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Alzheimer’s disease (AD) is the most common cause of progressive decline of memory function in aged humans. To study about a disease mechanism and progression, animal models for the specific disease are needed. For AD, although highly valid animal models exist, none of the existing models recapitulates all aspects of human AD. The pathogenic mechanisms involved in AD are diverse and thus it is difficult to recapitulate human AD in model organisms. Intracerebroventricular (ICV) injection of okadaic acid (OKA), a protein phosphatase 2A (PP2A) inhibitor, in rats causes neurotoxicity associated with neurofibrillary degeneration. However, this model lacks amyloid pathology as observed in AD. We aimed at combining two different treatments and hence producing a better animal model of AD which may mimic most of the neuropathological, neurobehavioral, and neurochemical changes observed in AD. For this, OKA (200 ng) was microinjected bilaterally into the hippocampus of male Wistar rats followed by exposure of same rats to hypoxic conditions (10%) for 3 days. The result of which, the combination model exhibited tau hyperphosphorylation along with Aβ upregulation as evident by western blotting and immunohistochemistry. The observed changes were accompanied with dysfunction of neurotransmitter system, i.e., decreased acetylcholine activity and expression. This combinatorial model also exhibited cognitive deficiency which was assessed by Morris water maze and avoidance tests along with enhanced oxidative stress which is thought to be a major player in AD pathogenesis. Taken together, we established an easily reproducible and reliable rat model for sporadic dementia of Alzheimer’s type in rats which allows effective testing of new therapeutic strategies.
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Okadaic Acid and Hypoxia Induced Dementia Model of Alzheimers
Type in Rats
Alka Kaushal
&Willayat Yousuf Wani
&Amanjit Bal
&Kiran Dip Gill
&Jyotdeep Kaur
Received: 11 February 2018 /Revised: 19 January 2019 /Accepted: 24 January 2019
#Springer Science+Business Media, LLC, part of Springer Nature 2019
Alzheimers disease (AD) is the most common cause of progressive decline of memory function in aged humans. To study about a
disease mechanism and progression, animal models for the specific disease are needed. For AD, although highly valid animal models
exist, none of the existing models recapitulates all aspects of human AD. The pathogenic mechanisms involved in AD are diverse and
thus it is difficult to recapitulate human AD in model organisms. Intracerebroventricular (ICV) injection of okadaic acid (OKA), a
protein phosphatase 2A (PP2A) inhibitor, in rats causes neurotoxicity associated with neurofibrillary degeneration. However, this
model lacks amyloid pathology as observed in AD. We aimed at combining two different treatments and hence producing a better
animal model of AD which may mimic most of the neuropathological, neurobehavioral, and neurochemical changes observed in AD.
For this, OKA (200 ng) was microinjected bilaterally into the hippocampus of male Wistar rats followed by exposure of same rats to
hypoxic conditions (10%) for 3 days. The result of which, the combination model exhibited tau hyperphosphorylation along with Aβ
upregulation as evident by western blotting and immunohistochemistry. The observed changes were accompanied with dysfunction of
neurotransmitter system, i.e., decreased acetylcholine activity and expression. This combinatorial model also exhibited cognitive
deficiency which was assessed by Morris water maze and avoidance tests along with enhanced oxidative stress which is thought to
be a major player in AD pathogenesis. Taken together, we established an easily reproducible and reliable rat model for sporadic
dementia of Alzheimers type in rats which allows effective testing of new therapeutic strategies.
Keywords Alzheimer disease .Amyloid beta .Neurodegeneration .Stereotaxic surgery .Okadaic acid .Hypoxia
AβBeta amyloid
NFTs Neurofibrillary tangles
OKA Okadaic acid
ROS Reactiveoxygenspecies
PHF Paired helical filament
-DCFHDA 2,7-Dichlorodihydrofluorescein diacetate
SOD Superoxide dismutase
Alzheimers disease (AD) is a neurodegenerative disorder
which is associated with loss of memory and cognition.
Pathological features of AD include neurofibrillary tangles
(NFT) composed of hyperphosphorylated tau and amyloid
plaques consisting of beta amyloid 42 (Aβ) aggregates which
lead to neuronal death associated with cerebral atrophy and
gliosis. These pathological changes are most prominent in the
hippocampus, entorhinal cortex, and association areas of the
neocortex and are believed to be responsible for the clinical
features of AD (Pratico et al. 2000). Apart from these patho-
logical abnormalities in AD, neurotransmitter disorders do
Okadaic acid and hypoxia induce plaque and tangle pathology in this rat
model of AD
Combination of okadaic acid and hypoxia produced enhanced oxidative
stress and associated neurodegeneration in rat brain
Okadaic acid and hypoxia exposure to rats mimic the neurobehavioral
and neurochemical alterations as observed in AD.
*Jyotdeep Kaur
Department of Biochemistry, Postgraduate Institute of Medical
Education and Research, Chandigarh 160012, India
The Ken and Ruth Davee Department of Neurology, Northwestern
University Feinberg School of Medicine, 303 E. Chicago Ave, Ward
12-369, Chicago, IL 60611, USA
Department of Histopathology, Postgraduate Institute of Medical
Education and Research, Chandigarh 160012, India
Neurotoxicity Research
occur in AD patients. Impairment of neurotransmitter systems
in AD is due to degeneration of cholinergic neurons with the
ensuing cholinergic hypofunction which is seen by decreased
choline acetyl transferase (ChAT) and acetyl cholinesterase
(AChE) activity in the cerebral cortex and hippocampus
(Francis et al. 1999). Cognitive and neural dysfunction occurs
due to the oxidative damage to nucleic acid, protein, and mi-
tochondria in the brain (Anand et al. 2012). Memory loss in
AD is mimicked by severe depression (Squire 1992). This
places an emotional toll on the patients caretakers, while
placing a financial burden of approximately $214 billion in
2014 alone on the American society (Alzheimers
Association 2015; Alzheimers Association 2016). The
number of those affected is expected to reach 115 million
worldwide by 2050. Thus, there is an ever-growing need to
find biomarkers for the early diagnosis of AD as well as to
predict disease progression.
To understand AD disease progression, animal studies can
be useful. But, the known animal models for AD depict one or
two pathological conditions of AD, instead of showing a full
blown symptomatology. A model depicting most of the dis-
ease features would be more informative in AD research. In
the recent past, transgenic animals attracted attention to serve
as AD models. However, very few cases of AD are known to
be genetic and majority of AD cases are known to be sporadic
in nature. Although some of the sporadically developed
models successfully mimic AD disease phenotypes, very
few animal models for AD are known which can develop most
of the neuropathological, neurobehavioral, and neurochemical
symptoms of AD.
Among the environmental factors causing SAD, the dam-
aging effects of hypoxia on neurodegeneration have been con-
sidered. It is reported that hypoxia lead to enhanced expres-
sion of amyloid precursor protein (APP) which is a substrate
for Aβ, upregulates cleavage of APP by increasing β-site
amyloid precursor protein cleaving enzyme 1 (BACE1) gene
expression, reduces Aβdegradation by decreasing production
of neprilysin, and also brings calcium (Ca
) dysfunction
(Choi and Zelig 1994). Chronic intermittent hypoxia (CIH)
exposure to rats for 3 days resulted in significant increase in
the production of Aβpeptides (Snyder et al. 2017; Zhang and
Le 2010). Also hypoxia has been found to increase plaque
production along with memory deficit in AD transgenic
mouse model (Gao et al. 2013). Various such studies have
mentioned the role of hypoxia in accelerating Aβpathology
in AD which led us to design hypoxic model for studying AD.
NFTs, the other major hallmark of AD, are mainly com-
prised of aggregates of a hyperphosphorylated microtubule-
associated protein called tau (Braak and Braak 1996). An im-
balance between tau phosphorylation and dephosphorylation
is a critical event in AD (Zhang and Simpkins 2010). Okadaic
acid (OKA) is a potent and selective inhibitor of protein phos-
phatases, PP2A, and protein phosphatase 1 (PP1). Inhibition
of PP2A by OKA induces an Alzheimer-like
hyperphosphorylation and accumulation of tau both in vivo
and in vitro (Kamat et al. 2014; Zhang et al. 2008). Apart from
inducing tau hyperphosphorylation, OKA also increases oxi-
dative damage in various parts of the brain. Experimental
studies have shown memory impairment induced by OKA
administration in rats (Zhang et al. 2008).
Along with Aβaggregation and intracellular NFTs
accumulation, the brain of an individual with AD ex-
hibits profound loss of basal forebrain cholinergic neu-
rons that innervate the hippocampus and the neocortex
(Kar et al. 2004; Price and Sisodia 1998). Biochemical
investigations of tissues from biopsy and autopsy indi-
cate that neurotransmitters and modulators like acetyl-
choline (ACh), serotonin, noradrenaline, and somatostat-
in are differentially altered in the AD brains. The early
and most consistently reproduced finding is a profound
zyme, choline acetyl transferase (ChAT), in the neocor-
tex, which correlates positively with the severity of de-
mentia (Ladner and Lee 1998). The oxidation of mito-
chondria and nuclear DNA has been observed in the
parietal cortex of AD patients (Mecocci et al. 1994).
Many studies have shown increased lipid peroxidation
in the temporal lobe of AD patients, where histopatho-
logic alterations are very noticeable (Marcus et al. 1998;
Palmer and Burns 1994).
The present study was planned to develop a dementia
model of Alzheimers type in rats by combining two dif-
ferent treatments, viz. stereotaxic injection of OKA and
exposure to hypoxic environment. We examined the effect
of OKA injection (200 ng) and hypoxic exposure (10%
) on male Wistar rats. The model was validated by
examining the pathological, neurochemical, and neurobe-
havioral changes associated with AD along with oxidative
stress generation.
Materials and Methods
Animals and Their Care
The study protocol was approved by Institutional Animal
Ethical committee (IAEC 285 dated 27 July 2011). Male
albino Wistar rats of approximately 2 months of age were
used for the study (Maddahi and Edvinsson 2008;Richer
et al. 2009; Unkruer et al. 2009). Animals were housed
under standard conditions of light and dark cycle with
free access to rat pellet diet (Ashirvad Industries,
Chandigarh) and water ad libitum. Animals were acclima-
tized to laboratory conditions at room temperature prior to
experimentation. OKA was obtained from Sigma
Chemical Co., St. Louis, MO, USA (Cat no. 459620).
Neurotox Res
Intrahippocampal Infusion of OKA
During surgical procedures, the animals were anesthetized
with ketamine/xylazine (90 and 4 mg/kg, respectively, intra-
peritoneally) and restrained in a stereotaxic apparatus. The
head was positioned in a frame, and a midline sagittal incision
was made in the scalp. Two holes were drilled in the skull for
the placement of the injection cannula in both lateral cerebral
ventricles. Coordinates for the i.c.v. cannula implantation
were 0.8 mm posterior to bregma, 1.8 mm lateral to the sagittal
suture, and 3.6 mm beneath the cortical surface. Rats were
infused i.c.v. with 200 ng of OKA dissolved in artificial cere-
brospinal fluid (aCSF) (147 mmol/L NaCl, 2.9 mmol/L KCl,
1.6 mmol/L MgCl
, 1.7 mmol/L CaCl
, and 2.2 mmol/L dex-
trose). Solution (10 μL) was injected using a Hamilton micro-
syringe positioned in the injection cannula which was left in
place for 3 min to allow diffusion of the drug (Watson and
Paxinos 2007). The scalp was then closed with a suture.
Gentamicin (5 mg/kg) was injected intraperitoneally into the
animals in order to prevent sepsis.
Hypoxia Exposure
Rats were placed in a hypoxic chamber containing 10% oxy-
gen for 22 h/day for a period of 3 consecutive days. The
desired oxygen levels were established by a mixture of room
air and nitrogen, which was monitored by an oxygen analyzer
(Chavez et al. 2000; Pichiule and LaManna 2002).
Experimental Groups
The rats were divided into following five groups namely, con-
trol, sham, OKA, hypoxia, and OKA + hypoxia groups. Each
group consisted of different number of animals (Table 1).
Control Group (C)
In this group, animals received no treatment.
Sham Group (S)
In this group, animals received aCSF intracerebroventricularly
Okadaic Acid-Treated Group (OKA)
In this group, animals received OKA (200 ng/10 μL) dis-
solved in freshly prepared aCSF intracerebroventricularly
once with the help of stereotaxic apparatus.
Hypoxia-Treated Group (Hyp)
In this group, animals received hypoxia (10% oxygen)
for 3 days.
Okadaic Acid + Hypoxia Group (OKA + Hyp)
In this group, animals received both OKA (200 ng/10 μL) and
hypoxia (10% oxygen).
After the completion of treatment, animals were fasted
overnight and sacrificed by decapitation using sodium pento-
thal. The brains were removed and rinsed in ice-cold physio-
logical saline (0.9% NaCl). Brain regions were processed im-
mediately for the assay of parameters related to oxidative
stress. One separate set of animals were employed for behav-
ioral studies. One set of animals was used for biochemical
assays. One set of animals were used to study the gene and
protein expression and another set of animals were used to
study the histology and immunohistochemistry.
Assessment of Short-Term Memory by Shuttle
Avoidance Tests
Shuttle box (Techno, India) was used to assess the conditioned
avoidance learning tasks (Piala et al. 1959)withslightmodi-
fications (Sharma et al. 2009). In brief, a shuttle box apparatus
consisting of a dark and an illuminated chamber separated by
a door was used in the study. The rats were placed in the floor
of the dark chamber, then the illuminated compartment and
allowed to explore both chambers of the apparatus for 5 min.
Table 1 Antibodies used for western blotting
Type Reactivity (isotype) Host Dilution for WB Manufacturer
Primary/polyclonal Anti-beta amyloid 1-42 antibody (ab10148) Rabbit 1:1000 Abcam
Primary/polyclonal Anti-tau (phospho T205) antibody (ab4841) Rabbit 1:1000 Abcam
Primary/polyclonal Anti-PHF1 antibody (ab66275) Rabbit 1:1000 Abcam
Primary/polyclonal Anti-tau antibody (ab39524) Rabbit 1:1000 Abcam
Primary/polyclonal Anti-caspase 3 antibody (H-277) Mouse 1:500 Santa Cruz Biotechnology
Primary/monoclonal Anti-acetylcholinesterase (ZR3) antibody Mouse 1:500 Sigma
Neurotox Res
On the second day, each animal was placed into the illuminat-
ed chamber of the apparatus. As soon as the rat entered the
dark chamber, 0.1 mA, 40 V was applied till the animal
crossed the door and came back into the illuminated chamber.
After the shock, animal was removed and returned to their
home cage. On day 3, each animal was placed into the
illuminated chamber, and the latency to enter into the dark
chamber was measured, which served as measure of re-
tention of avoidance response.
Active Avoidance Test
It was performed in the same way as passive avoidance test,
with the change that in case of active avoidance test, no shock
was applied and rats are placed in dark chamber and their
latency to enter in light chamber was measured. The mean
value of latency time of each session is shown in the results.
Assessment of Learning and Spatial Memory
by Morris Water Maze Test
This test was carried out by the method given by Morris
(Morris 1984) in which the animal is trained to escape from
water by swimming to a hidden platform. The Morris water
maze (MWM) is a circular pool filled with opaque water. Rats
or mice are trained to use extra-maze visual cues to locate an
escape platform hidden just below the surface of the opaque
water. The hidden platform version of the MWM is a test of
spatial memory which is sensitive to hippocampal damage. In
navigation tank, the place of the platform is the same on each
day but the starting point of the animal varies. This method
requires long-term spatial memory and learning. In our labo-
ratory, a video tracking system (Any-Maze software, USA),
installed on a Dell PC computer connected to a video camera,
was used to collect behavioral data. This test was performed
for 6 days. The first 4 days were acquisition training with
an invisible platform. Day 5 was reversal training, again
with an invisible platform, and the number of entries and
time spent in the quadrant containing hidden platform
were recorded. On the sixth day, a probe trial was con-
ducted with no escape platform and then the time spent in
the target area was recorded.
During acquisition training, the water tank was filled with
water up to the level so that the platform was covered by
1 cm of water (invisible platform). The platform was placed
in the center of the northwest quadrant. Each animal received
four trials of 60 s (max) per day. Rats were placed in all the
four quadrants (e.g., west, north, east, or south) during trials
per day. Each rat was taken out from its holding cage and then
placed into the water tank at the appropriate start position,
with its nose facing the center of the pool. The rat was then
permitted to explore the pool and to search for the hidden
escape platform for 60 s. Once on the platform, the rats were
permitted to stay on the platform for 20 s to visually explore
their surroundings and afterwards picked up and returned to
their home cage. Each animal completed four trials per day
over 4 days, for 16trials of acquisition training. On 5th day the
number of entries and time spent in quadrant containing hid-
den platform were recorded. The results were analyzed with
the help of video tracking system and Any-Maze software.
Retrieval Test
Reversal training began on day 56. The platform was re-
moved from the water tank. Each animal was placed in the
tank from one of the four possible start positions and allowed
to explore the pool for 90 s. The time spent in the target area
(where the platform had been positioned on days 15) was
Brain Tissue Collection
The rats were sacrificed with ether anesthesia at the end of
behavioral studies on the 27th day. The brain was removed
quickly and kept on an ice-cold plate immediately. The hip-
pocampus and cortex were dissected as per guidelines
(Glowinski and Iversen 1966) and proceeded for biochemical
and molecular studies. For immunohistochemistry, rats were
perfused intracardially with chilled normal saline followed by
10% paraformaldehyde and brains were removed (Fig. 1).
Preparation of Mitochondria
Rat brain mitochondria were isolated using a method in which
the hippocampal brain sections were placed in glass dounce
homogenizer containing homogenizing medium, with
0.1 mM ethylene glycol tetraacetic acid (EGTA), 0.32 M of
sucrose, and 10 mM of Tris HCl and pH adjusted to 7.4 with
potassium hydroxide (KOH) (Partridge et al. 1994). The tissue
homogenate was spun at 2100×gfor 15 min in a
microcentrifuge at 4 °C and the supernatant was transferred
to new tubes. The resulting supernatant was again spun at
14,000×gfor 15 min. The supernatant was discarded and the
pellet containing mitochondria was washed with homogeniz-
ing medium by centrifuging at 14,000×g. The pellet thus col-
lected was pure mitochondria which was further resuspended
in 300 μL of homogenizing buffer, and incubated with prote-
ase inhibitor on ice to carry out various experiments.
Reactive Oxygen Species Generation
Mitochondrial reactive oxygen species (ROS) generation was
assessed as described previously (Wasilewski and Wojtczak
Neurotox Res
2005) with modifications. In brief, mitochondria were added
to respiration buffer containing 5 mM pyruvate, 2.5 mM ma-
late, and 10 μM of dichlorodihydrofluorescein diacetate
DCFDA). Fluorescence was quantified after 20 min incu-
bation using a Cary Eclipse fluorimeter (Varian, Palo Alto,
USA) (excitation 488 nm, emission 525 nm) and related to
total protein content.
Lipid Peroxidation
malondialdehyde (MDA) and measured by thiobarbituric
acid(TBA)methodasdescribedbyWills(1966). Brain
tissue from the hippocampus (0.5 mL) was diluted to
1.0 mL using Tris HCl buffer (0.1 M, pH 7.4). The meth-
od involves heating of hippocampal samples with TBA
reagent for 10 min in a boiling water bath. TBA reagent
contained mixture of 20% TCA, 0.5% TBA, and 2.5 N
HCl. After heating of these samples with TBA reagent,
the mixture then is cooled at room temperature. The col-
ored supernatant was used to read absorbance at 532 nm.
AChE Assay
AChE activity was assayed in brain homogenates (forebrain
and midbrain sections) by the method of Ellman et al. wherein
the hydrolysis of acetylthiocholine to thiocholine and acetate
is measured. The thiocholine reacts with 5,5-
dithiobisnitrobenzoic acid (DTNB) to give a mixed disulfide
and 5-mercapto-2-nitrobenzoic acid, a yellow compound
which was measured spectrophotometrically at 412 nm.
Histological Staining
For hematoxylin and eosin (H&E) staining and immunohis-
tochemistry, rats were transcardially perfused with 0.1 M
phosphate-buffered saline (PBS) at pH 7.4, followed by
10% buffered formalin. Then the fixed brains were embed-
ded in paraffin and then paraffin-embedded blocks were
made. For histological experiments, the sectioning (4
5μm) was carried out on freshly prepared poly-L-lysine-
coated clean slides. H&E staining was performed and ex-
amined by light microscopy.
For immunohistochemistry, first deparaffinization was
done followed by dipping of slides in freshly prepared
blocking solution of 1% H
in methanol for 20 min in order
to quench endogenous peroxidase. Then slides were washed
thrice in PBS for 5 min each. The slides were then incubated in
primary antibodies overnight at 4 °C. After washing sections
with PBS, slides were incubated with HRP-labeled secondary
antibody (1:100) for 30 min. Sections were again washed
three times with PBS and covered with 3,3-diaminobenzidine
(DAB) solution for 10 min at room temperature followed by
washing in distilled water. Finally, slides were visualized by
counterstaining with hematoxylin and visualized.
For IHC, evaluation of the sections was undertaken by
assessing the intensity of staining (four grades) as follows:
B0^indicates very low density of positive staining; B1^indi-
cates moderate density of positive staining; B2^indicates a
higher but submaximal density of positive staining; and B3^
indicates the highest density of positive staining.
Protein Isolation and Western Blot Analysis
Brain tissue was homogenized with lysis buffer and pelleted
down in cytoplasmic, nuclear, and mitochondrial fractions
with the help of differential centrifugation. The rat brain hip-
pocampal tissue (2 g) was lysed in 4 mL of buffer A (50 mM
of 1 M Tris (7.8), 0.01% NP-40, 150 mM of NaCl, 2 mM of
EDTA, 0.1% SDS, 1 mM PMSF, and protease inhibitor in
50 mL of water). Lysate was centrifuged at 3000 rpm for
5 min at 4 °C to pellet unlysed cells. The supernatant formed
the extracellular fraction and was used for detection of amy-
loid beta plaques. Insoluble pellet (nuclear) were treated with
TNT buffer (50 mM of Tris, 150 mM NaCl, 0.1% of Triton
X-100, 1 mM PMSF, and protease inhibitor cocktail in 50 mL
of water). The lysate was centrifuged at 13,000 rpm at 4 °C for
90 min to pellet mitochondria. The pellet formed the mito-
chondrial fraction and contained mitochondrial proteins. The
supernatant contained the cytoplasmic fraction. Supernatant
was then used for protein estimation by bicinchoninic acid
assay (BCA) method.
For western blot analysis, 80 μg of protein from each sam-
ple was resolved by electrophoresis on a 10% polyacrylamide
gel. The proteins on the gel were transferred on a nitrocellu-
lose membrane electrically. After incubating with 5% bovine
Fig. 1 Schematic representation
of experimental procedures and
treatment schedule
Neurotox Res
serum albumin (BSA) for 1 h, the membrane was probed with
the primary antibodies listed in Table 1. Blots were developed
using metal enhanced DAB substrate kit followed by quanti-
fication of blots using AlphaEase software, Version 6.0.0
(Alpha Innotech Corporation, USA).
Statistical Analysis
All values are expressed as mean ± SEM of six animals in
each group. One-way ANOVA, with Holm-Sidak test was
used for analysis of the data and values with p<0.001,
p<0.01, p<0.05 were considered statistically significant.
All the calculations were carried out by Sigma Stat computer
software program 3.5 (TE subsystems, Inc., Germany).
OKA and Hypoxia Treatment in Rats Induce AD Type
Neuropathological Changes
To validate our model, the first approach was to evaluate the
levels of abnormally aggregated, amyloid beta, and tau pro-
teins. Amyloid plaques are formed by aggregation of Aβ40 or
42, which being hydrophobic, self-assemble, and form oligo-
mers and polymers. Western blotting and immunohistochem-
istry were performed to check for the amyloid deposition and
phospho-tau (p-tau) accumulation using anti-Aβ42 antibody
and paired helical filament 1 (PHF-1) antibody, respectively.
The protein expression of Aβ42 in the hippocampus of rat
brain was determined in all the groups. As shown in Fig. 2a, b,
densitometry analysis showed that hypoxia treatment signifi-
cantly elevated amyloid protein levels by 127% (p<0.01) in
hippocampal region of hypoxia exposed rats as compared to
control. Similarly, combined treatment of OKA and hypoxia
in rats showed 112% (p< 0.01) increase in β-amyloid levels.
OKA caused a small but significant upregulation (17%) in Aβ
levels compared to control rats. Further to confirm the pres-
ence of amyloid deposition with OKA and hypoxia treatment,
it was imperative to look for the expression of amyloid beta
protein by immunohistochemistry. Immunohistochemistry for
amyloid plaques was done by using anti-Aβ42 antibody. The
area observed mainly was the hippocampus, followed by cor-
tex. After 15 days of OKA injection, amyloid positivity was
observed in OKA-treated rats in comparison to control rats.
However, hypoxia-treated rats showed more number of dis-
tinct amyloid plaques. Similar to hypoxic rats, in combined
group of OKA + hypoxia (OH), high immunoreactivity for
Aβwas observed. As seen in the Fig. 2c, the amyloid plaques
are denser and compact in hypoxia-treated rats as compared to
OKA alone and OH.
We performed western blot analysis of phosphorylated tau
(P-tau) as it isthe component of paired helical filaments which
subsequently form NFTs. Anti P-tau antibody was used to
check for phosphorylation on tau protein (Fig. 2d).
Densitometry of P-tau protein was doneby normalizing itwith
unphosphorylated tau protein (Fig. 2e). Densitometry analysis
showed that P-tau levels were increased significantly
(p< 0.01) in OKA-injected rats by 96%, in hypoxic rats by
44%, and in OH-treated animals by 107% as compared to
control (p< 0.001). The combined treatment of OH resulted
in 5 and 43% increase in P-tau levels as compared to OKA and
hypoxia, respectively.
In order to assess the effect of OKA and hypoxia treatment
on PHF expression, western blotting was performed using
PHF-1 antibody (Fig. 2f, g). A significant upregulation in
the expression of PHF in OKA-, hypoxia-, and OH-treated
rats was observed in comparison to control rats. The upregu-
lation observed was 84% in OKA-injected rats, 29% in Hyp-
exposed rats, and 111% in rats given both OKA and Hyp
treatment as compared to control animals (p<0.01).Oncom-
parison to OKA alone, PHF levels were further increased by
15% in OH-treated rats.
After confirmation of tau hyperphosphorylation with OKA
and hypoxia treatment, we further checked PHF formation
following OKA and hypoxia exposure by immunohistochem-
istry using anti-PHF-1 antibody (Fig. 2h).
Immunohistochemistry showed increased PHF expression in
OKA- and hypoxia-treated rats as compared to control ani-
mals. PHF-1 antibody intensely stained cytoplasm of hippo-
campal neurons in the treated groups, i.e., OKA, hypoxia, and
OH group as compared to control rats. Along with the hippo-
campus, PHF positivity was also observed in the cortex of rat
brains injected with OKA.
OKA and Hypoxia Treatment Affect Learning
and Memory of Rats
The clinical outcome of AD is memory impairment which we
checked in our designed animal model, using avoidance tasks
and MWM test. Having observed AD like pathological and
neurochemical alterations in our rat model, it was imperative
to assess memory functions to check whether OKA and hyp-
oxia treatment had any effect on memory.
The active avoidance response increased significantly
in terms of latency time in OKA (11 s), hypoxia (11 s),
and OH (10 s) treated rats in comparison to control ani-
mals (3 s). The latency period of the OKA, hypoxia, and
OH animals was about 76, 75, and 73% higher than con-
trols, respectively (p<0.01) (Fig. 3a). The time taken to
remember not to enter the chamber where rats experi-
enced a foot pad shock was significantly less in OKA
(70%), hypoxia (78%), and OH (78%) treated rats as com-
pared to control rats (p<0.01) (Fig. 3b).
This experiment conveyed that OKA and hypoxia treat-
ment induced short-term memory loss as rats were taking
Neurotox Res
less time in entering the dark chamber where they had
experienced foot shock earlier.
The results indicated that the time spent in the quadrant
containing platform in the water maze by OKA- and
Fig. 2 Hypoxia and OKA + hypoxia significantly increases Aβ42
protein levels in the rat brain. aWestern blot analysis of Aβ42
(87 kDa). bGraph represents densitometric analysis of Aβ42 protein
levels done by ImageJ software 1.48e NIH, USA. Data are expressed as
means ± SD of three separate experiments. **p< 0.01 vs. respected
control, ##p< 0.01 vs. respected okadaic acid. cRepresentative IHC
images (×100 and ×400) of the hippocampal region of rat brain. d
Western blot analysis of p-tau (62 kDa) normalized with total tau
(79 kDa) from the brains of control and treated rats. Data is plotted as
mean ± S.D. of six animals. eSignificant difference was found between
control, sham vs. OKA, hypoxia, and OKA + hypoxia rats. fhWestern
blotting and immunohistochemistry assays for PHF-1 (62 kDa) in the
brains of control and treated rats. Magnifications are ×100 and ×400. C,
control; S, sham; O, okadaic acid; H, hypoxia; OH, okadaic acid +
Neurotox Res
hypoxia-treated rats was comparatively less, when com-
pared to control. However, the overall speed of all the
treated and control groups remain almost same at 0.20 ±
0.005 m/s. The plot curve (Fig. 3c) shows the path follow-
ed by animals of different groups, which depicts that rats
from OKA, hypoxia, and OH groups are confused and are
Neurotox Res
not able to locate the hidden platform. Thus, the animals
from OKA, hypoxia, and OH groups demonstrated signif-
icant memory impairments as shown by reduced ability to
perform the task as compared to controls.
After 24 h of acquisition test, time spent, number of entries,
and total distance traveled in the target quadrant (without plat-
form) were decreased in case of OKA- and hypoxia-treated
rats as compared to control rats (Fig. 3d, e). On the final day,
the time spent, number of entries, and distance traveled in
target quadrant for OKA-treated rats were 10 ± 0.43 s, 4.3 ±
0.17, 1.9 ± 0.22m/s, respectively, and for hypoxia-treated rats,
it was 9.3 ± 0.12 s, 4.6 ± 0.34, 2.45 ± 0.29 m/s and for OH it
was 7.6 ± 0.41 s, 5 ± 0.22, 1.89 ± 0.23 m/s (p< 0.01) (Fig. 3g
j). The OKA and hypoxia treatments alone and in combination
resulted in decreased ability of rats in recognizing the area
where platform was placed as compared to control animals.
OKA and Hypoxia Treatment Resulted in Decreased
Activity and Expression of AChE in Rats
To detect cholinergic deficits occurring in the rat brain fol-
lowing OKA and hypoxia exposure, AChE activity was
assessed separately in forebrain and midbrain regions of
rats. The effect of OKA and hypoxia on AChE activity
In forebrain region, OKA, hypoxia, and OH induced signif-
icant decrease of 33% (p<0.05),52% (p< 0.01), and 56%
(p< 0.01) in AChE activity respectively as compared to
controls (Fig. 4a). However, in midbrain tissue, there was
no change in the AChE activity (Fig. 4b).
In the next experiment, AChE immunohistochemistry
was performed to confirm the above spectrophotometric
findings. We determined AChE expression in hippocam-
pus region by staining sections with antibody against
AChE. Immunohistochemistry showed weak (1+) AChE
positivity in neurons of rat brain in OKA- and hypoxia-
treated animals as compared to controls which showed
high (3+) cytoplasmic positivity (Fig. 4c).
OKA and Hypoxia Exposure Induces Oxidative Stress
in Rat Brain
For estimation of pro-oxidant status of cell, we estimated ROS
levels and lipid peroxidation in the control and treated rat
brains. As compared to controls, the mitochondria isolated
from the hippocampus of rat brains of OKA- and hypoxia-
treated rats showed significant increase (p< 0.01) in signal
fluorescence suggesting higher ROS accumulation in mito-
chondria of these treated rats. ROS accumulation was seen
to be increased by 48% in OKA, 50% in hypoxia, and 51%
in OKA + Hyp-treated groups as compared to control
(p< 0.01) (Fig. 5a).
We measured lipid peroxidation by measuring MDA accu-
mulation in brains of treated rats and found that MDA levels
were significantly increased in the hippocampal region of
brains of OKA-injected rats by 46%, in hypoxia-exposed an-
imals by 90%, and after OKA + Hyp exposure by 110%
(p<0.01, Fig. 5b) in comparison to control rat hippocampal
brain. Similar to lipid peroxidation levels, protein carbonyla-
tion levels were also found to be increased with both OKA and
hypoxia treatment (Fig. 5c).
OKA and Hypoxia Treatment Resulted
in Neurodegeneration/Neuronal Death in Rats
Western blotting was performed to detect protein levels of
caspase 3 and procaspase 3 by using antibody against
caspase 3 (Fig. 6a, b). We found that procaspase 3 levels
were decreased in OKA, Hyp, and OH groups.
Simultaneously, caspase 3 levels were significantly higher
in OKA, Hyp, and OH groups respectively as compared
to control, showing procaspase cleavage on OKA and
hypoxia and combination treatment.
We performed H&E staining to check the pathological
changes at the neuronal level. In order to distinguish between
degenerating neuron and dark neuron artifact, all rat brains
were perfused. Histological examination of the hippocampal
region of different treatment groups demonstrated marked
neuronal damage as depicted by eosinophilic neurons (swol-
len cytoplasm) and shrunken and pyknotic hyperchromatic
nuclei (darkened nuclei) in the OKA-, hypoxia-, and OH-
treated groups when compared to the control group (Fig. 6c).
OKA was used to induce tau pathology. OKA inhibits protein
phosphatases 1 and 2A and thus induces hyperphosphorylation
of tau protein (Broetto et al. 2016; Kamat et al. 2013b). OKA-
treated rats also show memory impairment, oxidative stress,
and mitochondrial dysfunction, thereby sharing some features
of AD (Kamat et al. 2014). Hyperphosphorylation of tau
Fig. 3 Impairment of spatial learning and memory in rats with OKA and
hypoxia treatment. aTime of memory retention in the passive avoidance
test carried out in rats at day 15 after OKA i.c.v. injection and hypoxia
treatment. bDifferent groups of animals treated with vehicle (i.c.v.), OKA
(i.c.v.), hypoxia, and OKA + hypoxia. The results are expressed as
medians of n= 6 animals. cPlot curves showing the path followed by
animals of different groups in the Morris water maze test performed over
6days.dDuring acquisition, learning was expressed as the time spent in
the quadrant containing platform. eOverall average speed was calculated
to check for any motor impairment. fSpatial memory wasassessed during
the probe test on day 6 by measuring the time spent into the target and the
opposite quadrants. gThe latency to first cross the platform zone. iThe
overall speed of animals. h,jThe number of entries and total distance
traveled in the platform zone (Values are mean±SEM, n= 6 animals per
Neurotox Res
protein was evident by western blot analysis of p-tau protein
and immunohistochemical analysis for PHF in the combinato-
rial model. Previous studies on rats have also shown that OKA
induces tauopathic features (Kamat et al. 2013a; Kamat et al.
2013b) in rats. Cognitive defects were noted in the model along
with NFTs-like conformational changes in both the cortex and
hippocampal regions (Kamat et al. 2013a; Kamat et al. 2013b).
In the present study, OKA treatment caused a significant
increase in protein expression of phosphorylated tau and PHF
levels in the cerebral cortex and hippocampus.
Amyloid plaques deposition occurs either due to an over-
production or decreased clearance of Aβpeptides. To induce
Aβaggregation, various studies have suggested i.c.v. injec-
tion of Aβpeptide in various forms to induce amyloid plaque
formation in rats (Lecanu et al. 2006;Nakamuraetal.2001).
Aβinjection does induce cognitive deficits in animals with
Fig. 4 AChE activity and expression was decreased in rat forebrain in
response to OKA injection and hypoxic environment. aAChE activity in
rat forebrain and midbrain separately using Ellmans spectrophotometric
method for control and treated rat groups. There was no significant
change in AChE activity in midbrain tissue for all groups. b
Immunohistochemistry images showing AChE expression in the
hippocampal region of all rat groups. Magnification ×100, ×400. In the
OKA- and hypoxia-treated animals, hippocampal cells exhibited diffuse
and weak staining for AChE. Results are from three independent
experiments (n=6)
Neurotox Res
amyloid plaque formation (Gordon et al. 2009). However, the
pathology develops primarily at the site of injection; frequent-
ly, amyloid deposits were observed in the hippocampus and
neocortex but not cerebellum and brainstem (Forny-Germano
et al. 2014). Stroke is a well-known risk factor for AD (Honig
et al. 2003) and one of the pathological mechanisms is neuro-
nal hypoxia (Ogunshola and Antoniou 2009). Hence, hypoxia
was chosen since amyloid generation by this pathway could
closely mimic the pathophysiology of SAD. An earlier study
where 3-day chronic intermittent hypoxia was given to rats
resulted in increased beta amyloid generation (Ng et al.
2010). We adopted this strategy in our study to target
amyloidogenesis. Rats were kept in hypoxic conditions, fol-
lowing which they were sacrificed and brain tissue was exam-
ined for the presence of amyloid plaques. Three-day acute
exposure of 10% oxygen in rats resulted in upregulation of
amyloid beta levels as shown by western blotting and amyloid
plaque formation as evident by immunohistochemistry in the
present study.
xApart from amyloid plaques and NFTs, other neuropath-
ological alterations observed in AD are neuronal and synaptic
loss (Serrano-Pozo et al. 2011). Neuronal loss is the causative
factor behind clinical manifestation of AD (Cotman and Su
1996). In the present study, as depicted by H&E staining,
OKA- and hypoxia-treated sections showed significant degen-
eration of neurons. With OKA and hypoxia treatment, CA1
neurons appeared intensely hypereosinophilic with extensive
clumping of chromatin, particularly in the combined group
of OKA and hypoxia compared to the OKA and hypoxia
treatments alone. The final outcome of such pathological
and neurochemical changes along with enhanced oxida-
tive stress in rat brain is neuronal and synapse loss which
leads to memory impairment in rats. As depicted by H&E
staining, OKA- and hypoxia-treated sections showed
marked cell distortion with high level of degeneration in
the neurons as compared to control.
The result of OKA and hypoxia treatment thus is neurode-
generation in the subregions of the hippocampus (CA1, CA3,
and DH). However, the extent of degeneration was almost
similar for OKA and hypoxia treatments alone and in combi-
nation. Thus, it can be inferred that combined treatment of
OKA and hypoxia resulted in pathological alterations of am-
yloid plaques, NFTs, and neuronal degeneration. Neuronal
and hence synaptic loss results in cognitive dysfunction in
AD. It has also been suggested that in some cases, behavioral
changes may be more sensitive than neurochemical alter-
ations, as indicators of neurodegeneration and may be ob-
served early during disease progression.
Fig. 5 Oxidative stress parameters of rat brains. ROS levels, lipid
peroxidation, and protein carbonylation were evaluated after OKA and
hypoxia treatment. aROS levels in normal and treated rat brains; 200 ng
of OKA and 3 days of hypoxia increased ROS production when
compared to control rats; bMDA levels were significantly increased
with both OKA and hypoxia treatments compared to either of
treatments alone. cThe effects of OKA and hypoxia exposure on
protein oxidation were assessed from protein carbonyl contents in brain
homogenates. OKA and hypoxia treatment sharply increased carbonyl
contents in brain (p< 0.01). With combined treatment of OKA and
hypoxia, carbonyl content increased even further (p< 0.01 vs. OKA
and hypoxia alone treatment), indicating that combined treatment
inflicted appreciable oxidative damage on brain proteins. **p<0.01vs.
control group, ##p< 0.01 vs. respected okadaic acid, $$p<0.01 vs.
respected hypoxia. Data are shown as means ±SE (n=6)
Neurotox Res
To check, if significant neuronal shrinkage and degenerated
nuclei in the OKA and hypoxia rats lead to memory impair-
ment, we checked memory deficits in terms of short-term and
spatial memory. OKA- and hypoxia-treated rats showed de-
crease in the number of avoidances in the learning session and
retention which has been reported by others also (Dimitrova
and Getova-Spassova 2006; Dwivedi et al. 2013). MWM has
been performed to evaluate learning and memory deficits in
AD animals (Bromley-Brits et al. 2011). In the current study,
the effect of OKA on memory impairment was tested by the
avoidance and Morris water maze. The memory deficit in
OKA-treated rats was evident by significant changes in the
latency time and path length to reach the hidden platform.
Deficits in cholinergic neurotransmission are not the only
Fig. 6 OKA and hypoxia induces apoptosis in rat brain. aRepresentative
detection of caspase 3 protein by western blot. Procaspase 3 (35 kDa)
cleavage was increased in OKA- and hypoxia-treated rats compared to
control and sham rats. Western blotting was used to determine the protein
level of procaspase 3 and cleaved caspase 3 (17 kDa) in all groups. bWe
examined caspase 3 protein by western blotting and conducted
densitometry on the blots. We found that OKA and hypoxia treatment,
alone and in combination, significantly activated caspase 3. Results
shown are from six rats in each group. cRepresentative hematoxylin
and eosin images from the hippocampus of rat brain. Low
magnification, 100 μM; high magnification, 200 μM
Neurotox Res
cause of memory loss but this deficit clearly impairs memory
in AD. So we studied cholinergic neurotransmission in our
AD model by evaluating AChE activity and expression in
rat brain. Animal models of AD have been established based
on experimental cholinergic denervation (Muir et al. 1993).
We observed significant decrease in AChE activity in rat fore-
brain after OKA and hypoxia exposure. This finding is in
conformity with previous study showing OKA induced cho-
linergic dysfunction by decreasing AChE activity and mRNA
expression (Dwivedi et al. 2013). OKA also resulted in gen-
eration of oxidative stress which reduces cerebral blood flow
(CBF) in the brain. Decreased CBF impairs glucose and en-
ergy metabolism which lead to increased cellular dysfunction
(Olanow 1993). Disturbed glucose and energy metabolism
impairs cholinergic system, thus resulting in decreased level
of choline acetyl transferase and acetyl cholinesterase (Areosa
and Sherriff 2003). The role of hypoxia in decreasing AChE
activity has also been previously studied (Udayabanu et al.
2008). The cholinergic system is involved in many physiolog-
ical processes, including synaptic plasticity, learning, and
memory. Cholinergic agonists can facilitate memory, whereas
cholinergic antagonists can impair memory. Thus, OKA and
hypoxia affect AChE and ChAT enzyme activity and expres-
sion and interfere with cholinergic neurotransmission, affect-
ing learning, memory, and cognition functions.
Hence, we validated our induced model of AD on neuro-
pathological, neurobehavioral, and neurochemical parameters
and inferred that OKA and hypoxia treatment in the rat brain
resembles the changes observed in AD patients.
In AD, brain is under increased oxidative stress
which may have a role in neuronal degeneration and
death. Therefore, we looked for the presence of oxida-
tive markers viz. reactive oxygen species and lipid per-
oxidation levels. As compared to control rats, increased
mitochondrial ROS levels in rat brain after OKA and
hypoxia treatment were observed when given alone
and in combination. Several biological markers of oxi-
dative stress were found to be elevated in AD animal
models in different studies (Melov 2002). MPTP-treated
mice demonstrate elevated levels of ROS and lipid per-
oxidation (Sriram et al. 1997). Evidence for the involve-
ment of oxidative stress in AD has also been obtained
from AD patients. Increased lipid peroxidation and ox-
idative damage to DNA and proteins have been ob-
served in hippocampus of AD patients (Reist et al.
1998). The ROS molecules generated in AD might
cause neuronal damage by reacting with nucleic acids,
proteins, and lipids. Our data indicated increased ROS
levels and lipid peroxidation in brains of OKA- and
hypoxia-treated rats. With OKA and hypoxia treatment,
MDA levels were found to be significantly upregulated.
Increased ROS generation predisposes rats to enhanced
oxidative stress and consequently neuronal death.
To summarize, the present study proposes the use of combined
treatment of okadaic acid and hypoxia for developing a de-
mentia model of Alzheimers type in rats. The dementia model
described displays neurobehavioral impairments along with
pathological and neuorchemical changes as observed in AD.
Thus, a picture emerges that depicts a similar pathological
mechanisms between the experimental rat model and clinical
conditions. This novel rat model reliably simulates the main
pathological outcomes during Alzheimer disease. It represents
a promising and relevant preclinical model for testing new
therapeutic approaches.
Funding This study was funded by Council of Scientific and Industrial
Research (CSIR).
Compliance with Ethical Standards
Conflict of Interest No conflicts of interest are declared by the authors.
PublishersNoteSpringer Nature remains neutral with regard to jurisdic-
tional claims in published maps and institutional affiliations.
Alzheimers Association (2015) 2015 Alzheimers disease facts and fig-
ures. Alzheimers Dement 11:332384
Alzheimers Association (2016) 2016 Alzheimers disease facts and fig-
ures. Alzheimers Dement 12:459509
Anand R, Kaushal A, Wani WY, Gill KD (2012) Road to Alzheimers
disease: the pathomechanism underlying. Pathobiology 79:5571
Areosa SA, Sherriff F (2003) Memantine for dementia Cochrane
Database Syst Rev:CD003154
Braak H, Braak E (1996) Evolution of the neuropathology of Alzheimers
disease. Acta Neurol Scand Suppl 165:312
Broetto N, Hansen F, Brolese G, Batassini C, Lirio F, Galland F, dos
Santos JPA, Dutra MF, Gonçalves CA (2016)
Intracerebroventricular administration of okadaic acid induces hip-
pocampal glucose uptake dysfunction and tau phosphorylation.
Brain Res Bull 124:136143
Bromley-Brits K, Deng Y, Song W (2011) Morris water maze test for
learning and memory deficits in Alzheimers disease model mice. J
Vis Exp 20:2920
Chavez JC, Agani F, Pichiule P, LaManna JC (2000) Expression of
hypoxia-inducible factor-1alpha in the brain of rats during chronic
hypoxia. J Appl Physiol (1985) 89:19371942
Choi PM, Zelig MP (1994) Similarity of colorectal cancer in Crohns
disease and ulcerative colitis: implications for carcinogenesis and
prevention. Gut 35:950954
Cotman CW, Su JH (1996) Mechanisms of neuronal death in Alzheimers
disease. Brain Pathol 6:493506
Dimitrova DS, Getova-Spassova DP (2006) Effects of galantamine and
donepezil on active and passive avoidance tests in rats with induced
hypoxia. J Pharmacol Sci 101:199204
Dwivedi S, Nagarajan R, Hanif K, Siddiqui HH, Nath C, Shukla R (2013)
Standardized extract of Bacopa monniera attenuates okadaic acid
induced memory dysfunction in rats: effect on Nrf2 pathway. Evid
Based Complement Alternat Med 2013:294501
Neurotox Res
Forny-Germano L, Lyra e Silva NM, Batista AF, Brito-Moreira J, Gralle
M, Boehnke SE, Coe BC, Lablans A, Marques SA, Martinez AMB,
Klein WL, Houzel JC, Ferreira ST, Munoz DP, de Felice FG (2014)
Alzheimers disease-like pathology induced by amyloid-beta oligo-
mers in nonhuman primates. J Neurosci 34:1362913643
Francis PT, Palmer AM, Snape M, Wilcock GK (1999) The cholinergic
hypothesis of Alzheimers disease: a review of progress. J Neurol
Neurosurg Psychiatry 66:137147
Gao L, Tian S, Gao H, Xu Y (2013) Hypoxia increases Abeta-induced tau
phosphorylation by calpain and promotes behavioral consequences
in AD transgenic mice. J Mol Neurosci 51:138147
Glowinski J, Iversen L (1966) Regional studies of catecholamines in the
rat brain. 3. Subcellullar distribution of endogenous and exogenous
catecholamines in various brain regions. Biochem Pharmacol 15:
Gordon RY, Mugantseva EA, Khutzian SS, Podolski IY (2009)
Cycloheximide-induced inhibition of protein synthesis in hippocam-
pal pyramidal neurons is time-dependent: differences between CA1
and CA3 areas. Neurosci Lett 461:249251
Honig LS, Tang MX, Albert S, Costa R, Luchsinger J, Manly J, Stern Y,
Mayeux R (2003) Stroke and the risk of Alzheimer disease. Arch
Neurol 60:17071712
Kamat PK, Rai S, Nath C (2013a) Okadaic acid induced neurotoxicity: an
emerging tool to study Alzheimer's disease pathology.
Neurotoxicology 37:163172
Kamat PK, Rai S, Swarnkar S, Shukla R, Ali S, Najmi AK, Nath C
(2013b) Okadaic acid-induced Tau phosphorylation in rat brain: role
of NMDA receptor. Neuroscience 238:97113
Kamat PK, Rai S, Swarnkar S, Shukla R, Nath C (2014) Molecular and
cellular mechanism of okadaic acid (OKA)-induced neurotoxicity: a
novel tool for Alzheimers disease therapeutic application. Mol
Neurobiol 50:852865
Kar S, Slowikowski SPM, Westaway D, Mount HTJ (2004) Interactions
between β-amyloid and central cholinergic neurons: implications
for Alzheimers disease. J Psychiatry Neurosci 29:427441
Ladner CJ, Lee JM (1998) Pharmacological drug treatment of Alzheimer
disease: the cholinergic hypothesis revisited. J Neuropathol Exp
Neurol 57:719731
Lecanu L, Greeson J, Papadopoulos V (2006) Beta-amyloid and oxida-
tive stress jointly induce neuronal death, amyloid deposits, gliosis,
and memory impairment in the rat brain. Pharmacology 76:1933
Maddahi A, Edvinsson L (2008) Enhanced expressions of microvascular
smooth muscle receptors after focal cerebral ischemia occur via the
MAPK MEK/ERK pathway. BMC Neurosci 9:85
Marcus DL, Thomas C, Rodriguez C, Simberkoff K, Tsai JS, Strafaci JA,
Freedman ML (1998) Increased peroxidation and reduced antioxi-
dant enzyme activity in Alzheimers disease. Exp Neurol 150:4044
Mecocci P, MacGarvey U, Beal MF (1994) Oxidative damage to mito-
chondrial DNA is increased in Alzheimers disease. Ann Neurol 36:
Melov S (2002) Animal models of oxidative stress, aging, and therapeutic
antioxidant interventions. Int J Biochem Cell Biol 34:13951400
Morris R (1984) Developments of a water-maze procedure for studying
spatial learning in the rat. J Neurosci Methods 11:4760
Muir JL, Page KJ, Sirinathsinghji DJ, Robbins TW, Everitt BJ (1993)
Excitotoxic lesions of basal forebrain cholinergic neurons: effects on
learning, memory and attention. Behav Brain Res 57:123131
Nakamura S, Murayama N, Noshita T, Annoura H, Ohno T (2001)
Progressive brain dysfunction following intracerebroventricular in-
fusion of beta(1-42)-amyloid peptide. Brain Res 912:128136
Ng KM, Lau CF, Fung ML (2010) Melatonin reduces hippocampal beta-
amyloid generation in rats exposed to chronic intermittent hypoxia.
Brain Res 1354:163171
Ogunshola OO, Antoniou X (2009) Contribution of hypoxia to
Alzheimers disease: is HIF-1alpha a mediator of neurodegenera-
tion? Cell Mol Life Sci 66:35553563
Olanow CW (1993) A radical hypothesis for neurodegeneration. Trends
Neurosci 16:439444
Palmer AM, Burns MA (1994) Selective increase in lipid peroxidation in the
inferior temporal cortex in Alzheimers disease. Brain Res 645:338342
Partridge RS, Monroe SM, Parks JK, Johnson K, Parker WD Jr, Eaton
GR, Eaton SS (1994) Spin trapping of azidyl and hydroxyl radicals
in azide-inhibited rat brain submitochondrial particles. Arch
Biochem Biophys 310:210217
Piala JJ, High JP, Hassert GL Jr, Burke JC, Craver BN (1959)
Pharmacological and acute toxicological comparisons of
triflupromazine and chlorpromazine. J Pharmacol Exp Ther 127:5565
Pichiule P, LaManna JC (2002) Angiopoietin-2 and rat brain capillary
remodeling during adaptation and deadaptation to prolonged mild
hypoxia. J Appl Physiol (1985) 93:11311139
Pratico D, Clark CM, Lee VM, Trojanowski JQ, Rokach J, FitzGerald
GA (2000) Increased 8,12-iso-iPF2alpha-VI in Alzheimers disease:
correlation of a noninvasive index of lipid peroxidation with disease
severity. Ann Neurol 48:809812
Price DL, Sisodia SS (1998) Mutant genes in familial Alzheimersdisease
and transgenic models. Annu Rev Neurosci 21:479505
Reist M, Marshall KA, Jenner P, Halliwell B (1998) Toxic effects of
sulphite in combination with peroxynitrite on neuronal cells. J
Neurochem 71:24312438
Richer MJ, Lavallee DJ, Shanina I, Horwitz MS (2009) Toll-like receptor
3 signaling on macrophages is required for survival following
coxsackievirus B4 infection. PLoS One 4:e4127
Serrano-Pozo A, Frosch MP, Masliah E, Hyman BT (2011)
Neuropathological alterations in Alzheimer disease. Cold Spring
Harb Perspect Med 1:a006189
Sharma DR, Sunkaria A, Bal A, Bhutia YD, Vijayaraghavan R, Flora SJ,
Gill KD (2009) Neurobehavioral impairments, generation of oxida-
tive stress and release of pro-apoptotic factors after chronic exposure
to sulphur mustard in mouse brain. Toxicol Appl Pharmacol 240:
Snyder B, Shell B, Cunningham JT, Cunningham RL (2017) Chronic
intermittent hypoxia induces oxidative stress and inflammation in
brain regions associated with early-stage neurodegeneration.
Physiol Rep 5(9):e13258
Squire LR (1992) Memory and the hippocampus: a synthesis from find-
ings with rats, monkeys, and humans. Psychol Rev 99:195231
Sriram K, Pai KS, Boyd MR, Ravindranath V (1997) Evidence for gen-
eration of oxidative stress in brain by MPTP: in vitro and in vivo
studies in mice. Brain Res 749:4452
Udayabanu M, Kumaran D, Nair RU, Srinivas P, Bhagat N, Aneja R,
Katyal A (2008) Nitric oxide associated with iNOS expression in-
hibits acetylcholinesterase activity and induces memory impairment
during acute hypobaric hypoxia. Brain Res 1230:138149
Unkruer B et al (2009) Cellular localization of Y-box binding protein 1 in
brain tissue of rats, macaques, and humans. BMC Neurosci 10:28
Wasilewski M, Wojtczak L (2005) Effects of N-acylethanolamines on the
respiratory chain and production of reactive oxygen species in heart
mitochondria. FEBS Lett 579:47244728
Watson C, Paxinos G (2007) The rat brain in stereotaxic coordinates.
Academic Press, San Diego
Wills ED (1966) Mechanisms of lipid peroxide formation in animal tis-
sues. Biochem J 99:667676
Zhang X, Le W (2010) Pathological role of hypoxia in Alzheimersd
ease. Exp Neurol 223:299303
Zhang Z, Simpkins JW (2010) Okadaic acid induces tau phosphorylation
in SH-SY5Y cells in an estrogen-preventable manner. Brain Res
Zhang QG, Wang R, Khan M, Mahesh V, Brann DW (2008) Role of
Dickkopf-1, an antagonist of the Wnt/beta-catenin signaling path-
way, in estrogen-induced neuroprotection and attenuation of tau
phosphorylation. J Neurosci 28:84308441
Neurotox Res
... OA is a potent polyether marine toxin, which is responsible for diarrhetic shellfish poisoning around the world [33]. A growing body of evidence has supported that OA brings about neurotoxicity in AD, which may provide a novel tool to study AD [34][35][36]. In this study, OA was applied to establish PC12 cell model of AD and the experimental results revealed that the proliferation of PC12 cells was observably hampered in response to OA. ...
Full-text available
Alzheimer's disease (AD) is a neurodegenerative disorder with prominent individual morbidity and mortality among elderly people. Germacrone (Germ) has been reported to exert dominant protective roles in multiple human diseases, and neurological diseases are also included. The intention of this paper is to determine the impacts of Germ on okadaic acid (OA)-treated PC12 cells and confirm the hidden regulatory mechanism. First, PC12 cells were induced by OA in the absence or presence of Germ. Cell counting kit-8 assay was to monitor cell proliferation. Western blot was to test the protein levels of cholinergic muscarinic M1 receptor (CHRM1), Galphaq (Gq), phospholipase C beta (PLCβ) and protein kinase C (PKC). The levels of reactive oxygen species (ROS) and other oxidative stress markers were evaluated using corresponding kits. ELISA was used to estimate the levels of AD markers. RT-qPCR was used to examine the mRNA levels of beta-site amyloid-precursor-protein-cleaving enzyme 1 (BACE-1) and apolipoprotein E (APOE). The results uncovered that Germ enhanced the proliferation of OA-insulted PC12 cells, elevated the protein level of CHRM1 and activated the Gq/PLCβ/PKC signaling. Moreover, after OA-induced PC12 cells were administered with Germ, insufficiency of CHRM1 impeded cell proliferation, enhanced oxidative stress and neuron injury and inactivated the Gq/PLCβ/PKC signaling. Furthermore, the addition of Gq inhibitor UBO-QIC, PLCβ inhibitor U73122 or PKC inhibitor Go6983 reversed the enhanced proliferation, the reduced oxidative stress and neuron injury in OA-treated PC12 cells caused by Germ. Collectively, Germ modulated M1 muscarinic receptor-mediated Gq/PLCβ/PKC signaling, thereby alleviating OA-induced PC12 cell injury.
... In rat models of AD that present impairments in learning and memory, a loss of and damage to cholinergic neurons are observed (Klinkenberg and Blokland, 2010;Haider et al., 2015;Zhang et al., 2019). The degeneration of cholinergic neurons in AD promotes cholinergic hypofunction, which can result in a decrease in choline acetyltransferase and AChE activity in the hippocampus (Francis et al., 1999;Kaushal et al., 2019). Our data showed that Aβ promoted hippocampal tissue disorganization, vacuole formation, and neuronal loss, in addition to promoting a decrease in AChE activity. ...
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Alzheimer’s disease (AD) is the leading cause of dementia in the world, accounting for 50–75% of cases. Currently, there is limited treatment for AD. The current pharmacological therapy minimizes symptom progression but does not reverse brain damage. Studies focused on nonpharmacological treatment for AD have been developed to act on brain plasticity and minimize the neurotoxicity caused by the amyloid-beta (Aβ) peptide. Using a neurotoxicity model induced by Aβ in rats, the present study shows that physical (PE) and cognitive exercise (CE) reverse recognition memory deficits (with a prominent effect of long-term object recognition memory), decrease hippocampal lipid peroxidation, restore the acetylcholinesterase activity altered by Aβ neurotoxicity, and seems to reverse, at least partially, hippocampal tissue disorganization.
... In our previous research we have shown that PP2A expression and activity have been elevated in PACAP KO mice [7]. The activity and expression of this Ser/Thr phosphatase are reduced in AD [64], and its inhibition augmented Aβ accumulation in the CNS [65]. Although PP2A activity has been reduced in direct mechanical load [66], no data can be found about its systemic function during elevated physical activity. ...
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Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with protective functions in the central nervous system and various peripheral organs. PACAP has the highest expression level in the testes, among the peripheral organs, and has a positive regulative role in spermatogenesis and in sperm motility. In the present study, we explored testicular degenerative alterations in a mouse model of Alzheimer’s disease (AD) (B6C3-Tg(APPswe,PSEN1dE9)85Dbo/J) and demonstrated changes in PACAP-regulated signaling pathways. In addition, the effects of increased physical activity of AD (trained AD (TAD)) mice on testis were also followed. Reduced cell number and decreased thickness of basement membrane were detected in AD samples. These changes were compensated by physical activity. Expression of PACAP receptors and canonical signaling elements such as PKA, P-PKA, PP2A significantly decreased in AD mice, and altered Sox transcription factor expression was also detected. Via this signaling mechanism, physical activity compensated the negative effects of AD on the expression of type IV collagen. Our findings suggest that the testes of AD mice can be a good model of testis degeneration. Moreover, it can be an appropriate organ to follow the effects of various interventions such as physical activity on tissue regeneration and signaling alterations.
... OA, a PP2A inhibitor, triggers tau hyperphosphorylation. The literature reports that OA significantly decreased the expression of PP2A in mice, rats, zebrafish and some cell lines in vitro [33][34][35]. AD-like pathological changes, such as neuronal apoptosis, Ca 2+ overload, brain energy metabolic abnormalities, and decreases in brain-derived neurotrophic factors, have been found in OA models [32,36,37]. ICV-OA for approximately 4 weeks induced cognitive impairments in mice. ...
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Tolfenamic acid, a nonsteroidal anti-inflammatory drug, alleviated learning and memory deficits and decreased the expression of specificity protein 1 (SP1)-mediated cyclin-dependent kinase-5 (CDK5), a major protein kinase that regulates hyperphosphorylated tau, in Alzheimer's disease (AD) transgenic mice. However, whether tolfenamic acid can regulate the major tau protein kinase, glycogen synthase kinase-3β (GSK-3β), or tau protein phosphatase, protein phosphatase 2A (PP2A), further inhibiting hyperphosphorylation of tau, remains unknown. To this end, tolfenamic acid was administered i.p. in a GSK-3β overactivation postnatal rat model and orally in mice after intracerebroventricular (ICV) injection of okadaic acid (OA) to develop a PP2A inhibition model. We used four behavioural experiments to evaluate memory function in ICV-OA mice. In this study, tolfenamic acid attenuated memory dysfunction. Tolfenamic acid decreased the expression of hyperphosphorylated tau in the brain by inhibiting GSK-3β activity, decreasing phosphorylated PP2A (Tyr307), and enhancing PP2A activity. Tolfenamic acid also increased wortmannin (WT) and GF-109203X (GFX) induced phosphorylation of GSK-3β (Ser9) and prevented OA-induced downregulation of PP2A activity in PC12 cells. Altogether, these results show that tolfenamic acid not only decreased SP1/CDK5-mediated tau phosphorylation, but also inhibited GSK-3β and PP2A-mediated tau hyperphosphorylation in AD models.
Alzheimer's disease (AD) is an age-related neurodegenerative disease characterized by tau aggregating into neurofibrillary tangles. Targeting tau aggregation is one of the most critical strategies for AD treatment and prevention. Herein, a high-throughput screening of tau-aggregation inhibitors was performed by thioflavin T (ThT) fluorescence assay and tauR3 peptides. According to bioactivity-guided isolation, homoprejadomycin (1) was obtained from the marine bacterium Streptomyces tendae MCCC 1A01534. Two new stable derivatives, 2 and 3, were yielded in a one-step reaction. By ThT assay, transmission electron microscopy, and circular dichroism, we demonstrated that the angucyclinones 2 and 3 inhibited tau aggregation and disaggregated tau fibrils. In the presence of 2, native tauR3 peptides maintained the disorder conformation, whereas the tauR3 aggregates reduced β-sheet structures. And compound 2 was confirmed to inhibit the aggregation of full-length 2N4R tau protein. Furthermore, 2 with low cytotoxicity protected HT22 cells from okadaic acid-induced damage by suppressing tau aggregates. These results indicated that 2 was a promising lead structure with tau therapeutic potency for AD treatment.
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Alzheimer's disease (AD) is a progressive, fatal, neurodegenerative disorder for which only treatments of limited efficacy are available. Despite early mentions of dementia in the ancient literature and the first patient diagnosed in 1906, the underlying causes of AD are not well understood. This study examined the possible role of dopamine, a neurotransmitter that is involved in cognitive and motor function, in AD. We treated adult zebrafish (Danio rerio) with okadaic acid (OKA) to model AD and assessed the resulting behavioral and neurochemical changes. We then employed a latent learning paradigm to assess cognitive and motor function followed by neurochemical analysis with fast-scan cyclic voltammetry (FSCV) at carbon fiber microelectrodes to measure the electrically stimulated dopamine release. The behavioral assay showed that OKA treatment caused fish to have lower motivation to reach the goal chamber, resulting in impeded learning and decreased locomotor activity compared to controls. Our voltammetric measurements revealed that the peak dopamine overflow in OKA-treated fish was about one-third of that measured in controls. These findings highlight the profound neurochemical changes that may occur in AD. Furthermore, they demonstrate that applying the latent learning paradigm and FSCV to zebrafish is a promising tool for future neurochemical studies and may be useful for screening drugs for the treatment of AD.
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Background: The etiology of dementia may partly be underpinned by impaired lung function via systemic inflammation and hypoxia. Objective: To prospectively examine the association between chronic obstructive pulmonary disease (COPD) and subclinical impairments in lung function and the risk of dementia. Methods: In the Rotterdam Study, we assessed the risk of incident dementia in participants with Preserved Ratio Impaired Spirometry (PRISm; FEV1/FVC≥0.7, FEV1 < 80%) and in participants with COPD (FEV1/FVC < 0.7) compared to those with normal spirometry (controls; FEV1/FVC≥0.7, FEV1≥80%). Hazard ratios (HRs) with 95%confidence intervals (CI) for dementia were adjusted for age, sex, education attainment, smoking status, systolic blood pressure, body mass index, triglycerides, comorbidities and Apolipoprotein E (APOE) genotype. Results: Of 4,765 participants, 110 (2.3%) developed dementia after 3.3 years. Compared to controls, participants with PRISm, but not COPD, had an increased risk for all-type dementia (adjusted HRPRISm 2.70; 95%CI, 1.53-4.75; adjusted HRCOPD 1.03; 95%CI, 0.61-1.74). These findings were primarily driven by men and smokers. Similarly, participants with FVC%predicted values in the lowest quartile compared to those in the highest quartile were at increased risk of all-type dementia (adjusted HR 2.28; 95%CI, 1.31-3.98), as well as Alzheimer's disease (AD; adjusted HR 2.13; 95%CI, 1.13-4.02). Conclusion: Participants with PRISm or a low FVC%predicted lung function were at increased risk of dementia, compared to those with normal spirometry or a higher FVC%predicted, respectively. Further research is needed to elucidate whether this association is causal and how PRISm might contribute to dementia pathogenesis.
Alzheimer's (AD) and Parkinson's (PD) diseases are the two most prevalent age-related neurodegenerative diseases and currently no effective clinical treatments exist for either, despite decades of clinical trials. The failure to translate preclinical findings into effective treatments is indicative of a problem in the current evaluation pipeline for potential therapeutics. At present, there are no useful animal models for AD and PD research that reflect the entire biology of the diseases, specifically, the more common non-Mendelian forms. While the field continues to seek for suitable rodent models for investigating potential therapeutics for these diseases, rodent models have still been used primarily for preclinical studies. Here, we advocate for a paradigm shift towards the application of human induced pluripotent stem cells (hiPSC)-derived systems for PD and AD modelling and the development of improved human-based models in a dish for drug discovery and preclinical assessment of therapeutic targets.
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Sleep apnea is a common comorbidity of neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD). Previous studies have shown an association between elevated oxidative stress and inflammation with severe sleep apnea. Elevated oxidative stress and inflammation are also hallmarks of neurodegenerative diseases. We show increased oxidative stress and inflammation in a manner consistent with early stages of neurodegenerative disease in an animal model of mild sleep apnea. Male rats were exposed to 7 days chronic intermittent hypoxia (CIH) for 8 h/day during the light period. Following CIH, plasma was collected and tested for circulating oxidative stress and inflammatory markers associated with proinflammatory M1 or anti‐inflammatory M2 profiles. Tissue punches from brain regions associated with different stages of neurodegenerative diseases (early stage: substantia nigra and entorhinal cortex; intermediate: hippocampus; late stage: rostral ventrolateral medulla and solitary tract nucleus) were also assayed for inflammatory markers. A subset of the samples was examined for 8‐hydroxydeoxyguanosine (8‐OHdG) expression, a marker of oxidative stress‐induced DNA damage. Our results showed increased circulating oxidative stress and inflammation. Furthermore, brain regions associated with early‐stage (but not late‐stage) AD and PD expressed oxidative stress and inflammatory profiles consistent with reported observations in preclinical neurodegenerative disease populations. These results suggest mild CIH induces key features that are characteristic of early‐stage neurodegenerative diseases and may be an effective model to investigate mechanisms contributing to oxidative stress and inflammation in those brain regions.
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Alzheimer's disease (AD) is a devastating neurodegenerative disorder and a major medical problem. Here, we have investigated the impact of amyloid-␤ (A␤) oligomers, AD-related neurotoxins, in the brains of rats and adult nonhuman primates (cynomolgus ma-caques). Soluble A␤ oligomers are known to accumulate in the brains of AD patients and correlate with disease-associated cognitive dysfunction. When injected into the lateral ventricle of rats and macaques, A␤ oligomers diffused into the brain and accumulated in several regions associated with memory and cognitive functions. Cardinal features of AD pathology, including synapse loss, tau hyper-phosphorylation, astrocyte and microglial activation, were observed in regions of the macaque brain where A␤ oligomers were abun-dantly detected. Most importantly, oligomer injections induced AD-type neurofibrillary tangle formation in the macaque brain. These outcomes were specifically associated with A␤ oligomers, as fibrillar amyloid deposits were not detected in oligomer-injected brains. Human and macaque brains share significant similarities in terms of overall architecture and functional networks. Thus, generation of a macaque model of AD that links A␤ oligomers to tau and synaptic pathology has the potential to greatly advance our understanding of mechanisms centrally implicated in AD pathogenesis. Furthermore, development of disease-modifying therapeutics for AD has been hampered by the difficulty in translating therapies that work in rodents to humans. This new approach may be a highly relevant nonhuman primate model for testing therapeutic interventions for AD.
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Okadaic acid (OKA), a polyether C38 fatty acid toxin extracted from a black sponge Hallichondria okadaii, is a potent and selective inhibitor of protein phosphatase, PP1 and PP2A. OKA has been proved to be a powerful probe for studying the various regulatory mechanisms and neurotoxicity. Because of its property to inhibit phosphatase activity, OKA is associated with protein phosphorylation; it is implicated in hyperphosphorylation of tau and in later stages causes Alzhiemer's disease (AD)-like pathology. AD is a progressive neurodegenerative disorder, pathologically characterized by extracellular amyloid beta (Aβ) plaques and intracellular neurofibrillary tangles (NFTs). The density of tau tangles in AD pathology is associated with cognitive dysfunction. Recent studies have highlighted the importance of serine/threonine protein phosphatases in many processes including apoptosis and neurotoxicity. Although OKA causes neurotoxicity by various pathways, the exact mechanism is still not clear. The activation of major kinases, such as Ser/Thr, MAPK, ERK, PKA, JNK, PKC, CaMKII, Calpain, and GSK3β, in neurons is associated with AD pathology. These kinases, associated with abnormal hyperphosphorylation of tau, suggest that the cascade of these kinases could exclusively be involved in the pathogenesis of AD. The activity of serine/threonine protein phosphatases needs extensive study as these enzymes are potential targets for novel therapeutics with applications in many diseases including cancer, inflammatory diseases, and neurodegeneration. There is a need to pay ample attention on MAPK kinase pathways in AD, and OKA can be a better tool to study cellular and molecular mechanism for AD pathology. This review elucidates the regulatory mechanism of PP2A and MAPK kinase and their possible mechanisms involved in OKA-induced apoptosis, neurotoxicity, and AD-like pathology.
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The aim of the present study is to investigate the effect of standardized extract of Bacopa monnieri (memory enhancer) and Melatonin (an antioxidant) on nuclear factor erythroid 2 related factor 2 (Nrf2) pathway in Okadaic acid induced memory impaired rats. OKA (200 ng) was administered intracerebroventricularly (ICV) to induce memory impairment in rats. Bacopa monnieri (BM-40 and 80 mg/kg) and Melatonin (20 mg/kg) were administered 1 hr before OKA injection and continued daily up to day 13. Memory functions were assessed by Morris water maze test on days 13-15. Rats were sacrificed for biochemical estimations of oxidative stress, neuroinflammation, apoptosis, and molecular studies of Nrf2, HO1, and GCLC expressions in cerebral cortex and hippocampus brain regions. OKA caused a significant memory deficit with oxidative stress, neuroinflammation, and neuronal loss which was concomitant with attenuated expression of Nrf2, HO1, and GCLC. Treatment with BM and Melatonin significantly improved memory dysfunction in OKA rats as shown by decreased latency time and path length. The treatments also restored Nrf2, HO1, and GCLC expressions and decreased oxidative stress, neuroinflammation, and neuronal loss. Thus strengthening the endogenous defense through Nrf2 modulation plays a key role in the protective effect of BM and Melatonin in OKA induced memory impairment in rats.
This report discusses the public health impact of Alzheimer’s disease (AD), including incidence and prevalence, mortality rates, costs of care and the overall effect on caregivers and society. It also examines the challenges encountered by health care providers when disclosing an AD diagnosis to patients and caregivers. An estimated 5.3 million Americans have AD; 5.1 million are age 65 years, and approximately 200,000 are age <65 years and have younger onset AD. By mid-century, the number of people living with AD in the United States is projected to grow by nearly 10 million, fueled in large part by the aging baby boom generation. Today, someone in the country develops AD every 67 seconds. By 2050, one new case of AD is expected to develop every 33 seconds, resulting in nearly 1 million new cases per year, and the estimated prevalence is expected to range from 11 million to 16 million. In 2013, official death certificates recorded 84,767 deaths from AD, making AD the sixth leading cause of death in the United States and the fifth leading cause of death in Americans age 65 years. Between 2000 and 2013, deaths resulting from heart disease, stroke and prostate cancer decreased 14%, 23% and 11%, respectively, whereas deaths from AD increased 71%. The actual number of deaths to which AD contributes (or deaths with AD) is likely much larger than the number of deaths from AD recorded on death certificates. In 2015, an estimated 700,000 Americans age 65 years will die with AD, and many of them will die from complications caused by AD. In 2014, more than 15 million family members and other unpaid caregivers provided an estimated 17.9 billion hours of care to people with AD and other dementias, a contribution valued at more than $217 billion. Average per-person Medicare payments for services to beneficiaries age 65 years with AD and other dementias are more than two and a half times as great as payments for all beneficiaries without these conditions, and Medicaid payments are 19 times as great. Total payments in 2015 for health care, long-term care and hospice services for people age 65 years with dementia are expected to be $226 billion. Among people with a diagnosis of AD or another dementia, fewer than half report having been told of the diagnosis by their health care provider. Though the benefits of a prompt, clear and accurate disclosure of an AD diagnosis are recognized by the medical profession, improvements to the disclosure process are needed. These improvements may require stronger support systems for health care providers and their patients.
This report describes the public health impact of Alzheimer's disease, including incidence and prevalence, mortality rates, costs of care, and the overall impact on caregivers and society. It also examines in detail the financial impact of Alzheimer's on families, including annual costs to families and the difficult decisions families must often make to pay those costs. An estimated 5.4 million Americans have Alzheimer's disease. By mid-century, the number of people living with Alzheimer's disease in the United States is projected to grow to 13.8 million, fueled in large part by the aging baby boom generation. Today, someone in the country develops Alzheimer's disease every 66 seconds. By 2050, one new case of Alzheimer's is expected to develop every 33 seconds, resulting in nearly 1 million new cases per year. In 2013, official death certificates recorded 84,767 deaths from Alzheimer's disease, making it the sixth leading cause of death in the United States and the fifth leading cause of death in Americans age ≥65 years. Between 2000 and 2013, deaths resulting from stroke, heart disease, and prostate cancer decreased 23%, 14%, and 11%, respectively, whereas deaths from Alzheimer's disease increased 71%. The actual number of deaths to which Alzheimer's disease contributes is likely much larger than the number of deaths from Alzheimer's disease recorded on death certificates. In 2016, an estimated 700,000 Americans age ≥65 years will die with Alzheimer's disease, and many of them will die because of the complications caused by Alzheimer's disease. In 2015, more than 15 million family members and other unpaid caregivers provided an estimated 18.1 billion hours of care to people with Alzheimer's and other dementias, a contribution valued at more than $221 billion. Average per-person Medicare payments for services to beneficiaries age ≥65 years with Alzheimer's disease and other dementias are more than two and a half times as great as payments for all beneficiaries without these conditions, and Medicaid payments are 19 times as great. Total payments in 2016 for health care, long-term care and hospice services for people age ≥65 years with dementia are estimated to be $236 billion. The costs of Alzheimer's care may place a substantial financial burden on families, who often have to take money out of their retirement savings, cut back on buying food, and reduce their own trips to the doctor. In addition, many family members incorrectly believe that Medicare pays for nursing home care and other types of long-term care. Such findings highlight the need for solutions to prevent dementia-related costs from jeopardizing the health and financial security of the families of people with Alzheimer's and other dementias. © 2016 The Alzheimer's Association. Published by Elsevier Inc. All rights reserved.
Intraneuronal aggregates of neurofibrillary tangles (NFTs), together with beta-amyloid plaques and astrogliosis, are histological markers of Alzheimer's disease (AD). The underlying mechanism of sporadic AD remains poorly understood, but abnormal hyperphosphorylation of tau protein is suggested to have a role in NFTs genesis, which leads to neuronal dysfunction and death. Okadaic acid (OKA), a strong inhibitor of protein phosphatase 2A, has been used to induce dementia similar to AD in rats. We herein investigated the effect of intracerebroventricular (ICV) infusion of OKA (100 and 200ng) on hippocampal tau phosphorylation at Ser396, which is considered an important fibrillogenic tau protein site, and on glucose uptake, which is reduced early in AD. ICV infusion of OKA (at 200ng) induced a spatial cognitive deficit, hippocampal astrogliosis (based on GFAP increment) and increase in tau phosphorylation at site 396 in this model. Moreover, we observed a decreased glucose uptake in the hippocampal slices of OKA-treated rats. In vitro exposure of hippocampal slices to OKA altered tau phosphorylation at site 396, without any associated change in glucose uptake activity. Taken together, these findings further our understanding of OKA neurotoxicity, in vivo and vitro, particularly with regard to the role of tau phosphorylation, and reinforce the importance of the OKA dementia model for studying the neurochemical alterations that may occur in AD, such as NFTs and glucose hypometabolism.
Reports an error in the original article by L. R. Squire (Psychological Review, 1992[Apr], Vol 99[2], 195–231). The caption for Figure 7 was incorrect. The corrected caption is given. (The following abstract of this article originally appeared in record 1992-26428-001.) Considers the role of the hippocampus in memory function. A central thesis involving work with rats, monkeys, and humans (which has sometimes seemed to proceed independently in 3 separate literatures) is now largely in agreement about the function of the hippocampus and related structures. A biological perspective is presented that proposes multiple memory systems with different functions and distinct anatomical organizations. The hippocampus (together with anatomically related structures) is essential for a specific kind of memory, here termed declarative memory (similar terms include explicit and relational). Declarative memory is contrasted with a heterogeneous collection of nondeclarative (implicit) memory abilities that do not require the hippocampus (skills and habits, simple conditioning, and the phenomenon of priming). The hippocampus is needed temporarily to bind together distributed sites in the neocortex that together represent a whole memory. (PsycINFO Database Record (c) 2012 APA, all rights reserved)