Imaging and pharmacotherapy of blood-brain barrier ... · University of Veterinary Medicine Center for Systems Neuroscience Hannover Department of Pharmacology, Toxicology and Pharmacy

University of Veterinary Medicine

Center for Systems Neuroscience Hannover

Department of Pharmacology, Toxicology and Pharmacy

Hannover Medical School

Department of Nuclear Medicine, Preclinical Molecular Imaging

_______________________________________________________________

Imaging and pharmacotherapy

of blood-brain barrier impairment

during epileptogenesis

THESIS

Submitted in partial fulfillment of the requirements for the degree

DOCTOR OF PHILOSOPHY

-Ph.D.-

In the field of Pharmacology

awarded by the

University of Veterinary Medicine Hannover

by

Heike Breuer

Preetz

Hannover, Germany 2016

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Supervisor: Prof. Dr. Marion Bankstahl

Supervision Group: Prof. Dr. med. Martin Stangel

Prof. Dr. med. Xiao-Qi Ding

1st Evaluation: Prof. Dr. Marion Bankstahl

Department of Pharmacology, Toxicology and

Pharmacy

University of Veterinary Medicine Hannover

Prof. Dr. Martin Stangel

Department of Neurology


Prof. Dr. Xiao-Qi Ding

Institute of Diagnostic and Interventional

Neuroradiology


2nd Evaluation: Prof. Dr. rer. nat. Andreas Hess

Institute of Experimental and Clinical

Pharmacology and Toxicology

Friedrich-Alexander University Erlangen-Nürnberg

Date of final exam: 21.10.2016

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Parts of the thesis have been published previously in:

BREUER, H., MEIER, M., SCHNEEFELD, S., HÄRTIG, W., WITTNEBEN, A.,

Märkel, M., ROSS, T.L., BENGEL, F. BANKSTAHL, M., BANKSTAHL, J.P. (2016).

Multimodality Imaging Of Blood-Brain Barrier Impairment during Epileptogenesis.

Journal of Cerebral Blood Flow and Metabolism, DOI: 10.1177/0271678X16659672.

This thesis was accomplished within the PhD program “Systems Neuroscience“ of

the Center for Systems Neuroscience Hannover and parts of the work belong to the

European Union’s Seventh Framework Programme (FP7/2007-2013) under grant

agreement n°602102 (EPITARGET).

Heike Breuer was supported by a doctoral scholarship of the Studienstiftung des deutschen Volkes.

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Meiner Familie

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Table of Contents

SUMMARY ................................................................................................................. 1

ZUSAMMENFASSUNG ............................................................................................. 3

1 STATE OF RESEARCH ...................................................................................... 6

1.1 Epilepsy ................................................................................................................................. 6

1.1.1 Definition and significance ..................................................................................................... 6

1.1.2 Temporal lope epilepsy ......................................................................................................... 7

1.2 Epileptogenesis ..................................................................................................................... 7

1.3 The blood-brain barrier .......................................................................................................... 9

1.3.1 The role of blood-brain barrier in epileptogenesis ............................................................... 10

1.3.2 The blood-brain barrier and inflammation ........................................................................... 12

1.4 Treatment strategies targeting blood-brain barrier integrity ................................................ 13

1.5 Animal models of epileptogenesis ....................................................................................... 16

1.5.1 The pilocarpine post status epilepticus rat model ............................................................... 16

1.5.2 The intrahippocampal kainate mouse model ....................................................................... 17

1.6 Non-invasive in vivo imaging of blood-brain barrier leakage ............................................... 17

1.6.1 Positron Emission Tomography imaging ............................................................................. 18

1.6.2 Single Photon Emission Computed Tomography imaging .................................................. 21

1.6.3 Magnetic Resonance imaging ............................................................................................. 22

1.7 Aim of the studies ................................................................................................................ 26

1.7.1 Study 1: Multimodality imaging of blood-brain barrier impairment during epileptogenesis . 26

1.7.2 Study 2: Longitudinal in vivo evaluation and pharmacotherapy of blood-brain barrier leakage in a rat model of epileptogenesis ........................................................................... 26

1.7.3 Study 3: Dexamethasone protects blood-brain barrier integrity in a mouse model of epileptogenesis .................................................................................................................... 27

2 MULTIMODALITY IMAGING OF BLOOD-BRAIN BARRIER IMPAIRMENT

DURING EPILEPTOGENESIS .......................................................................... 28

3 LONGITUDINAL IN VIVO EVALUATION AND PHARMACOTHERAPY OF

BLOOD-BRAIN BARRIER LEAKAGE IN A RAT MODEL OF

EPILEPTOGENESIS.......................................................................................... 30

3.1 Abstract................................................................................................................................ 31

3.2 Introduction .......................................................................................................................... 32

3.3 Materials and Methods ........................................................................................................ 34

3.3.1 Animals ................................................................................................................................ 34

3.3.2 Status epilepticus ................................................................................................................ 34

3.3.3 Chemicals and drugs ........................................................................................................... 35

3.3.4 Study design ........................................................................................................................ 35

3.3.5 Longitudinal in vivo assessment of blood-brain barrier leakage ......................................... 36

3.3.6 Dexamethasone treatment .................................................................................................. 36

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3.3.7 Losartan treatment .............................................................................................................. 36

3.3.8 Assessment of blood-brain barrier integrity ......................................................................... 37

3.3.9 Image analysis ..................................................................................................................... 38

3.3.10 Histological analysis of albumin extravasation .................................................................... 39

3.3.11 Statistical analysis ............................................................................................................... 40

3.4 Results ................................................................................................................................. 40


3.4.2 Longitudinal in vivo assessment of blood-brain barrier leakage in saline-treated group .... 41

3.4.2.1 Development of T1 intensity during early epileptogenesis .......................................... 41

3.4.2.2 Development of T2 intensity during early epileptogenesis .......................................... 43

3.4.3 Pharmacotherapy with potentially blood-brain barrier protective drugs .............................. 44

3.4.3.1 Effects of dexamethasone on blood-brain barrier integrity .......................................... 45

3.4.3.2 Effects of losartan on blood-brain barrier integrity ....................................................... 47

3.4.4 Histological analysis of albumin extravasationJJJJJJJJJJJJJJJJJJ...49

3.5 Discussion ........................................................................................................................... 51

3.6 Conclusion ........................................................................................................................... 56

3.7 References .......................................................................................................................... 57

4 DEXAMETHASONE REDUCES BLOOD-BRAIN BARRIER LEAKAGE IN A

MOUSE MODEL OF EPILEPTOGENESIS ........................................................ 62

4.1 Abstract................................................................................................................................ 63

4.2 Introduction .......................................................................................................................... 64

4.3 Materials and methods ........................................................................................................ 66

4.3.1 Animals ................................................................................................................................ 66

4.3.2 Chemicals and drugs ........................................................................................................... 66


4.3.4 Study design ........................................................................................................................ 68

4.3.5 Magnetic Resonance imaging setup ................................................................................... 69

4.3.6 Evaluation of dexamethasone treatment on blood-brain barrier integrity during early epileptogenesis .................................................................................................................... 68

4.3.7 Image analysis ..................................................................................................................... 70

4.3.8 Statistical analysis ............................................................................................................... 70

4.4 Results ................................................................................................................................. 71

4.4.1 Tolerability of dexamethasone in the intrahippocampal kainate mouse model ................... 71

4.4.2 Longitudinal Magnetic Resonance imaging during epileptogenesis reveals blood-brain barrier leakage in epilepsy associated brain regions .......................................................... 72

4.4.3 Dexamethasone treatment protects blood-brain barrier integrity ........................................ 74

4.5 Discussion ........................................................................................................................... 77

4.5.1 Longitudinal in vivo assessment of blood-brain barrier leakage during epileptogenesis .... 77

4.5.2 Dexamethasone treatment significantly reduces blood-brain barrier leakage .................... 79

4.6 Conclusion ........................................................................................................................... 80

4.7 References .......................................................................................................................... 81

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5 GENERAL DISCUSSION .................................................................................. 85

5.1 Main findings ....................................................................................................................... 85

5.2 Multimodal imaging of blood-brain barrier leakage during epileptogenesis ........................ 85

5.3 Methodological issues ......................................................................................................... 88

5.4 Effects of losartan on blood-brain barrier integrity .............................................................. 89

5.5 Effects of dexamethasone on blood-brain barrier integrity .................................................. 89

5.6 Limitations of the studies ..................................................................................................... 91

5.7 Future directions .................................................................................................................. 91

5.8 Conclusion ........................................................................................................................... 92

6 REFERENCES ................................................................................................... 93

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List of abbreviations

µl: microliter

µm: micrometer

AG: Aktiengesellschaft

al.: alia

ANOVA: analysis of variance

b.i.d.: bis in die

BBB: blood-brain barrier

CA: contrast agent

cc: cubic centimeter

CNS: central nervous system

Co.: company

COX-2: cyclooxygenase-2

CT: computed tomography

d: day/s

Da: dalton

DAB-Ni: nickel-enhanced

diaminobenzidine

dex: dexamethasone

DOTA: 1,4,7,10-

tetraazacyclododecane-1,4,7,10-

tetraacetic acid

DTI: diffusion tensor imaging

DTPA: diethylenetriamine pentaacetic

acid

DWI: diffusion weighted imaging

ECoG: electrocorticography

EDTA: ethylenediaminetetraacetic acid

FDG: fluoro-2-deoxy-D-glucose

FOV: field of view

FP7: Seventh Framework Programme

FTY720: fingolimod

g: gram

Gd: gadolinium

GmbH: Gesellschaft mit beschränkter

Haftung

HEPES: ethanesulfonic acid

i.p.: intraperitoneal

i.v.: intravenous

ID: injection dose

IL-1ß: interleukin-1ß

ILAE: International League Against

Epilepsy

Inc.: incorporated

KA: kainate

KeV: kiloelectron volts

kg: kilogram

KG: Kommanditgesellschaft

l: liter

LLC: limited liability company

Ltd.: limited

lx: lux

m2: square meter

MBq: megabecquerel

MDEFT: modified driven equilibirum

fourier transformation

mg: milligram

MHz: megahertz

mm: millimeter

mm3: cubic millimeter

mmol: millimole

MR: magnetic resonance

MRI: magnetic resonance imaging

ms: millisecond

MSME: multi slice multi echo

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n: number

NMDA: N-methyl-d-aspartate

nmol: nanomole

OSEM: ordered subset expectation

maximization

p.o.: per os

PET: positron emission tomography

pH: power of hydrogen

PhD: Philosophiae Doctor

RARE: rapid acquisition refocusing

s: second

s.c.: subcutaneous

SE: status epilepticus

SEM: standard error of the mean

SPECT: single photon emission

computed tomography

SPM: service provider mechanism

T: tesla

TE: echo time

TGFßRII: transforming growth factor ß

receptor II

TLE: temporal lobe epilepsy

TR: repetition time

UCL: University College London

UK: United Kingdom

USA: United States of America

vs.: versus

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List of figures

Figure 1: Schematic illustration of epileptogenesis ................................................... 8

Figure 2: Blood-brain barrier associated mechanisms triggering epileptogenesis .. 11

Figure 3: Epileptogenesis and possible time window for antiepileptogenic therapy 12

Figure 4: Principle mechanism for detection of blood-brain barrier leakage ........... 17

Figure 5: Positron emission tomography imaging ................................................... 19

Figure 6: Weight development ................................................................................ 41

Figure 7: Quantification of changes in T1 and T2 intensities during

epileptogenesis ........................................................................................ 43

Figure 8: Longitudinal magnetic resonance imaging during epileptogenesis .......... 44

Figure 9: Comparative T2, T1 and leakage map images following saline,

dexamethasone and losartan treatment .................................................. 45

Figure 10: Quantification of BBB leakage and edema after dexamethasone

treatment ................................................................................................. 46

Figure 11: Quantification of blood-brain barrier leakage and edema after losartan

treatment ................................................................................................. 48

Figure 12: Histological evaluation of albumin extravasation ..................................... 50

Figure 13: Localization of kainate injection as assessed by magnetic resonance

imaging (MRI) .......................................................................................... 72

Figure 14: Quantification of blood-brain barrier leakage during epileptogenesis ...... 73

Figure 15: Blood-brain barrier leakage after dexamethasone treatment ................... 75

Figure 16: Serial magnetic resonance images in kainate, dexamethasone and sham

animals .................................................................................................... 76

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List of tables

Table 1: Representation of T1- and T2-contrast on MRI .......................................... 23

Table 2: Gd-DTPA infusion protocol rats .................................................................. 38

Table 3: Model-specific criteria for animal evaluation after surgery .......................... 68

Table 4: Gd-DTPA infusion protocol mice ................................................................ 69

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Summary

Heike Breuer

Imaging and pharmacotherapy of blood-brain barrier impairment during

epileptogenesis

Brain insults can result in the development of epilepsy, the most common chronic

neurologic disease worldwide. Epilepsy is a complex disease characterized by

unpredictably occurring spontaneous seizures, which cannot be treated efficiently in

approximately every third patient. The development of epilepsy is characterized by a

seizure-free latency period between the initiating brain insult and the occurrence of

spontaneous recurrent seizures, referred to as epileptogenesis. During this time,

functional and structural reorganization processes, including blood-brain barrier

(BBB) leakage, transform normal brain circuits into an epileptogenic network. To date

it is neither possible to predict which patient will develop epilepsy after a brain insult

nor to prevent epileptogenesis. Therefore, biomarkers for epileptogenesis are

urgently needed to identify at-risk patients. The latent period may offer a therapeutic

window for the prevention or modification of epileptogenesis. Given the increasing

evidence that BBB impairment following brain insults is one important trigger to set

the process of epileptogenesis in motion, non-invasive in vivo imaging of BBB

leakage might be a suitable biomarker for epileptogenesis. Moreover, strategies that

aim to restore the BBB integrity might consequently be beneficial for the prevention

of epilepsy after brain insults. However, the number of molecular imaging studies on

BBB leakage is limited and the presence of BBB leakage and its role in

epileptogenesis need to be further investigated.

Thus, the first objective of this study was to investigate whether dedicated small

animal [68Ga]-diethylenetriamine pentaacetic acid (DTPA) positron emission

tomography (PET), [99mTc]-DTPA single photon emission computed tomography

(SPECT) and contrast enhanced magnetic resonance imaging (MRI) following

infusion of gadobutrol, gadolinium (Gd)-DTPA, or Gd-albumin would be sensitive to

detect BBB leakage in animal models of epileptogenesis. Different imaging protocols

including [68Ga]-DTPA PET after bolus injection and after step-down tracer infusion

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were used and results were compared to histology as gold standard using

fluorescein-iso-thiocyanate (FITC)-labeled albumin. The second objective of this

study was to evaluate whether two potentially BBB-stabilizing drugs, losartan and

dexamethasone, can preserve BBB integrity and alleviate vascular edema during

early epileptogenesis. MRI protocols including step-down infusion of Gd-DTPA were

used to assess effects of dexamethasone and losartan in the pilocarpine post status

epilepticus (SE) rat model and of dexamethasone in the intrahippocampal kainate

mouse model.

We found that [68Ga]-DTPA PET, [99mTc]-DTPA SPECT and T1-weighted MRI

following infusion of Gd-DTPA, gadobutrol or Gd-albumin are sensitive to detect BBB

leakage in early epileptogenesis. Extravasation patterns are limited to specific,

typically epilepsy-associated brain regions. Contrast-enhanced MRI and

[99mTc]-DTPA SPECT are the most sensitive techniques for detecting BBB leakage

following SE. Moreover, our results show for the first time a successful reduction of

BBB leakage by dexamethasone in mice during epileptogenesis. However, in rats,

both dexamethasone treatment and losartan treatment had no BBB protective effects

at the used dose. Our data show that molecular imaging provides an evaluative

perspective to gain further insights into the dynamics of BBB leakage and its

association with epileptogenesis progression. Moreover, the study offers suitable

imaging protocols to further evaluate the role of BBB impairment as potential

biomarker for epileptogenesis and to evaluate new anti-epileptogenic treatments.

Such therapies could minimize the risk of epilepsy development or progression after

brain insults.

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Zusammenfassung

Heike Breuer

Bildgebung und Pharmakotherapie von Blut-Hirn-Schranken Veränderungen im

Verlauf der Epileptogenese

Hirninsulte können die Entwicklung einer Epilepsie initiieren, der häufigsten

chronischen neurologischen Erkrankung weltweit. Epilepsien sind komplexe

Erkrankungen, die durch unvorhersehbar auftretende spontane Anfälle

charakterisiert sind, welche bei etwa jedem dritten Patienten nicht effizient behandelt

werden können. Die Entwicklung von symptomatischen Epilepsien ist

gekennzeichnet durch eine anfallsfreie Latenzzeit, die sogenannte Epileptognese,

zwischen dem auslösenden Hirninsult und dem Auftreten wiederkehrender spontaner

Anfälle. Während dieser Zeit kommt es zu funktionellen und strukturellen

Reorganisationsprozessen im Gehirn, beispielsweise Veränderungen der

Blut-Hirn-Schranken (BHS)-Integrität. Diese tragen zur Entstehung von neuronaler

Hyperexzitabilität bei und initiieren den Prozess der Epileptogenese. Bis heute ist es

weder möglich vorherzusagen, welche Patienten nach einem Hirninsult eine

Epilepsie entwickeln werden, noch ist es möglich, die Entstehung von Epilepsien zu

verhindern. Daher besteht ein dringender Bedarf an diagnostisch nachweisbaren

Biomarkern für Epileptogenese, um Risikopatienten frühzeitig identifizieren zu

können. Die Latenzperiode nach einem Hirninsult bietet ein geeignetes Zeitfenster

für epilepsie-präventive Therapieansätze. Angesichts der zunehmenden Hinweise

darauf, dass Veränderungen der BHS-Integrität nach Hirninsulten sowohl an der

Initiierung als auch an der Progression der Epileptogenese beteiligt sind, könnte die

Visualisierung von Veränderungen der BHS-Integrität durch molekulare Bildgebung

einen geeigneten Biomarker für den Prozess der Epileptogenese darstellen. Mit Hilfe

dieser Bildgebungs-Biomarker könnte folglich ein möglicher Behandlungserfolg nach

BHS-protektiver Behandlung im Verlauf der Epileptogenese überprüft werden. Die

Zahl der bisher erfolgten molekularen Bildgebungs-Studien in diesem Bereich ist

stark limitiert, so dass weitere Untersuchungen nötig sind, um Fortschritte auf diesem

Gebiet zu erreichen.

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Somit war es das erste Ziel dieser These zu untersuchen, ob die Sensitivität

dedizierter Kleintier-[68Ga]-Diethylentriaminpentaessigsäure (DTPA) Positronen-

Emissions-Tomographie (PET), [99mTc]-DTPA Einzelphotonen-Emissions-

Computertomographie (SPECT) sowie kontrastverstärkter Magnet-Resonanz-

Tomographie (MRT) nach Infusion von Gadobutrol, Gadolinium (Gd)-DTPA oder

Gd-Albumin ausreichend ist, um Veränderungen der BHS-Integrität in Tiermodellen

für Epileptogenese nachweisen zu können. Hierzu wurden für [68Ga]-DTPA PET

verschiedene Bildgebungsprotokolle einschließlich Bolus-Injektion und Step-Down

Tracer-Infusion verwendet. Zusätzlich wurden als Vergleichsverfahren

immunhistologische Untersuchungen von Gehirnschnitten nach

Fluorescein-iso-thiocyanat (FITC)-Albumin-Infusion durchgeführt.

Das zweite Ziel der These lag darin festzustellen, ob die Behandlung mit zwei

verschiedenen potentiell BHS-stabilisierenden Medikamenten, Losartan und

Dexamethason, eine Protektion der BHS-Integrität während der auf einen Status

Epilepticus (SE) folgenden frühen Epileptogenese bewirken kann. Zur Beantwortung

dieser Frage wurden MRT-Untersuchungen nach Step-Down-Infusion von Gd-DTPA

durchgeführt, um die Effekte von Dexamethason und Losartan im Lithium-Pilocarpin-

Rattenmodell sowie die von Dexamethason im intrahippocampalen Kainat-Modell der

Maus zu beurteilen.

Wir konnten zeigen, dass [68Ga]-DTPA PET, [99mTc]-DTPA SPECT sowie

T1-gewichtetes MRT nach Infusion von Gadobutrol, Gd-DTPA oder Gd-Albumin

geeignet sind, Integritätsstörungen der BHS während der frühen Epileptogenese zu

detektieren. Die Extravasationsmuster von Tracern und Kontrastmitteln sind dabei

auf bestimmte, typischerweise epilepsie-assoziierte Hirnregionen beschränkt. MRT

und SPECT haben sich als die sensitivsten Methoden zur Erkennung von

BHS-Veränderungen nach SE erwiesen. Darüber hinaus konnten wir erstmals

während der Epileptogenese eine erfolgreiche Protektion der BHS-Integrität durch

Behandlung mit Dexamethason bei Mäusen nachweisen. Diese Erkenntnis könnte

zur weiteren Entwicklung anti-epiletogener oder epilepsie-modifizierender Therapien

beitragen. Im Lithium-Pilocarpin-Rattenmodell hingegen konnte mit dem von uns

verwendeten Behandlungsschema keine BHS-protektive Wirkung von

Dexamethason oder Losartan festgestellt werden.

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Unsere Daten zeigen, dass die molekulare Bildgebung eine wertvolle Methode ist,

um sowohl die Dynamik als auch die Pathogenese von BHS-Veränderungen

während der Epileptogenese zu untersuchen. Darüber hinaus können die in der

These etablierten Bildgebungsprotokolle genutzt werden, um in folgenden

Untersuchungen die Eignung der Detektion von BHS-Beeinträchtigungen als

potentiellen Biomarker für Epileptogenese weiter zu untersuchen und neue

anti-epileptogene Behandlungsansätze zu validieren. Diese Therapien könnten dann

zukünftig verwendet werden, um das Risiko der Entwicklung und der Progression

von Insult-assoziierten Epilepsien zu miniminieren.

State Of Research

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1 State Of Research

1.1 Epilepsy

1.1.1 Definition and significance

Epilepsy is defined as a "brain disorder which is characterized by a permanent

tendency to development of epileptic seizures, as well as by the neurobiological,

cognitive, psychological and social consequences of this condition" according to the

International League Against Epilepsy (ILAE) (Fisher et al., 2005). Epileptic seizures

are defined as the "transient occurrence of signs and/or symptoms due to abnormal

excessive or synchronous neuronal activity in the brain" (Fisher et al., 2005).

Epilepsies are among the most common chronic diseases of the brain, characterized

by an excessive and abnormal electrical activity of neurons resulting in spontaneous

and recurrent seizures (Chang & Lowenstein, 2003), often accompanied by a

progressive tendency and psychiatric comorbidities like depression, anxiety as well

as learning and memory deficits (Tellez‐Zenteno et al., 2007). Approximately 1-2 %

of the population are affected by this complex disease in the course of their lives

(Löscher & Schmidt, 2002; Bialer & White, 2010). Moreover, up to 5.7 % of dogs and

cats are afflicted (Volk et al., 2008; Volk & Loderstedt, 2011), making epilepsy the

most common neurological disease in domestic animals, too (Löscher, 2003). The

underlying causes of epilepsy are categorized as genetic, structural-metabolic or

unknown (Berg et al., 2010). An accurate differentiation and diagnosis of epilepsies –

based on the type of seizures, their etiology and triggering factors as well as

electroencephalographic findings and the patient’s age at onset – is essential for a

successful treatment (Engel, 2006; Berg et al., 2010). A classification issued by the

ILAE provides the basis for this (Engel, 2006). Focal seizures are distinguished from

generalized seizures. Focal epileptic seizures originate within networks limited to one

hemisphere and each seizure type has a consistent site of onset (epileptic focus).

Starting from this focus, they can spread to both hemispheres resulting in secondarily

generalized seizures. By contrast, primarily generalized epileptic seizures originate

from bilateral networks with localization and lateralization not being consistent

between seizures (Berg et al., 2010).

State Of Research

7

1.1.2 Temporal lope epilepsy

Structural-metabolic epilepsies are diagnosed in 30-40 % of patients, making them

the most common epilepsy subtype. They are initiated by brain insults triggering

diagnostically detectable structural lesions which ultimately lead to epilepsy

(Englander et al., 2003; Pitkänen & Lukasiuk, 2009). Underlying causes involve

traumatic brain insults as well as stroke, infections, brain tumors or status epilepticus

(SE). A SE can be characterized by tonic-clonic convulsions lasting longer than 5 min

and by a loss of consciousness (Engel, 2006; Lowenstein, 2009). SE is an acute life-

threatening event (Knake et al., 2009) and contributes to increased mortality rates

among epilepsy patients (Sperling et al., 1999). Temporal lobe epilepsy (TLE) is

localization-related and clinically accompanied by partial or secondarily generalized

tonic-clonic seizures, assigned to as complex partial seizures (Mattson et al., 1992).

These seizures generally last 1-2 min and can be associated with auras occurring

prior to seizure onset as well as with emotional alterations including anxiety (Engel,

1996; Chabardès et al., 2005). The epileptic focus is often located in the temporal

lobe, particularly in the hippocampus, the amygdala and in the adjacent

parahippocampal cortex (McNamara, 1994; Chang & Lowenstein, 2003; Bertram,

2009) which is why the resulting syndrome is referred to as TLE. It is the most

common form of partial epilepsy in humans (Engel, 1996) and one of the focal

epilepsies. A hallmark of TLE is hippocampal sclerosis, affecting approximately 65 %

of patients (Wieser, 2004). It is characterized by neuronal cell loss and synaptic

reorganization in hippocampal subregions (Chang & Lowenstein, 2003; Bertram,

2009) which can often be detected as hippocampal atrophy by magnetic resonance

imaging (MRI, Berkovic et al., 1991).

1.2 Epileptogenesis

The development of structural-metabolic epilepsies, called epileptogenesis (see

figure 1) is characterized by a latency period between the initiating brain insult and

the occurrence of complex partial seizures (Englander et al., 2003). The latency

period can last weeks to years. In TLE, the mean latency phase is two years in

human patients (Berkovic et al., 1991).

State Of Research

8

Brain insult First clinical seizure Chronic epilepsy

Epileptogenesis

Neurodegeneration, Blood-brain barrier impairment, Inflammation

Figure 1: Schematic illustration of epileptogenesis

The development of insult-associated epilepsy is characterized by a latency period between the initiating insult and the first occurrence of seizures, called epileptogenesis. During this time, multiple reorganization processes occur, amongst them blood-brain barrier leakage and inflammation.

During this period, cellular and structural reorganization processes occur in the brain

(Friedman et al., 2009). These include, but are not limited to, an increase in the

permeability of the blood-brain barrier (BBB), brain inflammation, neuronal loss and

sprouting of mossy fibers (Pitkänen & Lukasiuk, 2009; Löscher & Brandt, 2010).

Subsequently, surviving neuronal populations generate abnormal electrical activity.

This activity leads to recurrent focal or generalized seizures and results in chronic

epilepsy (Sloviter & Bumanglag, 2013).

To date, epilepsies cannot be cured with medication. With the exception of epilepsy

surgery, current treatment options are purely symptomatic. Furthermore, they are

unable to prevent the development or progression of epilepsy after brain insults

(Temkin, 2001; Löscher & Brandt, 2010). A hitherto remaining problem is that

50-75 % of TLE-patients cannot be adequately treated by anti-seizure drugs

(Spencer, 2002; Löscher & Potschka, 2005; van Vliet et al., 2014a). Despite a wide

range of approved anti-seizure drugs on the market, they remain unresponsive to

symptomatic therapy (Chang & Lowenstein, 2003) and suffer from unpredictably

occurring seizures. Cognitive impairment, side effects of therapy and other factors

which affect the quality of life are often very distressing for those affected and for

their families. The everyday and professional life of these people is severely

restricted. In addition, comorbidities like anxiety- or depression-associated behavioral

changes are more frequently found in drug-resistant patients (Shihab et al., 2011;

Bankstahl & Bankstahl, 2012). This is accompanied, among other things, with an

increased susceptibility to suicide (Löscher & Brandt, 2010).

State Of Research

9

These facts emphasize the importance of understanding epileptogenesis. There is a

strong interest to discover therapies that hold potential to prevent or to modify the

process of epileptogenesis (Löscher & Brandt, 2010). However, only a limited

number of individuals actually develop epilepsy as a consequence of brain insults.

Hence, diagnostic methods for identification of patients at risk are urgently needed.

Prognostic biomarkers for epileptogenesis are a prerequisite to identify patients at

risk at an early stage in epileptogenesis. The long-term goal is to provide an

antiepileptogenic treatment for at-risk patients to prevent the development of

epilepsy.

1.3 The blood-brain barrier

By excluding entry of many potentially harmful molecules from the blood, the BBB is

an important protective factor for the brain (Abbott et al., 2006). It consists of capillary

endothelial cells, having the apical membrane facing the lumen and the basolateral

membrane facing the brain parenchyma (Betz et al., 1980). Tight junctions between

adjacent endothelial cells with lack of fenestrations and little pinocytosis prevent the

paracellular passage of molecules into the brain (Abbott et al., 2006). Endothelial

cells, along with basal lamina, pericytes, perivascular astrocytic end-feet and

neurons, form the so-called "Neurovascular Unit" which is mandatory for a

physiological neuronal functioning (Abbott et al., 2010). The BBB achieves its

protective function by different mechanisms: tight junctions make it a physical barrier

and a variety of metabolizing enzymes an enzymatic barrier. Besides, uptake or

efflux transporters enable active transportation of diverse compounds (van Vliet et

al., 2014a). Thereby, the BBB allows delivery of essential nutrients as well as

discharge of toxic metabolites out of the central nervous system (CNS) (Pardridge,

2007), but still protects the brain from potentially harmful substances (Abbott &

Friedman, 2012). Moreover, the BBB is the place of interactions between central and

peripheral immune systems (Engelhardt & Coisne, 2011). It is present through the

whole of the developed brain’s vasculature. Exceptions are the circumventricular

organs where secretion of neuropeptides and hormones as well as chemoreception

require a less restrictive BBB (Abbott et al., 2010) .

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1.3.1 The role of blood-brain barrier in epileptogenesis

Accumulating evidence strongly suggests that insult-associated BBB impairment is a

key step in the initiation of epileptogenesis and may contribute to disease

progression (Janigro, 1999; Oby & Janigro, 2006; van Vliet et al., 2007b; Friedman

et al., 2009; Friedman & Heinemann, 2010; Ndode-Ekane et al., 2010; Friedman,

2011; Abbott & Friedman, 2012). Therefore, BBB-protective treatment might prevent

or attenuate epileptogenesis (Dedeurwaerdere et al., 2007; Friedman et al., 2009;

Friedman & Heinemann, 2010).

Changes in BBB integrity during epileptogenesis were found at the structural, cellular

and molecular level in animal models and in human brain tissue (van Vliet et al.,

2014a), suggesting that BBB leakage triggers a chain of events causing epilepsy

(Janigro, 1999; Seiffert et al., 2004; Ivens et al., 2007; Marchi et al., 2007a). After

kainate-induced SE in rats, increased extracellular levels of the excitatory

neurotransmitter glutamate were observed (Ueda et al., 2002). These changes were

accompanied by generation of free radicals. Subsequent excessive stimulation of

BBB endothelial N-methyl-d-aspartate (NMDA) receptors results in BBB impairment

early after SE (Sharp et al., 2005) and leads to further oxidative stress. Moreover, the

neuronal hyperexcitability accompanying seizures causes an increased glucose and

oxygen demand and thus an increased blood volume in the affected brain regions.

The resulting hypertension in the brain capillaries initiates an increased BBB

permeability and a metabolic mismatch (Lothman, 1990). Increased vascular

oxidative stress, along with increased blood pressure as well as hypoxia and

reduction of blood pH (Stanimirovic & Friedman, 2012) further impair BBB integrity.

The extravasation of blood components like albumin and immune cells into the brain

through a leaky BBB, alterations in electrolyte concentrations as well as loss of

glutamate and extracellular homeostasis favor an increased neuronal excitability

(Oby & Janigro, 2006; Ndode-Ekane et al., 2010, see figure 2) which may trigger

epileptogenesis (Ivens et al., 2007; Friedman et al., 2009). Albumin is incorporated

by astrocytes and binds to the transforming growth factor ß receptor II (TGFßRII).

Subsequently, the TGF-ß signaling cascade is activated within the neurovascular

unit, resulting in proepileptogenic alterations including astrocytic transformation and

dysfunction (Ivens et al., 2007; Friedman et al., 2009).

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Figure 2: Blood-brain barrier associated mechanisms triggering epileptogenesis

Different mechanisms affecting parts of the neurovascular unit contribute to blood-brain barrier (BBB) leakage. Leucocyte-endothelial interactions directly affect endothelial cells. Potassium entering the brain lowers the seizure-threshold. Extravasation of serum components like albumin into the brain parenchyma causes astrocytic responses. Albumin is taken up into astrocytic end-feet via transforming growth factor (TGF)-ß receptors and activates the TGF-ß signaling cascade. Subsequently, the potassium channel Kir4.1 and glutamate transporters are downregulated. This mechanism results in impaired potassium- and glutamate buffering. Moreover, pro-inflammatory cytokines are released by astrocytes and microglia, resulting in downregulation of endothelial zonula occludens-1, further increasing BBB permeability. Consequently, neuronal hyperexcitability occurs, resulting in seizures. Figure modified from (Obermeier et al., 2013) and (Abbott et al., 2006).

The occurrence of increased BBB permeability is limited to specific brain regions in

epilepsy patients (Bradbury & Lightman, 1990). These regions are congruent with

regions of increased metabolic activation after SE as assessed by 2-deoxyglucose

autoradiography (Van Landingham & Lothman, 1991). Moreover, they often

anatomically overlap with those regions associated with the development and spread

of epileptic seizures, such as hippocampus, amygdala and piriform cortex. Hence, it

was suggested that a metabolic over-activation, resulting in increased glucose

demand and subsequent increase in blood-pressure in cerebral capillaries during SE

causes BBB leakage (van Vliet et al., 2014a). Conversely it was shown that the

extent of BBB impairment correlates with seizure duration (Cornford & Oldendorf,

1986). The aforementioned findings, and the fact that BBB leakage was detected in

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the early phase of epileptogenesis around the time of the brain insult (Rigau et al.,

2007; van Vliet et al., 2007b; Ndode-Ekane et al., 2010), suggest that BBB-protective

therapies are promising strategies for the prevention of epilepsy (Marchi et al., 2012).

However, preclinical studies propose that the therapeutic time window after brain

insults is rather narrow (Herman, 2002). This underlines the need of suitable

biomarkers to identify patients at risk as early as possible during epileptogenesis

(figure 3). BBB leakage might be such a biomarker for epileptogenesis as it plays a

key role in the induction of epileptogenesis and in seizure generation (Vezzani &

Friedman, 2011; van Vliet et al., 2014b). Despite, the number of studies investigating

the presence of BBB leakage in this context is limited. Thus, the occurence of BBB

leakage and its role in epileptogenesis need to be further investigated. Studies are

needed to better delimit the occurrence of BBB impairment and to identify the most

suitable time window for selected BBB-protective treatment approaches. Molecular

imaging modalities provide the possibility for this by obtaining longitudinal in vivo

information on BBB integrity.

Brain insult First clinical seizure Chronic epilepsy

Blood-brain barrier disturbance

Epileptogenesis only in minority of patiens → predicitive biomarkers

urgently needed

Latency phase

Possible time window for

antiepileptogenic treatment

Epileptogenesis

Figure 3: Epileptogenesis and possible time window for antiepileptogenic therapy

As only a limited number of brain insults result in epilepsy, biomarkers for epileptogenesis are urgently needed to identify at-risk patients as early as possible during epileptogenesis. Blood-brain barrier leakage is strongly associated with the initiation and progression of epileptogenesis, thus being a candidate biomarker. The latency period between initial brain insult and first clinical seizure offers a time window for future antiepileptogenic treatments in identified patients at risk with the long-term goal to prevent the development of epilepsy.

1.3.2 The blood-brain barrier and inflammation

Interactions between brain endothelium and astrocytes can influence BBB integrity in

both physiological and pathological conditions (Abbott et al., 2006). Recent studies

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point to an involvement of BBB impairment and subsequent albumin extravasation in

TGFßRII-mediated astrocytic transformation and its role in epileptogenesis (Ivens et

al., 2007). Astrocytes are involved in neuronal hyper-synchronicity and excitability

(Friedman et al., 2009). Activated Astrocytes and microglia cells can release pro-

inflammatory mediators (Allan et al., 2005) in response to seizures (Vezzani et al.,

2011). In the pilocarpine post SE rat model, increased interleukin-1ß (IL-1ß) and IL-1

receptor expression is present in parallel to BBB leakage (Ravizza et al., 2008).

Accumulating evidence suggests that IL-1ß is related to increased seizure

susceptibility and epileptogenesis (Vezzani & Baram, 2007; Ravizza et al., 2008).

Besides brain inflammation, peripheral inflammation is related to BBB impairment,

too. For example, an overexpression of BBB adhesion molecules was found to occur

in response to epileptiform neuronal activity in an experimental in vitro guinea pig

model (Librizzi et al., 2007). Circulating leucocytes bind to adhesion molecules at the

apical endothelium, a mechanism which can result in decreased BBB integrity and

subsequent brain edema as well as extravasation of blood components. These

components again trigger microglia activation (Rivest et al., 2000; Riazi et al., 2010).

Thus, a pharmacological treatment targeting inflammation and leucocyte-BBB

interactions might stabilize BBB integrity following SE.

1.4 Treatment strategies targeting blood-brain barrier integrity

Starting from the assumption (see 1.3.1) that a dysfunctional BBB integrity plays a

central role in epileptogenesis (Oby & Janigro, 2006; van Vliet et al., 2007b; Marchi

et al., 2011b; Vezzani et al., 2011; Abbott & Friedman, 2012), pharmacotherapeutic

approaches to restore or to maintain BBB integrity may help to modify or to prevent

epileptogenesis (Dedeurwaerdere et al., 2007). Possible therapeutic options include

targeting inflammation along with peripheral immune responses, changes in

metabolic equilibrium, glutamate receptor activation, increased blood pressure or

angiogenesis.

In a rat model of focal cerebral ischemia, cyclooxygenase-2 (COX-2) inhibition using

nimesulide led to decreased BBB impairment and reduced leukocyte infiltration

(Candelario‐Jalil et al., 2007). Another target for protection of BBB integrity

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(see 1.3.2) are leukocyte-endothelial interactions (Fabene et al., 2008). For example,

BBB leakage was prevented by blockade of leukocyte-vascular adhesion via α4

integrin specific antibodies after pilocarpine induced SE in mice (Fabene et al.,

2008). In a mouse model of cerebral malaria, increased BBB permeability, amongst

other things, was reduced by fingolimod (FTY720), a modulator of the sphingosine-1-

phosphate signaling cascade, in combination with the tyrosine kinase inhibitor

imatinib (Nacer et al., 2012). Another possible therapeutic approach is directed to

nitric oxide which can contribute to ischemia-induced inflammation and deterioration

of BBB integrity. Curcumin is a polyphenol which balances different cell signaling

pathways (Gupta et al., 2013). A single injection of curcumin following focal

ischemia/reperfusion in rats led to a reduced extravasation of Evans blue (Jiang et

al., 2007). As underlying mechanism, the authors suggest BBB protection, mediated

by a reduced incidence of nitric oxide and subsequently a reduced damage to

endothelial cells (Jiang et al., 2007).

Another interesting approach to prevent BBB leakage is treatment with an

angiotensin II type 1 receptor antagonist, losartan, which is clinically approved for

patients with hypertension. Different studies have shown that hypertension has

detrimental effects on BBB integrity (Bauer et al., 2011) and that blood pressure

influences seizure-induced BBB impairment (Nitsch & Klatzo, 1983; Bauer et al.,

2011). In diabetic rats with epinephrine-induced hypertension, losartan treatment

resulted in a significant reduction of BBB permeability (Kaya et al., 2003).

Ivanova et al. (2013) found that in the systemic kainate rat model, all animals that

were chronically treated with losartan (treatment started during SE) developed

spontaneous seizures. However, the latent period was significantly longer in

losartan-treated rats as compared to controls, suggesting epileptogenesis-modifying

effects of losartan. Moreover, anxiety levels were reduced and diurnal rhythms in

locomotor activity were absent in losartan-treated animals.

Tchekalarova et al. (2014a) compared the onset of kainate-induced seizures in

hypertensive and normotensive rats following 14 d of subcutaneous (s.c.) losartan

application. Losartan did not affect the seizure intensity but slowed down seizure

onset in the hypertensive group. Moreover, kainate-induced lipid peroxidation was

weakened in both groups. Thus, the authors suggest losartan to be beneficial as an

adjunctive treatment in SE by limiting oxidative stress and neurotoxicity.

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Pereira et al. (2010) showed that angiotensin II type 1 receptors are upregulated in

the brain of a rat epilepsy model and that the receptors are implicated in seizure

susceptibility. Losartan treatment resulted in a significant decrease in both frequency

and severity of epileptic seizures. In a recent study (Bar‐Klein et al., 2014), losartan

treatment was shown to prevent the occurrence of delayed recurrent seizures by

blocking albumin-induced TGF-ß activation in a rat model of vascular injury-induced

epileptogenesis. Tchekalarova et al. (2014b) found that losartan treatment after

kainate-induced SE alleviated seizure activity, extended the seizure-free latency

phase and decreased the frequency of seizures. Moreover, epilepsy-associated

behavioral changes like anxiety and depression were positively influenced by

losartan.

Other studies have shown that, inter alia, inflammatory reactions at the BBB are

relevant for the development of seizures (see 1.3.2). Thus, treatment with

glucocorticoids such as dexamethasone, which is widely used as an anti-

inflammatory and brain edema-reducing drug (Kaal & Vecht, 2004; Abbott et al.,

2006), may positively affect BBB integrity (Oby & Janigro, 2006; Marchi et al., 2012).

Dexamethasone increases the tightness of endothelial tight junctions (Cucullo et al.,

2004). Moreover, it can change BBB properties by regulation of efflux pumps like

p-glycoprotein which is highly expressed in endothelial brain cells (Bauer et al., 2005)

as well as by modulating gene transcription (Cucullo et al., 2004). In specific

neoplastic and inflammatory processes of the brain, glucocorticoids are BBB

protective and alleviate brain edema (Friedman & Heinemann, 2010). In a rat model

of allergic encephalomyelitis, increased BBB permeability could be counteracted

dose-dependently by short-term treatment with the broad-spectrum glucocorticoid

dexamethasone (Paul & Bolton, 1995). On the other hand, glucocorticoids are

ineffective or even harmful in patients with acute ischemic stroke (Kleinschnitz et al.,

2011). Adverse effects after dexamethasone treatment were published recently in the

lithium-pilocarpine post SE rat model, too. Duffy et al. (2014) found a deterioration of

both brain injury and survival rate following dexamethasone treatment.

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1.5 Animal models of epileptogenesis

Despite major research on alternative methods, it is so far impossible to dispense the

use of rodent models in epilepsy research as appropriate in vitro models allowing for

investigation of the complex underlying pathological mechanisms of epileptogenesis

are hitherto not available (Raol & Brooks-Kayal, 2012; Stewart et al., 2012).

Thus, chronic epilepsy models with spontaneously occurring seizures following a

primary brain insult are commonly used to investigate epileptogenesis (Löscher,

2002). These include chemical and electrical post-SE models, traumatic brain insult

models, the kindling model, febrile convulsion models as well as stroke models (Ben-

Ari et al., 1979; Honchar et al., 1983). These models allow for examinations of

epileptogenesis over the entire course of time. The post SE models are characterized

by a model-dependent latency phase preceding the occurrence of seizures (see 1.2).

The post SE models are particularly recommended for anti-epileptogenesis studies

(Stables et al., 2003) because these models of epileptogenesis display different

phenotypic features, for example a latent period varying in length (Galanopoulou et

al., 2012; White & Löscher, 2014). Chemical post-SE models are characterized by a

relatively short latency period and a high seizure frequency. Models that do not

exhibit these characteristics might abet false positive or false negative results.

1.5.1 The pilocarpine post status epilepticus rat model

Pilocarpine is an excitatory acetylcholine receptor agonist that evolves

parasympathomimetic effects via binding to the muscarinic subtype of acetylcholine

receptors (Hamilton et al., 1997). Subsequently, seizures are supposed to be

maintained via activation of NMDA receptors (Smolders et al., 1997). This leads to

increased hippocampal glutamate levels, triggering limbic seizures and SE. After

pilocarpine injection, animals display akinesia, ataxia, facial automatisms and

complex partial seizures that generalize secondarily (Turski et al., 1984; Turski et al.,

1989). SE is followed by a latent period, characterized by structural reorganization

processes and later by the appearance of spontaneous recurrent seizures (Curia et

al., 2008).

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1.5.2 The intrahippocampal kainate mouse model

Kainate is an agonist at glutamate receptors. It induces sustained neuronal

depolarization, resulting in SE. In the intrahippocampal kainate model, kainate is

administered via microinjection directly into the hippocampus of mice (Suzuki et al.,

1995; Bouilleret et al., 1999; Gröticke et al., 2008; Klein et al., 2014) or rats (Bragin et

al., 1999; Dudek et al., 2006). Animals develop a non-convulsive SE that persists

over several hours (Bouilleret et al., 1999; Gröticke et al., 2008; Maroso et al., 2011).

Generalized tonic-clonic seizures occur only rarely in mice (Riban et al., 2002). After

a latency period, frequent spontaneous and focally limited seizures occur. This model

is regarded one of the best models for TLE due to its resemblance to the clinical

situation (Sharma et al., 2007).

Still, the neurohistopathological changes occurring in the pilocarpine and focal

kainate models are very similar to those of patients with TLE, too. Histopathological

findings include neurodegeneration in hippocampal subregions, hippocampal

sclerosis as well as aberrant mossy fibre sprouting in the inner molecular layer of the

dentate gyrus in both models (Turski et al., 1983; Bouilleret et al., 1999; Pernot et al.,

2011). Moreover, behavioral, learning and memory deficits occur in the pilocarpine

model (Curia et al., 2008). As there is no model which simulates all relevant aspects

of disease, it can be useful to apply different models.

1.6 Non-invasive in vivo imaging of blood-brain barrier leakage

Figure 4: Principle mechanism for detection of blood-brain barrier leakage

The blood-brain barrier (BBB) functions as interface between blood and brain. Intravenously administered exogenous markers or endogenous markers deriving from the blood are excluded from entry into the brain under physiological conditions (left). A leaky BBB allows entry of these molecules, which can subsequently be detected in affected brain regions (right).

Blood vessel

Brain

BBB

Tracer / Contrastagent

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Different markers and methods have been used to visualize changes in BBB integrity

in both pre-clinical and clinical settings. The mechanism underlying them all is the

detection of blood-derived endogenous molecules or injected exogenous tracers that

cannot pass the intact BBB (see

figure 4) due to their polarity or molecular weight (Friedman et al., 2009). In the event

of BBB impairment, tracer extravasation into specific brain regions can be detected

and quantified (van Vliet et al., 2014a). Suitable in vivo imaging techniques for this

purpose include positron emission tomography (PET), single photon emission

computed tomography (SPECT), MRI and optical imaging.

Nuclear medicine imaging is an evolving field in small animal imaging. Until the

1950s, hand-held Geiger-Müller tubes were in general use to detect biodistribution of

radioactivity in patients (Allen Jr et al., 1951). The tubes reacted only to about 2 % of

total gamma ray emission; images were not generated. Since then, the technology

has evolved remarkably. Recent advances in small animal imaging technology have

facilitated the application of clinical PET and SPECT radiotracers for pre-clinical

small animal imaging. The continuing improvement in resolution and sensitivity has

permitted to study biomolecular changes in the brain during epileptogenesis

(Dedeurwaerdere et al., 2007). Both PET and SPECT as well as MRI allow for serial

non-invasive measurements in the same animal, supporting accurate assessment of

pathogenesis and pharmacological agent efficacy (Roselt et al., 2004). Thus, modern

non-invasive imaging methods can be valuable diagnostic tools for in vivo

investigation of BBB permeability changes during epileptogenesis (Wunder et al.,

2012). A great advantage of these methods is the fact that the same protocols can be

used for animal research and clinical applications. This facilitates the translation of

pre-clinical research into medical practice. In addition, the statistical power of

preclinical studies can be increased if each animal serves as its own control (Cherry

& Gambhir, 2001), resulting in reduced numbers of required experimental animals

(Cherry & Gambhir, 2001; Bankstahl & Bankstahl, 2012).

1.6.1 Positron Emission Tomography imaging

PET is a dynamic and non-invasive molecular imaging technique that allows in vivo

visualization of the spatial and temporal distribution of radiolabeled biological or

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pharmacological ligands or metabolic substrates, so-called radiotracers (Cherry &

Gambhir, 2001). Strengths of PET imaging involve high sensitivity, depth of

penetration and quantification. The spatial resolution is 5-7 mm in clinical and 1-2

mm in preclinical setups; the temporal resolution is seconds to minutes (James &

Gambhir, 2012). The method is based on decay of positron-emitting radionuclides

(see figure 5). After intravenous (i.v.) administration to the subject or animal, the

isotope is subjected to ß+-decay. It releases a positron from the nucleus which is

positively charged and has the mass of an electron. Upon collision with an electron

originating from the surrounding tissue, the positron annihilates, generating two

anti-parallel high energy photons of 511 keV. The emitted γ-radiation is measured

noninvasively using a PET camera as external detector. The camera comprises a

ring of detectors which are able of coincidence timing detection of the emitted

photons. Afterwards, the photons are converted into an electrical signal. Within the

field of view, iterative and non-iterative reconstruction algorithms create a map of

activity distribution over time. Thereby, dynamic images as well as time-activity

curves can be generated for regions of interest (Koeppe, 2002; Thompson, 2002;

Cherry & Dahlbom, 2004).

Radiotracer

e+

e+ e-511 keV

photon

511 keV

photon

*

*Coincidence

count

A B

Figure 5: Positron emission tomography imaging

A tracer labeled with a positron-emitting radioisotope is injected. The tracer circulates and binds to or accumulates in target structures. (A) Emitted positrons collide with electrons from the surrounding target tissue. The positron annihilates and generates two anti-parallel photons (511 keV). (B) Two photons, that strike opposing detectors at the same time, are counted as coincidences. The location of annihilations is calculated by observing multiple events. Electrical signals are converted into sinograms which are subsequently reconstructed to images. Figure modified from (Herschman, 2003).

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In vivo studies of epileptogenesis can benefit from PET as it enables the study of

functional metabolic processes with high sensitivity and accurate quantification. PET

has been used to study receptor-ligand interactions, enzyme activity, protein-protein

interactions, gene expression as well as cell- and gene-therapy (Herschman, 2003).

For example, PET allows the examination of glucose metabolism, protein synthesis

and neuro-receptor status in different cerebral diseases. Suitable PET tracers and

imaging protocols would permit for early assessment of BBB leakage as a potential

biomarker for epileptogenesis and for monitoring BBB-protective pharmacological

agent efficacy (Bankstahl & Bankstahl, 2012; Neal et al., 2013).

2-[18F]-fluoro-2-deoxy-D-glucose ([18F]-FDG) is currently the most widely used tracer

in epilepsy diagnosis (Dedeurwaerdere et al., 2007). It allows an indirect statement

on neuronal activity by quantitative determination of regional cerebral glucose

metabolism. [18F]-FDG is transported into cells by glucose transporters equivalent to

normal glucose (Phelps, 2000). Inside the cell, the tracer is converted to the

phosphorylated form by hexokinase. This form cannot be further metabolized and

gets "trapped" inside the cells. Its accumulation displays regional glucose utilization

patterns which can be measured by PET. In human medicine, [18F]-FDG PET scans

are used in epilepsy patients in whom MRI cannot clearly locate the epileptic focus

prior to surgery (Bankstahl & Bankstahl, 2012). Interictally, the epileptic focus is

usually characterized by a relative glucose hypometabolism. The regional extent of

hypometabolism was shown to increase with seizure frequency and duration of

disease and extends beyond the epileptic focus (Goffin et al., 2008). The reason for

this is so far unknown (Goffin et al., 2008). Preclinical [18F]-FDG PET studies after

acute kainate-induced SE demonstrated a glucose hypermetabolism primarily in the

hippocampus and entorhinal cortex (Kornblum et al., 2000; Mirrione et al., 2006). In

analogy to the situation in patients, longitudinal [18F]-FDG PET examinations in

chronic epilepsy models revealed hypometabolic changes in the course of

epileptogenesis. These changes began shortly after the initial insult and partially

persisted in the chronic phase (Goffin et al., 2009; Guo et al., 2009; Jupp et al., 2012;

Lee et al., 2012).

In addition to [18F]-FDG PET, which contributes to a better understanding of the local

brain metabolic activity, PET radiotracers are of great interest as they might enable

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investigation of changes in BBB permeability during epileptogenesis. Imaging BBB

permeability changes requires peripheral administration of non-permeable tracers

(Friedman et al., 2009). 68Gallium-ethylenediaminetetraacetate ([68Ga]-EDTA) is a

hexadentate chelator which cannot pass the intact BBB due to its molecular weight.

[68Ga]-EDTA PET was successfully used to assess BBB permeability in multiple

sclerosis patients (Pozzilli et al., 1988). So far, no PET tracers for detecting BBB

leakage have been used in epilepsy research. PET with a new tracer, [68Ga]

combined with the chelating agent diethylenetriamine pentaacetic acid

([68Ga]-DTPA), might be another appropriate approach (see manuscript 1, chapter 2).

A similar tracer, [99mTc]-DTPA, is a standard SPECT tracer for detecting BBB leakage

in patients (see 1.6.2). Moreover, a gadolinium (Gd) formulation combined with DTPA

as chelate (Gd-DTPA) is frequently used as contrast agent (CA) for detecting BBB

leakage on MRI in pre-clinic and clinic (see 1.6.3).

Other possible tracer candidates for imaging of BBB function include [68Ga]-albumin,

as albumin can only cross the BBB in pathological conditions (see 1.3.1),

[13N]-glutamate or [82Rb]-chloride (Saha et al., 1994; Wunder et al., 2012). Yen et al.

have demonstrated osmotically evoked BBB opening in rhesus monkeys using

[82Rb]-chloride (Yen & Budinger, 1981). Comparable studies in epilepsy models have

not yet been performed.

1.6.2 Single Photon Emission Computed Tomography imaging

SPECT is the most frequently used nuclear imaging technique in the clinic (Lecchi et

al., 2007). It is based on scintigraphy technique. The used nuclides emit single γ-rays

with different energies (James & Gambhir, 2012). A gamma camera captures data

from diverse positions by rotating around the subject or animal. The higher the

metabolic activity, the more accumulation of the radiolabeled tracer occurs in the

tissue. As it is not possible to obtain information on the photons origin as it is in PET,

a collimator is used which rejects diagonally incident photons. Hence, as many

photons are rejected, SPECT imaging is less sensitive than PET imaging. Its spatial

resolution is 8-10 mm in clinical and 1-2 mm in preclinical setups; its temporal

resolution is minutes (James & Gambhir, 2012). Nevertheless, SPECT tracers

generally have a longer half-life compared with PET tracers and are more readily

available. Advantages of SPECT include depth of penetration and multiplexing

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capabilities, meaning that different targets can be imaged simultaneously. Moreover,

SPECT is less expensive than PET. As PET and SPECT detect biochemical changes

and these usually occur prior to anatomical changes, they have a diagnostic

advantage in imaging acute processes compared to computed tomography (CT) and

MRI (James & Gambhir, 2012). However, by combining MRI and highly sensitive

nuclear medicine molecular imaging, the advantages of both techniques can be

combined (Blamire, 2014).

The SPECT tracer [99mTc]-DTPA was developed in 1969 for the study of brain,

kidneys and vascular dynamics (Hauser et al., 1970). The aims were to combine the

physical properties of the radionuclide [99mTc] with the advantageous biological

properties of the chelating agent DTPA. About 90 % of the tracer is excreted in 24 h

via the kidneys (Hauser et al., 1970). A preclinical study has shown that ultrasound-

induced (Lin et al., 2009) increased BBB permeability can be detected and quantified

using [99mTc]-DTPA SPECT. The same is true for BBB leakage following seizures

arising in connection with stroke in humans (Gilad et al., 2012). Moreover,

[99mTc]-DTPA was used for the detection of brain tumors in an animal model and

revealed subtle BBB leakage (Singh et al., 1991). Another tracer that might be used

for SPECT-imaging of BBB leakage is [99mTc]-albumin (Saha et al., 1994). Both

tracers cannot cross the intact BBB due to their molecular weight (Lampl et al.,

2005). These facts make [99mTc]-DTPA and [99mTc]-albumin promising tracers for the

study of BBB leakage in epileptogenesis models.

1.6.3 Magnetic Resonance imaging

MRI technique is based on electromagnetic effects of rotating hydrogen nuclei in

organic compounds. When placed inside a magnet, unpaired nuclear spins inside the

body function like magnetic dipoles (James & Gambhir, 2012). A radio frequency

electromagnetic pulse is applied, followed by a return to equilibrium (Gröhn &

Pitkänen, 2007). The dipoles arrange parallel or anti-parallel to the magnetic field,

thus producing a small net difference between numbers of parallel and anti-parallel

aligned dipoles. From this difference, the nuclear magnetic resonance (NMR) signal

is generated. Magnetic field gradients can be used to locate and assign the signals

source (Gröhn & Pitkänen, 2007), providing adequate signal-to-noise ratio and

images with excellent resolution. The MR signal is primarily dependent upon three

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factors: the concentration of nuclei and their constant spin around the axes, the

gyromagnetic ratio, which can be determined from the magnetization as well as from

the angular momentum (Greiner & Müller, 1994), and the polarization (James &

Gambhir, 2012).

Compared with nuclear medicine techniques, MRI stands out due to its excellent

spatial resolution (pre-clinical 25-100 µm, clinical ~ 1 mm, James & Gambhir, 2012)

and a high soft tissue contrast which makes it irreplaceable in particular for high

resolution in vivo visualization of anatomical changes in the brain and in cerebral

blood flow. The temporal resolution is minutes till hours. MRI is a very versatile

imaging technique (Blamire, 2014). However, due to its limited sensitivity, the ability

of MRI to display functional changes is limited (James & Gambhir, 2012).

Nevertheless, during recent years, spatial resolution has continuously improved in

MRI, allowing for better imaging protocols for BBB leakage in patients (Chassidim et

al., 2015) and providing the basis for evaluation of epileptic progression (Marchi et

al., 2012). Moreover, since MRI does not require exposure to radiation, it can be

performed repeatedly in both animals and patients (Runge et al., 1985; Neumann-

Haefelin et al., 2000). This is true for pre-clinical PET and SPECT as well. However,

radiation protection regulations need to be taken into consideration for personnel and

patients. Despite these advantages, MRI is still much less used in pre-clinical

epilepsy research than used in the clinic (Marchi et al., 2012).

Different changes in the brain can be assessed using MRI during epileptogenesis.

Cytotoxic edema, resulting from a breakdown of sodium-calcium pumps and

subsequent water imbalances between intracellular and extracellular compartments

(BBB is intact), occurs during and shortly after SE and can be detected using

diffusion-weighted MRI (Gröhn et al., 2011). Subsequent vasogenic edema, a

consequence of BBB leakage, can be assessed with T2-weighted MRI sequences.

T2 (transversal relaxation time) and T1 (longitudinal relaxation time) define the return

of hydrogen nuclei towards equilibrium after they were excited by radio frequency

pulses (Gröhn & Pitkänen, 2007) which are influenced by water dynamics. Thus, the

focus of contrast can be drawn for example on T2 or T1 relaxation or water diffusion

by modifying pulse sequences.

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Table 1: Representation of T1- and T2-contrast on MRI

Contrast Repetition time (TR)

=Transverse magnetization

Echo time (TE)

=Selective magnetization

Representation

T1 short short (< T2) Tissue with short T1 = bright

Tissue with long T1 = dark

T2 long (> T1) long Tissue with short T2 = dark

Tissue with long T2 = bright

T1-weighted MR sequences have a short repetition time and a short echo time (TE). Therefore, proton density and T2 relaxation have only low influences. Differences in signal intensities largely depend on the T1 relaxations of the tissue. T2-weighted MR sequences have a long TE. With increasing TE, signals diverge and the signal intensity of spin echoes is dependent from the T2 relaxations of the tissue.

Water and fat appear hyperintense in T2-weighted MRI. T2-weighted MR sequences

can detect edema formation. Contrast-enhanced MRI is a method to study BBB

leakage. Extravasated CA shortens the T1-relaxation time in affected brain regions

which are subsequently represented by bright signals on MRI images. An exogenous

way of contrast modification is i.v. injection of MRI CA. The utilization of Gd-based

MRI CA, such as Gd-DTPA, is considered the gold standard for non-invasive

assessment of BBB leakage (Tofts & Kermode, 1991). Gd-DTPA was the first

paramagnetic CA and clinically approved in 1988. It is excluded from brain entry due

to its polarity and molecular weight of 550 dalton (Da) in physiological conditions

(Kassner & Thornhill, 2011), has a half-life of 1.5 h and is excreted via the kidneys.

Other MRI CA used for detecting BBB leakage include gadobutrol and Gd-albumin.

Gd injections result in T1 hyperintensity via shortening of the T1 relaxation time, thus

improving image quality.

In epilepsy models, MRI was first applied in 1988. Karlik et al. (1991) examined rats

with an epileptic focus induced via penicillin application and King et al. (1991) found a

difference in image contrast between edematous and normal brain tissue in the

kainate rat model using diffusion tensor imaging (DTI). Until today, diverse studies

were performed in animal models, including T1- und T2-weighted MRI, functional

MRI, diffusion weighted imaging (DWI) as well as DTI (Marchi et al., 2012). However,

only few longitudinal MRI studies targeting structural reorganization processes during

State Of Research

25

epileptogenesis have been performed whilst most MRI studies in epilepsy models

have concentrated on neuronal loss (Gröhn & Pitkänen, 2007).

Different studies demonstrated the usability of MRI to localize brain regions with

increased BBB permeability and to quantify the extent of BBB leakage in

experimental models and in humans (Dijkhuizen, 2011; Kassner & Thornhill, 2011).

Using bolus injections of CA in the pilocarpine post SE rat model, acute BBB leakage

was only detected in the thalamus at 2 h after SE (Roch et al., 2002). Yet, it was not

possible to detect subtle BBB leakage occurring later in disease as shown by

histology results (van Vliet et al., 2007b; van Vliet et al., 2014b). In a rat model of

stroke following unilateral reperfused cerebral ischemic infarct, Nagaraja et al. (2007)

showed that a step-down infusion of Gd-DTPA reveals subtle BBB leakage that was

not detected in the same model using bolus injection. Bolus injection caused a sharp

increase in CA concentration with peak at around 10 s after administration which

declined after a short period of time. Step-down infusion resulted in a sharp rise and

subsequently almost steady-state of CA concentration over the entire period of scan

time. The authors concluded that, using the infusion protocol, higher plasma

concentrations result in increased CA extravasation into the brain parenchyma

compared to bolus injection, thus providing the possibility to detect even subtle BBB

leakage that cannot be observed using bolus injections. The suitability of infusion

protocols was confirmed in another stroke model (Knight et al., 2009) and in an

epilepsy rat model after systemic kainate-induced SE using a dynamic and a post-pre

approach (van Vliet et al., 2014b).

State Of Research

26

1.7 Aim of the studies

1.7.1 Study 1: Multimodality imaging of blood-brain barrier impairment during

epileptogenesis

The aim of this study was to determine [68Ga]-, [99mTc]- and gadolinium Gd-labelled

DTPA as well as Gd-albumin and gadobutrol uptake for the detection of BBB leakage

after lithium-pilocarpine induced SE as an epileptogenic brain insult using small

animal PET, SPECT and MRI in rats.

The working hypotheses were:

1. [68Ga]-DTPA PET, [99mTc]-DTPA SPECT and contrast-enhanced MRI allow for

early detection of BBB leakage following lithium-pilocarpine induced SE.

2. [68Ga]-DTPA PET is the most sensitive of the evaluated molecular imaging

techniques for detection of BBB leakage during early epileptogenesis.

3. PET detects BBB leakage more sensitive after step-down infusion of [68Ga]-DTPA

compared to bolus injection of [68Ga]-DTPA.

1.7.2 Study 2: Longitudinal in vivo evaluation and pharmacotherapy of blood-

brain barrier leakage in a rat model of epileptogenesis

The first objective of this study was to assess a non-invasive theranostic imaging

marker for BBB leakage longitudinally during early epileptogenesis. The second

objective was to evaluate whether two potentially BBB-protective treatments, losartan

and dexamethasone, can alleviate vascular edema and preserve BBB integrity during

early epileptogenesis in the lithium-pilocarpine post SE rat model. To this end, the

MRI protocol for Gd-DTPA from study 1 was used.


1. Longitudinal MRI following step-down infusion of the CA Gd-DTPA allows

detection of the peak increase in BBB leakage during early epileptogenesis.

2. Dexamethasone and losartan possess BBB-integrity stabilizing properties which

can be detected using the aforementioned MRI protocol.

State Of Research

27

1.7.3 Study 3: Dexamethasone protects blood-brain barrier integrity in a

mouse model of epileptogenesis

The aim of this study was to assess a non-invasive theranostic imaging marker for

BBB leakage longitudinally during early epileptogenesis in the intrahippocampal

mouse model using in vivo 7 T MR imaging following a step-down Gd-DTPA infusion

protocol. Dexamethasone treatment was evaluated with regard to BBB protection at

2 d and 4 d post SE.


1. MRI following step-down CA infusion is a suitable imaging biomarker for the

detection of BBB leakage in the intrahippocampal kainate mouse model.

2. Dexamethasone treatment improves BBB integrity following intrahippocampal

kainate injection as assessed by MRI.

Multimodality Imaging Of Blood-Brain Barrier Impairment During Epileptogenesis

28

2 Multimodality Imaging Of Blood-Brain Barrier Impairment During Epileptogenesis

Heike Breuer1,2, Martin Meier3, Sophie Schneefeld1, Wolfgang Härtig4, Alexander

Wittneben1, Martin Märkel4, Tobias L. Ross1, Frank M. Bengel1, Marion Bankstahl2,

Jens P. Bankstahl1*

1 Department of Nuclear Medicine, Hannover Medical School, Germany

2 Institute of Pharmacology, Toxicology and Pharmacy, University of VeterinaryMedicine Hannover and Center for Systems Neuroscience, Germany

3 Preclinical Imaging Labs, Central Laboratory Animal Facility & Institute for Laboratory Animal Science, Hannover Medical School, Germany

4 Paul Flechsig Institute for Brain Research, University of Leipzig, Germany

* Corresponding author: Jens P. Bankstahl, PhD, Department of Nuclear Medicine,

Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. Tel.:

+49-511-532-3504; Fax: +49-511-532-3761; Email: [email protected]

Published in

Journal of Cerebral Blood Flow and Metabolism (Epub ahead of print)

DOI: 10.1177/0271678X16659672

Financial support: This study was funded by the European Union Seventh’s

Framework Programme (FP7/2007-2013) under grant agreement n°602102

(EPITARGET). H. Breuer was supported by a scholarship from the Studienstiftung

des Deutschen Volkes.

Running headline: Multimodal BBB imaging during epileptogenesis

Multimodality Imaging Of Blood-Brain Barrier Impairment During Epileptogenesis

29

Abstract

Objective: Insult-associated blood-brain barrier (BBB) leakage is strongly suggested

to be a key step during epileptogenesis. In this study, we used three non-invasive,

translational imaging modalities, i.e. positron emission tomography (PET), single

photon emission computed tomography (SPECT), and magnetic resonance imaging

(MRI), to evaluate BBB leakage after an epileptogenic brain insult.

Materials and methods: Sprague-Dawley rats were scanned during early

epileptogenesis initiated by status epilepticus (SE). PET and SPECT scans were

performed using the novel tracer [68Ga]DTPA or [99mTc]DTPA, respectively. MRI

included T2 and post-contrast T1 sequences after infusion of Gd-DTPA, gadobutrol

or Gd-albumin.

Results: All modalities revealed increased BBB permeability 48 h post SE, mainly in

epileptogenesis-associated brain regions like hippocampus, piriform cortex,

thalamus, or amygdala. In hippocampus, Gd-DTPA-enhanced T1 MRI was increased

by 199%, [68Ga]DTPA PET by 37%, and [99mTc]DTPA SPECT by 56%. Imaging

results were substantiated by histological detection of albumin extravasation.

Conclusions: Comparison with quantitative PET and SPECT shows that MRI

sequences successfully amplify the signal from a moderate amount of extravasated

DTPA molecules, enabling sensitive detection of BBB disturbance in epileptogenesis.

Imaging of the disturbed BBB will give further pathophysiologic insights, will help to

stratify anti-epileptogenic treatment targeting BBB integrity, and may serve as a

prognostic biomarker.

Key words: Blood-brain barrier, epilepsy, imaging, MRI, PET

Longitudinal In Vivo Evaluation And Pharmacotherapy Of Blood-Brain Barrier Leakage In A Rat Model Of Epileptogenesis

30

3 Longitudinal In Vivo Evaluation And Pharmacotherapy Of Blood-Brain Barrier Leakage In A Rat Model Of Epileptogenesis

H. Breuer1,2, M. Meier3, W. Härtig4, J.P. Bankstahl2, M. Bankstahl1 1 Institute of Pharmacology, Toxicology and Pharmacy, University of Veterinary

Medicine Hannover and Center for Systems Neuroscience, Germany

2 Preclinical Molecular Imaging, Department of Nuclear Medicine, Hannover Medical School, Germany

3 Preclinical Imaging Labs, Central Laboratory Animal Facility & Institute for Laboratory Animal Science, Hannover Medical School, Germany

4 Paul Flechsig Institute for Brain Research, University of Leipzig, Germany

The extent of author’s contributions to this work:

A) Design of project: J.P.B., M.B.

B) Design of experiments: H.B., J.P.B., M.B., M.M., W.H.

C) Performance of experiments: H.B., M.M., M.B., W.H.

D) Analysis of experiments: H.B., M.B.

E) Writing and revision of manuscript: H.B., M.B., J.P.B.


31

3.1 Abstract

Objective: Blood-brain barrier (BBB) leakage is a frequent finding in association with

brain insults and may contribute to both initiation and progression of epilepsy. Hence,

BBB leakage might be a suitable biomarker for epileptogenesis and BBB-stabilizing

treatment might prevent or attenuate epileptogenesis. In this study, longitudinal

contrast-enhanced in vivo magnetic resonance imaging (MRI) was performed for

evaluation of BBB impairment during early epileptogenesis. Moreover, BBB integrity

and edema formation, as assessed by MRI, were used as surrogate markers for the

evaluation of potentially BBB-protective effects of losartan and dexamethasone.

Materials and methods: As primary brain insult, a status epilepticus (SE) was

induced by fractionated pilocarpine injection in 27 adult female Sprague-Dawley rats.

For longitudinal assessment of BBB leakage, one group of saline-treated (SE-saline)

rats was scanned before and 5 h, 48 h, 10 d (n = 5) and 4 d (n = 6 additional rats)

following SE. Six rats were treated with dexamethasone (SE-dexamethasone, 3 and

24 h post SE, 8 mg/kg i.p.; 48 h post SE, 4 mg/kg i.p.) and 5 rats with losartan

[SE-losartan, 30 min post SE, 50 mg/kg i.p.; 24 h till 5 d post SE, 5 mg/kg i.p. twice

daily; day 6 till 14, 80.4±38.1 mg/d, mean±standard error of the mean (SEM)],

respectively. Five additional rats (SE) which received no treatment after SE served

as controls for the SE-dexamethasone group. Animals of treatment groups were

scanned baseline and 48 h after SE and additionally 5 h and 10 d after SE in the

losartan-treated group. The MRI setup included a T2 sequence, followed by infusion

of gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA) and post-contrast T1

modified driven equilibrium fourier transformation (MDEFT). MR images were

analyzed by co-registration with a rat brain atlas and calculation of T1 and T2 signal

increase, normalized to pons. For histological evaluation of BBB integrity, animals

were infused with FITC-labelled albumin (100 mg/kg) and perfused thereafter.

Results: In SE-saline rats, BBB leakage was detected in hippocampus, amygdala,

thalamus, entorhinal cortex, piriform cortex, substantia nigra and frontal cortex at

48 h post SE. Moreover, BBB leakage was detected in the amygdala and in the

piriform cortex at 4 d post SE as well as in the entorhinal and piriform cortex at 10 d


32

post SE. Neither dexamethasone nor losartan protected BBB integrity. The spatial

extent of T1 intensity increase was comparable between SE-losartan and SE-saline

groups 48 h post SE. At 10 d, increased T1 values were only present in SE-losartan

animals (entorhinal cortex and piriform cortex). T2 signals, indicative of brain edema,

were significantly increased in SE-losartan vs. SE-saline rats at 48 h post SE in

substantia nigra and, like T1 values, present only in SE-losartan animals (entorhinal

cortex) at 10 d after SE. Dexamethasone treatment did not influence T1 values but

significantly increased T2 signals in the piriform cortex 48 h post SE vs. SE-saline.

Histological analysis revealed a significant increase of extravasation in the SE group

in amygdala, piriform and entorhinal cortex. Dexamethasone treatment resulted in

significantly more severe extravasation compared to SE-saline and to baseline in

amygdala, piriform and entorhinal cortex at all investigated section levels and in

thalamus vs baseline at section level -3.72 mm relative to bregma.

Conclusions: Gd-DTPA-enhanced MRI is sensitive to detect BBB leakage 48 h, 4 d

and 10 d after SE which could not be counteracted by two potentially BBB-protective

treatments. Furthermore, our data suggest that that losartan may exacerbate BBB

leakage and that losartan and dexamethasone can exacerbate SE-associated

cerebral edema at the chosen dosing regimen in specific brain regions in the

pilocarpine post SE rat model.

Key words: epilepsy, MRI, losartan, dexamethasone

3.2 Introduction

Status epilepticus (SE) and other brain insults can result in structural and functional

brain alterations associated with an increased risk of developing epilepsy (Herman,

2002; van Vliet et al., 2007b; Friedman et al., 2009; Pitkänen & Lukasiuk, 2009;

Löscher & Brandt, 2010). Since not all individuals develop epilepsy after brain insults

(Sloviter & Bumanglag, 2013), there is an urgent need for diagnostically detectable

biomarkers for epileptogenesis as a prerequisite to identify patients at risk as early as

possible in epileptogenesis. Recent findings strongly suggest that blood-brain barrier

(BBB) leakage as well as inflammation may contribute to both initiation and


33

progression of epileptogenesis (Janigro, 1999; Oby & Janigro, 2006; Friedman &

Heinemann, 2010; Ndode-Ekane et al., 2010; Friedman, 2011; Marchi et al., 2011b).

Therefore, early detection of BBB leakage by magnetic resonance imaging (MRI)

may be a suitable biomarker for epileptogenesis. Moreover, imaging biomarkers can

be used to evaluate effects and efficacy of potentially antiepileptogenic treatments in

the latency period after an epileptogenesis-triggering brain insult. Starting from the

assumption that increased BBB permeability plays a causative role in

epileptogenesis, pharmacotherapeutic approaches to restore or to maintain BBB

integrity are promising antiepileptogenic strategies (Friedman et al., 2009; Friedman

& Heinemann, 2010). In the presence of a leaky BBB, blood serum constituents

extravasate into the brain parenchyma, resulting in transforming growth factor ß

receptor II (TGF-ß)-mediated impaired astrocyte function, inflammatory signalling

responses and altered potassium distribution (Janigro et al., 1997; Heinemann et al.,

2000; Seiffert et al., 2004; Ivens et al., 2007; van Vliet et al., 2007b; Cacheaux et al.,

2009; David et al., 2009; Friedman et al., 2009; Duffy et al., 2014). It was reported by

Bar-Klein et al. (2014) that the angiotensin II type 1 receptor antagonist losartan

blocks albumin-induced TGF-ß activation in the brain and prevents delayed recurrent

seizures in a rat model of vascular injury-initiated epileptogenesis. In a recent study,

intracerebroventricular administration of losartan preceding pilocarpine-induced SE in

rats was found to inhibit SE-induced cognitive impairment and microglia-mediated

inflammation (Sun et al., 2015).

Another approach which may positively affect the recovery of BBB integrity is

treatment with glucocorticoids. Glucocorticoids exert BBB stabilizing effects and can

alleviate edema formation in specific neoplastic and inflammatory diseases of the

central nervous system (CNS, Friedman & Heinemann, 2010). Bertorelli et al. (1998)

showed that dexamethasone administration following cerebral ischemia appears to

reduce infarct volume in rats.

In the present study a non-invasive, potentially theranostic imaging marker for BBB

leakage was assessed longitudinally during early epileptogenesis. Furthermore,

gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA)-enhanced MRI was used

to evaluate whether two potentially BBB-protective compounds, losartan and


34

dexamethasone, can preserve BBB integrity and alleviate vascular edema during

early epileptogenesis in the pilocarpine post SE rat model.

3.3 Materials and Methods

3.3.1 Animals

All animal procedures were approved by the institutional animal care and use

committee and performed in accordance with German research regulations. Female

Sprague-Dawley rats (230-270 g, n = 27, Harlan Laboratories, Udine, Italy) were

housed in individually ventilated cages under controlled climate conditions (22±1°C,

humidity 45-55 %) and a 10/14 h dark light cycle. Food (Altromin 1324; Altromin

Spezialfutter GmbH, Lage, Germany) and autoclaved water were accessible ad

libitum. Animals were allowed to adapt to the new environment for a minimum of one

week and were handled daily before study initiation.

3.3.2 Status epilepticus

As epileptogenic brain insult, a SE was induced by fractionated pilocarpine injection.

The animals were pre-treated with lithium chloride (p.o., 127 mg/kg in 3 ml/kg 0.9 %

saline) 12-14 h and methyl-scopolamine (i.p., 1 mg/kg in 2 ml/kg 0.9 % saline) 30 min

before pilocarpine injection. Pilocarpine was injected every 30 min (bolus 30 mg/kg in

1 ml/kg, subsequently 10 mg/kg i.p.) till SE onset and limited to a maximum of three

injections per rat. Seizure onset and activity were monitored until rats developed

repetitive generalized stage 4 or 5 seizures according to Racine’s scale (Racine,

1975), which marked SE onset. This method was previously described (Glien et al.,

2001; Curia et al., 2008). Using the aforementioned protocol, all rats developed SE.

Ninety min after onset of SE, rats received two diazepam injections (10 mg/kg in

2 ml/kg i.p.) at intervals of 15-20 min to terminate seizure activity. If needed, a third

dose (5 mg/kg i.p.) was injected. Standard feed mash was fed by hand three times

daily over the first 4 d after SE. Fluid balance was stabilized with subcutaneous (s.c.)

saline and electrolyte injections (NaCl 0.9 % B. Braun and Sterofundin/G5, B. Braun


35

Melsungen, Germany; 5 ml s.c. each daily). This regimen was continued until

animals showed normal feed and water intake again at about 4 d after SE.

3.3.3 Chemicals and drugs

Unless otherwise stated, the chemicals used in this study (lithium-chloride, methyl-

scopolamine, pilocarpine, bovine albumin-fluorescein isothiocyanate conjugate

[FITC-albumin] and chloral hydrate) were obtained from Sigma-Aldrich Co. LLC

(Schnelldorf, Germany). Isoflurane (Isofluran Baxter) was purchased at Baxter Inc.

(Unterschleißheim, Germany) and diazepam (Diazepam-ratiopharm) from

Ratiopharm GmbH (Ulm, Germany). Gd-DTPA (Magnevist) was obtained from Bayer

HealthCare (Leverkusen, Germany), dexamethasone (Dexamethasone inject

JENAPHARM, 8 mg/2 ml) from Jena Pharm (Jena, Germany), losartan from Santa

Cruz Biotechnology, Inc. (Dallas, Texas, USA) and Sterofundin from B. Braun

Melsungen AG (Melsungen, Germany). Pilocarpine and chloral hydrate were diluted

in 0.9 % saline (B. Braun Melsungen AG). Losartan was dissolved in aqua ad

injectionem and FITC-albumin was dissolved in 0.1 M phosphate buffered saline

(PBS) prior to each administration.

3.3.4 Study design

Rats were randomly assigned to one of the following three groups: Saline treatment

after status epilepticus (SE-saline, n = 5-6), dexamethasone treatment after status

epilepticus (SE-dexamethasone, n = 6), Status epilepticus without treatment

(SE, n = 5) and losartan treatment after status epilepticus (SE-losartan, n = 5).

Animal numbers before SE are higher because baseline scans were performed on

naive animals (SE-saline and SE-losartan, n = 13; SE-dexamethasone and SE,

n = 11). For longitudinal in vivo assessment of BBB leakage during early

epileptogenesis, animals of the vehicle group (SE-saline) were scanned before and

at 5 h, 48 h, 4 d and 10 d after SE. SE-dexamethasone, SE and SE-losartan groups

were scanned baseline and 48 h after SE and additionally at 5 h and 10 d after SE in

the SE-losartan group.


36

3.3.5 Longitudinal in vivo assessment of blood-brain barrier leakage

Firstly, the time course of BBB leakage after SE was assessed using longitudinal 7 T

MRI at baseline (n = 11-13) and during early epileptogenesis at 5 h (n = 5), 48 h

(n = 5), 4 d (n = 6) and 10 d (n = 5) post SE in SE-saline rats. Less animals than at

baseline were used for subsequent scans. Saline treatment was started 30 min after

the first diazepam injection. Rats received 0.9 % saline in a volume of 1 ml/kg i.p.

twice daily at intervals of 12-14 h for the subsequent 5 d. This dosing regimen was

chosen to match injection times and volumes of the losartan-treated group.

3.3.6 Dexamethasone treatment

Results of MRI examinations following SE were compared to extravasation patterns

and edema formation in the SE-dexamethasone group as measured by 7 T MRI at

48 h post SE. This time point was chosen based on previous work from our group

(see chapter 2) which revealed prominent BBB leakage at 48 h post SE. Rats

received 8 mg/kg dexamethasone i.p. 3 h after the first diazepam injection, followed

by two dexamethasone injections at a dose of 4 mg/kg at 24 h and 48 h after SE.

Dexamethasone was administered 1.5 h before the MRI scan 48 h after SE. Dose

selection and dosing intervals were chosen based on personal communication with

C. Stegmayr and K.J. Langen (Research Center Jülich, Institute of Neuroscience and

Medicine, Jülich, Germany and Department of Nuclear Medicine and Neurology,

University of Aachen, Aachen, Germany), who detected a reduction of tumor-

associated BBB leakage at the used dose in a glioma rat model as assessed by

Evans blue extravasation.

3.3.7 Losartan treatment

Losartan is a clinically approved angiotensin II-receptor (type AT1)-antagonist which

selectively blocks the AT1-receptor and thereby the TGF-ß signal cascade (Bar‐Klein

et al., 2014). To evaluate the efficacy of losartan in protecting BBB integrity during

early epileptogenesis, five rats were treated with a bolus of 50 mg/kg losartan in a

volume of 1 ml/kg aqua ad injectionem i.p. 30 min after SE termination.

Subsequently, losartan was administered at a dose of 5 mg/kg i.p. twice daily from

24 h till 5 d post SE, followed by 9 d of oral administration (2 g/l in drinking water,


37

diluted freshly every day) from day 6 till 14 post SE. Losartan doses were chosen

based on a previous study (Bar‐Klein et al., 2014) in which systemic losartan

treatment in a rat model of vascular injury has shown anti-epileptogenic effects.

Animal weights and water intake were measured daily to calculate the individual

losartan uptake (80.4±38.1 mg/d, mean±standard error of the mean [SEM]).

3.3.8 Assessment of blood-brain barrier integrity

Small animal MRI setup included a T2 (multi slice multi echo) MSME sequence,

followed by intravenous (i.v.) infusion of Gd-DTPA and post-contrast T1 modified

driven equilibrium fourier transformation (MDEFT). For contrast agent (CA) infusion,

a catheter was fixed in a lateral tail vein and connected to a syringe pump (model

PHD Ultra; Harvard Apparatus Inc.). A 20 min step down infusion schedule was used

to infuse 1.78 ml of Gd-DTPA (0.5 mmol/ml) in order to quickly achieve and obtain a

plateau level of CA in the blood (Nagaraja et al., 2007; Knight et al., 2009; van Vliet

et al., 2014b, see Table 2 and chapter 2). All MRI scans were performed on a 7 T

(300 MHz) small animal MR system (Bruker BioSpin MRI GmbH) with a 16-cm

horizontal bore (Bruker BioSpec 70/16) under isoflurane anesthesia (3 % in air/O2

[2:1] for induction of anesthesia, 1-2.5 % in air/O2 [2:1] for maintenance of

anesthesia) with continuous control of body temperature and respiration. The system

was controlled by a Bruker BioSpec console (ParaVision 5.1) equipped with a 9 cm

inner diameter gradient insert (Bruker BGA-9S) and a 38-mm circular polarized rat

head volume coil with a rat brain receive-only coil array 11483V3 in combination with

a quadrature MRI transmit-only coil with active decoupling T11070. To ensure

identical positioning of rat and coil within the magnet bore between separate

measurements, the receive coil was placed at a fixed position on the animal bed with

the rat head fixed with an incisor bar. For registration purposes, a TRIPILOT scan

was acquired. Subsequently, regions subjected to brain edema were located using a

T2 weighted MSME sequence [Repetition Time (TR) = 2500 ms, Echo Time

(TE) = 11 ms]. The geometric parameters of the 2D scan were: 96 slices of 0.8 mm,

matrix 256 x 256 and field of view [FOV] 35 x 35 x 25.6 mm, resulting in a voxel size

of 0.136 x 0.136 mm. To evaluate time-dependant changes in T1 intensity during CA

infusion, dynamic short T1 weighted rapid acquisition refocusing (RARE) sequences


38

were obtained simultaneously (TR = 1500 ms, TE = 9 ms, 16 slices of 0.8 mm, slice

gap 1 mm, matrix 256 x 256 and FOV 35 x 35 x 25 resulting in a voxel size of

0.136 x 0.136). The sequence was repeated eleven times in a total of 30 min and

additionally after 50 min. Brain regions of interest were assessed by MDEFT method

with MDEFT preparation 30 min after CA infusion. The following geometric

parameters were chosen for the high resolution 3D scans: matrix 256 x 256 x 32 and

FOV 35 x 35 x 25.6 mm, resulting in a voxel size of 0.136 x 0.136 x 0.8 mm. A slice

thickness of 0.8 mm was selected to obtain detailed information of anatomical

structures.

Table 2: Gd-DTPA infusion protocol rats

Duration (min) Rate (ml/min) Volume (ml)

0.5 0.340 0.17

0.5 0.265 0.13

1.0 0.195 0.20

1.0 0.140 0.14

1.0 0.110 0.11

1.0 0.095 0.10

2.0 0.085 0.17

3.0 0.070 0.21

5.0 0.060 0.30

5.0 0.050 0.25

Infusion time = 20 min Total volume = 1.78 ml

3.3.9 Image analysis

For data evaluation, the image sets obtained at baseline and 5 h, 48 h, 4 d and 10 d

after SE were reconstructed in transaxial, coronal and sagittal images and displayed

using PMOD 3.5 software. The PMOD fusion tool was used to co-register T1 and

T2 weighted MR images to a rat brain atlas (Schwarz et al., 2006). T1 and T2 signals

were calculated and normalized to pons as a reference region. A total of eight brain

regions was analyzed: hippocampus, piriform cortex, entorhinal cortex, frontal cortex,

amygdala, thalamus, substantia nigra and cerebellum. Gd-leakage maps were


39

calculated using MATLAB software (MathWorks, Natick, Massachusetts, USA) and

SPM12 (UCL, London, UK).

3.3.10 Histological analysis of albumin extravasation

To complement the imaging results, histology as the gold-standard for detecting BBB

leakage was performed in SE-saline and SE-dexamethasone groups. Control rats

(n = 7) were used from a previous study (see chapter 2). After MR imaging (48 ± 6 h

post SE), bovine albumin-fluoresceinisothiocyanate conjugate (FITC-albumin) was

infused i.v. (100 mg/kg in 10 ml/kg, diluted in 0.1 M phosphate buffered saline [PBS];

infusion rate 1 ml/min) under isoflurane anesthesia. Two hours after FITC-albumin

application, rats were perfused through the left ventricle in a deep anesthetic state.

The vascular system was flushed at a speed of 16.6 ml/min with 125 ml 0.01 M PBS,

followed by fixative (250 ml 4 % paraformaldehyde) using a tubing pump (Ismatec

Reglo, Cole-Parmer, Wertheim, Germany). The brains were removed one hour after

perfusion and post fixed in 4 % paraformaldehyde for 24 h before being transferred to

and stored in 30 % sucrose in 0.1 M PBS with 0.2 % sodium azide at 4 °C. Coronal

sectioning (30 µm slice thickness) was performed using a freezing microtome

(Frigomobil 1205; Jung, Heidelberg, Germany). Brain sections were transferred into

0.1 M Tris-buffered saline (TBS, pH 7.4) containing 0.2 % sodium azide. For

conversion into a nickel-enhanced diaminobenzidine (DAB-Ni) reaction product

(Michalski et al., 2010), series of brain sections were rinsed with TBS and washed

with distilled water. The sections were subsequently exposed to 0.6 % H2O2 for 30

min to abolish endogenous peroxidase activity, extensively rinsed with TBS and

blocked with TBS containing 2 % bovine serum albumin and 0.3 % Triton X-100

(TBS-BSA-T) for 1 h. Afterwards, brain sections were incubated with peroxidase-

conjugated anti-FITC IgG (1:2000 in TBS-BSA-T; Dianova, Hamburg, Germany) for

16 h and stained with the chromogen DAB-Ni as previously described (Härtig et al.,

1995). Images of whole brain sections were acquired using a light microscope

(Keyence, Neu-Isenburg, Germany) and stained areas were calculated by the total

number of pixels in the stained area relative to the total area of the respective brain

section.


40

3.3.11 Statistical analysis

Differences in CA leakage and T2 intensities were compared by one-way analysis of

variance (ANOVA) with Dunnett’s post hoc test for individual differences.

Comparisons of two groups were performed using Bonferroni's multiple comparison

post hoc tests. GraphPad Prism 5 software (GraphPad Software Inc., La Jolla, CA,

USA) was used for statistical analysis and a p-value < 0.05 was considered

statistically significant. For MRI leakage maps, differences between baseline and

48 h post SE were calculated for SE-saline, SE-dexamethasone and SE-losartan

groups using a two-sample unpaired t-test, a significance level threshold of 0.001,

and a minimum cluster size of 100 voxels. All results are presented as mean

± standard error of the mean (SEM).

3.4 Results


All rats exhibited stage five seizures according to the Racine scale (Racine, 1975)

and subsequently developed SE. The latency to SE onset was not different between

the three study groups (44 ± 6 min SE-saline, 27 ± 4 min SE-dexamethasone, and

38 ± 4 min SE-losartan). Survival rate after SE was 97.3 %. All rats survived

treatment with dexamethasone or losartan, one rat (belonging to the SE-saline

group) died 12 h after SE. However, adverse effects in forms of gastric ulcers and

tympany were observed in one of the SE-dexamethasone rats during perfusion 48 h

post SE. Body weights significantly decreased after SE in all groups (SE-saline

group at day 1 and day 2, p < 0.05; SE-losartan group at day 3 and day 4, p < 0.05;

SE-dexamethasone group at day 2, p < 0.001) but returned to baseline levels in

SE-saline and SE-losartan groups at about 10 d post SE (see figure 6).


41

Figure 6: Weight development

Weight development in saline-treated animals post status epilepticus (SE-saline, n = 3-14), SE-dexamethasone (n = 5-6) and SE-losartan (n = 3-5) groups (mean ± SEM, one-way ANOVA, Dunnett’s post-hoc test vs. day 0, i.e. before SE induction, *p < 0.05).

3.4.2 Longitudinal in vivo assessment of blood-brain barrier leakage in saline-

treated group

To investigate the time course of BBB impairment during early epileptogenesis in the

lithium-pilocarpine post SE rat model, SE-saline animals were scanned longitudinally

at five time points during epileptogenesis: at baseline and at 5 h, 48 h, 4 d and 10 d

post SE. To improve the sensitivity for detection of BBB leakage we used the

previously adjusted step-down infusion protocol for Gd-DTPA (Nagaraja et al., 2007,

see chapter 2).

3.4.2.1 Development of T1 intensity during early epileptogenesis

Gd-DTPA leakage, resulting in elevated T1 values and being indicative of BBB

impairment, was absent at baseline and at 5 h post SE (see figure 7 A and figure 8,

only selected brain regions are shown). Significantly increased T1 values vs.

baseline were present in both limbic and cortical brain structures 48 h post SE. The

maximum permeability increase occurred in the amygdala with a mean T1 intensity

increase of 221 % (p < 0.001). Moreover, significantly increased T1 intensities were

detected in the dorsal hippocampus as well as in thalamus, entorhinal cortex, piriform

cortex, substantia nigra (p < 0.001 for all brain regions) and frontal cortex (p < 0.05).

0 1 2 3 4 5 6 7 8 9 10100

150

200

250

300

350 SE-saline

SE-dexamethasoneSE-losartan

**

**

*

Days post SE

Weig

ht

(g)


42

Baseline5h post SE48 h 4 d 10d Baseline5h post SE48 h 4 d 10d Baseline5h post SE48 h 4 d 10d Baseline5h post SE48 h 4 d 10d0

1

2

3

4

5

* * *

Amygdala Frontal Cortex Substantia nigraPiriform cortex

B 5h 48h 10d4d 10d4d B 5h 48h 10d4d B 5h 48h 10d4d

*

B 5h 48h 10d4d B 5h 48h 10d4d B 5h 48h 10d4d B 5h 48h 10d4d

*

T1

in

ten

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reg

ion

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ns

]

* * *

At 4 d post SE, Gd-DTPA leakage was present in the amygdala and in the piriform

cortex, proposing a partial recovery of BBB integrity in the other formerly affected

brain regions. The time point 10 d post SE was characterized by BBB leakage

occurring solely in cortical subregions (entorhinal cortex and piriform cortex). Visual

observation of dynamic T1 rapid acquisition refocusing (RARE) magnetic resonance

images before and after contrast agent infusion during epileptogenesis showed

similar results (see figure 8) but was not quantified.

A

Baseline5h post SE48 h 4 d 10d Baseline5h post SE48 h 4 d 10d Baseline5h post SE48 h 4 d 10d Baseline5h post SE48 h 4 d 10d0

1

2

3

4

5

**

*

BaselineSE-saline

Hippocampus Entorhinal Cortex CerebellumThalamus

*

B 5h 48h 10d4d B 5h 48h 10d4d B 5h 48h 10d4d B 5h 48h 10d4dT1

in

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43

Figure 7: Quantification of changes in T1 and T2 intensities during epileptogenesis

(A) T1 intensities as surrogate marker for blood-brain barrier (BBB) leakage measured pre (n = 13), 5 h (n = 5), 48 h (n = 5), 4 d (n = 6) and 10 d (n = 5) post status epilepticus (SE). Exemplary brain regions after step-down infusion of gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA). At 48 h post SE, significantly increased T1 values vs. baseline are present in all investigated brain regions. The greatest permeability increase occurs in the amygdala at 48 h. Moreover, T1 values are significantly increased at 4d (amygdala and piriform cortex) and 10 d post SE (entorhinal and piriform cortex). (B) T2 values, indicative of brain edema, measured pre (n = 13), 5 h (n = 4), 48 h (n = 5), 4d (n = 6 ) and 10 d (n = 5) post SE. T2 values are increased at 48 h and 4 d in hippocampus, thalamus and entorhinal cortex (all regions p < 0.001), at 48 h post SE in amygdala, piriform cortex and cerebellum and at 4 d after SE in substantia nigra. Mean ± SEM, one-way ANOVA, Dunnett’s post-hoc test vs. B, *p < 0.05. B = baseline).

3.4.2.2 Development of T2 intensity during early epileptogenesis

At 48 h, T2 values, indicative of brain edema, were significantly increased in seven

examined brain regions: the anterodorsal hippocampus, thalamus, entorhinal cortex,

cerebellum, amygdala and piriform cortex (all regions p < 0.001, see figure 7 B and

figure 8). At 4 h post SE, T2 values were still increased in hippocampus, thalamus

and entorhinal cortex as well as in substantia nigra. Subsequently, values decreased.

No increased T2 values were present 10 d after SE.

B

B 5h 48h 4d 10d Baseline5h post SE48 h 4 d 10d Baseline5h post SE48 h 4 d 10d Baseline5h post SE48 h 4 d 10d0

1

2

3

4

5

*

Amygdala Frontal Cortex Substantia NigraPiriform Cortex

B 5h 48h 10d4d B 5h 48h 10d4d B 5h 48h 10d4dB 5h 48h 10d4d

*

B 48h 4d 10d5h B 48h 4d 10d5h B 48h 4d 10d5h

* *

T2

in

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]

B 5h 48h 4d 10d Baseline5h post SE48 h 4 d 10d Baseline5h post SE48 h 4 d 10d Baseline5h post SE48 h 4 d 10d0

1

2

3

4

5

** *


B 5h 48h 10d4d B 5h 48h 10d4d B 5h 48h 10d4d

*

B 5h 48h 10d4d

**

B 48h 4d 10d5h B 48h 4d 10d5h B 48h 4d 10d5h

*

T2

in

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]


44

Figure 8: Longitudinal magnetic resonance imaging during epileptogenesis

Coronal T2 (left); high-resolution T1 modified driven equilibirum fourier transformation (MDEFT, center left) after contrast agent infusion and dynamic T1 rapid acquisition refocusing (RARE) magnetic resonance images before (center right) and after contrast agent infusion (right) during epileptogenesis. Changes are represented as bright enhancement in brain regions affected by blood-brain barrier leakage or cerebral edema. B = baseline.

3.4.3 Pharmacotherapy with potentially blood-brain barrier protective drugs

The results of the longitudinal examinations were subsequently compared to

extravasation patterns and edema formation in SE-dexamethasone and SE-losartan

groups.

B

5 h

48 h

4 d

10 d

T2 MSME T1 MDEFT T1 RARE pre CA T1 RARE post CA

0 166


45

Figure 9: Comparative T2, T1 and leakage map images following saline, dexamethasone and

losartan treatment

(A) Coronal T2 weighted MR images, (B) gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA)-enhanced T1 MR images, and (C) SPM leakage maps indicate edema formation and blood-brain barrier leakage 48 h post status epilepticus (SE) in SE-saline as well as in SE-dexamethasone and in SE-losartan groups. (A) Edematous brain tissue and (B) blood-brain barrier leakage are represented as bright enhancement in hippocampus, thalamus, amygdala, entorhinal and piriform cortex. (D) T-maps resulting from comparisons of Gd-DTPA extravasation between baseline and 48 h post SE (n = 5) confirm the results (unpaired t-test, p < 0.001, minimum cluster size of 100 voxels).

3.4.3.1 Effects of dexamethasone on blood-brain barrier integrity

Dexamethasone treatment failed to attenuate or reverse BBB leakage and edema

formation during early epileptogenesis (see figure 9 and figure 10). The extent of

BBB leakage in SE-dexamethasone rats was comparable to SE rats. T1 intensities

were significantly increased at 48 h in the hippocampus, thalamus, entorhinal cortex,

amygdala, piriform cortex, frontal cortex and substantia nigra of both groups.

Saline


46

Figure 10: Quantification of BBB leakage and brain edema after dexamethasone treatment

Dexamethasone treatment does not attenuate blood-brain barrier (BBB) leakage and edema formation during early epileptogenesis. Mean (A) T1 intensities as surrogate marker for BBB leakage measured pre (n = 11) and 48 h following status epilepticus (SE) in dexamethasone (SE-dexamethasone, n = 6) and status epilepticus (SE, n = 5) groups. T1 intensities are increased at 48 h in hippocampus, thalamus, entorhinal cortex, amygdala, piriform cortex, frontal cortex and substantia nigra of both groups. (B) T2 intensities. T2 values are significantly increased at 48 h in SE-dexamethasone vs. SE groups in the piriform cortex and vs. baseline in most investigated brain regions (Mean ± SEM, one-way ANOVA and Bonferroni’s post test, *p < 0.05, significance vs. baseline. #p < 0.05 SE-dexamethasone vs. SE. B = baseline).

B

A

B 48 48 dex B 48 48 dex B 48 48 dex B 48 48 dex0

1

2

3

4 CerebellumHippocampusanterodorsal

Thalamus

*

B 48h B 48h B 48h B 48h

SESE-dexamethasone

Baseline

Entorhinal cortex

*

B 48h B 48h B 48h B 48h

T2 in

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B 48 48 Dexa B 48 48 Dexa B 48 48 Dexa B 48 48 Dexa0

1

2

3

4 Substantia nigraAmygdala Piriform Cortex

**

B 48h B 48h B 48h B 48h

Frontal Cortex

B 48h B 48h B 48h B 48h

#

T2

in

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]

B 48 48 dex B 48 48 dex B 48 48 dex B 48 48 dex0

2

4

6 CerebellumHippocampus

anterodorsalThalamus

**

**

B 48h B 48h B 48h B 48h

SESE-dexamethasone

Baseline

Entorhinal cortex

**

B 48h B 48h B 48h B 48h

*

T1 i

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B 48 48 Dexa B 48 48 Dexa B 48 48 Dexa B 48 48 Dexa0

2

4

6 Substantia nigraAmygdala Piriform Cortex

**

*

B 48h B 48h B 48h B 48h

Frontal Cortex

**

B 48h B 48h B 48h B 48h

*

*

* *

T1 in

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47

T2 values were significantly increased in SE-dexamethasone vs. SE animals in the

piriform cortex 48 h after SE. Moreover, T2 values were significantly increased only

in SE-dexamethasone animals at 48 h in thalamus, entorhinal cortex, amygdala,

piriform cortex and substantia nigra (see figure 10 B). The mean T2 increase in the

anterodorsal hippocampus at 48 h was 37 % vs. 19 % for SE-dexamethasone and

SE groups, respectively; 38 % vs. 18 % in thalamus, 54 % vs. 23 % in the entorhinal

cortex, 59 % vs. 13 % in the piriform cortex, 52 % vs. 15 % in the amygdala and

23 % vs. 11 % in substantia nigra. Edema formation and SPM mapping are displayed

in figure 9.

3.4.3.2 Effects of losartan on blood-brain barrier integrity

Furthermore, we aimed to elucidate whether treatment with losartan, a clinically

approved angiotensin II receptor (type AT1) antagonist, preserves BBB integrity and

reduces brain edema formation following SE. We found that the CA distribution in

terms of affected brain regions as detected by high-resolution T1 MDEFT at 48 h

after SE did not differ between SE-losartan and SE-saline groups. Hippocampus,

thalamus, entorhinal cortex, amygdala, piriform cortex, substantia nigra (all regions

p < 0.001) and frontal cortex (p < 0.05) exhibited significant increases in T1 intensity

vs. baseline (see figure 11 A). At 10 d post SE increased T1 values vs. baseline were

only present in SE-losartan animals (entorhinal cortex and piriform cortex). Contrast

agent extravasation into affected brain regions and Gd-DTPA leakage maps

confirming these results are shown in figure 9.

A

Baseline5h post SE5 h post SE Losartan48 h48 h Losartan10d10d LosartanBaseline5h post SE5 h post SE Losartan48 h48 h Losartan10d10d LosartanBaseline5h post SE5 h post SE Losartan48 h48 h Losartan10d10d Losartan0

1

2

3

4

5

T1

in

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] Hippocampus Entorhinal Cortex Cerebellum

* *

* * SE-saline

Thalamus

Baseline

* * SE-losartan

*

B 5h 48h 10d B 48h 10d B 48h 10d B 48h 10d5h 5h 5h


48

Figure 11: Quantification of blood-brain barrier leakage and edema after losartan treatment

T1 and T2 intensities before (n = 13), 5 h (n = 4 - 5), 48 h (n = 5) and 10 d (n = 5) post status epilepticus (SE) and losartan (SE-losartan) or saline treatment (SE-saline) in different brain regions during epileptogenesis. (A) Shows T1 intensities as surrogate marker for blood-brain barrier (BBB) leakage, exemplified for different brain regions after step-down gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA) infusion. T1 intensities are significantly increased vs. baseline in the same brain regions of both groups. However, brain uptake of Gd-DTPA vs. baseline is increased only in SE-losartan animals at 10 d after SE (entorhinal and piriform cortex). (B) T2 values are significantly higher in SE-losartan vs. SE-saline groups at 48 h in substantia nigra. Increased T2 values vs. baseline are present only in SE-losartan rats at 48 h in hippocampus, thalamus and substantia nigra and at 10 d in the entorhinal cortex. Mean ± SEM, one-way ANOVA, Bonferroni's post hoc test. * p < 0.05, significance SE-losartan or SE-saline vs. baseline, # significance SE-losartan vs. SE-saline. B = baseline.

B

0

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]

B 5h 48h 10d

Amygdala Frontal Cortex Substantia NigraPiriform Cortex

*

B 5h 10d48h B 5h 48h 10d B 5h 48h 10d

* *** *__#

0

1

2

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]

B 5h 48h 10d


*

B 5h 10d48h B 5h 48h 10d B 5h 48h 10d

**

Baseline

SE-salineSE-losartan*

* *

Baseline5h post SE5 h post SE Losartan48 h48 h Losartan10d10d LosartanBaseline5h post SE5 h post SE Losartan48 h48 h Losartan10d10d LosartanBaseline5h post SE5 h post SE Losartan48 h48 h Losartan10d10d LosartanBaseline5h post SE5 h post SE Losartan48 h48 h Losartan10d10d Losartan0

1

2

3

4

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] Amygdala Frontal Cortex Substantia nigra

*

*

* *

Piriform cortex

* *

*

B 5h 48h 10d B 48h 10d B 48h 10d B 48h 10d5h 5h 5h

* * *


49

48 h post SE – Saline 48 h post SE – Dexa48 h post SE – Control

Significantly higher T2 values were detected in the SE-losartan group vs. SE-saline

in the substantia nigra at 48 h post SE (p < 0.05, see figure 11). Increased T2 values

vs. baseline were present only in SE-losartan rats at 48 h in hippocampus, thalamus

and substantia nigra and at 10 d in the entorhinal cortex. (p < 0.05; see figure 11).

Edema formation is displayed in figure 9 A.

3.4.4 Histological analysis of albumin extravasation

Histological analysis revealed albumin extravasation at ~48 h post SE (see figure

12). Dexamethasone treatment resulted in significantly increased extravasation in the

thalamus vs. baseline at section level -3.72 mm relative to bregma. In amygdala,

piriform and entorhinal cortex, dexamethasone treatment led to significantly more

severe extravasation compared to both baseline and SE-saline animals at all

investigated section levels. Visual inspection of the cerebellum by an experienced

experimenter did not reveal any albumin extravasation.

A


50

B

Thalamus

0

2

4

6

+0.36 mm -3.72 mm-1.56 mm

SE-dexamethasone

Control

SE-saline

Section level relative to bregma

*

Sta

ined

are

a [

%]

Piriform/entorhinal cortex and amygdala

0

2

4

6

8

10

+0.36 mm -3.72 mm-1.56 mm

Section level relative to bregma

*

**

#

#

#

Sta

ined

are

a [

%]

Figure 12: Histological evaluation of albumin extravasation

(A) Exemplary brain sections of a control rat, a saline-treated rat 48 h post status epilepticus (SE), and a dexamethasone-treated rat 48 h post SE after conversion of FITC-albumin signal into a light-microscopy reaction product at about -3.72 mm relative to bregma. (B) Quantification of stained area relative to the total section area at +0.36, -1.56 and -3.72 mm relative to bregma. Data is presented as mean ± SEM. Asterisk indicates statistical significance compared to control (n = 4-7), # indicates statistical significance of SE-dexamethasone (n = 6) compared to SE (n = 10) after calculation by one-way ANOVA followed by Bonferroni’s multiple comparison test (p < 0.05).


51

3.5 Discussion

The aims of this study were to (i) longitudinally assess BBB leakage following lithium-

pilocarpine-induced SE using 7 T T1 MRI with step-down infusion of Gd-DTPA and to

(ii) investigate BBB integrity stabilizing properties of dexamethasone and losartan.

BBB leakage and edema formation as assessed by 7 T MRI served as surrogate

markers for BBB stabilizing therapeutic effects of dexamethasone and losartan

compared with rats treated with saline or without treatment.

The main findings from this study are:

1) MRI sensitively detects BBB leakage as well as edema formation during

epileptogenesis in the lithium-pilocarpine rat model.

2) BBB leakage and edema following SE are limited to specific brain regions and

subject to changes in temporal occurrence and intensity.

3) Pharmacotherapy with dexamethasone or losartan revealed no BBB protective

effect. On the contrary, we found evidence that losartan treatment may

exacerbate BBB leakage and that both treatments increase edema formation

in certain brain regions after SE.

This study longitudinally detects and quantifies BBB leakage during epileptogenesis

using high-field MRI after step-down infusion of Gd-DTPA. The time course of

T1 intensity increase, indicative of BBB leakage, was found to be different dependent

on brain regions. Quantification and spatial distribution of BBB leakage were highest

48 h after SE, affecting both limbic and cortical brain regions. A previous study using

step-down gadobutrol infusion detected increased T1 intensities 24 h post SE limited

to limbic brain regions and postulated that the motor cortex was not affected (van

Vliet et al., 2014b). In the present study we used 7 T MRI after step-down CA

infusion (Nagaraja et al., 2007; Knight et al., 2009; van Vliet et al., 2014b) to detect

BBB leakage. This approach combines high-field MRI, an imaging protocol with high

spatial resolution (Nagaraja et al., 2007; van Vliet et al., 2014b) and nearly steady-

state CA levels improving contrast enhancement throughout imaging, thus allowing

for sensitive detection of subtle changes of BBB leakage in vivo throughout


52

epileptogenesis. Significantly increased T1 values vs. baseline were present in both

limbic and cortical brain structures 48 h post SE with the amygdala being the region

of the highest increase in T1 intensity. By day 4, BBB leakage was still present in the

amygdala and in the piriform cortex, suggesting a partial recovery of BBB integrity in

formerly affected brain regions like hippocampus, entorhinal cortex and thalamus. At

10 d after SE, BBB leakage was detected anew, limited to the entorhinal and piriform

cortex and lower compared to 48 h. Similar results were also found in models of

traumatic brain insults (Shapira et al., 1993; Başkaya et al., 1997). A second opening

of the BBB occurred 3-7 d following traumatic brain injury and was not accompanied

by edema formation, as assessed by quantitative Evans blue measurement (Başkaya

et al., 1997). Van Vliet et al. (2014b) detected BBB leakage in the chronic phase six

weeks following kainate-induced SE in all investigated limbic brain regions in the

systemic kainate rat model. The reappearance of BBB leakage at later time points

may be caused by recurrent seizure activity.

Peak values of T2 intensities were detected 48 h post SE. This finding corresponds

to those of previous pre-clinical studies by Roch et al. (2002), Choy et al. (2010) and

Duffy et al. (2014) who detected peaks of T2 intensity around 48 h following brain

insults. Furthermore, hippocampal edema was shown to occur within 48 h after SE in

human subjects, too (VanLandingham et al., 1998; Scott et al., 2002). Cerebral

edema was limited to hippocampus, thalamus, entorhinal cortex, cerebellum,

amygdala and piriform cortex and had returned to baseline levels in all investigated

brain regions by d 10 in the present study, suggesting cerebral edema to appear

predominantly early after SE and to resolve within 10 d thereafter. These findings are

in accordance with Choy et al. (2010) who used T1 and T2 weighted sequences as

well as apparent diffusion coefficient MR imaging and found changes in MR signals

returning to baseline levels around day 7 post SE.

The second aim of this study was to evaluate effects of dexamethasone and losartan

treatment on BBB integrity following SE. Inflammation in the CNS is correlated to

impaired BBB integrity which again triggers seizures and epileptogenesis (Seiffert et

al., 2004; Ivens et al., 2007; Marchi et al., 2007b; Van Vliet et al., 2007a).

Inflammatory processes following brain insults are complex and were observed


53

“inside” in the brain parenchyma and “outside” at endothelial cells of the BBB

(Vezzani & Granata, 2005; Ravizza et al., 2008). Therefore, treatment with broad-

spectrum anti-inflammatory and immunosuppressive drugs like dexamethasone

(McMaster & Ray, 2007) may be beneficial in antiepileptogenic trials.

Dexamethasone acts on the glucocorticoid receptor (Auphan et al., 1995), inducing

target genes and interfering between glucocorticoid receptor and transcription factors

like NF-kappa-B, inhibiting its activity. However, the exact mechanisms of

dexamethasone-effects on BBB integrity are not known (Duffy et al., 2014).

Pilocarpine-induced seizures and SE are accompanied by activation of receptors and

adhesion molecules triggering immunologic reactions (Fabene et al., 2008; Marchi et

al., 2009). Fabene et al. (2008) showed that BBB leakage was prevented by blocking

leucocyte-endothelial adhesion. The same molecules that trigger immunologic

reactions in the lithium-pilocarpine model are target structures of corticosteroids

(Marchi et al., 2011a). Therefore, we hypothesized that dexamethasone treatment

might reveal BBB-stabilizing effects. In the present study, unexpectedly,

dexamethasone and losartan treatment failed to attenuate or reverse BBB leakage

and edema formation at the used treatment regimen during early epileptogenesis in

the pilocarpine post SE rat model.

Previous studies that evaluated effects of dexamethasone on BBB integrity have

reported conflicting findings (Duffy et al., 2014; Holtman et al., 2014). In different

inflammatory and neoplastic conditions of the central nervous system (CNS),

glucocorticoids had BBB stabilizing effects and counteracted edema formation

(Friedman & Heinemann, 2010). In specific pediatric epilepsies, for example the

Landau-Kleffner syndrome, glucocorticoids are used as treatment in clinics (Vezzani

et al., 2011). In drug-resistant pediatric epilepsies, dexamethasone can interrupt SE

and reduce seizure occurrence (Marchi et al., 2011a). However, like in the present

study, dexamethasone treatment was ineffective to harmful in a post-SE rat model as

assessed by Duffy et al. (2014) and also in patients with acute ischemic stroke

(Kleinschnitz et al., 2011). Moreover, Smith-Swintosky et al. (1996) showed that

neuronal loss in hippocampal CA3 and CA1 regions following kainate-induced SE

can be reduced by metyrapone administration which inhibits endogenous

glucocorticoid production.


54

Interestingly, we found no differences in T1 intensities between SE-dexamethasone

and SE animals 48 h after SE. Both affected brain regions and extent of BBB leakage

after triple dexamethasone treatment were comparable between groups. On the

contrary, T2 values were significantly higher in the SE-dexamethasone group

compared with the SE-group in the piriform cortex, suggesting an exacerbation of

cerebral edema by dexamethasone. Besides, edema was detected in thalamus,

entorhinal cortex and amygdala only in the SE-dexamethasone group. Our findings

are consistent with a study from Duffy et al. (2014) who showed increased T2

alterations 48 h and 4 d after SE even at lower doses of dexamethasone (2 mg/kg)

compared with the present study. Moreover, the authors found that dexamethasone

deteriorates hippocampal injury three weeks following SE. Maintaining a balance

between damaging and rehabilitating processes is important for successful treatment

strategies (Gröhn & Pitkänen, 2007). Brain-protective effects of early inflammation

after brain insults (Serrano et al., 2011) which may be counteracted by

dexamethasone were suggested (Duffy et al., 2014) as a possible reason for the

worsening of edema formation. Moreover, dexamethasone was found to attenuate

cerebral edema in a non-acidotic state whilst aggravated brain edema was detected

after dexamethasone treatment under acidotic conditions in a study on rats following

traumatic brain injury (Tran et al., 2010). Thus, we cannot rule out that acid-base

homeostasis has had an effect on the result of dexamethasone treatment in this

study. Besides, the treatment protocol may have influenced results. The effective

therapeutic time window after brain insults can be narrow in pre-clinical studies

(Herman, 2002), and we tested only one dosing regimen. Thus, different dosing and

application intervals may reveal a different outcome. Characteristics of the lithium-

pilocarpine rat model resemble those of human temporal lobe epilepsy (TLE; Leite et

al., 2002). One example is the presence of a latency phase which is following SE and

preceding the occurrence of spontaneous recurrent seizures as well as structural

reorganization processes. However, there is no model which simulates all relevant

aspects of human TLE. Thus, the used animal model may play a role in the outcome

of BBB protective treatment, too. It can be advisable to study pathophysiological

processes in different animal models. Therefore, we re-investigated effects of


55

dexamethasone on BBB integrity using a different animal model and dosing regimen

(see chapter 4).

Losartan acts on angiotensin II type 1 receptors in astrocytes and blocks albumin-

induced TGF-ß activation (Bar‐Klein et al., 2014). Thus we expected that losartan

might prevent a further damaging of BBB integrity by antagonizing TGF-ß mediated

transcriptional changes, impaired astrocyte function, inflammatory signalling

responses and altered potassium equilibrium (Janigro et al., 1997; Heinemann et al.,

2000; Seiffert et al., 2004; Ivens et al., 2007; van Vliet et al., 2007b; Cacheaux et al.,

2009; David et al., 2009; Friedman et al., 2009; Duffy et al., 2014). This is the first

study to investigate BBB integrity following losartan-treatment in the lithium-

pilocarpine rat model during early epileptogenesis. Surprisingly, losartan-treatment

was found to exacerbate brain edema in substantia nigra 2 d after SE and to favor

BBB leakage at 10 d after SE.

Pereira et al. (2010) found that angiotensin II type 1 receptors were upregulated in

the brains of audiogenically induced seizure models. Losartan treatment significantly

decreased frequency and severity of epileptic seizures. In a recent study,

intracerebroventricular administration of losartan before pilocarpine induced SE in

rats was found to inhibit SE-induced cognitive impairment and microglia-mediated

inflammation (Sun et al., 2015). However, the clinical relevance is arguable as

losartan administration preceded the insult, suggesting an insult-modification.

The present study was not designed to determine the mechanism for exacerbation of

edema and BBB leakage after losartan treatment. Angiotensin II type 1 receptors are

implicated in seizure susceptibility in different epilepsy models (Tchekalarova &

Georgiev, 2005; Pereira et al., 2010). In previous studies, losartan-treatment delayed

the onset of chronic seizures in rat models of vascular injury and after kainate-

induced SE (Ivanova et al., 2013; Bar‐Klein et al., 2014; Tchekalarova et al., 2014a).

These studies have in common that they evaluated effects of losartan-treatment in

the chronic epileptic phase. Bar‐Klein et al. (2014) acquired electrocorticography

(ECoG) recordings starting from day 28 after the initial insult and one week after drug

withdrawal.


56

In the present study, we evaluated changes in BBB-integrity during early

epileptogenesis (until day 10 post SE). Effects of losartan on acute alterations of BBB

integrity after SE have not been investigated before. We cannot rule out that losartan,

despite the lack of BBB protective effects during early epileptogenesis, may have

revealed disease modifying effects if animals had been monitored in the chronic

phase. Such modifications may include the number of animals developing epilepsy,

seizure onset or seizure frequency.

Another factor that might be considered is that albumin uptake into astrocytes seems

to be mediated not only by TGF-ß mediated changes but also by other pathways

including caveolae-mediated albumin-endocytosis (Bar‐Klein et al., 2014), indicating

that losartan might only partially block albumin uptake into astrocytes.

3.6 Conclusion

Longitudinal changes in T1 and T2 values following SE can be detected via high-field

MRI following step-down infusion of Gd-DTPA. Post SE treatment with losartan or

dexamethasone does not reduce vascular edema or BBB leakage during early

epileptogenesis in the lithium-pilocarpine rat model. Rather, both treatments can

exacerbate SE-associated cerebral edema. However, we could not rule out in this

study whether losartan or dexamethasone had epileptogenesis modifying effects.

Thus, more studies are needed to further investigate the antiepileptogenic potential

of dexamethasone and losartan.

ACKNOWLEDGEMENTS: C. Bergen and M. Märkel are gratefully acknowledged for their

technical assistance. This study is funded by the European Union’s Seventh Framework

Programme (FP7) under grant agreement n°602102 (EPITARGET). H.B. was supported by a

scholarship from the Studienstiftung des Deutschen Volkes.

CONFLICTS OF INTEREST: none


57

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Dexamethasone Reduces Blood-Brain Barrier Leakage in a Mouse Model of Epileptogenesis

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4 Dexamethasone Reduces Blood-Brain Barrier Leakage in a Mouse Model of Epileptogenesis

H. Breuer1,2, M. Meier3, J.P. Bankstahl2, M. Bankstahl1

1 Institute of Pharmacology, Toxicology and Pharmacy, University of Veterinary

Medicine Hannover and Center for Systems Neuroscience, Germany

2 Preclinical Molecular Imaging, Department of Nuclear Medicine, Hannover

Medical School, Germany

3 Preclinical Imaging Labs, Central Laboratory Animal Facility & Institute for

Laboratory Animal Science, Hannover Medical School, Germany

The extent of author’s contributions to this work:

Design of project: J.P.B., M.B.

Design of experiments: H.B., J.P.B., M.B., M.M.

Performance of experiments: H.B., M.B., M.M.

Analysis of experiments: H.B.

Writing and revision of manuscript: H.B., M.B., J.P.B.


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4.1 Abstract

Objective: Epileptogenesis can be initiated by primary brain insults and results in

chronic epilepsy, characterized by the occurrence of spontaneous recurrent seizures.

Growing evidence suggests that brain insult-associated blood-brain barrier (BBB)

impairment is a key step in the initiation of epileptogenesis and may contribute to

disease progression. Therefore, BBB-protective treatment might prevent or attenuate

the process of epileptogenesis. In this study, longitudinal in vivo 7 T small animal

magnetic resonance imaging (MRI) was used to detect changes in BBB leakage

during epileptogenesis and to evaluate potentially BBB-protective effects of

dexamethasone treatment.

Materials and methods: As primary brain insult, status epilepticus (SE) was induced

by intrahippocampal injection of kainate in 13 male NMRI mice. Seven sham animals

received intrahippocampal injections of saline. Following SE, mice were treated with

dexamethasone [SE-dex, n = 5; 6, 24, 48, 72 h post SE, 2 mg/kg intraperitoneally

(i.p.)] or vehicle saline (SE-KA, n = 8). Dexamethasone treated mice were scanned

before (baseline), 48 h and 4 d after SE. Vehicle and sham groups were scanned

additionally on d 16 and 37 after SE or sham surgery, respectively. High field 7 T

small animal MRI consisted of a T2 map for anatomical co-localization and a T1

modified driven equilibirum fourier transformation (MDEFT) sequence after step-

down infusion of gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA). Images

were analyzed by co-registration with a standard mouse brain atlas. T1 signal

increase in dorsal and ventral hippocampus, amygdala, thalamus and cortex was

normalized to cerebellum and calculated using PMOD software (PMOD Technologies

Ltd., Zurich, Switzerland).

Results: High field MRI revealed BBB leakage in epilepsy-associated brain regions

during epileptogenesis. In the hippocampus of SE-KA mice, T1 signal after contrast

agent (CA) infusion increased up to 274 % in the ipsilateral hippocampus vs.

baseline 48 h after SE (p < 0.05). The ipsilateral ventral hippocampus as well as

amygdala, thalamus and cortex were subjected to BBB leakage at 2 d and 4 d after

SE. T1 values subsequently decreased during the five weeks of follow-up. Sixteen


64

days post SE, T1 values were increased in the ipsilateral dorsal hippocampus and

thalamus. At 37 d post SE, T1 values were increased in hippocampus, thalamus and

cortex but had returned to baseline levels in all other investigated brain regions. In

SE-KA vs. sham animals, significantly increased T1 signals were present 48 h after

SE in dorsal and ventral hippocampus, amygdala and thalamus. Dexamethasone

treatment resulted in significantly reduced BBB leakage in hippocampus and

thalamus. Contrast agent extravasation into the dorsal hippocampus was reduced by

20 % on day 2 (p < 0.05) in the ipsilateral brain hemisphere and by 24 % on day 4 in

the contralateral brain hemisphere in SE-dex vs. SE-KA mice (p < 0.05).

Conclusion: Contrast-enhanced MRI is sensitive to detect BBB leakage in the

intrahippocampal kainate mouse model. Our results show for the first time a

successful reduction of BBB leakage by a dosing regimen of dexamethasone in mice

during epileptogenesis using in vivo MR imaging. A potentially beneficial impact on

chronic seizure development will be tested.

Key words: antiepileptogenesis, epilepsy, imaging, pharmacotherapy

4.2 Introduction

Temporal lobe epilepsy (TLE) is the most common form of epilepsy in adults

(Sloviter, 2005). It is usually induced by a primary brain insult triggering a cascade of

structural and functional changes in the brain, termed epileptogenesis (Herman,

2002; Friedman et al., 2009; Pitkänen & Lukasiuk, 2009; Löscher & Brandt, 2010).

So far, the development of TLE in at-risk patients cannot be predicted or prevented

and no antiepileptogenic therapies have been identified (Sloviter & Bumanglag,

2013). The current antiepileptic medication for chronically epileptic patients is solely

symptomatic in terms of seizure suppression and has no anti-epileptogenic effects.

Small animal models have been comprehensively used to study epileptogenesis,

providing an abundant record of histological data (Jupp et al., 2007). In contrast to

histological methods which are performed post mortem, non-invasive in vivo

magnetic resonance imaging (MRI) enables longitudinal evaluation of changes in

blood-brain barrier (BBB) integrity on an individual basis in the same animal (Hsu et


65

al., 2007; Jupp et al., 2007). This provides the opportunity to further examine the

process of epileptogenesis and favours the development of early diagnostic

biomarkers and disease-modifying or anti-epileptogenic therapies. One suitable

model for in vivo imaging of BBB leakage might be the intrahippocampal kainate

mouse model. This model is suitable for the study of epileptogenesis and

antiepileptogenesis, for research on drug resistance and to evaluate antiepileptic

therapies (Stables et al., 2002; Stables et al., 2003). It was used in the present study

because it is characterized by a fast development of spontaneous seizures with high

seizure frequency, allowing for a statement on the effectiveness of potentially BBB-

stabilizing drugs early after the initial status epilepticus (SE). As epileptogenesis-

initiating brain insult, the glutamate receptor agonist kainate is injected into the dorsal

hippocampus (Suzuki et al., 1995). Subsequently, mice develop a non-convulsive SE

(Bouilleret et al., 1999; Gröticke et al., 2008).

Several neurologic diseases, including epilepsies, are accompanied by pathological

changes in BBB integrity (Abbott et al., 2006). Accumulating evidence suggests that

BBB leakage influences both initiation and progression of epileptogenesis (Oby &

Janigro, 2006; Friedman & Heinemann, 2010; Marchi et al., 2011b). Extravasation of

blood components initiates a signalling cascade resulting in increased neuronal

excitability (Oby & Janigro, 2006; Ndode-Ekane et al., 2010). Thus, pharmacotherapy

aiming to restore or maintain BBB integrity may reveal antiepileptogenic or

epileptogenesis-modifying effects (Friedman et al., 2009; Friedman & Heinemann,

2010).

Leucocyte-endothelial interactions were shown to be involved in BBB impairment

(Rivest et al., 2000; Riazi et al., 2010). Treatment with anti-inflammatory drugs like

glucocorticoids were shown to be BBB protective in brain tumors and in specific

inflammatory brain diseases in humans (Friedman & Heinemann, 2010). In rats,

dexamethasone reduced the infarct volume when administered following cerebral

ischemia (Bertorelli et al., 1998). Effects of dexamethasone on BBB integrity of

healthy mice were studied by Hedley-Whyte et al. (1986). Dexamethasone treatment

resulted in decreased BBB permeability for the macromolecule horseradish


66

peroxidase. Thus, dexamethasone treatment may provide BBB stabilizing effects

during epileptogenesis, too.

In the present study, we longitudinally evaluated BBB leakage during epileptogenesis

in the intrahippocampal kainate mouse model using a high field strength (7 T)

magnet. The MRI protocol was subsequently used to follow and to evaluate effects of

dexamethasone treatment on BBB integrity.

4.3 Materials and methods

4.3.1 Animals

Adult male NMRI mice (n = 20) were purchased from Charles River Laboratories, Inc.

(Sulzheim, Germany) at six weeks of age and housed in separately ventilated cages

under a 14/10 h dark light cycle and controlled climate conditions (22 ± 1 °C, humidity

45 – 55 %). Standard feed (Altromin 1324; Altromin Spezialfutter GmbH & Co. KG,

Lage, Germany) and autoclaved water were freely accessible. All mice were allowed

to adapt to the new environment for at least two weeks before being used in

experiments. Animals were handled at least three times before start of experiments.

All animal procedures were performed in accordance to the EU council directive

86/609/EWG and the German Law on Animal Protection and approved by the

institutional animal care and use committee. Greatest efforts were made to minimize

animal suffering and to reduce the number of animals used.

4.3.2 Chemicals and drugs

Chloral hydrate and kainate monohydrate were obtained from Sigma-Aldrich

Co. LLC. (Schnelldorf, Germany). Isoflurane (Isofluran Baxter) was purchased from

Baxter Inc. (Unterschleißheim, Germany), gadolinium-diethylenetriamine pentaacetic

acid (Gd-DTPA, Magnevist) was obtained from Bayer HealthCare (Leverkusen,

Germany), dexamethasone (Dexamethasone inject JENAPHARM, 8 mg/2 ml) from

Jena Pharm (Jena, Germany) and electrolyte solution (sterofundin) and saline

(NaCl 0.9 % B. Braun) from B. Braun Melsungen AG (Melsungen, Germany).


67


A SE was induced by unilateral intrahippocampal kainate injection in 13 mice as

epileptogenesis-triggering primary brain insult. For surgery, animals were

anesthetized with chloral hydrate (400 mg/kg in 10 ml/kg saline i.p.). Body

temperature, heart rate and respiratory rate were monitored continuously. Kainate

was diluted in saline (10 mg kainate in 2345 µl saline) and 50 nanoliters of the

solution, equivalent to 213,2 nanograms kainate, were injected stereotaxically

(Stoelting Co, Wood Dale, Illinois, USA) into the cornu ammonis 1 region of the right

dorsal hippocampus as previously described by Gröticke et al. (2008). Coordinates

for kainate injection were: anterior-posterior: -2.1 mm, laterolateral: -1.6 mm and

dorsoventral: -2.3 mm; relative to bregma. A micro syringe (Hamilton Company,

Franklin, Massachusetts, USA) was lowered to the correct position and left in place

for 2 min prior to injecting 0.05 µl kainate solution slowly over one minute. Sham

animals (n = 7) were injected with 0.05 µl saline instead of kainate. The needle was

left in the injection site for another 2 min to prevent reflux. After surgery, animals

were kept on a heating mat to prevent hypothermia until they were fully awake. Any

seizure activity or abnormal behavior was recorded. All mice received sterofundin

injections [0.5 ml subcutaneous (s.c.) and 0.5 ml i.p.] and body temperature was

controlled before returning to home cage. During subsequent days, body weight, feed

and water intake, movement and body posture as well as grooming and fur condition

were evaluated according to a scoring protocol twice daily (see Table 3). None of the

experiments resulted in scores above 1. Additionally, mice received sterofundin

injections twice a day (0.5 ml s.c.) and were offered standard feed mash for the first

3 d after surgery.


68

Table 3: Model-specific criteria for animal evaluation after surgery

Parameter Feed and water intake and body weight

Movement and body posture

Grooming and fur

Score

0 normal quick and typical movement, curious, normal body posture

normal

1 reduced feed/water intake, loss of body weight < 10 %

reduced activity, normal reaction to stimuli

normal grooming behavior, reduced gloss of the fur

2 severely reduced feed/water intake, loss of body weight < 20 %

no spontaneous activity, reduced reaction to stimuli

reduced grooming behavior, reddish fur around eyes and nose; penis prolapse

3 no feed/water intake, loss of body weight > 20 %

no spontaneous activity, no reaction to stimuli and/or humpy back

no grooming, shaggy fur, red exudate around eyes and nose; penis prolapse

4.3.4 Study design

BBB integrity and response to dexamethasone treatment were assessed by MRI

(Bruker BioSpin, Ettlingen, Germany). Mice were randomly assigned to one of three

study groups prior to surgery: (1) kainate plus vehicle treatment (SE-KA, n = 8),

(2) sham (intrahippocampal saline, n = 7) and (3) kainate plus dexamethasone

treatment (SE-dex, n = 5). Mice in sham and SE-KA groups were scanned before

(baseline) and at 48 h, 4 d, 16 d and 37 d after surgery for longitudinal in vivo

evaluation of BBB leakage during epileptogenesis. To compare BBB leakage

patterns with and without dexamethasone treatment, SE-dex mice were scanned at

baseline and additionally at 48 h and 4 d after SE.

4.3.5 Evaluation of dexamethasone treatment on blood-brain barrier integrity

during early epileptogenesis

Treatment with the broad spectrum anti-inflammatory drug dexamethasone was

evaluated for BBB protective effects during the acute phase of epileptogenesis in the

intrahippocampal kainate mouse model. T1 intensities after CA infusion in SE-dex

mice were compared with extravasation patterns in SE-KA mice as measured by

MRI. Dexamethasone was used as a commercial solution and injected at a dose of

2 mg/kg i.p. five times following kainate injection (at 6 h, 24 h, 48 h, 72 h and 96 h).

Dose selection and dosing intervals were chosen based on literature (Hedley‐Whyte


69

& Hsu, 1986; Bertorelli et al., 1998; Campolo et al., 2013; Al-Shorbagy et al., 2012).

Saline treatment was started 6 h after kainate injection in the SE-KA group. Mice

received 0.9 % saline in a volume of 0.5 ml/kg i.p. once daily for the subsequent 4 d.

This dosing regimen was chosen to match injection times and volumes of the SE-dex

group.

4.3.6 Magnetic Resonance Imaging setup

Anesthesia for MRI was induced by isoflurane inhalation [3 % in air/O2 (2:1) for

anesthesia induction, 1-1.5 % in air/O2 (2:1) for maintaining anesthesia]. A catheter

was fixed in a lateral tail vein to allow contrast agent (CA) administration via infusion

pump (model PHD Ultra; Harvard Apparatus Inc., South Natick, Massachusetts,

USA). All investigations were performed on a 7 T (300 MHz) small animal MR system

(Bruker) with a 16-cm horizontal bore (Bruker BioSpec 70/16) and ParaVision 5.1

(Bruker) acquisition software. The MRI sequences included a T2 map for anatomical

co localization and a post-contrast high resolution T1 modified driven equilibirum

fourier transformation (MDEFT) sequence which was started 30 min after intravenous

(i.v.) infusion of gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA) to assess

BBB leakage. Step-down infusion of CA was previously reported to increase MRI

sensitivity for changes in BBB integrity and their delineation in rats (Nagaraja et al.,

2007; Knight et al., 2009). In this study, we modified our previously adjusted rat

infusion-protocol (Table 4) for imaging BBB leakage post SE in mice.

Table 4: Gd-DTPA infusion protocol mice

Step Duration (min) Rate (ml/min) Volume (ml)

1 0-0.5 0.056 0.028

2 0.5-1 0.044 0.022

3 1-2 0.033 0.033

4 2-3 0.023 0.023

5 3-4 0.018 0.018

6 4-5 0.016 0.016


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Step Duration (min) Rate (ml/min) Volume (ml)

7 5-7 0.014 0.028

8 7-10 0.012 0.036

9 10-15 0.010 0.050

10 15-20 0.008 0.040

Infusion time = 20 min

Total volume = 0.294 ml

Geometric imaging parameters of the 3D scan were 25 x 25 x 13.6 mm field of view

and 256 x 256 x 34-pixel matrix, resulting in a voxel size of 0.098 x 0.098 x 0.4 mm

[repetition time (TR) = 40 ms, echo time (TE) = 3.5 ms, flip angle = 18°,

ID = 1100 ms]. As radiofrequency coil, a mouse brain receive-only coil array

11765V3 was used in combination with a quadrature MRI transmit-only coil with

active decoupling T11070. The receive coil was placed on the animal bed in a

defined position and mice were fixed with an incisor bar to allow comparable

positioning between scan time points and animals.

4.3.7 Image analysis

Accurate segmentation of epilepsy-associated brain regions was achieved by co-

registration of MR images to a mouse brain atlas (Mirrione et al., 2006) using PMOD

3.5 software (see chapter 2). The following brain regions were analyzed: dorsal

hippocampus, ventral hippocampus, amygdala, thalamus and cortex. T1 signal

increase was calculated and normalized to cerebellum as the reference region.

4.3.8 Statistical analysis

Results are displayed as mean ± standard error of the mean (SEM). Multiple

comparisons of Gd-DTPA leakage were calculated by one-way analysis of variance

(ANOVA) with or with Bonferroni’s post hoc test for individual differences using

GraphPad Prism 5 software (GraphPad Software Inc., La Jolla, CA, USA). A p-

value < 0.05 was considered statistically significant.


71

4.4 Results

4.4.1 Tolerability of dexamethasone in the intrahippocampal kainate mouse

model

Kainate injection resulted in characteristic behavioral changes in all mice. Mild

forelimb clonus, circling, and freezing behavior as well as persisting immobility were

observed. One mouse developed generalized clonic seizures. No mouse from the

sham group displayed seizure-like behavior. Survival rate after surgery was 85.7 %

(6 out of 7 mice), 100 % (8 out of 8 mice) and 100 % (5 out of 5 mice) in sham,

SE-KA and SE-dex groups. Following surgery or SE no significant differences in

body weight were found between groups. The mean scores for movement and body

posture as well as for grooming were found to range between 0 and 1 in the first 3 d

following surgery in the SE-KA group. A score of 0 represents quick and typical

movement, curious behavior, a normal body posture and normal grooming behavior.

A score of 1 represents a reduced activity but normal reaction to stimuli and normal

grooming behavior but reduced gloss of the fur (see Table 3). In the sham group,

mice were scored 0 concerning movement and body posture and grooming from

day 1 post surgery. SE-dex mice were scored 1 in the first 2 d following SE. Score 2

or 3 did not occur in any mouse. No adverse effects of dexamethasone were

observed during the study or at necropsy thereafter.


72

A B

D

C

Figure 13: Localization of kainate injection as assessed by magnetic resonance imaging (MRI)

(B) Kainate was injected into the cornu ammonis 1 region of the right hippocampus according to Paxinos (2001). Localization was visualized by (A) coronal, (C) sagittal and (D) horizontal MR imaging of the injection site.

4.4.2 Longitudinal Magnetic Resonance imaging during epileptogenesis

reveals blood-brain barrier leakage in epilepsy associated brain regions

Longitudinal in vivo MRI was used to assess BBB leakage before and at d 2, d 4, d

16 and d 37 following focal kainate or saline injections into the right hippocampus.

MRI revealed significantly increased T1 values, indicative of BBB leakage, in limbic

brain regions of SE-KA mice vs baseline. In the hippocampus of SE-KA mice, T1

signal after contrast agent (CA) infusion increased up to 274 % in the ipsilateral

hippocampus 48 h after SE vs. baseline (p < 0.001 , see figure 15). The ipsilateral

ventral hippocampus as well as amygdala, thalamus and cortex were subjected to

BBB leakage, too. T1 hyperintensity was still present in the ipsilateral hippocampus,

the amygdala, thalamus and cortex at 4 d post SE. T1 values subsequently

decreased during the five weeks of follow-up. Sixteen days post SE, T1 values of the

ipsilateral dorsal hippocampus and thalamus were increased. At 37 d post SE, T1


73

values were increased in hippocampus, thalamus and cortex but had returned to

baseline levels in all other investigated brain regions.

Moreover, longitudinal in vivo MR imaging revealed significantly increased T1 values,

indicative of BBB leakage, in limbic brain regions of SE-KA vs. sham groups

(see figure 14). Statistical analyses between groups for each time point revealed

significantly higher T1 values 2 d following surgery in the SE-KA group vs. sham in 4

of the examined brain regions: the ipsilateral dorsal hippocampus, the ipsilateral

ventral hippocampus, the ipsilateral amygdala and the ipsilateral thalamus.

Hippocampus dorsal

B li kainatB re kainatSham li 4848 li kainatSham re 4848 re kainatSham li 4d4d li kainatSham re 4d4 re kainatSham li 1616d li kainatSham re 1616 re kainatSham li B3737d li kainatSham re B3737 re kainat0

1

2

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4 Baseline contralateral

Baseline ipsilateral

2 4 16 37

__*

* significance kainate vs. sham

Baseline

#

#

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Sham contralateralKainat contralateralSham ipsilateralKainat ipsilateral

# significance kainate vs. baseline

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1

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Figure 14: Quantification of blood-brain barrier leakage during epileptogenesis

Comparison of T1 intensities between kainate (SE-KA) and sham groups for hippocampus, amygdala, thalamus and cortex before (n = 6) and at 2 d (n = 5-6), 4 d (n = 4-5), 16 d (n = 4-5) and 37 d (n = 3-5) after surgery. Longitudinal in vivo magnetic resonance imaging (MRI) analyses between groups for each time point reveal significantly higher T1 values in the SE-KA group vs. sham in the ipsilateral dorsal hippocampus, ventral hippocampus, amygdala and thalamus at 2 d following surgery. One-way ANOVA, Bonferroni’s post hoc test, data marked with * indicate a significant difference vs. sham. All values are presented as mean ± standard error of the mean (SEM).

4.4.3 Dexamethasone treatment protects blood-brain barrier integrity

Dexamethasone treatment was found to significantly reduce BBB leakage in epilepsy

associated brain regions in mice post kainate-induced SE (see figure 15 and figure

16). (1) In the dorsal hippocampus, dexamethasone treatment reduced the T1 signal

intensity by 20.43 % 2 d after SE and by 24.07 % on day 4 as compared to SE-KA

mice (ipsilateral, p < 0.05). (2) In the ventral hippocampus, dexamethasone treatment

reduced the T1 signal intensity by 33.79 % 2 d after SE as compared to SE-KA mice

(ipsilateral, p < 0.05). (3) In thalamus, dexamethasone treatment reduced the T1

signal intensity by 13.34 % 2 d after SE and by 15.31 % on day 4 as compared to

SE-KA mice (ipsilateral, p < 0.05). Moreover, significantly increased T1 values vs.

baseline were present at 2 d and 4 days, respectively, after SE only in the amygdala

of SE-KA animals. Interestingly, signal intensities in the cortex were very similar over

time in both SE-dex and SE-KA groups. BBB leakage occurred at day 2 in this region

and persisted until the end of the dexamethasone study on day 4.

Thalamus


1

2

3

4

2 4 16 37

*__

Baseline

## # #

T1 in

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1

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2 4 16 37Baseline

# ##

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75

Figure 15: Blood-brain barrier leakage after dexamethasone treatment

T1 intensities in kainate animals before and after status epilepticus (SE-KA, red) compared with dexamethasone treated (SE-dex) animals (green) for the ipsilateral and contralateral brain hemisphere. Dexamethasone treatment was found to significantly reduce blood-brain barrier leakage in typically epilepsy associated brain regions: dorsal hippocampus, ventral hippocampus and thalamus (all brain regions ipsilateral) on day 2 following SE. Furthermore, significantly lower T1 intensities were present in the dorsal hippocampus and in thalamus of SE-dex animals vs SE-KA 4 d after SE. (Mean ± SEM, baseline (n = 6), d 2 (n = 5), d 4 (n = 5, one-way ANOVA, Bonferroni’s post hoc test, p < 0.05. B = baseline).

Hippocampus dorsal

B left KainatB right Kainat48 h l Kainat48 h left Dexa48 h r Kainat48 h right Dexa4 d l Kainat4 d left Dexa4 d r Kainat4 d right Dexa0

1

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Hippocampus ventral

B left KainatB right Dexa48 h l Kainat48 h left Dexa48 h r Kainat48 h right Dexa4 d l Kainat4 d left Dexa4 d r Kainat4 d right Dexa0

1

2

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#

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1

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1

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B left KainatB right Kainat48 h l Kainat48 h left Dexa48 h r Kainat48 h right Dexa4 d l Kainat4 d left Dexa4 d r Kainat4 d right Dexa0

1

2

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##

# # # significance vs. baseline

* significance SE-KA vs SE-dex

B left right

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Baseline

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T1 in

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Figure 16: Serial magnetic resonance images in kainate, dexamethasone and sham animals

Coronal magnetic resonance (MR) images in kainate induced status epilepticus (SE-KA, left), kainate-SE + dexamethasone (KA-dex, center) and sham (saline, right) mice. BBB leakage was assessed by longitudinal T1 weighted 7 T MRI after contrast agent infusion. BBB leakage is indicated by contrast enhancement in affected brain regions (white areas, exemplary indicated by arrows in SE-KA 4 d post SE). The comparison of SE-KA and KA-dex mice 48 h and 4 d after SE demonstrates lower T1 signals in different brain regions of the dexamethasone treated group. Comparison between SE-KA and sham mice reveals less pronounced contrast enhancement in specific brain regions of sham animals.

Kainate Dexamethasone Sh am

B

48 h

4 d

16 d

37 d


77

4.5 Discussion

We performed a longitudinal MR imaging study and found that in vivo MRI during

epileptogenesis is a suitable imaging biomarker for the early detection of changes in

BBB leakage in the intrahippocampal kainate mouse model. Moreover, we used the

imaging protocol to evaluate effects of anti-inflammatory treatment on BBB integrity

and showed for the first time that dexamethasone treatment following

intrahippocampal kainate injection induces a significant protection of BBB integrity in

mice as assessed by MRI following step-down infusion of Gd-DTPA.

4.5.1 Longitudinal in vivo assessment of blood-brain barrier leakage during

epileptogenesis

MRI is accepted as one of the most sensitive in-vivo molecular imaging techniques

for detection of brain lesions present in human TLE (Bouilleret et al., 2000;

Schoknecht & Shalev, 2012). Moreover, MRI is the most commonly used technique

for non-invasive imaging of BBB leakage in pre-clinical animal models, too (Rebeles

et al., 2006; Wunder et al., 2012).

BBB leakage was detected throughout epileptogenesis in the ipsilateral (kainate

injection) hemisphere of SE-KA mice vs. baseline. Two d and 4 d after SE, affected

brain regions included hippocampus, amygdala, thalamus and cortex; brain regions

which are associated with epilepsy. Intensities and spatial extent of BBB impairment

subsequently declined in the five weeks of follow-up. Sixteen days post SE,

increased T1 intensities was present in the ipsilateral dorsal hippocampus and in

thalamus. BBB leakage was still present in the ipsilateral hippocampus, in thalamus

and in the cortex in the chronic epileptic phase. Moreover, comparison between

groups for each time point revealed significantly higher T1 values in the SE-KA group

vs. sham in 4 of the examined brain regions 2 d after surgery: the ipsilateral dorsal

hippocampus, the ventral hippocampus, the amygdala and in thalamus.

The injection cannula produces a mechanical lesion (Bouilleret et al., 2000) in focal

kainate models. We saw hyperintense tissue in the cortex above the

intrahippocampal injection site which occurred solely in the ipsilateral hemisphere of


78

the brain at d 2 and d 4 in all investigated groups. These findings suggest that, to a

large proportion, the T1 hyperintensity along the injection track is a result of this

lesion rather than of excitotoxic effects of kainate or epileptogenesis-associated BBB

leakage. Nevertheless, diffusion of kainate into the injection track was limited as

much as possible by leaving the needle in situ for additional 2 min following kainate

injection (see 4.3.3). We observed the highest BBB permeability of all investigated

brain regions in the dorsal hippocampus of the SE-KA group at day 2 following SE.

These results are in agreement with the finding that hippocampal CA1 and CA3

subfields are particularly vulnerable to injury associated with SE (Fujikawa, 1996).

Moreover, we observed hyperintense T1 signals in the ventral hippocampus,

amygdala and thalamus on day 2 and 4. Subsequently, T1 intensities progressively

decreased during the five weeks of follow-up and significantly increased values were

present in less brain regions in the chronic epileptic phase, 37 d after SE. These

results support the assumption that BBB leakage is not static but a dynamic process

with varying degrees depending on the progress of epileptogenesis and occurring in

different brain regions during focal kainate-induced epileptogenesis.

An increase in T1 intensity was shown in the cortex (Bouilleret et al., 2000) in a

previous study. This signal appeared in the first hour and lasted until 3 d after kainate

injection. However, in other brain regions, only changes in T2 intensity but not in T1

intensity were detected. The discrepancies compared to our study presumably result

from differences in MRI technique and protocols. In our study, 7 T MRI using a high

resolution 3D T1 MDEFT imaging sequence following step-down infusion of Gd-

DTPA was applied to detect BBB leakage. Step-down infusion protocols for MRI CA

were previously shown to improve sensitivity for detection of BBB leakage compared

with bolus injection of CA (Nagaraja et al., 2007; Knight et al., 2009). Bouilleret et al.

(2000) used 4.7 T MRI after bolus injection of Gd-DOTA. They found that T2 signal

increase 7 d after kainate injection was limited to the ipsilateral hippocampus and

concluded that the reason might be absent spreading of seizure activity.


79

4.5.2 Dexamethasone treatment significantly reduces blood-brain barrier

leakage

We found a significant reduction of BBB leakage after dexamethasone treatment in

typically epilepsy associated brain regions during early epileptogenesis. SE-KA

treated and SE-dex mice were scanned 2 d and 4 d following intrahippocampal

kainate injection. Analysis revealed that mice being treated with dexamethasone after

SE showed significantly less BBB leakage in the ipsilateral dorsal hippocampus,

ventral hippocampus and thalamus on day 2 compared with saline treated animals.

BBB leakage was reduced by dexamethasone treatment in the ipsilateral dorsal

hippocampus and thalamus on day 4, too. Thus, we conclude that dexamethasone

protects BBB integrity in the most affected brain regions after KA-induced SE. T1

hyperintensity in the cortex was not reduced or modified by dexamethasone

treatment. It is considered that the increase of T1 intensities is caused by the lesion

of the injection cannula along the injection track rather than by excitotoxic effects of

kainate or by epileptogenesis-associated BBB leakage. The cortex, therefore, may

not be suitable to assess BBB leakage in the focal kainate model as discussed

above.

Dexamethasone is a broad-spectrum anti-inflammatory drug which acts on

glucocorticoid receptors and reduces the expression of adhesion molecules,

inflammatory chemokines and cytokines (Barnes, 2006; Laurila et al., 2009). The

molecular mechanisms, underlying possible therapeutic effects on the BBB, are not

known in detail. In a recently published study, BBB-restorative effects were found to

be mediated by upregulation of ZO-1 tight junction proteins and occurred via binding

of dexamethasone to glucocorticoid receptors in an vitro model of primary blast injury

(Hue et al., 2015). The mechanism of action was confirmed by application of

mifepristone (RU-486), a steroid antagonist which prevented BBB-restorative effects

of dexamethasone. Another suggested mechanism of action is the inhibition of NF-

kappa-B activity by dexamethasone (Auphan et al., 1995). NF-kappa-B can impair

BBB function by alteration of tight junction protein expression (Brown et al., 2003).

Dexamethasone significantly reduced TNF production and reduced the infarct size by

50 % in a rat model of focal cerebral ischemia (Bertorelli et al., 1998).


80

However, as inflammatory processes themselves play a role in restoring brain

homeostasis following brain insults (Abbott, 2002; Serrano et al., 2011), finding the

right balance between aggravation and rehabilitation of inflammatory processes is

important (Gröhn & Pitkänen, 2007). Disturbing BBB homeostasis could entail side

effects (Abbott, 2002). Glucocorticoids are widely used in the clinic, for example for

treatment of the Landau-Kleffner syndrome, a specific form of pediatric epilepsy

(Vezzani et al., 2011). However, glucocorticoids can be harmful in patients with acute

ischemic stroke (Kleinschnitz et al., 2011). Moreover, deterioration of brain injury and

survival rate were the outcome of dexamethasone treatment in a recent study in the

lithium-pilocarpine post SE rat model (Duffy et al., 2014). These findings suggest that

the efficacy of glucocorticoids can be very different dependent on the underlying

disease. Hence, effects of dexamethasone on specific alterations occurring in the

process of epileptogenesis need to be further evaluated.

4.6 Conclusion

We demonstrate for the first time that dexamethasone protects BBB integrity in the

intrahippocampal kainate mouse model. Moreover, longitudinal in vivo MRI detects

changes in BBB leakage during epileptogenesis in this model. Restoration of

impaired BBB integrity may contribute to prevention of epilepsies following brain

insults as BBB leakage is strongly associated with the process of epileptogenesis

(Gorter et al., 2015). We hence hypothesize that BBB-protective dexamethasone

treatment may reveal antiepileptogenic or epileptogenesis-modifying effects in future

studies that include evaluation of the chronic epileptic phase.

ACKNOWLEDGEMENTS: We thank Christian Bergen and Friederike Twele for skillful technical

assistance. The study is funded by the European Union’s Seventh Framework Programme (FP7)

under grant agreement n°602102 (EPITARGET). H.B. was supported by a scholarship from the

Studienstiftung des Deutschen Volkes.

CONFLICTS OF INTEREST: none


81

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General discussion

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5 General Discussion

5.1 Main findings

The studies were set out to investigate suitable molecular imaging methods and

protocols for evaluation of blood-brain barrier (BBB) integrity as a prerequisite to

identify BBB leakage as a potential biomarker for epileptogenesis and as a target for

antiepileptogenic treatment which may contribute to the prevention of epilepsy

development in at-risk patients after brain insults in the future.

The main findings of the studies presented in this work are that (1) small animal

[68Ga]-diethylenetriamine pentaacetic acid (DTPA) positron emission tomography

(PET) imaging, (2) [99mTc]-DTPA single photon emission computed tomography

(SPECT) imaging and (3) magnetic resonance (MR) imaging following tracer or

contrast agent (CA) infusion allow for in vivo detection of BBB leakage in a post

status epilepticus (SE) rat model during early epileptogenesis, (4) magnetic

resonance imaging (MRI) being the most sensitive molecular imaging method.

Moreover we showed that (5) the same is true for MR imaging of the

intrahippocampal mouse model throughout the process of epileptogenesis. (6) The

pharmacological intervention with losartan may adversely affect BBB integrity and

edema formation after SE in rats. (7) Dexamethasone treatment exacerbates edema

formation in the lithium-pilocarpine rat model but (8) significantly reduces BBB

leakage following intrahippocampal kainate injections in mice.

These are the first studies showing BBB protective effects of dexamethasone in the

intrahippocampal mouse model and the first studies to use [68Ga]-DTPA PET and

[99mTc]-DTPA SPECT for pre-clinical detection of BBB leakage in the lithium-

pilocarpine rat model.

5.2 Multimodal imaging of blood-brain barrier leakage during

epileptogenesis

Dedicated small animal PET, SPECT and MR imaging in vivo enabled us to

characterize and to compare the spatial and temporal extent of BBB leakage during

General discussion

86

early epileptogenesis in the lithium-pilocarpine rat model. All imaging modalities

detect BBB leakage 48 h post SE in the hippocampus and cortex; brain regions

which are known to be connected with epilepsy. Because PET and SPECT have

higher sensitivity, specificity and signal-to-noise ratio compared with MRI (Skotland,

2012; Wunder et al., 2012), one might expect that nuclear imaging is more suitable

for detecting BBB leakage. However, our data revealed that contrast-enhanced MRI

is the most appropriate technique for this purpose. Despite its limitations in sensitivity

(Skotland, 2012) and quantification, MRI detected CA extravasation in both limbic

and cortical brain regions 48 h after SE, including the hippocampus, amygdala,

entorhinal cortex, piriform cortex, motor cortex, substantia nigra, thalamus and

different cortical sub-regions. SPECT detected BBB leakage in the hippocampus,

motor cortex, piriform cortex, cingulate cortex and septum.

Furthermore, longitudinal imaging allowed us to examine temporal patterns of BBB

leakage. No BBB leakage was detected at 5 h after SE. The highest increase in BBB

leakage was detected by both MRI and SPECT at 48 h. Moreover MRI was sensible

to detect BBB leakage at 4 d in the amygdala and in a cortical subregion. At 10 d,

SPECT did not detect BBB leakage whilst MRI found increased tracer extravasation

into hippocampal subregions. Recently, an MRI study has shown maximal MR signal

intensities in hippocampus and piriform cortex 48 h after SE which returned to

baseline levels around day 7 (Choy et al., 2010). Moreover, van Vliet et al. (2014b)

detected BBB leakage in limbic brain regions 24 h and six weeks after SE. Our

results support these findings and extend them by showing that at 48 h post SE

cortical brain regions, besides limbic structures, are subjected to BBB impairment

and by implementing the time course of T2 intensities as indicators for cerebral

edema.

MRI has the highest spatial resolution. Of the three evaluated techniques it is the

only one that can display structural changes occurring during epileptogenesis, like

hippocampal sclerosis and associated loss of hippocampal volume (Spencer, 1994).

T2 intensities reached peak values at 2 d post SE. Accordingly, edema formation

occurs early after SE, like detectable BBB leakage does. Fast edema formation is

followed by a quick resolution by day 10. The results are consistent to studies by

General discussion

87

Roch et al. (2002), Choy et al. (2010) and Duffy et al. (2014) who found that edema

formation following brain insults peaks around day 2.

The intrahippocampal mouse model was included in the studies besides the lithium-

pilocarpine rat model to take interspecies and model-specific differences into

consideration. MRI following Gd-DTPA infusion allowed for longitudinal investigation

of BBB integrity despite the small brain size of mice. SE-KA animals were scanned

baseline and at 2 d, 4 d, 16 d and 37 d after SE. BBB leakage was present in the

ipsilateral dorsal hippocampus, ventral hippocampus, cortex, amygdala and thalamus

2 d and 4 d after SE vs. baseline. Moreover, BBB leakage was found in the ipsilateral

hippocampus and thalamus at 16 d post SE. At 37 d after SE, BBB leakage was

detected in the ipsilateral hippocampus, thalamus and cortex of SE-KA mice vs.

baseline. These results show that BBB leakage is present in both the acute and the

chronic phase of epileptogenesis in mice. In agreement with the study on rats, we

observed the highest BBB permeability of all investigated brain regions in the dorsal

hippocampus at day 2 and day 4 following SE. These results are in agreement with

the finding that hippocampal CA1 and CA3 subfields are particularly vulnerable to

injury associated with SE (Fujikawa, 1996). The time course of BBB leakage is

divergent whilst affected brain regions are similar in both models. However, identical

time points for investigations in rats and mice would be needed to fully evaluate

matches in the time course of BBB leakage in both models. BBB leakage was

present in both brain hemispheres in the pilocarpine post SE rat model whilst

changes were limited to the ipsilateral brain hemisphere in the intrahippocampal

kainite mouse model. This may be explained by the fact that compared to the

systemic pilocarpine rat model, intrahippocampal kainate injection in mice induces a

focal injury.

PET detected BBB impairment 48 h post SE in rats in hippocampus and motor

cortex. A tendency towards increased tracer uptake was present in the entorhinal

cortex. Thus, unexpectedly, PET is the least sensitive imaging modality. Firstly, the

brains microvasculature may contribute to this result. The adult human blood-brain

interface amounts to an average between 12 and 18 m2 (Nag & Begley, 2005).

Hence, the background signal deriving from surrounding blood vessels may be high.

Consequently, the specific tracer signal could be too low to be distinguished from the

General discussion

88

background signal in the presence of only subtle BBB leakage. Secondly, the tracer

itself may play a role. This is the first study to use [68Ga]-DTPA for PET imaging of

BBB leakage. Thus, not much is known about the tracer kinetics in combination with

the chelator DTPA. Our proof-of-principle study showed that [68Ga]-DTPA detects

BBB leakage. However, further in vivo-research that goes behind the scope of this

study is needed to evaluate whether the tracer characteristics correspond to those of

an ideal tracer for BBB imaging. However, a structurally similar tracer, [68Ga]-

ethylenediamine tetraacetic acid, has been successfully used to detect BBB integrity

in multiple sclerosis and glioma patients (Pozzilli et al., 1988; Keith L. Black et al.,

1997). Beyond, very little literature is available on PET studies of BBB leakage. So

far most PET studies aimed at the BBB targeted multidrug transporters like

p-glycoprotein (Bankstahl et al., 2008; Bankstahl et al., 2011) or BBB penetration of

tracers (Waterhouse, 2003).

5.3 Methodological issues

The use of bolus injections of [68Ga]-DTPA in one group and step-down infusion of

tracer in another group allowed us to compare differences between the two

approaches. PET imaging following step-down infusion of [68Ga]-DTPA detects BBB

leakage in the hippocampus whilst PET imaging following bolus injection of the tracer

does not. It thus seems that PET imaging following tracer infusion is more sensitive

in detecting BBB leakage compared with injection as a bolus. These data agree with

those of previous MRI studies targeting BBB leakage after CA infusion in rat models

of transient cerebral ischemia or systemic kainate-induced SE (Nagaraja et al., 2007;

Knight et al., 2009; van Vliet et al., 2014b). These studies found that MRI following

step-down infusion of Gd-DTPA and gadobutrol, respectively, yielded greater

contrast enhancement than bolus injections. It can be assumed that these

dissimilarities result from differences in plasma-concentrations of CA. CA

concentration reaches a quick peak and subsequently decreases continuously after

bolus injection (Knight et al., 2009). Thus, spatial limits were found to be about 2 mm

for detecting BBB leakage after bolus injection of Gd-DTPA (Knight et al., 2005).

Step-down infusion allows maintenance of constant CA concentrations in the blood

(Hamilton et al., 1997). These lead to quantitatively and spatially accurate

General discussion

89

estimations of Gd-DTPA concentrations as shown in cerebral ischemia for volumes

as low as 0.5 mm3 (Knight et al., 2009). The accuracy of estimations was confirmed

by comparison with Gd-[14C]-DTPA, a radio-labeled analog of Gd-DTPA and

quantitative autoradiography.

5.4 Effects of losartan on blood-brain barrier integrity

MRI has proven to be the most sensitive imaging modality for detecting BBB

impairment. Thus, MR imaging of BBB leakage and edema formation were used as

imaging biomarkers to evaluate effects of a potentially BBB-protective losartan

treatment schedule in rats after SE. In the SE-losartan group, BBB leakage was

present at day 10 whilst it was absent in the SE-saline group. Furthermore, edema

formation was increased in SE-losartan vs. SE-saline rats in distinct brain regions. It

thus seems that losartan does not stabilize BBB integrity or attenuate cerebral

edema in the pilocarpine post SE rat model. On the contrary, losartan may adversely

affects BBB integrity and edema formation in the chosen setup. In previous studies,

losartan treatment resulted in delayed seizure onset or the prevention of seizures,

respectively (Ivanova et al., 2013; Bar‐Klein et al., 2014). A possible explanation for

the apparent discrepancy may be the study setup. We evaluated BBB leakage after

losartan treatment during early epileptogenesis. Previous epilepsy-related studies

were designed to evaluate antiepileptogenic effects of losartan. Hence, losartan was

administered chronically and the focus of examinations was monitoring the onset and

characteristics of spontaneous recurrent seizures Bar‐Klein et al. (2014) started

electrocorticography recordings from day 28. Monitoring animals until the chronic

epileptic phase was beyond the scope of this study. Therefore we cannot rule out

that losartan would have revealed disease modifying effects in the chronic epileptic

phase.

5.5 Effects of dexamethasone on blood-brain barrier integrity

The establishment of suitable imaging protocols (see chapter 2) for imaging BBB

leakage enabled us to evaluate effects of dexamethasone treatment on BBB integrity

in both rats and mice. To test the working hypothesis that dexamethasone is BBB

General discussion

90

protective, rats and mice were subjected to SE, treated with dexamethasone and

subsequently scanned on MRI.

MR imaging performed at 48 h after SE in rats revealed BBB leakage in a spatial and

quantitative extent that was comparable to SE-saline animals. However, T2 values

were found to be higher in specific brain regions of SE-dexamethasone rats. Our

results suggest that edema formation is favored by dexamethasone treatment in the

lithium-pilocarpine rat model.

As described in manuscript 3 (chapter 4), dexamethasone treatment induces a

significant reduction of BBB leakage in typically epilepsy associated brain regions in

mice. Thus, dexamethasone treatment leads to controversial results in the two

different animal models. Different conceivable reasons can be discussed for these

distinctions.

First, the dosing regimen needs to be taken into account. Rats received three

dexamethasone injections, starting 3 h after SE (8 mg/kg i.p) and 24 h (8 mg/kg i.p)

and 48 h thereafter (4 mg/kg i.p.). Mice received 2 mg/kg i.p. five times following

kainate injection (at 6 h, 24 h, 48 h, 72 h and 96 h). Thus, mice were treated with

lower but more doses of dexamethasone. The higher doses in rats may induce side

effects. Duffy et al. (2014) found that a dose of 10 mg/kg sodium phosphate

dexamethasone injected twice resulted in increased T2 values at day 2 and day 4

after SE and was accompanied by a high mortality in rats. However, also low doses

of dexamethasone (2 mg/kg i.p.) administered once resulted in increased T2 values.

This leads to a second possible explanation for the apparent discrepancy on

dexamethasone treatment in rats and mice, namely differences in animal models.

Rats after SE seem to be more sensitive to side effects deriving from

dexamethasone treatment than mice. Our finding that one rat developed visible side

effects in form of gastric ulcerations support this assumption. An accumulative effect

of more frequent dexamethasone application can be ruled out as dexamethasone

revealed BBB protective effects in mice at day 2 following SE, a time point that was

investigated in both models.

General discussion

91

Positive outcomes of glucocorticoid treatments are shown by previous reports on

cerebral ischemia. Reduced infarct volumes and tumor necrosis factor levels were

found following methyl-prednisolone or dexamethasone treatment (Bertorelli et al.,

1998; Slivka & Murphy, 2001). Moreover, a combination therapy with dexamethasone

and melatonin was neuro-protective and reduced cerebral edema in a mouse model

of traumatic brain injury (Campolo et al., 2013); agreeing with the BBB protective

results we found in mice. Further studies on epilepsy models have been performed

(Marchi et al., 2011a; Al-Shorbagy et al., 2012). However, dexamethasone was given

prior to SE. Therefore, such studies may have observed brain insult-modifying effects

of dexamethasone rather than its impact on epileptogenesis-associated mechanisms.

The use of glucocorticoids is a controversial issue and remains unsolved to date. Our

studies in two different animal models indicate that the outcome of dexamethasone

treatment is dependent on the dosing regimen and on the animal model. We showed

BBB protective effects in mice which recommend dexamethasone for further studies

of the relationship with the process of epileptogenesis.

5.6 Limitations of the studies

The present studies investigated treatment effects on acute changes in BBB integrity.

Further research may implicate continuous video/ECoG monitoring for determination

of changes during the latency phase as well as during seizure onset, seizure

frequency and seizure duration and thus further evaluate the relationship between

BBB integrity and antiepileptogenesis.

5.7 Future directions

Tracer injection as a bolus is the standard procedure in (preclinical) PET (Carson,

2000). Our results suggest that radiotracer infusion may be beneficial for detecting

BBB leakage during epileptogenesis not only in MRI but in nuclear medicine

techniques, too. Furthermore, a slower but longer lasting application as in infusion vs.

bolus provides a lesser impact on the brains endothelium. It may be taken into

consideration that a quickly administration of a volume which is high in relation to the

General discussion

92

bodyweight may per se influence BBB integrity (Nagaraja et al., 2007) in epilepsy

models.

Even though all imaging techniques reveal changes in BBB integrity, MRI detects

BBB leakage that is missed by PET and SPECT. Thus, MRI is superior to PET and

SPECT in the used study design. However, every imaging modality has its specific

limitations (James & Gambhir, 2012). A combinational use of PET, SPECT and MRI

could therefore benefit from the strength of each imaging technique.

The results of the present studies are important as they provide further insights into

the dynamics of BBB leakage and cerebral edema formation during epileptogenesis.

Moreover, they offer suitable imaging protocols to further evaluate the role of BBB

impairment as potential biomarker for epileptogenesis. A combination of MRI and

nuclear imaging may be beneficial for further investigating BBB during

epileptogenesis in the future.

5.8 Conclusion

The study has offered an evaluative perspective on multimodal molecular imaging of

changes in BBB integrity during epileptogenesis.

In the current studies, we present evidence that pre-clinical nuclear imaging is a

valuable tool for the evaluation of BBB integrity. While contrast-enhanced MRI is a

generally accepted and widely used tool for detecting BBB impairment, our

observations that [68Ga]-DTPA PET and [99mTc]-DTPA SPECT can contribute to

evaluation of BBB integrity during epileptogenesis are novel.

Our results show for the first time a successful reduction of BBB leakage by a dosing

regimen of dexamethasone in mice during epileptogenesis which may contribute to

epileptogenesis-modifying pharmacotherapies. The use of in vivo MR imaging

enables the evaluation of pharmacotherapeutic effects on BBB integrity. Thus,

assessment of patients responding to BBB-protective therapy may be feasible in the

future.

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