entry chamber (A), the time taken by the animal to

reach the reward chamber (B) (TL) was recorded. A

maximum of 10 min was given for the animal to explore

the reward chamber (B), the animal who failed to explore

the B within 10 min was given a score of 600 and the

animals were returned to its home cage. Behavioural

study was carried for 14 days, where the animals were


p.o.). TL was recorded for all animals one hour after the

administration of test compounds, animals were allowed

to explore the maze.

Actophotometer: This method aims to evaluate the

locomotor activity of the control and treated animals.The

locomotor activity will be measured using actophotometer

(Joshi H &Parle M, 2006). Each animal will be placed

individually in the actophotometer for 3 min and the basal

activity score will be obtained. Subsequently, the animals

were divided into 11 groups, each group consisting of six

animals. The training trials were carried out for three days

before initiation of behavioural study, the average was

taken as basal activity score. It was followed by

behavioural study which was conducted for 14 days and

recording of activity score was noted as described earlier

(PragatiKhare et al., 2014). The animals were


p.o.) on daily basis for 14 days. Basal activity score was

recorded for all animals one hour after the administration

of test compounds, animals were allowed to explore the

maze.

Estimation of cholinesterase enzyme: The cholinergic

marker, acetylcholinesterase was estimated in the whole

brain. Briefly, the brains of the rats were removed over

ice and the brains were separated using fine forceps. The

tissue was then homogenized in 0.03 M sodium phosphate

buffer, pH 7.4. 25 µl of this homogenate was incubated

for 5 min with 75 µl of TrisHCl and 75 µl of DTNB (Kirti

Kulkarni S et al., 2010; Ellman GL et al., 1961). Then,

0.1ml of freshly prepared acetyl thiocholine iodide, pH 8

was added and the absorbance was read at 412 nm.

Estimation of serum biochemical parameters: Total

cholesterol was estimated by CHOD-PAP method,

triglycerides by GPO method and HDL, LDL & VLDL

were estimated by phosphotungstic acid precipitation

method.

Brain histopathology: After the treatment and behavioral

studies, two animals in each group were sacrificed by

excessive ether anesthesia and the brains were isolated

and were kept in 10% formaldehyde solution. The brain

was stained with cresylviolet, cerebellum and basal

ganglia were studied under light microscope (Pramodinee

Kulkarni D et al., 2011).

Statistical Analysis: All the results were expressed as

mean ± SEM. The data were analyzed using two way

analysis of variance (ANOVA) followed by Dunnett’s

test. Significance was observed at *P<0.05, **P<0.01 &

***P<0.001.

RESULTS

Effect of hypocholesterolemic drugs on Body weight:

There was a significant (P<0.001) increase in the body

weight of animals over the period of 12 weeks in rats

receiving high fat diet, when compared to the body

weights of rats on day 1. The rats in all treatment groups

except donepezil -treated group showed marked reduction

in body weight. There was substantial decrease in body

weight of the animals treated with simvastatin (Group 4),

rosuvastatin (Group 5), fenofibrate (Group 6) and

nicotinic acid (Group 7). The reduction in body weight

obtained with rosuvastatin was found to be significant

(P<0.05) than that of simvastatin, fenofibrate and

nicotinic acid. The reduction in TL observed with the

combination of above compounds with donepezil was

12% better than that of compounds alone. The reduction

in body weight obtained with simvastatin (Group 8) and

fenofibrate (Group 10) along with donepezil was found to

be significant (P<0.01) than that of rosuvastatin(Group 9)

and nicotinic acid (Group 11) with donepezil.

Effect of hypocholesterolemic drugs on high fat diet

induced amnesia in rats using Elevated plus-maze:

HFD rats (rats receiving high fat diet for 12 weeks

successively), when, treated with simvastatin (Group 4),

rosuvastatin (Group 5) and fenofibrate (Group 6) for 14

days successively produced a significant (P<0.001)

decrease in TL when compared to TL of HFD rats

respectively. The attenuating effect of simvastatin was

relatively better than that of rosuvastatin and fenofibrate.

The reduction in TL observed with the combination of

above compounds with donepezil was 8% better than that

of compounds alone. The administration of simvastatin

(Group 8), rosuvastatin (Group 9) and nicotinic acid

(Group 11) along with donepezil showed significant

(P<0.001) reduction in TL when compared to that of

fenofibrate (Group 10) with donepezil. These


observations suggested that hypocholesterolemic drugs

had attenuated HFD induced amnesia.


induced amnesia in rats using Rectangular maze: The

learning scores (Transfer latency) obtained by each group

were suggestive of the fact that rats took lesser time on

day 14 compared to high fat diet group. The transfer

latency score obtained by Group 1 (Positive control) was

higher than those afforded by Groups 4-7 indicating better

and efficient learning in them, as compared to the positive

control. Group 2 (Negative control group) showed an

increase (P<0.001) in transfer latency score due to the

memory deficit induced by high fat diet. The transfer

latency scores observed for simvastatin (Group 4),

rosuvastatin (Group 5) and fenofibrate (Group 6) afforded

a significant (P<0.001) memory compared to high fat diet

group. The reduction in TL observed with the

combination of above compounds with donepezil was

36% better than that of compounds alone. Administration

of simvastatin (Group 8) and nicotinic acid (Group 11)

along with donepezil afforded better (P<0.01) learning

scores than that of fenofibrate (Group 10) and

rosuvastatin (Group 9) with donepezil.


induced amnesia in rats using actophotometer: Rats

subjected to high fat diet (Group 2) showed a prominent

reduction in basal activity score when compared to

normal (Group 1) rats. Hypocholesterolemic drugs, when

administered after high fat diet, simvastatin (Group 4),


nicotinic acid (Group 7) significantly (P<0.001)

augmented basal activity score compared to high fat diet.

The enhancement in locomotor activity produced by

simvastatin, rosuvastatin, and fenofibrate was far better

than that of nicotinic acid and comparable to that of

standard donepezil (Group 3). The enhancement of basal

activity score when simvastatin (Group 8), rosuvastatin

(Group 9), fenofibrate (Group 10) and nicotinic acid

(Group 11) were administered along with donepezil was

16% better than that of drugs when administered alone.

Effect of hypocholesterolemic drugs on

acetylcholinesterase activity: High fat diet treated

animals (Group 2) showed elevated levels of

acetylcholinesterase indicating its memory reducing

potential. The animals of positive control group treated

with standard drug donepezil (Group 3) produced

significant (P<0.01) reduction of acetylcholinesterase

enzyme activity in comparison with normal control

(Group 1). In the treatment group, the animals exposed to

high fat diet and treated with simvastatin (Group 4)

significantly (P<0.05) decreased the acetylcholinesterase

activity in comparison with negative control. The activity

of this compound was found to be better than that of


nicotinic acid (Group 7). The reduction of

acetylcholinesterase activity when simvastatin (Group 8),


nicotinic acid (Group 11) were administered along with

donepezil was 10% better than that of above drugs when

administered alone. There was significant (P<0.01)

reduction in acetylcholinesterase activity when

simvastatin (Group 8) were administered along with

donepezil and it was better than that of rosuvastatin


(Group 11) with donepezil.

Effect of hypocholesterolemic drugs on lipid profile:

Rats subjected to high fat diet for 12 weeks (Group 2)

showed significant (P<0.001) increase in their total

cholesterol, triglycerides, LDL, VLDL and decreased

HDL, when compared to normal diet (Group 1) rats.

Treatment with donepezil (Group 3) showed significant

(P<0.001) alteration in lipid profile compared to high fat

diet rats (Group 2). Simvastatin (Group 4), rosuvastatin


(Group 7) treatment produced a significant (P<0.001)

change in lipid profile. Similarly, administration of

simvastatin (Group 8), rosuvastatin (Group 9), fenofibrate

(Group 10) and nicotinic acid (Group 11) along with

donepezil produced significant (P<0.001) decrease in

lipid profile. The change in serum lipid profile with

hypocholesterolemic drugs administered along with

donepezil was 36% better than that of above drugs

administered alone.

Brain Histopathology: Microscopic examination of brain

sections of negative control (Group-1) showed normal

cerebellum. The molecular layer and purkinjiec cells were

unremarkable. The basal ganglion showed normal

morphology. There was no neuronal edema or

degeneration or gliosis. Micrographs of brain section of

amnesia-induced group showed severe neuronal edema

with marked gliosis and neuronal degeneration (Group-2).

Micrograph of brain section of amnesia-induced

rats treated with donepezil (Group-3) showed very mild

histopathological alteration in the basal ganglion.

Micrograph of brain sections of amnesia-induced rats

treated with simvastatin (Group-4) and rosuvastatin

(Group-5) showed mild gliosis of the basal ganglia. While

amnesia-induced rats treated with fenofibrate (Group-6)

and nicotinic acid (Group-7) showed moderate edema,

gliosis and degeneration in basal ganglion.

Micrograph of brain sections of amnesia-induced

rats treated with simvastatin (Group 8) and rosuvastatin

(Group 9) along with donepezil showed mild gliosis

otherwise normal in basal ganglia. Amnesia-induced rats

treated with fenofibrate (Group 10) and nicotinic acid

(Group 11) moderate neuronal edema and gliosis in basal

ganglia.


Table 1. Effect of hypocholesterolemic drugs on Body Weights

Group

No.

Treatment & Dose Initial (gms.) Week-12 (gms.) Week-14 (gms.)

1 Control (1% Na CMC p.o) 215.38± 9.33 ND 201.02± 12.69

2 Only High Fat Diet (HFD) 126.00±7.98 285.08±19.25*** 278.32±19.62

3 Donepezil 5 mg/kg 154.17±7.96 279.83±9.14*** 266.00.6±7.45

4 Simvastatin 5 mg/kg 116.67±10.98 280.48±11.41*** 276.00±11.52

5 Rosuvastatin 5 mg/kg 108.5±7.42 281.77±21.87*** 303.05±17.04A

6 Fenofibrate 65 mg/kg 125.5±6.54 279.17±11.82*** 281.50±7.42

7 Nicotinic Acid 85 mg/kg 105.83±3.94 264.3±19.90*** 272.52±15.07

8 Donepezil 5 mg/kg + Simvastatin 5 mg/kg 95.5±5.20 228.68±6.66*** 239.33±14.21B

9 Donepezil 5 mg/kg + Rosuvastatin 5 mg/kg 121.67±10.29 287.8±15.37*** 278.00±14.48

10 Donepezil 5 mg/kg + Fenofibrate 65 mg/kg 137.00±5.88 206.18±17.41*** 206.83±21.44BB

11 Donepezil 5 mg/kg + Nicotinic Acid 85 mg/kg 127.00±5.53 264.05±10.71*** 270.67±11.63 Each group (n=6), each value represents Mean±SEM.

a) Denotes *P<0.05, **P<0.01, and ***P<0.001 compared to initial body weight. b) Denotes Ap<0.05 compared with HFD-group and Bp<0.05 and BBP<0.01 compared with Donepezil 5 mg/kg at week-14, ANOVA followed by dunnett’s

test. NA - Not determined.

Table 2. Effect of hypocholesterolemic drugs on transfer latency using Elevated plus-maze Group

No.

Treatment & Dose Initial (Sec.) Day-1

(Sec.)

Day-7 (Sec.) Day-14 (Sec.)

1 Control (1% Na CMC p.o) 39.89± 7.76 23.5± 6.48 9.17± 2.33 15.33± 3.78


3 Donepezil 5 mg/kg 46.06±8.33 35.50±4.13 55.00±6.63 43.00±5.44








11 Donepezil 5 mg/kg + Nicotinic Acid 85 mg/kg 28.72±2.51 47.67±6.21 77.67±3.57 AA 74.50±7.50+++,BBB Each group (n=6), each value represents Mean±SEM. a) Denotes *P<0.05, **P<0.01, and ***P<0.001 (Day-7) &+P<0.05, ++P<0.01, and +++P<0.001 compared to initial total latency (Day-14).

b) Denotes Ap<0.05 compared with HFD-group and Bp<0.05 compared with Donepezil 5 mg/kg at week-14, ANOVA followed by dunnett’s test.

Table 3. Effect of hypocholesterolemic drugs on transfer latency using Rectangular maze Group

No.

Treatment & Dose Initial

(Sec.)

Day-1 (Sec.) Day-7 (Sec.) Day-14 (Sec.)

1 Control (1% Na CMC p.o) 53.56±22.79 117.00±52.39 145.67±21.84 63.33±11.95


3 Donepezil 5 mg/kg 69.11±10.07 127.67±16.73 204.33±14.57*** 176.17±13.93+++

4 Simvastatin 5 mg/kg 60.11±8.53 210.00±13.80 253.67±16.35*** 257.17±16.66+++, BB

5 Rosuvastatin 5 mg/kg 63.39±16.98 316.67±21.74 288.67±15.26***, AA 255.33±13.29+++, BB

6 Fenofibrate 65 mg/kg 36.11±6.82 252.83±8.08 373.50±15.68**, AAA 444.83±25.32+++, BBB

7 Nicotinic Acid 85 mg/kg 44.56±6.41 280.00±7.94 405.67±21.04AAA 487.83±15.5 BBB

8 Donepezil 5 mg/kg+Simvastatin 5 mg/kg 35.17±10.23 106.17±15.01 108.67±17.45***, AAA 111.33±15.12+++, B

9 Donepezil 5 mg/kg+Rosuvastatin 5mg/kg 75.39±12.45 173.67±14.36 154.00±14.44*** 160.00±10.66+++

10 Donepezil 5 mg/kg+Fenofibrate 65 mg/kg 48.33±10.48 252.33±13.55 237.83±18.24*** 213.67±10.92+++

11 Donepezil 5 mg/kg+Nicotinic Acid

85mg/kg

85.50±21.88 295.33±34.52 311.17±26.55***, AAA 436.5±22.22+++, BBB

Each group (n=6), each value represents Mean±SEM. a) Denotes *P<0.05, **P<0.01, and ***P<0.001 (Day-7) &+P<0.05, ++P<0.01, and +++P<0.001 compared to initial total latency (Day-14).



Table 4. Effect of hypocholesterolemic drugs on locomotor activity using Actophotometer

Group

No.

Treatment & Dose Initial (Nos.) Day-1 (Nos.) Day-7 (Nos.) Day-14 (Nos.)

1 Control (1% Na CMC p.o) 303.22±24.40 287.33±38.24 342.00±20.00 377.17±9.46


3 Donepezil 5 mg/kg 325.67±28.03 298.00±15.07 223.00±20.07*** 429.00±27.71+++

4 Simvastatin 5 mg/kg 286.67±14.52 284.00±24.91 270.00±26.62*** 328.00±22.89+++

5 Rosuvastatin 5 mg/kg 421.00±28.69 380.00±34.87 380.00±25.09*** 320.00±27.23+++, B

6 Fenofibrate 65 mg/kg 330.67±12.24 260.00±18.34 261.00±13.28** 320.00±28.24+++, B

7 Nicotinic Acid 85 mg/kg 314.67±9.96 242.00±6.44 232.00±9.93 218.00±7.51+++, BBB

8 Donepezil 5 mg/kg + Simvastatin 5 mg/kg 357.33±18.92 298.00±19.55 218.00±41.58***, A 310.00±28.80+++



11 Donepezil 5 mg/kg + Nicotinic Acid 85

mg/kg

350.33±11.72 291.00±14.31 320.00±22.63*** 487.00±26.03+++

Each group (n=6), each value represents Mean±SEM. a) Denotes *P<0.05, **P<0.01, and ***P<0.001 (Day-7) &+P<0.05, ++P<0.01, and +++P<0.001 compared to initial basal activity (Day-14).


Table 5. Effect of hypocholesterolemic drugs on Brain Acetylcholinesterase levels


1 Control (1% Na CMC) 0.028±0.00065


3 Donepezil 5 mg/kg 0.014±0.0009**,AAA





8 Donepezil 5 mg/kg + Simvastatin 5 mg/kg 0.017±0.0004*,AA

9 Donepezil 5 mg/kg + Rosuvastatin 5 mg/kg 0.019±0.0005

10 Donepezil 5 mg/kg + Fenofibrate 65 mg/kg 0.022±0.0008

11 Donepezil 5 mg/kg + Nicotinic Acid 85 mg/kg 0.020±0.0008 Each group (n=2), each value represents Mean±SEM.

a) Denotes *P<0.05, **P<0.01, and ***P<0.001 (Day-7) &+P<0.05, ++P<0.01, and +++P<0.001 compared to initial basal activity (Day-14).


Table 6. Effect of hypocholesterolemic drugs on serum lipid profile Group no. Treatment & dose Test Initial Post induction1 Final

1 Control

(1% Na CMC)

HDL mg dl 98.60±2.24 - 103.15±0.99

LDL mg/dl 73.37±7.45 - 131.33±7.82

TC mg/dl 47.63±2.11 - 56.55±0.52

TG mg/dl 36.29±0.8 - 20.33±1.33

VLDL mg/dl 14.67±1.49 - 26.27±1.56

2 HDL mg/dl 106.10±3.41 214.90±6.90*** 217.5±7.85

Only High Fat Diet (HFD) LDL mg/dl 91.92±10.71 197.98±22.55*** 190.98±20.33

TC mg/dl 56.88±3.88 27.55±1.33*** 31.48±1.36

TG mg/dl 30.83±2.84 147.75±7.03*** 147.82±9.6

VLDL mg/dl 18.38±2.14 39.60±4.51*** 38.20±4.07

3 Donepezil 5 mg/kg HDL mg/dl 103.82±4.25 172.92±10.7*** 176.9±9.51AAA

LDL mg/dl 70.90±4.87 119.77±4.49*** 99.95±2.66AAA

TC mg/dl 55.23±2.78 41.28±2.70*** 46.42±2.98AAA

TG mg/dl 34.40±4.47 107.68±11.73*** 110.49±10.60AAA,BBB

VLDL mg/dl 14.18±0.97 23.95±0.90*** 19.99±0.53AAA

4 Simvastatin 5 mg/kg HDL mg/dl 99.30±2.52 204.97±2.29 128.73±2.54A,BB

LDL mg/dl 89.25±2.71 176.60±6.77 129.07±6.88 AAA,BBB

TC mg/dl 46.23±1.16 30.43±1.04*** 38.05±1.39AAA, BBB

TG mg/dl 35.22±2.70 139.21±1.77 64.87±2.17AAA,BBB


VLDL mg/dl 17.85±0.54 35.32±1.35 25.81±1.38AAA,BBB

5 Rosuvastatin 5 mg/kg HDL mg/dl 102.87±0.75 207.08±1.03 129.88±1.95AAA,BB

LDL mg/dl 75.87±0.77 155.15±2.08 103.10±1.63AAA,BBB

TC mg/dl 57.68±1.11 42.77±0.9*** 54.13±1.41AAA, BBB

TG mg/dl 30.01±1.2 133.29±1.5*** 55.13±2.72AAA

VLDL mg/dl 15.17±0.15 31.03±0.42 20.62±0.33AAA

6 Fenofibrate 65 mg/kg HDL mg/dl 103.92±1.28 210.63±0.91 131.23±1.75AAA,B

LDL mg/dl 73.75±1.22 144.52±1.47 95.42±2.21AAA,BBB

TC mg/dl 55.67±1.24 39.33±0.91*** 52.48±1.20AAA, BBB

TG mg/dl 33.50±1.99 142.4±1.66*** 59.67±2.65AAA

VLDL mg/dl 14.75±0.24 28.90±0.29 19.08±0.44AAA

7 Nicotinic Acid 85 mg/kg HDL mg/dl 104.17±1.5 217.53±2.21 196.47±2.55AAA

LDL mg/dl 83.62±1.86 169.43±4.51 144.85±6.17AAA

TC mg/dl 55.08±1.89 41.37±2.83*** 50.92±3.76AA, BBB

TG mg/dl 32.36±2.98 142.28±3.97*** 116.58±3.89 AAA,BBB

VLDL mg/dl 16.72±0.37 33.89±0.90 28.97±1.23AAA,BBB


Simvastatin 5 mg/kg

HDL mg/dl 101.47±1.06 204.02±4.31 158.77±2.9AAA

LDL mg/dl 63.72±4.03 125.68±7.16 80.43±1.66AAA,B

TC mg/dl 59.95±1.37 37.13±1.44*** 47.00±1.36AAA, BB

TG mg/dl 28.77±1.32 141.75±5.97 95.68±3.97AAA,B

VLDL mg/dl 12.74±0.81 25.14±1.43 16.09±0.33AAA,B


Rosuvastatin 5 mg/kg

HDL mg/dl 104.73±1.76 204.23±3.31 147.78±3.74AAA

LDL mg/dl 69.75±3.57 138.25±5.25 104.00±2.53AAA,BBB

TC mg/dl 56.78±1.34 41.4±1.28*** 50.78±1.57AAA, BBB

TG mg/dl 34.00±1.22 135.18±2.98 76.20±3.51AAA

VLDL mg/dl 13.95±0.71 27.65±1.05 20.80±0.51AAA


Fenofibrate 65 mg/kg

HDL mg/dl 102.05±0.97 211.18±1.07 143.15±2.76AAA

LDL mg/dl 92.90±7.44 148.15±3.23 110.15±3.21AAA,BBB

TC mg/dl 54.73±0.71 39.38±0.97*** 47.08±0.90AAA, BBB

TG mg/dl 28.74±1.68 142.17±0.77 74.04±2.05AAA

VLDL mg/dl 18.58±1.49 29.63±0.65 22.03±0.64AAA


Nicotinic Acid 85 mg/kg

HDL mg/dl 106.42±2.25 218.63±3.28 150.28±4.4 AAA

LDL mg/dl 72.44±9.03 141.43±4.60 94.57±3.92AAA,BBB

TC mg/dl 55.83±1.54 40.92±1.53*** 47.22±1.21AAA, BBB

TG mg/dl 36.09±2.77 149.43±4.21 84.15±4.54AAA

VLDL mg/dl 14.49±1.81 28.29±0.92 18.91±0.78AAA Each group (n=6), each value represents Mean±SEM.

a) Denotes *P<0.05, **P<0.01, and ***P<0.001 compared to initial lipid profile. b) Denotes Ap<0.05 compared with HFD-group and Bp<0.05 and BBP<0.01 compared with Donepezil 5 mg/kg at week-14, ANOVA followed by Dunnett’s test.

Figure 1. Effect of hypocholesterolemic drugs on Body

Weights in animals receiving high fat diet (HFD).


Figure 2. Effect of hypocholesterolemic drugs on transfer

latency using Elevated plus-maze in animals receiving high fat

diet (HFD).



Figure 3. Effect of hypocholesterolemic drugs on transfer

latency using Rectangular maze in animals receiving high fat

diet (HFD).


Figure 4. Effect of hypocholesterolemic drugs on locomotor

activity using Actophotometer in animals receiving high fat diet

(HFD).


Figure 5. Effect of hypocholesterolemic drugs on Brain

Acetylcholinesterase levels in animals receiving high fat diet

(HFD).


Figure 6. Effect of hypocholesterolemic drugs on serum lipid

profile in animals receiving high fat diet (HFD).


Brain Histopathology pictures of high fat diet (HFD) induced amnesia in Rats Figure 7. Basal ganglia of normal rat

Figure 8. Basal ganglia of HFD induced rat

Figure 9. Basal ganglia of rat treated with Donepezil in HFD induced

amnesia

Figure 10. Basal ganglia of rat treated with Simvastatin in HFD

induced amnesia


Figure 11. Basal ganglia of rat treated with Rosuvastatin in HFD

induced amnesia

Figure 12. Basal ganglia of rat treated with Fenofibrate in HFD

induced amnesia

Figure 13. Basal ganglia of rat treated with Nicotinic acid in HFD

induced amnesia

Figure 14. Basal ganglia of rat treated with Donepezil+Simvastatin in

HFD induced amnesia

Figure 15. Basal ganglia of rat treated with Donepezil+Rosuvastatin in

HFD induced amnesia

Figure 16. Basal ganglia of rat treated with Donepezil+Fenofibrate in

HFD induced amnesia

Figure 17. Basal ganglia of rat treated with Donepezil+Nicotinic acid in HFD induced amnesia

DISCUSSION Amnesia is inability to remember past

experiences or loss of memory. Anterograde amnesia is

impairment of memory for events occurring after the

accident/drug treatment. In such a case, new memories are

not formed. Whereas, retrograde amnesia is impairment

of memory of events, which have occurred before the

accident/drug treatment. In such a case, new memories

can be formed, but old memories are lost.

In the present study, chronic administration (12

weeks) of high fat diet (HFD) not only produced

significant increase in the total serum cholesterol levels,

but also impaired memory. This impairment of memory

might be due to the increased CNS cholesterol pool

because there is a cross talk between CNS and peripheral

cholesterol levels. The increased CNS cholesterol might

lead to deposition of amyloid peptide in brain (Ashutosh

Agarwal et al., 2002). Statins exerted cognitive benefits in

AD and were reported to affect CNS cholesterol

homeostasis (Haley RW & Dietschy JM, 2000). This

contention is further confirmed by the present study,

wherein, simvastatin and rosuvastatin significantly

prevented HFD induced memory deficits indicated by

augmentation of transfer latency and reduction in basal


activity score. In addition to statins, fenofibrate and

nicotinic acid also ameliorated HFD induced memory

deficits.

The current study revealed that HFD caused

elevation of serum LDL, VLDL, total cholesterol,

triglycerides and reduction of HDL levels. Simvastatin,

rosuvastatin, fenofibrate and nicotinic acid reverted back

the altered lipid profile. These drugs might have improved

the memory by reducing the serum cholesterol level with

subsequent reduction of CNS cholesterol and deposition

of β-amyloid peptide. However, anti-inflammatory and

anti-oxidant actions of statins to enhance memory cannot

totally be ignored at this point and might have contributed

to the beneficial effect on memory.

The tested drugs were also found to decrease the

acetylcholinesterase levels. This activity of the above

compounds might also contribute to their memory

enhancing effect by decreasing the metabolism of

acetylcholine.

Histopathological study further corroborated the

result. Standard donepezil successfully reversed the

marked gliosis and neurodegenaration of basal ganglia

induced by scopolamine. The photomicrographs of brain

sections treated with hypocholesterolemic drugs showed

mild gliosis and lack of neurodegenaration of basal

ganglia. It indicated neuroprotective role of

hypocholesterolemic drugs.

Hypocholesterolemic drugs from various classes

were found to reverse memory deficits induced by high

fat diet through their cholesterol dependent effects.

CONCLUSIONS

Treatment of amnesia-induced rats with

hypocholesterolemic drugs significantly ameliorates the

cholinergic dysfunction and inflammation-induced

neurodegeneration characterizing Alzheimer’s disease.

Noteworthy, statins revealed more pronounced

modulatory effect on most of the measured physical and

biochemical parameters as well as histological feature of

the brain than fibrates and nicotinic acid. The successful

reversal of memory deficits induced by high fat diet might

be through their cholesterol dependent effects. However,

anti-inflammatory and antioxidant actions of

hypocholesterolemic drugs can not totally be ignored at

this point and might have contributed to the beneficial

effect on memory. The selected drugs may represent new

therapeutic approaches for intervention of the progressive

neurological damage associated with Alzheimer’s disease.

ACKNOWLEDGEMENT: None

CONFLICT OF INTEREST:

The authors declare that they have no conflict of interest.

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EFFECT OF HYPOCHOLESTERIMIC DRUGS FOR MEMORY … · 2016. 10. 23. · I, Srinivasa Rao Talasila declare that the thesis entitled “Effect of Hypocholesteremic Drugs for Memory Enhancement