Int J Clin Exp Pathol 20158(7):7710-7728 [600132]

Int J Clin Exp Pathol 2015;8(7):7710-7728
www.ijcep.com /ISSN:1936-2625/IJCEP0010095
Original Article
Protective effect of chronic caffeine intake
on gene expression of brain derived neurotrophic
factor signaling and the immunoreactivity of glial
fibrillary acidic protein and Ki-67 in Alzheimer’s disease
Fatma M Ghoneim1, Hanaa A Khalaf1, Ayman Z Elsamanoudy2, Salwa M Abo El-khair2, Ahmed MN Helaly3,
El-Hassanin M Mahmoud4, Saad H Elshafey1
Department s of 1Histology and Cell Biology, 2Medical Biochemistry and Molecular Biology, 3Forensic Medicine and
Toxicology, 4Psychiatry, Faculty of Medicine, Mansoura University, Egypt
Received May 11, 201 5; Accepted June 26, 2015; Epub July 1, 2015; Published July 15, 2015
Abstract: Alzheimer’s disease (AD) is a neurodegenerative disorder with progressive degeneration of the hippocam –
pal and cortical neurons. This study was designed to demonstrate the protective effect of caffeine on gene expres –
sion of brain derived neurotrophic factor (BDNF) and its receptor neural receptor protein-tyrosine kinase-β (TrkB) as
well as glial fibrillary acidic protein (GFAP) and Ki-67 immunoreactivity in Aluminum chloride (AlCl3) induced animal
model of AD. Fifty adult rats included in this study were classified into 5 group (10 rats each); negative and positive
control groups (I&II), AD model group (III), group treated with caffeine from the start of AD induction (IV) and group
treated with caffeine two weeks before AD induction (V). Hippocampal tissue BDNF and its receptor (TrkB) gene
expression by real time RT-PCR in addition to immunohistochemical study of GFAP and Ki67 immunoreactivity were
performed for all rats in the study. The results of this study revealed that caffeine has protective effect through im –
proving the histological and immunohistochemical findings induced by AlCl3 as well as BDNF and its receptor gene
expression. It could be concluded from the current study, that chronic caffeine consumption in a dose of 1.5 mg/kg
body weight daily has a potentially good protective effect against AD
Keywords: Caffeine, BNDF signaling, glial fibrillary acidic protein, Ki-67, Alzheimer’s disease
Introduction
Alzheimer’s disease (AD) is a neurodegenera –
tive disorder with progressive degeneration of
the hippocampal and cortical neurons, and
thus impairment of memory and cognitive abil –
ity. The pathological hallmarks of AD are spheri –
cal accumulations of b-amyloid protein (senile
plaques) and degenerating neuronal processes
as well as neurofibrillary tangles (paired helical
filaments and other proteins) [1, 2] Many bio –
chemical cellular changes induce neuronal pro –
grammed cell death. Among these cellular
changes in AD are metabolic disturbances, dis –
ruption of Ca2+ homeostasis, oxidative stress,
inflammation, and accumulation of unfolded/
mis-folded proteins [3].
Brain derived neurotrophic factor (BDNF), a
member of the neurotrophin family, is essential for growth, survival and neuronal cell differen –
tiation. Moreover, it is involved in learning and
memory by binding to its main functional recep –
tor (neural receptor protein-tyrosine kinase-β;
TrkB), present in the hippocampus, cortex and
basal forebrain. The BDNF signaling, through
binding to TrkB, is involved in the pathophysiol –
ogy of AD [4, 5].
Astrocytes are critical for the survival of neu –
rons in the central nervous system (CNS) by
playing a role in the energy metabolism, mainte –
nance of the blood-brain barrier, vascular reac –
tivity, regulation of extracellular glutamate lev –
els and finally protection from reactive oxygen
species. These cells react to the neuronal dam –
age, resulting from physical or chemical insults,
by over expression of the glial fibrillary acidic
protein (GFAP), an intermediate cytoskeletal fil –
ament protein specific for astrocytes [6]. Ki-67

Histological and molecular study of the protective effect of caffeine in AD
7711 Int J Clin Exp Pathol 2015;8(7):7710-7728is a reliable marker of cell proliferation
expressed during all active phases of the cell
cycle [G(1), S, G(2)] but is absent in resting cells
[G(0)] [7]. During interphase, Ki-67 antigen can
be exclusively detected in the nucleus, whereas
in mitosis most of the protein is relocated to the
chromosomal surface. Previously, Smith and
Lippa [8] reported that Ki-67 may be involved in
the pathogenesis of neurofibrillary degenera –
tion in AD, other neurodegenerative disorders,
normal aging, and neuronal neoplasms as
ganglioglioma.
Santos et al. [9] have demonstrated that caf –
feine intake, one of the most widely consumed
psychoactive substances, may reduce the risk
of AD and cognitive impairment in elderly
patients. Other studies have indicated that caf –
feine intake (1.5 mg/day) may reverse cognitive
impairment and decrease brain amyloid beta
(Aβ) levels in aged AD mice [10]. However, epi –
demiologic studies in humans came to be
inconclusive, with some suggesting a reduced
risk of dementia with higher coffee intake in
midlife, and others pointing to a lack of associa –
tion [11].
So, the present study was designed to clarify
the role of chronic habitual use of caffeine
intake as a protective agent in AD and to inves –
tigate its effect on gene expression of BNDF
and TrkB as well as GFAP and Ki-67 immunore –
activity in aluminum chloride induced animal
model of AD.
Materials and methods
Animals
A total of 50 adult male albino Sprague-Dawley
rats (200-250 gm) were used in this study.
Used drugs
Aluminum chloride (AlCl3, MW = 133.34) was
purchased from Sigma-Aldrich Co. (Munich,
Germany) and Caffeine was purchased from
Pratap chemical industry Pvt Ltd (India).
Induction of Alzheimer’s disease (AD) in rats
Induction of AD in rats was carried out by
administering AlCl3 orally at a dose of 17 mg/kg
body weight daily for 4 successive weeks,
according to the procedure described by Karam
et al. [12]. Experimental protocol
The experimental protocol of the study was
approved by the ethical committee of Medical
Faculty of Mansoura University. Animals were
used in accordance with the Animal Welfare Act
and Guide for Care Use of MERC (Mansoura
Experimental Research Center) prepared by
Mansoura University. In this study 50 adult
male rats weighing (200-250 gm) each were
housed in a quite non-stressful environment for
one week before study. They were fed ad libi –
tum and allowed free access to water during
the experimental period. Animals were divided
into five equal groups (10 rats each):
Group I: Each animal received 1 ml normal
saline 0.9% by gavage for 4 successive weeks
and served as the negative control group.
Group II: Each animal received 1.5 mg/day of
caffeine [5] by gavage for 4 successive weeks
and served as the positive control group.
Group III: Each animal received 17 mg/kg body
weight of aluminum chloride once daily [12] by
gavage for 4 successive weeks.
Group IV: Each animal received a combination
of caffeine (1.5 mg/day) with AlCl3 (17 mg/kg
body weight) for 4 successive weeks.
Group V: Rats were given caffeine for 2 succes –
sive weeks, followed by a combination of caf –
feine with AlCl3 for 4 successive weeks.
Sampling
At the end of each experiment, all animals were
sacrificed by cervical dislocation. Craniotomy
was performed to dissect out the intact brains
for histological, immunohistochemical and
molecular study. Brains were removed and sag –
ittaly divided into right and left hemispheres
using a sharp blade for isolating the hippocam –
pal tissues. Total RNA was extracted from
25-30 mg hippocampus tissue from one hemi –
sphere immediately after shock freeze in liquid
nitrogen and real time RT-PCR for BDNF and its
couple receptor TrkB was performed [13].
Histological study
For histological study, the brain specimens (the
other hemisphere) were fixed in Bouin’s solu –
tion. After fixation, specimens were dehydrated

Histological and molecular study of the protective effect of caffeine in AD
7712 Int J Clin Exp Pathol 2015;8(7):7710-7728in an ascending series of alcohol, cleared in
two changes of xylene and embedded in mol –
ten paraffin. Sections of 5 microns thickness
were cut using rotary microtome and mounted
on clean slides. For histological examination,
sections were stained with hematoxylin and
eosin (H&E) [14].
Immunohistochemical (IHC) study
The other paraffin-embedded sections were
immunohistochemically stained with Anti-Ki-67
and with GFAP.
a)→Brain sections were immunohistochemi –
cally stained with proliferating cell nuclear anti –
gen (Mouse Monoclonal Anti-Ki-67, Clone
GM010, from Genemed Biotechnology Inc.,
458 Carlton Ct. South San Francisco, CA
94080, USA), at dilution 1:50 or 1:100. Tonsil
was used as a positive control.
b)→Brain sections were immunohistochemi –
cally stained with GFAP mouse monoclonal
antibody (Mouse Monoclonal anti-human clo –
ne6F2 code No. M 0761, from DakoCytomation.
Produktionsvej 42 DK-2600 Glostrup Denmark)
at dilution 1:50 or 1:100. Brain was used as a
positive control.
Sections were taken on positive slides and
immunostained using avidin-biotin technique.
Sections were deparaffinized in xylene, rehy –
drated and pretreated with 0.01% hydrogen
peroxide (H2O2) for blocking endogenous peroxi –
dise activity and unmasking of the antigenic
site was carried out by transmitting sections
into 0.01 M citrate buffer (ph 6) for 10 minutes
in ethanol for 10 minutes. Microwave -assisted
antigen retrieval was then performed for 20
minutes. Sections were incubated overnight at
40C with the diluted primary antibody at dilu –
tion 1/500 & 1/100 monoclonal mouse anti –
bodies for GFAP and Ki-67. Sections were incu –
bated with the avidin-biotin complex (ABC)
reagent for 60 minutes then incubated in per –
oxidase substrate solution for 6-10 minutes.
Finally, haematoxylin was used as a counter
stain, dehydration in absolute alcohol, clearing
and mounting were done. Immunoreactivity
was visualized as dark brown cytoplasmic stain –
ing for GFAP while for Ki-67 it was visualized in
the nucleus [15]. For the negative control slide,
the specific 1 ry antibody was replaced by phos –
phate buffer saline. Quantitative analysis
Astrocyte counting in CA1 and CA3 regions:
Slides were digitized using Olympus® digital
camera installed on Olympus® microscope with
1/2 X photo adaptor, using 40 X objective. The
result images were analyzed on Intel® Core I3®
based computer using VideoTest Morphology®
software (Russia) with a specific built-in routine
for immunohistostaining analysis and stain
quantification. The system measured the area
percentage of GFAP positive expression.
Ki67-positive cells counting in the dentate
gyrus
The numbers of Ki-67 positive cells in samples
from all groups were determined using an
image analysis system equipped with a com –
puter-based charge-coupled device (CCD) cam –
era (Optimas 6.5; Media Cybernetics, USA).
Ki67-positive cells in the dentate gyrus of each
section were counted using Optimas 6.5 soft –
ware (Media Cybernetics). Cell counts for all the
sections from every rat were averaged and are
presented as a percentage.
Total RNA extraction from the hippocampal tis –
sue samples: According to the manufacturer’s
instructions, total RNA was extracted from
25-30 mg hippocampal tissue samples. The
tissue samples were snap frozen in liquid nitro –
gen and then used immediately for RNA extrac –
tion using the TriFast TM reagent (PeqLab.
Biotechnologie GmbH, Carl-Thiersch St. 2B
91052 Erlongen, Germany, Cat. No. 30-2010).
Any remained DNA was removed by digestion
with DNase I (Sigma). The concentration and
purity of the isolated RNA was determined
using NanoDrop™ 2000 Spectrophotometer
(Thermo Scientific., USA). Purity of the extract –
ed RNA was also, confirmed using 2% formalde –
hyde agarose gel electrophoresis with ethidium
bromide staining, to find the 2 sharply purified
bands that represent 28S and 18S ribosomal
RNA.
Quantitative real-time PCR
According to the manufacturer’s instructions,
Reverse Transcription (RT) of the extracted RNA
was performed using Maxima First Strand
cDNA Synthesis Kit for RT-qPCR provided by
ThermoScientific, U.S.A, cat No. #K1641. A
control reaction, with 2 μl of purified RNA, was

Histological and molecular study of the protective effect of caffeine in AD
7713 Int J Clin Exp Pathol 2015;8(7):7710-7728run without reverse transcription to ensure
absence of any DNA contamination in the
extracted RNA samples. The synthesized cDNA
is stored at -20°C until use for real time RT-PCR.
Gene-specific real time RT-PCR primers (pur –
chased from Oligo™ Macrogen): Oligonucleo-
tide primers (specific for the coding region of
exon 5) were designed to amplify rat BDNF.
Sequences of BDNF primers [16] were
5’-CAGGGGCATAGACAAAAG-3’ (forward); and
5’-CTTCCCCTTTTAATGGTC-3’ (reverse) (PCR
product: 167 bp); TrkB-specific primers were
5’-CTGCCAGCCCTCTCCACCACAT-3’ (forward);
and 5’-GCAAGGACTTTCATCGCGAAGC-3’ (re-
verse) (TrkB-product: 315 bp) [17], while for
β-actin, used as internal control, 5’-CCTG-
TATGCCTCTGGTCGTA-3’ (forward) and 5’-CCAT-
CTCTTGCTCGAAGTCT-3’ (reverse) (PCR prod –
uct: 260 bp) [18].
PCR reactions for optimization of primer anneal –
ing temperature were carried out using
DreamTaq™ Green PCR Master Mix (2X)
(Thermo Scientific, USA, cat No. 1081). Also,
optimization of the primer concentration is per –
formed, for use in real time RT-PCR reactions,
to determine the primer concentration giving
the lowest CT (threshold cycle) and minimizing
nonspecific amplification. Briefly, amplifications
(25 μL reaction) in duplicates, containing 12.5
μL 2X SYBR green Master mix, 2 μL cDNA and a
variable concentration of forward and reverse
primers, were run with thermal cycling param –
eters as an initial step of holding at 95°C for 10
min followed by 40 cycles of denaturation (hold
at 95°C, 30 sec) and annealing (at 60°C, 45
sec). Then, RT-PCR products were analyzed on
2% agarose gel electrophoresis.
Quantitative real-time PCR analysis: The Real
time PCR reactions were performed using real
time PCR equipment (Applied Biosystem 7500,
USA) with 96-well plates, and SybrGreen
reagent [SYBR® Green PCR Master Mix (Applied
Biosystem, USA, cat. No. 4344463)].
Amplification were performed, in duplicates, in
a 25 μL total reaction containing 12.5 μL Power
Sybr® Green PCR Master Mix reaction buffer
(Applied Biosystems), forward and reverse gene
specific primers (10 pmol) and 2 μL cDNA. The
cycling parameters included 40 cycles (each of
hold at 95°C for 30 sec, 60°C for 45 sec) after
one initial step of 95°C for 10 min, to activate AmpliTaq Gold polymerase. The PCR reactions
were monitored by measuring the increase in
fluorescence caused by binding of SYBR Green
Dye to the double stranded DNA and CT values
(cycle threshold) were determined. To confirm
the specificity of the PCR products, melting
curve analysis and agarose gel electrophoresis
2% were performed. In each experiment, nega –
tive control reaction with no template was run.
The relative quantification of the BDNF and
TrkB genes in different hippocampal tissue
samples was performed by the comparative
method. β actin was used as an endogenous
control gene to standardize the cDNA amount
added to the reaction. Each analysis requires 4
reactions (2 for analysis of the target gene
(BDNF or TrkB) and 2 for analysis of the internal
standard gene, β actin). The ΔCT for each sam –
ple is calculated and linearized using 2-ΔCT.
Finally ΔΔCT between experimental and control
samples were calculated and linearized using
2-ΔΔCT formula for overall change. Thus, the
amount of target gene (BDNF or TrkB) in the
experimental groups, normalized to the endog –
enous reference gene (β actin) and relative to
control group, is calculated by an arithmetic
formula.
Statistical analysis
Data were tabulated, coded then analyzed
using the computer program SPSS (Statistical
package for social science) version 17.0.
Descriptive statistics were calculated in the
form of Mean and Standard deviation (± SD). In
the statistical comparison between the differ –
ent groups, the significance of difference was
tested using ANOVA (analysis of variance) to
compare between more than two groups of
numerical (parametric) data followed by post-
hoc for multiple comparisons. Pearson correla –
tion coefficient test was used correlating differ –
ent parameters. A P value <0.05 was consid –
ered statistically significant in all analyses.
Results
Histological results
Group I : Microscopic examination of H&E.
stained sections of all specimens in this group
(negative control group) was similar and
revealed the normal histological structure spe –
cific for the hippocampus. These were the hip –

Histological and molecular study of the protective effect of caffeine in AD
7714 Int J Clin Exp Pathol 2015;8(7):7710-7728pocampus proper, dentate gyrus and subicu –
lum. The hippocampus proper was formed of
the Cornu Ammonis (CA) as CA1, CA2, CA3 &
CA4 regions, and was continued as subiculum.
Dentate gyrus appeared as a dark C shaped
structure enclosing CA4. Areas inside concavity
of CA and dentate gyrus comprised the molecu –
lar layer ( Figure 1 ).
Examination of CA1 & CA3 regions showed that
it was formed of three layers; molecular, pyra –
midal and polymorphic layers. The main cellular
layer was the pyramidal layer which was formed
of 4-5 compact layers of small pyramidal neu –
rons with vesicular nuclei in CA1 ( Figure 2A )
and many large pyramidal neurons also with
vesicular nuclei in CA3 ( Figure 2B ). Both the
molecular and polymorphic layers were rela –
tively cell-free layers. They contained sparse
nuclei of neuroglial cells as astrocytes in addi –
tion to blood vessels on a pink neuropil back –
ground consisted of neuronal and glial cell pro –
cesses ( Figure 2A and 2B).
The dentate gyrus consisted of molecular, gran –
ular and polymorphic layers. The granular layer
constituted the principal layer. It had the great –
est cell density and was formed of dense col –
umns of granule cells that appeared rounded
with vesicular nuclei. It was noticed that small
dark cells variable in shape and size were pres -large pyramidal cells respectively with some
cell loss. The pyramidal cells lost their triangu –
lar shape, showed darkened nuclei and were
surrounded with pericellular haloes. As regard
the granular layer it showed an apparent
increase in the number of the granule cells with
decreasing in their diameter. There was marked
shrinkage in size of granular cells with some
cell loss and marked vacuolation. Moreover,
there was a marked decrease and sometimes
absence of the dark cells which were previously
observed in the sub-granular zone in the con –
trol sections ( Figure 3C ). Molecular & polymor –
phic layers revealed enlarged and excess astro –
cytes and widened blood capillaries ( Figure
3A-C ).
Group IV: Examination of hippocampus sec –
tions of this group (caffeine with AlCl3 for 4 suc –
cessive weeks) revealed less prominent histo –
pathological changes when compared with alu –
minum chloride group. Preservation of most
pyramidal cells and markedly decreased apop –
tosis of cells were observed in CA1 ( Figure 4A )
and CA3 ( Figure 4B ) regions. Less vacuolations
and fewer apoptotic cells persisted in the gran –
ular layer ( Figure 4C ) with persistence of
enlarged astrocytes and widened capillaries in
many fields ( Figure 4A-C ).
Group V : Examination of hippocampus sections
of this group (caffeine for 2 successive weeks,
Figure 1. A photomicrograph of H&E stained sections of the rat hippocam –
pus of group I (negative control group) showing the different areas of the
hippocampus formation where the hippocampus proper is formed of the
CornuAmmonis (CA) as CA1, CA2, CA3 & CA4 regions, and is continued as
subiculum (S). Dentate gyrus (DG) is seen surrounding CA4 by its upper &
lower limbs. Notice the presence of molecular layer (M) inside concavity
of CA and DG .ent in the sub-granular zone.
The molecular layer which is the
outermost one was a relatively
cell-free layer. It contained few
neuroglial cells as astrocytes in
addition to blood vessels. The
polymorphic layer contained
various types of cells including
pyramidal cells and astrocytes
in addition to blood vessels
(Figure 2C ). Compared with the
control specimens, the animals
of group II, which received 1.5
mg/day of caffeine orally by
gavage for 4 weeks revealed the
same general picture of the
hippocampi.
Group III: Animals received alu –
minum chloride for 4 successive
weeks showed distinct histologi –
cal changes. The pyramidal lay –
ers in CA1 ( Figure 3A ) and CA3
(Figure 3B ) showed marked
shrinkage in size of small and

Histological and molecular study of the protective effect of caffeine in AD
7715 Int J Clin Exp Pathol 2015;8(7):7710-7728followed by a combination of caffeine with AlCl3
for 4 successive weeks) showed return of hip –
pocampus tissue towards normal morphology
Figure 2. Photomicrographs of H&E stained sections
of the rat hippocampus of group I (A) showing the
three layers of CA1 region of hippocampus proper;
molecular layer (M), pyramidal layer (P) & polymor –
phic layer (Pr). The pyramidal layer (P) shows 4-5
compact layers of small pyramidal cells (arrow) most
with vesicular nuclei (B) showing the three layers of
CA3 region of hippocampus proper; molecular layer
(M), pyramidal layer (P) & polymorphic layer (Pr).
The pyramidal layer (P) shows few layers of large py –
ramidal cells (arrow) also with vesicular nuclei. (C)
Showing dentate gyrus forms of molecular layer (M),
granular layer (G) and polymorphic layer (Pr) contains
pyramidal cells (arrow head). The granular layer (G)
contains granule cells (arrow) arranged in dense col –
umns, each appears rounded with vesicular nuclei.
Dark cells are seen in the subgranular zone (tailed
arrow). Notice: both molecular & polymorphic layers
contain astrocytes (*) and blood capillaries (crossed
arrow) .Figure 3. Photomicrographs of H&E stained sec –
tions of the rat hippocampus of group III (17 mg/
kg body weight aluminum chloride once daily for 4
successive weeks) (A) showing the three layers of
CA1 region of hippocampus proper; molecular layer
(M), pyramidal layer (P) & polymorphic layer (Pr). The
pyramidal layer (P) shows marked shrinkage in size
of small pyramidal cells (arrow) with some cell loss
(curved arrow). The pyramidal cells (arrow) show
darkened nuclei and surrounded with pericellular
haloes. (B) Showing the three layers of CA3 region of
hippocampus proper; molecular layer (M), pyramidal
layer (P) & polymorphic layer (Pr). The pyramidal layer
(P) shows marked shrinkage in size of large pyrami –
dal cells (arrow) with some cell loss (curved arrow).
The pyramidal cells (arrow) show darkened nuclei
and surrounded with pericellular haloes. (C) Showing
dentate gyrus forms of molecular layer (M), granu –
lar layer (G) and polymorphic layer (Pr). The granular
layer (G) shows marked shrinkage in size of granule
cells (arrow) with some cell loss (curved arrow) and
marked vacuolation (V). Notice: both molecular (M)
& polymorphic (Pr) layers contain enlarged and ex –
cess astrocytes (*) and widened blood capillaries
(crossed arrow).

Histological and molecular study of the protective effect of caffeine in AD
7716 Int J Clin Exp Pathol 2015;8(7):7710-7728as evidenced by preservation of small pyrami –
dal cells ( Figure 5A ) large pyramidal cells (Figure 5B ) and granular cells ( Figure 5C ).
Molecular & polymorphic layers contained nor -Figure 4. Photomicrographs of H&E stained sections
of the rat hippocampus of group IV (a combination of
caffeine at a dose of 1.5 mg/day with AlCl3 for 4 suc –
cessive weeks) (A) showing the three layers of CA1
region of hippocampus proper; molecular layer (M),
pyramidal layer (P) & polymorphic layer (Pr). Some
small pyramidal cells appear with vesicular nuclei
(arrow) and apoptotic cells appear (zigzag arrow) (B)
showing the three layers of CA3 region of hippocam –
pus proper; molecular layer (M), pyramidal layer (P)
& polymorphic layer (Pr). Some large pyramidal cells
appear with vesicular nuclei (arrow) and apoptotic
cells (zigzag arrow) appear (C) showing dentate gyrus
forms of molecular layer (M), granular layer (G) and
polymorphic layer (Pr). Granule cells show less vacu –
olation (V). Most cells appear normal (arrow) and
some cells show apoptosis (thick arrow) Notice: wid –
ened blood capillaries (crossed arrow), some normal
astrocytes (*) and others enlarged (arrow head) ap –
pear in the molecular (M) & polymorphic (Pr) layers.Figure 5. Photomicrographs of H&E stained sections
of the rat hippocampus of group V (caffeine for 2 suc –
cessive weeks, followed by a combination of caffeine
with AlCl3 for 4 successive weeks) (A) showing the
three layers of CA1 region of hippocampus proper;
molecular layer (M), pyramidal layer (P) & polymor –
phic layer (Pr). The pyramidal layer (P) shows pres –
ervation of small pyramidal cells (arrow) (B) show –
ing the three layers of CA3 region of hippocampus
proper; molecular layer (M), pyramidal layer (P) &
polymorphic layer (Pr). The pyramidal layer (P) shows
preservation of large pyramidal cells (arrow) (C)
showing dentate gyrus forms of molecular layer (M),
granular layer (G) & polymorphic layer (Pr). The gran –
ule cells (arrow) appear nearly similar to the control.
Notice: both molecular (M) & polymorphic (Pr) layers
contain normal astrocytes (*) and blood capillaries
(crossed arrow).

Histological and molecular study of the protective effect of caffeine in AD
7717 Int J Clin Exp Pathol 2015;8(7):7710-7728mal astrocytes and blood capillaries ( Figure
5A-C ).
Immunohistochemical stains and Morphomet –
ric analysis
GFAP immunostaining: Immunohistochemical
staining for GFAP showed its normal distribu –
tion in the control group as a mild positive reac –
tion in the immuno-reactive astrocytes and glial
fibers. The immuno-reactive astrocytes ap-
peared as star-shaped cells and the fibers were
thin and had a regular course ( Figure 6A ).
Group II revealed the same results as group I. In
group III, there was an apparent increase in the
amount and intensity of GFAP immuno-reactive
astrocytes. The glial fibers appeared twisted
with an irregular course and were thickened
with increase in their staining intensity ( Figure
6B). An apparent decrease in the amount and intensity of GFAP immuno-reactive astrocytes
together with decreased intensity of staining of
glial fibers were noticed in group IV (caffeine
with AlCl3 for 4 successive weeks) ( Figure 6C ).
Weak GFAP immuno-reaction nearly similar to
the control was seen in the star-shaped astro –
cytes and the thin regular glial fibers in group V
(caffeine for 2 successive weeks, followed by a
combination of caffeine with AlCl3 for 4 succes –
sive weeks) ( Figure 6D ).
In Table 1 ; GFAP positive areas (%) are signifi –
cantly higher in group III (AD disease model)
than that of group I (negative control) as well as
group II (positive control). It starts to be lower in
group IV (AD + Caffeine) but still significantly
higher than that of both control groups (I&II). In
group V, GFAP positive areas (%) showed the
lowest expression in experimental AD groups; it
showed significant decrease than that of group
Figure 6. Photomicrographs of GFAP immune-stained sections in the rat hippocampus. (A) Group I showing mild
positive reaction in the immuno-reactive astrocytes (s) and glial fibers (arrow). Note the GFAP immuno-reactive
astrocytes (s) appear as star-shaped cells and the fibers (arrow) are thin and have a regular course (B) Group III
showing an apparent increase in the number of GFAP immuno-reactive astrocytes (s) and the glial fibers (arrow) ap –
pear twisted, thickened and intensely stained as compared to the control group (C) Group IV showing an apparent
decrease in the number of GFAP immuno-reactive astrocytes (s) and the glial fibers (arrow) appear less twisted and
thickened as compared to the control group (D) Group V showing weak GFAP immuno-reaction in the star-shaped
astrocytes (s) and glial fibers (arrow).

Histological and molecular study of the protective effect of caffeine in AD
7718 Int J Clin Exp Pathol 2015;8(7):7710-7728III and IV with no significant difference than that
of group I&II.
Ki-67 immunostaining
Ki67-positive nuclei were detected in the sub –
granular zone of the dentate gyrus in the con –
trol group ( Figure 7A ). Group II revealed the
same results as group I. The number of Ki67 immunoreactive nuclei was significantly de-
creased in group III as compared to control
groups (group I&II) ( Figure 7B ; Table 1 ). An
apparent increase in the number of Ki67-
positive nuclei was observed in group IV ( Figure
7C) when compared to group III (AD model) but
still significantly lower than that of the control
groups (I&II) as shown in Table 1 . In group V the Table 1. Ki67 and GFAP immunoreactivity as well as BDNF and its receptor gene expression in the
different studied groups
GroupKi67-positive cells
(%) Mean ± SDGFAP positive areas
(%) Mean ± SDBDNF gene expression
(2-∆∆CT) Mean ± SDTrkB gene expression
(2-∆∆CT) Mean ± SD
Control (I) 95.26 ± 10.55 9.79 ± 2.77 1.00 ± 0.10 1.00 ± 0.10
Control + Caffeine (II) 96.63 ± 12.01 9.09 ± 2.93 1.00 ± 0.11 1.08 ± 0.24
AD disease model (III) 22.28a,b ± 5.92 28.93a,b ± 6.18 0.35a,b ± 0.07 0.32a,b ± 0.07
AD + Caffeine (IV) 58.88a,b,c ± 8.36 19.50a,b,c ± 3.46 0.53a,b,c ± 0.08 0.55a,b,c ± 0.12
AD + Caffeine Before (V) 84.91c,d ± 10.76 11.95c,d ± 2.34 0.85b,c,d ± 0.13 0.77a,b,c,d ± 0.13
P < 0.0001 < 0.0001 < 0.0001 < 0.0001
SD: standard deviation; P: Probability Test used: ANOVA followed by post-hoc Tukey for multiple comparisons; a: significance
relative to Control Group; b: significance relative to Control + caffeine Group; c: significance relative to AD disease model Group;
d: significance relative to AD + Caffeine Group.
Figure 7. Photomicrographs of Ki67 immune-stained sections in the rat hippocampus. (A) Group I showing Ki67-pos –
itive nuclei in the subgranular zone of the dentate gyrus (arrow). (B) Group III showing an apparent decrease in the
number of Ki67 immunoreactive nuclei (C) Group IV showing an apparent increase in the number of Ki67-positive
nuclei compared to group III (D) Group V showing Ki67-positive nuclei in the subgranular zone of the dentate gyrus
(arrow) nearly similar to the control group.

Histological and molecular study of the protective effect of caffeine in AD
7719 Int J Clin Exp Pathol 2015;8(7):7710-7728number of Ki67-positive nuclei was nearly simi –
lar to the control group ( Figure 7D ). Moreover, it
shows significant increase than that of group III
and group IV ( Table 1 ).
The correlation between the percentage of
GFAP and Ki67 immunoreactive positive cells is
presented in Figure 8 in the form of significant
negative correlation (r = -0.871 & P < 0.0001).
Real time RT-PCR study of BDNF and its recep –
tors TrkB genes expression
BDNF and TrkB genes expression by real time
RT-PCR study showed statistically significant
decrease in group III when compared to control
groups (I&II) which started to increase in group
IV but still significantly lower than that of the
control groups. Group V became significantly
higher than that of group III and IV with BDNF
gene expression has no significant difference
when compared to group I (negative control
group) ( Table 1 ).
In Figure 8 hippocampal BDNF gene expression
shows significant positive correlation with the percentage of Ki67 immunoreactive positive
cells (r = 0.869 & P < 0.0001). While, it shows
significant negative correlation with the per –
centage of GFAP immunoreactive positive cells
(r = -0.834 & P < 0.0001).
Discussion
In the current study, aluminum chloride (AlCl3)
was used for induction of AD in a dose of 17
mg/kg body weight orally as a single daily dose
through a gastric tube as described by Karam
et al. [12]. The choice of aluminum chloride was
based on the fact that aluminum (Al) has the
potential to be neurotoxic in humans and ani –
mals. It is present in many manufactured foods
and medicines and is also, added to drinking
water for purification purposes [19], one of the
ingredients of antacid drugs, in food additives
and tooth paste [20].
Aluminum chloride is considered to be an ideal
substance to be used for induction of AD model
as a result of being able to cross the blood
brain barrier as an L-glutamate complex and it
deposits in a rat’s brain [21]. It is documented
Figure 8. Correlations between the BDNF gene
expression, Ki-67 and GFAP immunoreactivity in
all studied groups. r: Pearson Correlation coef –
ficient; P: Probability.

Histological and molecular study of the protective effect of caffeine in AD
7720 Int J Clin Exp Pathol 2015;8(7):7710-7728to induce AD as it enhances neuroinflammatory
events in the brain by many proposed mecha –
nisms. It could exacerbate amyloid beta (Aβ)
deposition and plaque formation in the brain of
transgenic mice [22], in addition to oxidative
stress potentiated by both Aβ and aluminum
that will lead to genotoxicity and DNA damage
[23]. This oxidative damage may lead to the for –
mation of amyloid plaques and hyperphosphor –
ylated tau that polymerizes to form neurofibril –
lary tangles which are hallmarks of Alzheimer’s
[24].
In the current study, histological examination of
the hippocampus of rats in group III (AD model
group) revealed that the pyramidal layers in
CA1 and CA3 regions of the hippocampus
showed marked shrinkage in size of small and
large pyramidal cells respectively with some
cell loss. Choosing hippocampus for studying
the pathological findings of AD comes from the
fact that the hippocampus remains one of the
most vulnerable brain regions to AD, and its
degeneration may directly underlie memory
deficit, the earliest symptom of AD [25, 26].
These histological results are in agreement
with Padurariu et al. [27] and Nirmala et al.
[28]. They demonstrated in their study that the
cytoplasm of neurons was shrunken, the nuclei
were side-moved and dark-stained, neurofibril –
lary degeneration and neuron loss were
observed in hippocampus of rat received
ALCL3. Moreover, Yassin et al. [29] observed
that sections of rat brains receiving only AlCl3
(17 mg/kg) for 4 weeks showed brain necrosis,
spongy appearance, plaques with loss of nor –
mal structure, outlines, and nuclei of cells.
The pyramidal cells lost their triangular shape,
showed darkened nuclei and were surrounded
with pericellular haloes. Similar results were
reported by Abd El-Rahman [30]. He demon –
strated that Al administration causes the
appearance of neuritic plaques with a dark cen –
ter in the hippocampus and he stated that it is
typical for AD. It is confirmed by Aly et al. [31]
and explained it by presence of β-amyloid
plaques in the cerebral cortex and the hippo –
campus. Slight disorganization of the pyramidal
cell layer, little degeneration of pyramidal cells
and slight spongiosis were reported by a finding
of AD model studied by Abo El-Khair et al. [32].
As regard the granular layer it showed an appar –
ent increase in the number of the granule cells
with decreasing in their diameter. There was marked shrinkage in size of granule cells with
some cell loss and marked vacuolation. This
indicates a clear evidence of chronic inflamma –
tion and oxidative damage [33] and the same
result was confirmed later by Abo El-Khair et al.
[32] and Lynch [34]. Inflammatory changes are
features of AD. Several studies have reported
this observation and have shown that activated
cells cluster around Aβ-containing plaques
[35]. An increase in expression of inflammatory
cytokines, interleukin-1β (IL-1β), IL-6 and tumor
necrosis factor-α (TNF-α) has been detected in
brain of AD model animal and IL-1-positive
microglia present with Aβ-containing plaques, a
similar event occurs in activated astrocytes
[34].
During this work it was observed that the con –
trol animals showed small dark cells in the sub-
granular zone of the dentate gyrus. These cells
were previously described by Song et al. [36] as
neural stem cells that are present both in devel –
oping nervous system and in the adult nervous
system of all mammals, including human. The
number of these dark cells was apparently
decreased after exposure to aluminum chlo –
ride. This might be due to their differentiation
into granule cells, in order to compensate for
their loss, caused by exposure to aluminum
chloride. This might account for an apparent
increase in the number of granule cells com –
pared to the control groups. Our finding coin –
cides with finding of Nobakht et al. [37] as they
reported that there was reduction in neuronal
population in hippocampus of rat model with
AD. Serrano-Pozo et al. [38] mentioned that
neuronal loss is the main pathological sub –
strate of the cortex and hippocampus which is
evident in sections stained with hematoxylin
and eosin, it can be more readily shown with a
Nissl staining or a NeuN immunohistochemis –
try. It is reported that the neuronal loss is a
common pathway for a large number of degen –
erative processes in AD and can be triggered by
various factors, such as β amyloid plaques,
perturbed calcium regulation, glutamate, isch –
emia, inflammatory processes or oxidative
stress [27, 39]. Moreover, neuronal loss in the
hippocampus due to β-amyloid deposition
might induce glucose dysregulation leading to
hepatic insulin resistance which is one of the
mechanisms of cognitive dysfunction in AD
[40]. Molecular & polymorphic layers revealed
enlarged and excess astrocytes and widened
blood capillaries. This confirmed a result

Histological and molecular study of the protective effect of caffeine in AD
7721 Int J Clin Exp Pathol 2015;8(7):7710-7728detected previously by Hashem et al. [41] as
they found numerous astrocytes and microglial
cells in the dentate gyrus granule cell layer of
the hippocampus of their AD model. Numerous
dilated blood capillaries were also, observed by
Abo El-Khair et al. [32].
In the present study, immunohistochemical
staining for GFAP showed its normal distribu –
tion in the control group and group II as a mild
positive reaction in the immuno-reactive astro –
cytes and glial fibers. In AD model group, an
increase in the amount and intensity of GFAP
immuno-reactive astrocytes was detected as
the glial fibers appeared twisted with an irregu –
lar course and were thickened with increase in
their staining intensity. As GFAP is an intermedi –
ate cytoskeletal filament protein specific for
astrocytes [42], this astrocyte processes with
the pre- and postsynaptic elements envelope
neuronal synapses forming the tripartite syn –
apse, these astrocytes are actively involved in
modulating synaptic transmission [43]. As-
trocytes changed into reactive state in response
to damage of the central nervous system. This
transition is termed astrogliosis and character –
ized by an increase in the expression of their
main intermediate filament (IF), glial fibrillary
acidic protein (GFAP), by morphological altera –
tions (hypertrophy) and by functional changes
[44]. GFAP expression is highly associated with
amyloid plaque load and to a lesser extent, with
the number of neurofibrillary tangles [45]. In
human study, it is reported that GFAP expres –
sion significantly increased, reaching a 1.5- to
3.2-fold increase at end stage AD, this gradual
increase of GFAP transcription is parallel to the
progression of AD which is reflected by the
number of plaques, amyloid scores, and Braak
stages [46].
Exposure of astrocytes in culture to different
forms of Amyloid β (Aβ) resulted in GFAP upreg –
ulation and various other astroglial alterations
such as the production of inhibitory extracellu –
lar matrix proteins and a change of morphology
without compromising viability [47] these astro –
cytes are able to remove Aβ which may reduce
the amyloid deposition in the brain [48]. So, it
may be hypothesized that the increase in GFAP
in reactive astrocytes influences plaque Aβ
load [45]. It is reported that the number of
these GFAP expressing astrocytes significantly
increases during the progress of AD which is confirmed in the current study, suggesting that
elevated Aβ level in AD is an enhancing factor
[46]. So, GFAP is not only a marker for AD patho –
genesis, as it is found throughout the hippo –
campus as well as in reactive astrocytes, but it
is also considered as a defense mechanism
against AD progression. This could be explained
and proved by the important role of astrocytes
in the formation of the blood-brain barrier as it
confers a protective role against hypoxia and
aglycemia, the cleavage of cytoskeletal pro –
teins, such as GFAP and tau, may be one of
many factors that contribute to the compro –
mised blood-brain barrier observed in AD [49].
The present study showed that Ki-67 positive
nuclei were detected in the subgranular zone
(SGZ) of the dentate gyrus in the control group.
Ki-67 protein is a reliable biomarker present
during all active phases of the cell cycle and is
commonly used as an indicator to detect cellu –
lar proliferation [50, 51]. It is detected also, in
subgranular zone of the dentate gyrus in a
study of Yoo et al. [7] in agreement with our
study. The subgranular zone of the hippocam –
pal dentate gyrus constitutes one of the only
two neurogenic niches of the adult brain [52].
Adult neurogenesis has been observed in all
mammalian species including humans and
results in the formation of new neurons in the
olfactory bulb and the dentate gyrus of the hip –
pocampus. Radial glia-like (RGL) neural stem
cells (NSCs) that reside in the SGZ of the den –
tate gyrus, proliferate and give rise to transit-
amplifying progenitors (TAP) expressing the
T-box brain gene 2 (Tbr2) antigen which then
give rise to dou- blecortin (DCX)-expressing
immature neurons [53]. Neurogenesis is regu –
lated by different physiological effects espe –
cially exposure to environmental enrichment,
learning, aging and stress [36]. Several signal –
ing molecules and pathways have been
described to be essential for the maintenance,
self-renewal and proliferation of NSCs and
hence differentiation into mature functional
neurons [54].
In our study, the number of Ki-67 immunoreac –
tive nuclei was significantly decreased in group
III (AD model group) as compared to control
groups (group I&II) which indicates decreased
hippocampal neurogenesis in AD that coincides
with Ihunwo et al., [26]. This finding indicates
that neurogenesis is down regulated in AD

Histological and molecular study of the protective effect of caffeine in AD
7722 Int J Clin Exp Pathol 2015;8(7):7710-7728which is reported previously by Verret et al. [55]
in AD of aged rat. It has been suggested that
altered hippocampal neurogenesis (HN) might
be an integral part of AD progression [56]. This
is confirmed in animal and human study by
Gomez-Nicola et al. [57]. Also, Varela-Nallar et
al. [58] demonstrated the same result and
explained it by chronic hypoxia as a main mech –
anism of AD pathogenesis. The importance of
altered hippocampal neurogenesis (HN) was
summarized by Maruszak et al. [52]. They
reported that HN is an integral part of AD pro –
gression. A variety of important biomolecules
involved in AD pathogenesis as presenilin 1
(PS1), amyloid-β precursor protein (Aβ-PP) and
its metabolites, play either a positive or nega –
tive role in adult HN [59]. In addition, many
growth factors, such as brain derived neuro –
trophic factor (BDNF), vascular endothelial
growth factor, fibroblast growth factor, and cys –
tatin C, have been reported to be upregulated
in amyloid plaques. These factors are also
known to be potent modulators of neural stem
cell activity [52].
In the current study, brain derived neurotrophic
factor (BDNF) and its main functional receptor
(neural receptor protein-tyrosine kinase-β;
TrkB) genes expression showed significant
decrease in group III (AD model group) when
compared to control groups (I&II). The impor –
tance of studying BDNF came from its role in
development and maintenance of normal neu –
ronal circuits in the brain. It is reported that
appropriate intracellular processes including
transcription from BDNF gene, translation to
protein, BDNF protein sorting to secretory vesi –
cles, BDNF-containing vesicle transport, and
BDNF secretion are essential to achieve normal
BDNF functions as well as to activate the sig –
naling pathways after TrkB phosphorylation
[60].
BDNF acts as a neurite outgrowth and elonga –
tion factor, pro-survival factor, and synaptic
regulator in the CNS through multiple mecha –
nisms. Firstly, it promotes axon initiation
through two distinct signaling pathways; it
induces TrkB-dependent local elevation and
stabilization of cAMP/PKA activity that are
essential for axon initiation in undifferentiated
neurites of hippocampal neurons [61].
Secondly, BDNF/TrkB-induced Akt phosphory –
lation reduces GSK-3 activation which in turn
decreases production of the active form of col -lapsin response mediator protein-2 that plays a
critical role in microtubule assembly during
axon elongation and branching in rat hippo –
campal neurons [60, 62]. Thirdly, it enhances
the insulin sensitivity and plays significant roles
in the regulation of glucose metabolism, as
impaired insulin sensitivity and signaling path –
way is one of the characteristic features in AD
[63].
The reduction of BDNF and its receptors TrkB
genes expression in our work supports previ –
ous studies that demonstrated a decreased
expression levels of BDNF protein and mRNA
have been consistently reported in the hippo –
campus and cortex of individuals with AD [64-
66]. This reduction was explained by that Aβ
disturbs normal synaptic and cognitive function
[67] which is associated with loss of neuro –
trophins, as nerve growth factor (NGF),brain-
derived neurotrophic factor (BDNF), neuro –
trophin 3 (NT3) and NT4/5, in addition to sup –
pression of molecular transduction responsible
for learning and memory as mitogen-activated
protein kinases (MAPK) and cAMP response
element-binding protein (CREB) [68]. The
effects of reduced expression of BDNF or dis –
ruption of its signaling pathway were summa –
rized by Poon et al. [69]. They reported that
reduced BDNF signaling leads to defective hip –
pocampal and cortical synaptic plasticity and
they proved this by an experimental study that
concluded that mice lack either BDNF or TrkB
exhibited arborization impairment, defective
synaptic sprouting, lowered synapse number
and impaired hippocampal long term potentia –
tion (LTP). In contrast to the current study, there
was higher BDNF serum levels in preclinical
stages of Alzheimer’s disease as well as a sig –
nificant increase of BDNF concentration in hip –
pocampus and parietal cortex and of its recep –
tor TrkB in astrocytes and senile plaques as
reported by Baglio et al. [70]. They explained
this by that BDNF seems to have a protective
role for brain with normal morphology in early
preclinical stages of AD through its ability to
induce nitric oxide production by astrocytes
and promote cytokine production so, it is con –
sidered as a defensive molecule expressed
early trying to suppress disease progression
[70].
In the current work, studying the protective
effect of caffeine in group IV (caffeine adminis –
trated with AlCl3 for 4 successive weeks in a

Histological and molecular study of the protective effect of caffeine in AD
7723 Int J Clin Exp Pathol 2015;8(7):7710-7728dose of 1.5 mg/day) showed obvious improve –
ment of the histological picture, increase in the
number of Ki-67 positive nuclei, decrease in
the amount and intensity of GFAP immuno-
reactive astrocytes together with decreased
intensity of staining of glial fibers in addition to
the increase in BDNF/B-actin and TrkB/B-actin
genes expression when compared to the AD
model group (group III) but still lower than that
of the control groups. The histopathological
changes in this study are presented by preser –
vation of most pyramidal cells and markedly
decreased apoptosis of cells were observed in
CA1 and CA3 regions. Less vacuolations and
fewer apoptotic cells persisted in the granular
layer with persistence of enlarged astrocytes
and widened capillaries in many fields.
The caffeine dose in our study was in agree –
ment with many studies who used different
doses of caffeine in their study and concluded
that caffeine intake in a dose of 1.5 mg/day
may reverse cognitive impairment and decrease
brain Aβ levels in aged AD mice [5, 71] and the
response to treatment was in a dose depen –
dent manner. The mechanism by which caf –
feine could be a good protective agent used in
treatment of AD comes from the following
effects; caffeine and other adenosine receptor
antagonists could prevent the accumulation of
amyloid-β-peptide (Aβ) in and around cerebral
blood vessels [72]. Another possible mecha –
nism is through its stimulating effect of prosur –
vival cascades and inhibiting of pro-apoptotic
pathways in the different brain regions [73, 74].
Caffeine and other adenosine receptor antago –
nists could lead to down regulation of the
inflammatory response which is an obvious
hallmark of AD [75]. It also, can perform its pro –
tective effect through prevention of beta amy –
loid (Aβ)-induced synaptotoxicity and by induc –
tion of the production of interleukin-10 (IL-10)
[74].
It is reported also, that caffeine can reduce hip –
pocampal Aβ, presenilin 1 (PSEN1), and γ- and
β-secretase levels in AD transgenic mice, in
addition to increased plasma level of granulo –
cyte-colony stimulating factor (GCSF) and IL-6
[76] as well as TNF-α [77]. The anti-inflammato –
ry and antioxidant effects of caffeine could be
beneficial both for hippocampal neurogenesis
and for protection against AD [52]. It is reported
that caffeine is associated with reduced oxida –
tive stress as demonstrated by reduction in ROS production, glutathione depletion, isopros –
tane production, and markers of endoplasmic
reticulum stress levels this would reduce Aβ
accumulation [78]. Lastly, Caffeine has a role in
stabilization of blood-brain barrier (BBB) integ –
rity and has been implicated in modulating BBB
functions through blocking cellular surface
adenosine receptors, inhibition of cAMP phos –
phodiesterase (PDE) activity, and by affecting
calcium release from intracellular stores [79].
In agreement with the results of the current
study, it has been reported that chronic caf –
feine administration to mice from adulthood to
old age increases hippocampus levels of BDNF
and TrkB [80]. In addition, Sallaberry et al. [81]
Chen et al., [82] and Mioranzza et al., [83]
reported that caffeine modulates the balance
between cell survival and cell death which
depends upon the mature/proneurotrophin
ratio, as caffeine induced increase in proBDNF,
reducing neurotrophin signaling abnormalities
of TrkB receptor as well as relevant morphologi –
cal changes in hippocampus histology as it is
known that BDNF signaling through its receptor
TrkB can influence the morphology and synap –
tic connectivity of hippocampal neurons.
Moreover, the effects of caffeine on prolifera –
tion of neuronal precursors had been investi –
gated and it is reported that it increases hip –
pocampal neuronal proliferation [83, 84].
The early mid-life protective effect of caffeine
in the present study was studied in group V in
which rats received caffeine in a dose of 15
mg/Kg body weight/day for 2 successive
weeks, followed by a combination of caffeine
with AlCl3 for 4 successive weeks. The results
of the current study revealed marked improve –
ment of histological picture of hippocampus
which nearly returned to normal morphology,
weak GFAP immuno-reaction with GFAP posi –
tive areas (%) showed the lowest expression,
the number of Ki-67 positive nuclei was nearly
similar to the control group. BDNF/β-actin and
TrkB/β-actin genes expression became signifi –
cantly higher than that of group III and IV with
no significant difference when compared to
group I&II (control groups). These results are in
agreement with previous study that reported
that chronic caffeine consumption reverses
cognitive impairment and decreases brain Aβ
levels in AD mice [85]. Moreover, Eskelinen and
Kivipelto [86] stated that drinking of 3 -5 cups
of coffee per day at midlife is associated with

Histological and molecular study of the protective effect of caffeine in AD
7724 Int J Clin Exp Pathol 2015;8(7):7710-7728decreased risk of dementia/AD in later life
nearly by 65%, suggesting its protective role
against AD. The experimental findings of the
current study come in accordance with the
results of Sallaberry et al. [81] Han et al. [5]
Mioranzza et al. [83] Rivera-Oliver and Díaz-
Ríos M [74].
Recently, In 2015, It is reported that besides
the short-term effect of caffeine, epidemiologi –
cal and experimental studies indicate that caf –
feine, the main psychoactive component of cof –
fee and tea, when ingested chronically, has
protective effects against a number of acute
and chronic neurological diseases including
stroke, Parkinson’s disease, amyotrophic later –
al sclerosis, dementia, and AD through inhibi –
tion of Aβ production in brain of rodents [87].
Conclusion
It could be concluded from the current study,
that chronic caffeine consumption in a dose of
1.5 mg/kg body weight daily in early midlife has
a potentially good protective effect against AD
through modulation of the hippocampus tissue
gene expression of some important molecules;
BDNF and TrkB and modifying Ki-67 a well as
GFAP immunoreactivity. Moreover, it could have
neuroprotective effect on tissue morphology
including histological preservation of the hippo –
campus. Further research studies are needed
to prove and validate our results as well as to
study other possible pathophysiological mech –
anisms by which caffeine can perform its pro –
tective effects on hippocampus tissue against
AD and to study the potential therapeutic effect
of caffeine in AD.
Recommendations
The daily consumption of caffeine in a dose of
1.5 mg/kg body weight daily in early midlife has
a potentially good protective effect against the
possibility of AD with the advance of age, so it is
recommended for its habitual intake. Further
investigations are needed for finding more
mechanisms that explain; prove the protective
effect of caffeine and study of its potential ther –
apeutic effect.
Limitations of the study
This study has a limitation, It didn't evaluate the
potential therapeutic effect of caffeine aginst AD which would be performed in further future
study.
Disclosure of conflict of interest
None.
Address correspondence to: Dr. Ayman Z Elsa-
manoudy, Department of Medical Biochemistry and
Molecular Biology, Faculty of Medicine, Mansoura
University, Egypt. E-mail: ayman.elsamanoudy@
gmail.com
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