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 zone
Rehab Ahmed Rifaai, Nashwa Fathy El-Tahawy and Entesar Ali Saber
Department of Histology, Faculty of Medicine, Minia
University, Egypt
Correspondence to Rehab Ahmed Rifaai, Department
of Histology, Faculty of Medicine, Minia University,
Egypt
Tel: + 103358376; fax: + 86 2342813;
e-mail: rehabrifaai@yahoo.com
Received 24 January 2011
Accepted 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 Histology
1110-0559
Introduction
Adult 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 methods
Animals
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 30min 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 Â 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 300 mm 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 300 mm.
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.
Results
The 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 1
showing the dentate gyrus (DG). (b) A photomicrograph of group 1
dentate gyrus showing its different layers: molecular (M) layer, granule
cell layer (GCL), and polymorphic layers (PL). Observe the hilus (H) and
the subgranular zone (SGZ).
HE, A =Â100, B = Â400.
Figure 2.
(a) A photomicrograph of group 1 subventricular zone throughout
the lateral wall of the lateral ventricle (LV). (b) A photomicrograph
of group 1 subventricular zone showing the different layers of the
subventricular zone: ependymal cells with microvilli (1), hypocellular
gap of astrocytic processes (2), a ribbon of astrocyte cell bodies (3),
and the transition zone (4).
HE, A = Â100, B = Â1000.
Omega 3 and neurogenesis Rifaai et al. 3

Figure 6.
Bromodeoxyuridine-labeled cells in the subventricular zone of the control
group showing numerous immunopositive cells 2 days after the last BrdU
injection (arrow). Observe the initial migration along the rostromigratory
stream (RMS) ( Â 100). Inset showing immunopositive cell in the
anaphase stage of mitosis (circle).
Â 1000.
Figure 8.
Bromodeoxyuridine-labeled cells in the subventricular zone of the
control group showing few immunopositive cells seen 4 weeks after the
last bromodeoxyuridine injection (arrow)
Â100.
Figure 4.
Bromodeoxyuridine-labeled cells in the dentate gyrus of the control
group 2 weeks after the last bromodeoxyuridine injection showing many
immunopositive cells in the subgranular zone (SGZ) and granule cell
layer (GCL) (arrow).
Â 400.
Figure 5.
group 4 weeks after the last bromodeoxyuridine injection showing many
layer (GCL) (arrow).
Â 400.
Figure 7.
Bromodeoxyuridine-labeled cells in the subventricular zone of the control
group showing numerous immunopositive cells 2 weeks after the last
bromodeoxyuridine injection (arrow) (Â 100). Observe that the migrating
cells have reached the olfactory bulb (OB) along the rostromigratory
stream (RMS) (inset).
Â40.
Figure 3.
group 2 days after the last BrdU injection showing many
layer (GCL) (arrow) (Â 400). Inset showing the immunopositive cells
Â1000.

Figure 9.
Bromodeoxyuridine-labeled cells in the dentate gyrus of the soft diet-
fed group 2 days after the last bromodeoxyuridine injection showing
immunopositive cells in the subgranular zone (SGZ) (arrow)
Â400.
Figure 10.
Bromodeoxyuridine-labeled cells in the dentate gyrus of the soft diet-fed
group 2 weeks after the last bromodeoxyuridine injection showing
apparent decreased immunoreactive cells compared with the control
group (arrow).
Â 400.
Figure 11.
Bromodeoxyuridine-labeled cells in the dentate gyrus of the soft-diet
fed group 4 weeks after the last bromodeoxyuridine injection showing
apparent decreased immunoreactive cells in the SGZ and the GCL
compared with the control group (arrow).
Â 400.
Figure 12.
Bromodeoxyuridine-labeled cells in the subventricular zone of the soft
diet-fed group 2 days after the last bromodeoxyuridine injection
showing decreased reaction compared with the control group (arrow)
Â 100.
Figure 13.
diet-fed group 2 weeks after the last bromodeoxyuridine injection
showing decreased reaction compared with the control group (arrow).
Â 100.
Figure 14.
diet-fed group 4 weeks after the last bromodeoxyuridine injection
showing decreased reaction compared with the control group (arrow).
Â 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 (P0.05) 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 bromodeoxyuridine
injection showing many immunopositive cells in the subgranular zone
(SGZ) and granule cell layer (GCL) (arrow).
Â400.
Figure 15.
fortified soft diet-fed group 2 days after the last bromodeoxyuridine
Â 400.
Figure 17.
fortified soft diet-fed group 4 weeks after the last bromodeoxyuridine
Â400.
Figure 18.
Bromodeoxyuridine-labeled cells in the subventricular zone of the omega
3-fortified soft diet-fed group showing that a large number of immuno-
positive cells are seen in the subventricular zone and along the
rostromigratory stream (RMS) 2 days after the last bromodeoxyuridine
injection (arrow).
Â100.
Figure 19.
Figure 19.
omega 3-fortified soft diet-fed group showing that many
immunopositive cells are present 2 weeks after the last
bromodeoxyuridine injection (arrow) ( Â 100). Observe that the
migrating cells reaching the olfactory bulb (OB) along the
rostromigratory stream (RMS) (inset).
Â40.

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 (P0.05) 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).
Discussion
This 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.
omega 3-fortified soft diet-fed group showing that many
immunopositive cells are present 4 weeks after the last
bromodeoxyuridine injection (arrow) (Â 400). Observe some
immunopositive cells grouped into nests (upper inset). Lower inset
showing the immunopositive cells incorporated among cells in the
granular cell layer of the olfactory bulb (OB)
Â1000.
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 of
bromodeoxyuridine-positive cells in the omega 3-fortified soft diet-fed
group 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].
Conclusion
In 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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