2 neurogenesis

Immunohistochemical study on the effect of soft diet and

omega 3-fortified soft diet on neurogenesis in the rat dentate

gyrus and the subventricular zoneRehab Ahmed Rifaai, Nashwa Fathy El-Tahawy and Entesar Ali Saber

Department of Histology, Faculty of Medicine, MiniaUniversity, Egypt

Correspondence to Rehab Ahmed Rifaai, Departmentof Histology, Faculty of Medicine, Minia University,EgyptTel: + 103358376; fax: + 86 2342813;e-mail: [email protected]

Received 24 January 2011Accepted 23 April 2011

The Egyptian Journal of Histology

2011, 00:000–000

Background

Adult brain neurogenesis persists in the subventricular zone (SVZ) and in the

subgranular zone (SGZ) of the dentate gyrus. Modulation of neurogenesis by diet is a

mechanism by which nutrition affects memory, learning, and mood.

Aim of the study

To study the effect of the soft diet with or without omega 3 fatty acids on neurogenesis.

Materials and methods

Thirty weaned male albino rats (3 weeks) were divided into three groups. Group 1

(control group) were fed on hard diets, group 2 were fed on soft diets, and group 3

were fed on soft diets plus omega 3 fatty acids for 3 months. Nerve cell proliferation in

the SVZ and the SGZ was detected immunohistochemically using thymidine analog

bromodeoxyuridine (BrdU). The results were statistically analyzed.

Results

In the dentate gyrus, there was a significant increase in the number of BrdU-positive

cells in groups 1 and 3 compared with group 2. Meanwhile in the SVZ, there was a

significant increase in the number of BrdU-positive cells in group 3 compared with

group 1. In group 1, the newly formed cells in the SGZ reached the granular cell layer

of the dentate gyrus. The newly formed cells in the SVZ reached the olfactory bulb

(OB) after 2 weeks but failed to survive for 4 weeks in the OB. In group 2, few newly

formed cells reached the granular cell layer of the dentate gyrus, but they failed to

reach the OB. In group 3, the newly formed cells reached their destination in the

granular cell layer of the dentate gyrus and the OB. In the OB, the cells succeeded to

survive for 4 weeks and were incorporated among the granular cells of OB.

Conclusion

Hard diet and omega 3-fortified soft diet had a stimulatory effect on the process of

neurogenesis in the dentate gyrus. Meanwhile in the SVZ, fortified soft diet had more

stimulatory effect on proliferation and improvement of the survival rate of the newly

formed cells than the hard diet.

Keywords:

dentate gyrus, diet, immunocytochemical, neurogenesis, subventricular zone

Egypt J Histol 00:000–000�c 2011 The Egyptian Journal of Histology1110-0559

IntroductionAdult stem cells are present in many tissues, including

bone marrow, skin, gastrointestinal tract, muscle, adipose

tissue, and brain [1]. Neurogenesis is a life-long

occurrence that is limited to specific sites within the

brain, namely, the subventricular zone (SVZ) and the

subgranular zone (SGZ) of the hippocampus [2].

Neurogenesis is a complex multistage and multiweek

process involving proliferation, neuronal differentiation

and, ultimately, survival and integration into circuitry [3].

The integration of adult-born neurons into the circuitry

of the adult hippocampus suggests an important role for

adult hippocampal neurogenesis (AHN) in learning and

memory [4]. Nutrition affects brain function [5].

Modulation of AHN by diet emerges as a possible

mechanism by which nutrition impacts mental health.

Reduction of masticatory afferent stimuli due to long-

term soft diet fed may induce neuronal loss in the

hippocampus and reduce memory/learning ability [6].

The omega 3 fatty acids are found in the diet as doco-

sahexaenoic acid (22 : 6n-3, DHA), a-linolenic acid (18 : 3

omega-3), and eicosapentaenoic acid (20 : 5 omega-3).

Salmon, flax seeds, and walnuts are excellent sources of

omega 3 fatty acids. Very good sources of these healthy

fats include cauliflower, cabbage, cloves, and mustard

seeds. Good sources of these fats include halibut, shrimp,

cod, tuna, soybeans, tofu, kale, collard greens, and

brussels sprouts [7]. Each of the omega 3 fatty acids

has different functions in different cells. DHA is found

predominantly in neuronal membranes in the gray matter

and constitutes a major component of the brain [8]. It

plays important roles functionally and structurally [9].

Original article 1

1110-0559 �c 2011 The Egyptian Journal of Histology DOI: 10.1097/01.EHX.0000399683.51243.c2

Depletion of DHA from brain and retina interferes with

normal neurogenesis and neurological function [10].

Bromodeoxyuridine (BrdU) is a thymidine analog that

incorporates DNA of dividing cells during the S-phase of

the cell cycle. As such, BrdU is used for birth dating and

for monitoring cell proliferation. BrdU can be detected by

immunohistochemistry, using a monoclonal antibody

directed against single-stranded DNA containing BrdU

[11]. BrdU immunohistochemistry has been instrumental

for the study of the development of the nervous system

and to confirm that neurogenesis occurs in the adult

mammalian brain, including human [12]. It was

interesting to study the effect of fortification of the soft

diet with omega 3 fatty acid on neurogenesis in

comparison with the hard diet.

Materials and methodsAnimals

This study was carried out according to the protocols

approved by the Animal Care and Use Committee at the

Minia University Animal House (Egypt). This study was

carried out on 30 male albino pups, which were weaned at

3 weeks after birth. The pups were divided into three

groups of 10 pups each.

Group 1: the control group (hard diet-fed group)

The animals were fed on pelleted chow for 3 months.

Group 2 (soft diet-fed group)

The animals were fed on powdered and wetted chow with

the same ingredients as that of the previous group for 3

months.

Group 3 (omega 3-fortified soft diet-fed group)

The animals were fed on powdered chow containing the

same ingredients but with the addition of omega 3 fatty

acid (35 mg/kg) for 3 months [13].

Bromodeoxyuridine administration

At the end of experiment, rats of groups 1, 2, and 3

received intraperitoneal injection with BrdU (50 mg/kg)

dissolved in PBS every 12 h for 3 consecutive days [14].

The animal were killed 2 days, 2 weeks, and 4 weeks after

the last BrdU injection. The number of BrdU-positive

cells after 2 days indicates the proliferation rate. The

number of BrdU-positive cells after 2 weeks shows the

migration of the newly formed cells, whereas the number

of BrdU-positive cells after 4 weeks indicates the survival

rate [15].

Immunohistochemistry

Brains were removed and fixed in 4% paraformaldehyde

and then processed for paraffin sectioning. Sections were

cut at 10 mm. The sections were immunostained with a

monoclonal BrdU antibody (1 : 500; Biodesign Inc., Sigma

Aldrich, Egypt). Immunohistochemical staining was

performed according to a previously published protocol

[16] as follows:

(1) Sections were deparaffinized, hydrated and then

washed in phosphate buffered saline (PBS)

(0.1 mol/l);

(2) Sections were pretreated with hydrogen peroxide (1%

H2O2 for 2 min) to eliminate endogenous peroxidase;

(3) Sections were incubated in 4 N HCl for 30 min at

room temperature (for DNA denaturation);

(4) Sections were immersed in trypsin and PBS (1 mg/

ml) for 10 min at 371C. After the acid washes, borate

buffer (0.1 mol/l) was added to buffer the cells for

12 min at room temperature;

(5) Sections were incubated with 1% Triton X-100 (0.1 M

PBS, pH = 7.4) solution containing rat anti-BrdU

antibody (1 : 500). Sections were incubated overnight

at room temperature. After incubation with the

primary antibodies, the sections were rinsed with

PBS and subsequently incubated in a biotinylated

goat anti-rat IgG secondary antibody (Vector Labora-

tory, 1 : 2000) for 1 h at room temperature;

(6) Sections were then incubated for 30 min in the Vecta-

stain ABC reagent. Diaminobenzidine was used as a

chromagen [17].

Bromodeoxyuridine-positive cells counts

The morphometric measurements were taken using

a Leica Quin 500C image analyzer computer system

(Leica Imaging system Ltd., Cambridge, England). In

the dentate gyrus, cells were counted in the field of a

40� objective using light microscope; BrdU-positive

cells were counted throughout the entire SGZ and the

granule cell layer of the dentate gyrus. In each animal, six

sections were counted and the distance between sections

was 300mm to scan through the depth of the hippocampus.

In the SVZ, cells were counted in the field of the oil

immersion lens. Cells were counted in 10 adjacent

nonoverlapping fields. In each animal, six sections were

counted and the distance between sections was 300mm.

Statistical analysis

Statistical analysis was carried out using the Statistics

Package instat. Statistical significance of the experiments

was determined using the one-way analysis of variance test

followed by the Tukey–Cramer posthoc test. A P value less

than 0.05 was considered as statistically significant.

ResultsThe dentate gyrus is one component of the hippocampal

formation (Fig. 1a). The dentate gyrus consists of the

molecular layer, the GCL, and the polymorphic layers.

The concavity of the dentate gyrus is termed the hilus.

Some ectopic granule cells are located within the hilus.

The SGZ is the area of two cell bodies’ width between

the GCL and the hilus (Fig. 1b).

The SVZ is a paired brain structure situated throughout

the lateral walls of the lateral ventricles (Fig. 2a). It has

four distinct layers. The innermost layer consisted of a

single layer (monolayer) of ependymal cells with

microvilli lining the ventricular cavity. The second layer

2 The Egyptian Journal of Histology

consisted of a network of astrocytic processes forming a

hypocellular gap. The third layer was formed of a ribbon

of astrocyte cell bodies. The fourth layer served as a

transition zone containing oligodendrocytes separating

between the third layer with its ribbon of astrocytes and

the brain parenchyma (Fig. 2b).

Immunohistochemical results

Group 1 (control group)

Immunopositive cells were numerous in the SGZ after

2 days, 2 weeks, and 4 weeks. Many immunopositive

cells appeared in the GCL (Figs 3–5). High expression

was observed in the SVZ and along the rostromigratory

stream (RMS) 2 days after the last BrdU injection

(Fig. 6). Two weeks later, the immunopositive cells were

observed reaching the olfactory bulb (OB) along the

RMS (Fig. 7). Four weeks after the last BrdU injection,

most of the immunopositive cells disappeared and the

immunoreactivity became localized to few cells in the

SVZ (Fig. 8).

Group 2 (soft diet-fed group)

Strong immunopositive reactions were observed in the

SGZ and the SVZ after 2 days, whereas weak reaction was

observed after 2 and 4 weeks both in the SGZ (Figs 9–11)

and in the SVZ (Figs 12–14).

Group 3 (omega 3-fortified diet-fed group)

The immunopositive cells were more numerous in the

SGZ and the GCL 2 days, 2 weeks, and 4 weeks after the

last BrdU injection (Figs 15–17).

High expression was observed in the SVZ and along the

RMS at 2 days after the last BrdU injection (Fig. 18). Two

weeks after the last BrdU injection, the immunopositive

cells were observed reaching the OB along the RMS

(Fig. 19). Still many immunopositive cells were observed

4 weeks after the last BrdU injection. At this time, the

immunopositive cells were seen incorporated among the

granular cells of the OB (Fig. 20).

Changes in BrdU-positive cell count in the dentate

gyrus

Quantitative assessment of the number of BrdU-immuo-

positive cells within the SGZ was significant (P < 0.005)

for all groups. Post-hoc comparisons showed that at all

Figure 1.

(a) A photomicrograph of the hippocampal formation of group 1showing the dentate gyrus (DG). (b) A photomicrograph of group 1dentate gyrus showing its different layers: molecular (M) layer, granulecell layer (GCL), and polymorphic layers (PL). Observe the hilus (H) andthe subgranular zone (SGZ).

H&E, A =�100, B = �400.

Figure 2.

(a) A photomicrograph of group 1 subventricular zone throughoutthe lateral wall of the lateral ventricle (LV). (b) A photomicrographof group 1 subventricular zone showing the different layers of thesubventricular zone: ependymal cells with microvilli (1), hypocellulargap of astrocytic processes (2), a ribbon of astrocyte cell bodies (3),and the transition zone (4).

H&E, A = �100, B = �1000.

Omega 3 and neurogenesis Rifaai et al. 3

Figure 6.

Bromodeoxyuridine-labeled cells in the subventricular zone of the controlgroup showing numerous immunopositive cells 2 days after the last BrdUinjection (arrow). Observe the initial migration along the rostromigratorystream (RMS) (�100). Inset showing immunopositive cell in theanaphase stage of mitosis (circle).

�1000.

Figure 8.

Bromodeoxyuridine-labeled cells in the subventricular zone of thecontrol group showing few immunopositive cells seen 4 weeks after thelast bromodeoxyuridine injection (arrow)

�100.

Figure 4.

Bromodeoxyuridine-labeled cells in the dentate gyrus of the controlgroup 2 weeks after the last bromodeoxyuridine injection showing manyimmunopositive cells in the subgranular zone (SGZ) and granule celllayer (GCL) (arrow).

�400.

Figure 5.

Bromodeoxyuridine-labeled cells in the dentate gyrus of the controlgroup 4 weeks after the last bromodeoxyuridine injection showing manyimmunopositive cells in the subgranular zone (SGZ) and granule celllayer (GCL) (arrow).

�400.

Figure 7.

Bromodeoxyuridine-labeled cells in the subventricular zone of the controlgroup showing numerous immunopositive cells 2 weeks after the lastbromodeoxyuridine injection (arrow) (�100). Observe that the migratingcells have reached the olfactory bulb (OB) along the rostromigratorystream (RMS) (inset).

�40.

Figure 3.

Bromodeoxyuridine-labeled cells in the dentate gyrus of the controlgroup 2 days after the last BrdU injection showing manyimmunopositive cells in the subgranular zone (SGZ) and granule celllayer (GCL) (arrow) (�400). Inset showing the immunopositive cells

�1000.


Figure 9.

Bromodeoxyuridine-labeled cells in the dentate gyrus of the soft diet-fed group 2 days after the last bromodeoxyuridine injection showingimmunopositive cells in the subgranular zone (SGZ) (arrow)

�400.

Figure 10.

Bromodeoxyuridine-labeled cells in the dentate gyrus of the soft diet-fedgroup 2 weeks after the last bromodeoxyuridine injection showingapparent decreased immunoreactive cells compared with the controlgroup (arrow).

�400.

Figure 11.

Bromodeoxyuridine-labeled cells in the dentate gyrus of the soft-dietfed group 4 weeks after the last bromodeoxyuridine injection showingapparent decreased immunoreactive cells in the SGZ and the GCLcompared with the control group (arrow).

�400.

Figure 12.

Bromodeoxyuridine-labeled cells in the subventricular zone of the softdiet-fed group 2 days after the last bromodeoxyuridine injectionshowing decreased reaction compared with the control group (arrow)

�100.

Figure 13.

Bromodeoxyuridine-labeled cells in the subventricular zone of the softdiet-fed group 2 weeks after the last bromodeoxyuridine injectionshowing decreased reaction compared with the control group (arrow).

�100.

Figure 14.

Bromodeoxyuridine-labeled cells in the subventricular zone of the softdiet-fed group 4 weeks after the last bromodeoxyuridine injectionshowing decreased reaction compared with the control group (arrow).

�100.


time points the number of BrdU-immuopositive cells was

significantly (P < 0.05) increased in the hard diet and

omega 3-fortified soft diet-fed groups compared with the

soft diet-fed group. The number of the BrdU-labeled

cells in the SGZ of the omega 3-fortified soft diet-fed

group was also significantly higher (P < 0.05) than that of

the hard diet-fed group 2 days, 2 weeks, and 4 weeks after

the last BrdU injection (Graph 1a).

Figure 16.

Bromodeoxyuridine-labeled cells in the dentate gyrus of the omega 3-fortified soft diet-fed group 2 weeks after the last bromodeoxyuridineinjection showing many immunopositive cells in the subgranular zone(SGZ) and granule cell layer (GCL) (arrow).

�400.

Figure 15.

Bromodeoxyuridine-labeled cells in the dentate gyrus of the omega 3-fortified soft diet-fed group 2 days after the last bromodeoxyuridineinjection showing many immunopositive cells in the subgranular zone(SGZ) and granule cell layer (GCL) (arrow).

�400.

Figure 17.

Bromodeoxyuridine-labeled cells in the dentate gyrus of the omega 3-fortified soft diet-fed group 4 weeks after the last bromodeoxyuridineinjection showing many immunopositive cells in the subgranular zone(SGZ) and granule cell layer (GCL) (arrow).

�400.

Figure 18.

Bromodeoxyuridine-labeled cells in the subventricular zone of the omega3-fortified soft diet-fed group showing that a large number of immuno-positive cells are seen in the subventricular zone and along therostromigratory stream (RMS) 2 days after the last bromodeoxyuridineinjection (arrow).

�100.

Figure 19.

Figure 19.

Bromodeoxyuridine-labeled cells in the subventricular zone of theomega 3-fortified soft diet-fed group showing that manyimmunopositive cells are present 2 weeks after the lastbromodeoxyuridine injection (arrow) (�100). Observe that themigrating cells reaching the olfactory bulb (OB) along therostromigratory stream (RMS) (inset).

�40.


Changes in BrdU-positive cell count in the SVZ

The BrdU-labeled cells in the SVZ of the hard and the

omega 3-fortified soft diet-fed groups were significantly

higher (P < 0.05) than the soft diet-fed group 2 days, 2

weeks, and 4 weeks after the last BrdU injection. On

comparing the number of the immunopositive cells in the

hard diet and the omega 3-fortified soft diet-fed groups, it

was found that the number of the immunopositive cells was

significantly higher (P < 0.05) in the omega 3-fortified soft

diet-fed group than in the hard diet-fed group 2 days, 2

weeks, and 4 weeks after the last BrdU injection (Graph 1b).

DiscussionThis study demonstrated that neurogenesis persists in the

SVZ and in the SGZ of adult rat brain. More BrdU-

immunopositive cells were observed in both areas 2 days

after the last BrdU injection than after 2 weeks and 4 weeks

in all the examined groups. The reduction in the number of

the immunopositive cells along the time course might be as

a result of the death of some newly born cells. Hard diet and

omega 3-fortified soft diet increased neuronal proliferation

and survival in both areas in comparison with the soft

diet alone. Both the proliferation and the survival rate of the

newly born cells were better in the omega 3-fortified soft

diet-fed group. In the omega 3-fortified soft diet-fed group,

the newly born neurons succeeded in reaching their

destination either in the GCL or in the OB.

Neuronal precursors cells are self-renewing, with the

potential to differentiate into all three basic cell types in

the central nervous system (CNS), including neurons, oligo-

dendrocytes, and astrocytes [18]. Neuronal progenitors in

the SVZ migrate to the OB along the RMS. After the

newborn neurons reach the middle of the OB, they detach

from the chain and migrate radially. They differentiate into

granule and periglomerular neurons [19]. These modulate

the tuning of bulbar activity to enhance olfactory

discrimination performance and potentially regulate the

categorization of novel odorants [20].

Many precursors in the SGZ die within 2 weeks [21].

Living precursors in the SGZ migrate into the dentate GCL

where the majority ultimately acquire morphological

characteristics of granule cells and express neuron-specific

markers [22]. In the GCL, they develop synapses and axonal

projections to receive and deliver signals, respectively, and

hence they can execute their function [23].

Food texture has an impact on AHN, rats fed with a soft

diet, as opposed to a solid diet, exhibit decreased

hippocampal progenitor cell proliferation [24]. The

decrease of neurogenesis in the soft diet-fed rats had

been explained by the emotional stress observed in those

rats that led to increased level of corticosterone [25].

Corticosterone is a common downregulator of neurogenesis

[26]. Corticosterone reduces the level of brain-derived

neurotrophic factor (BDNF) mRNA through the

glucocorticoid receptor located in the dentate gyrus [27].

BDNF is a member of a family of related neurotrophic

proteins. It is a positive regulator of both proliferation and

survival of neurons. It also prevents neurons from dying

Figure 20.

Bromodeoxyuridine-labeled cells in the subventricular zone of theomega 3-fortified soft diet-fed group showing that manyimmunopositive cells are present 4 weeks after the lastbromodeoxyuridine injection (arrow) (�400). Observe someimmunopositive cells grouped into nests (upper inset). Lower insetshowing the immunopositive cells incorporated among cells in thegranular cell layer of the olfactory bulb (OB)

�1000.

Graph 1.

Mean + standard deviation (SD) bar graphs of bromodeoxyuridine(BrdU) immunopositive cell counts of (a) subgranular zone and (b)subventricular zone showing significant increase in the number ofbromodeoxyuridine-positive cells in the omega 3-fortified soft diet-fedgroup when compared with both soft diet and hard diet-fed groups.


during development [28]. BDNF level increased in the

brain after voluntary physical activity, including masticatory

activity. This might explain why hard diet enhances

neurogenesis. BDNF infusion in the lateral ventricles was

found to augment SVZ neurogenesis [29]. In contrast,

administration of BDNF into the lateral ventricles led to a

decrease in SVZ neurogenesis in rat [30].

DHA is an omega 3 fatty acid highly enriched in the CNS

and is critical for brain development and function. DHA

improves both neuronal proliferation and survival. This

observation is consistent with the findings of recent studies

in rats fed with DHA [31]. DHA significantly enhances

hippocampal neurogenesis in the transgenic fat-1 mice rich

in endogenous DHA. DHA can influence cell function

through multiple mechanisms. DHA esterified into

phospholipids of the plasma membrane bilayer signi-

ficantly alters many basic membrane properties, including

fluidity, flexibility, permeability, electrostatic behavior, and

consequently regulates the neurotransmission and signal

transduction [32]. However, the unesterified free-DHA

exerts complex changes in gene expression in the brain,

including the expression of genes involved in neurogenesis

[33]. Omega 3 regulates corticotrophin factor, increases

seretonergic function, increases dentritic arborization,

prevents neural apoptosis, improves cerebral blood flow,

and regulates gene expression [34]. Omega 3 fatty acids are

the most efficient for the development of adequate brain

cell membranes and intercellular neuronal connections [35].

AHN affects learning and memory [36]. Newborn neurons

that are young when events occur have a specialized role in

encoding, in storage, and in temporally relating one event to

another, explaining a possible requirement of newborn

neurons in the process of learning and memory [37].

Neurodegenerative diseases such as Alzheimer’s disease and

Parkinson’s disease affect AHN either by stimulation or by

inhibition. AHN is also influenced by pathological

conditions. For example, it is increased in epilepsy and

stroke and decreased in HIV infection. CNS inflammation

affects the integration of newborn neurons into circuits [38].

ConclusionIn conclusion, the following observations are presented:

(1) Hard diet has a stimulatory effect on the process of

neurogenesis through the masticatory activity;

(2) In contrast, soft diet alone has an inhibitory effect on

the process of neurogenesis;

(3) If there is no escape from using soft diet, it might be

fortified with omega 3 fatty acid to obtain a better

effect on neurogenesis.

(4) The omega 3-fortified soft diet is suggested to

replace the insufficient masticatory activity.

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