Biomedicine & Pharmacotherapy 110 (2019) 47–58

Contents lists available at ScienceDirect

Biomedicine & Pharmacotherapy

journal homepage: www.elsevier.com/locate/biopha

Neuroprotective role of astaxanthin in hippocampal insulin resistance T

induced by Aβ peptides in animal model of Alzheimer’s disease
Syed Obaidur Rahmana, Bibhu Prasad Pandab, Suhel Parvezc, Madhu Kaundald, Salman Hussaina,
⁎
Mohd. Akhtard, Abul Kalam Najmid,
a
Pharmaceutical Medicine, Department of Pharmacology, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi, 110062, India
b
Pharmaceutical Biotechnology Laboratory, Department of Pharmacognosy & Phtyochemistry, School of Pharmaceutical Education and Research, Jamia Hamdard, New
Delhi, 110062, India
c
Department of Toxicology, School of Chemical and Life Sciences, Jamia Hamdard, New Delhi, 110062, India
d
Department of Pharmacology, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi, 110062, India

A R T I C LE I N FO A B S T R A C T

Keywords: With the constant failure of the clinical trials continuous exploration of a therapeutic target against Alzheimer’s
Alzheimer’s disease disease (AD) is the utmost need. Numerous studies have supported the hypothesis that central insulin resistance
Astaxanthin plays a significant role in AD. Serine phosphorylation of Insulin Receptor Substarte-1 (IRS-1) has been found to
Neuronal insulin resistance be a contributing factor in neuronal insulin resistance. Astaxanthin (ASX) is xanthophyll carotenoid which has
Type-3 diabetes
previously demonstrated significant antidiabetic and neuroprotective actions. In the present study, AD was in-
Aβ (1-42) peptides
Insulin receptor substarte-1
duced by i.c.v administration of Amyloid-β (1–42) peptides in Wistar rats. After 7 days of recovery, rats were
treated with 0.5 mg/kg and 1 mg/kg of ASX orally for 28 days. Behavioral analysis was done in the last week of
our experimental study. On the 36th day, rats were sacrificed and their hippocampus were separated from the
whole brain, then homogenized and stored for biochemical estimations. ASX significantly and dose-dependently
reversed the cognitive and memory impairment, assessed by Morris water maze test and Novel object
Recognition test, Aβ (1–42) peptides infused Wistar rats. ASX also significantly attenuated soluble Aβ (1–42)
level, IRS-S307 activity, GSK-3β activity, TNF-α level, AChE level, nitrite level and oxidative stress in the hip-
pocampus. Histopathological evaluation, done through H&E and Congo red staining, also demonstrated neu-
roprotective and anti-amyloidogenic effects of ASX in hippocampus. Our study concludes preventive action of
Astaxanthin against hippocampal insulin resistance and Alzheimer’s disease complications, supporting potential
role of hippocampal insulin resistance targeting against AD.

1. Introduction production of unstable molecules called free radicals, and mitochon-

Global estimated cost for treating 44 million people suffering from
Alzheimer’s disease or a related dementia is approx. $605 billion. This
amount is equivalent to 1% of the entire world’s gross domestic product
[1]. Alzheimer’s disease (AD) is the most common type of dementia that
results from age-associated, progressive and chronic neurodegenera-
tion. Causes of AD are not yet fully understood but advances in brain
imaging have allowed researchers to see the development and spread of
abnormal amyloid and tau proteins in the cerebral cortex including
hippocampus (part of temporal lobe of the brain responsible for pro-
cessing of memory), as well as shrinkage in brain structure and change
in its function [2]. These affects worsens with age and also includes
atrophy (shrinking) of certain parts of the brain [3], inflammation [4],
drial dysfunction [5].
Numerous in vivo studies have demonstrated that chronic hyper-
insulinemia negatively modulates memory, impairs blood-brain barrier
(BBB) function, insulin receptor activity and reduces insulin transport
to the brain. Interestingly, neuronal insulin has been correlated with
cognitive ability since more insulin receptors are found in the areas of
the brain that regulate cognition [6]. Serine phosphorylation of Insulin
Receptor Substrate (IRS-1) was found to be a key feature in neuronal
insulin resistance. The high density of IRS-1 serine phosphorylation in
neurons has been identified during an autopsy in the brain of AD-af-
fected patients [7]. When insulin binds to its receptor, it instigates in-
tracellular signaling cascades thereby activating Insulin Receptor Sub-
strate-1 and 2 (IRS1 & 2), PI3K/AKT pathway and extracellular signal-

⁎
Corresponding author.
E-mail address: aknajmi@jamiahamdard.ac.in (A.K. Najmi).

https://doi.org/10.1016/j.biopha.2018.11.043
Received 27 June 2018; Received in revised form 6 November 2018; Accepted 10 November 2018
0753-3322/ © 2018 Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
S.O. Rahman et al.
related kinase/mitogen-activated protein kinase (ERK/MAPK). Upre-
gulation of these pathways inhibits glycogen synthase kinase-3 (GSK-3)
activation and leads to consolidation of memory and synaptic plasticity
[8].
The overall increase in Aβ in hippocampus disrupts Insulin Receptor
signaling either by binding to insulin receptor or by over-activation of
JNK [9]. Studies have reported that insulin resistance mediated AKT
proteins downregulation activates GSK-3β. GSK-3β over-activation po-
tentiates tau hyperphosphorylation at serine residues leading to the
formation of neurofibrillary tangles. GSK-3β is also activated by in-
creased Aβ oligomers [10]. The AKT down-regulation due to insulin
resistance also inhibits GLUT-4 translocation causing hindrance in
glucose absorption. This interference with ATP production in mi-
tochondria thereby promoting the release of reactive oxygen species
(ROS) followed by increased oxidative stress [11]. On the other hand,
pro-inflammatory cytokine TNF-α was also indicated for IRS-1 inhibi-
tion in the insulin signaling pathway through stimulation of its p55
receptor (Peraldi et al., 1996) and activation of JNK1 and IKKβ [12].
Astaxanthin (3, 3′-dihydroxy-β, β′-carotene-4, 4′-dione) is a xan-
thophyll carotenoid. It is found in the aquatic animals and the marine
world of algae as a dark-red pigment. It is also present in different forms
of seafood, including trout, shrimp, red sea bream, salmon, and lobster,
and also in birds such as quail and flamingo [13]. ASX is has been
approved as a food colorant by both the United States Food and Drug
Administration (USFDA) [14] and the European Commission [15]. The
commercial astaxanthin is extracted mainly from Phaffia rhodozyma
(yeast), and the same was used in this study [16,17]. Astaxanthin ex-
hibits potential pharmacological activity, like immunomodulation, an-
tioxidants, anti-inflammatory, anti-cancer and more importantly anti-
diabetic actions [18]. Neuroprotective effects of ASX have been verified
by previous in vitro [19], in vivo [20,21] and clinical studies [22]. The
use of astaxanthin as a nutritional supplement has been rapidly growing
in foods, feeds, nutraceuticals and pharmaceuticals [18].
This is the first study to demonstrate the activity of ASX on AD
complications mediated via neuronal insulin resistance. Astaxanthin
had shown convincing effects against peripheral insulin resistance
[23,24] and thus holds a promising approach towards preventing cen-
tral insulin resistance and also Alzheimer’s disease-related complica-
tions (Fig. 1).
2. Materials and methods
2.1. Drugs and chemicals
Astaxanthin was extracted and graciously provided by B.P. panda
and team [17]. Amyloid beta (1–42) peptides (Abcam, India), Aβ
(1–42) ELISA kit (GenxBio, India), IRS-s307 ELISA kit (GenxBio, India),
GSK-3β ELISA kit (GenxBio, India), TNF- α ELISA kit (Krishgen Bio-
systems, India) were procured for the study. All chemicals and reagents
used in this study were of AR grade only.
2.2. Animal procurement
The study was approved by the Institutional Animal Ethics
Committee (IAEC) [Reg. No. and Date of Reg: 173/GO/Re/S/2000/
CPCSEA, 28th January 2000], Jamia Hamdard (Delhi, India). This
committee follows the guidelines for Committee for Purpose of Control
and Supervision of Experiments on Animals (CPCSEA). 40 female
Albino rats (Wistar strain, 250–300 g) were procured from Central
Animal House facility of Jamia Hamdard (Delhi, India). The animals
were acclimatized in polypropylene cages under standard laboratory
conditions (12- hour light/dark cycles) and had free access to a com-
mercial pellet diet and water ad libitium. The animals were maintained
under a controlled room temperature of 25 ± 2 °C and relative hu-
midity of 60 ± 5% degree.
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Biomedicine & Pharmacotherapy 110 (2019) 47–58
2.3. Drug preparation
Astaxanthin (0.5 mg/kg and 1 mg/kg) was dissolved in canola oil
for its oral administration. 4 μg of Aβ (1–42) peptides were dissolved in
artificial cerebrospinal fluid (aCSF) to form a dilution of 4 μg/4 μl.
2.4. Surgical procedure
On the 1st day of our experiment protocol, animals underwent
surgery for i.c.v administrations. Rats were anesthetized with ketamine
hydrochloride (100 mg/kg) and xylazine (5 mg/kg). Rats were then
placed on stereotaxic apparatus followed by a midline sagittal incision
for exposing the skull. Bregma and lambda were identified and taking
bregma as the center point, bilateral holes were drilled into rat’s skull at
the coordinates; -3.5 mm anteroposterior, 2.0 mm lateral to sagittal
suture and 2.7 mm dorsoventral. Using 5 μl of Hamilton syringe (26-
gauge), Aβ (1–42) peptides dissolved in aCSF (4 μg/4 μl) was slowly
injected into the each cerebral ventricle (ICV) and then the needle was
left in the place for 120 s (post-infusion period) to allow proper infusion
of the peptides into the injection site [25]. Drilled holes were filled with
bone wax and the incised area was sutured properly. Animals were then
kept in the cages for 7 days for recovery.
2.5. Experimental protocol
Rats were acclimatized for 1 week and fed with the standard diet.
Animals were randomly divided into 5 groups, containing 8 rats each.
Group 1 served as sham control group and received artificial cere-
brospinal fluid (aCSF) in a volume of 4 μl bilaterally on 1st day, Group 2
served as Aβ (1–42) infused group and was intracerebroventricularly
administered with Aβ (1–42) dissolved in aCSF in a concentration of
4 μg/4 μl bilaterally on 1st day, Group 3 and group 4 received 0.5 mg/
kg/d and 1 mg/kg/d p.o. of Astaxanthin respectively from 8th day of
ICV-Aβ(1–42) for a period of 28 days, Group 5 served as Astaxanthin
per se group and received 1 mg/kg p.o. of ASX for 28 days starting from
day 8th of i.c.v. aCSF infusion (Fig. 2).
2.6. Behavioral assessment
2.6.1. Morris water maze test
Spatial learning and memory of animals in Morris water maze
(MWM) was tested by the method described [26]. MWM was performed
in rats from 29th to 33rd day of our experimental protocol which
consisted of 4 days of acquisition i.e. training trials and after 24 h i.e. on
the 33rd-day probe trial was performed. In this procedure, a water tank
of 130 cm diameter and 50 cm of height was used. It was filled up to the
depth of 30 cm with water maintained at a temperature of 26 ± 2 °C.
Division of pool into hypothetical 4 quadrants and the measurement of
time in both acquisition and probe trial were done using Any-maze
software (ANY MAZE, Video tracking Software, US).
During the acquisition phase, a rectangle shaped platform of 8 × 6
cm2 area was submerged underwater in the 4rth quadrant placed at a
fixed position. Animals were trained for 4 trials of the maximum time of
120 s each for 4 consecutive days. Time taken by each rat to find and
climb the hidden platform was noted as escape latency period. After
climbing the hidden platform, rats were allowed to stay there for 30 s. If
a rat couldn’t locate the platform, it was placed manually on the plat-
form.
During probe trials, the platform was removed and each rat was
given 60 s for the searching of the hidden platform. % time spent by the
rat in the target quadrant in the search of the hidden platform and
platform crossing frequency was noted.
2.6.2. Novel object recognition (NOR) test
Novel Object recognition (NOR) test was performed for analyzing
non-spatial and short-term memory in rats. We followed the protocol
S.O. Rahman et al. Biomedicine & Pharmacotherapy 110 (2019) 47–58

Fig. 1. Flowchart representation of the rationale behind the study.

previously described by Bevins and Besheer, 2006 [27]. A rectangular
box of volume 40 × 35 × 40 cm3 was used for the test. Our protocol
consisted of habituation, familiarization and retention phase. Ob-
servation of animal’s activity and time measurement was done through
Any-maze software (ANY MAZE, Video tracking Software, US).
Each rat was exposed to the box environment for 10 min for accli-
matization. After 24 h, the familiarization phase was commenced in
which two identical objects (FO1 & FO2) were placed in two opposite
and parallel corners of the box. Each rat was allowed to explore both
the objects for 5 min. After 1 h of this phase, the animal’s spontaneous
behavior was assessed in the test phase where one of the objects was
replaced by a novel object (F & N). Each rat was again allowed to ex-
plore both the objects for 5 min.
2.7. Brain homogenate preparation
After completion of 5 weeks of our study, rats were sacrificed on the
36th day under light ether anesthesia. Blood was completely removed
from the brain tissues using perfusion technique with phosphate buffer
through the heart to avoid any interference with the homogenate
readouts. The brain was carefully removed from the skull and rinsed
with ice-cold isotonic saline. Hippocampal tissues were separated from
the whole brain and then homogenized in ice-cold phosphate buffer
(0.1 M; pH 7.4) at 10,000 g for 15 min at (4 °C). Supernatants were
separated and stored at −80 °C for performing biochemical estimations.
Hippocampal Protein was measured by the method of Lowry et al. [28]
using bovine serum albumin (1 mg/ml) as a standard.

Fig. 2. Diagrammatic Scheme of experimental procedure.

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S.O. Rahman et al.
2.8. Biochemical assessment
2.8.1. Determination of hippocampal soluble Aβ (1-42), IRS-S307, GSK-
3β, and TNF-α
Soluble Aβ (1–42), IRS-S307, GSK-3β and TNF-α level in the hip-
pocampal homogenate of rats were measured using commercial ELISA
kits as per the manufacturer’s instructions.
2.8.2. Determination of acetylcholinesterase level
Acetylcholinesterase (AChE) level in the hippocampus was de-
termined using Ellman’s method [29]. 0.05 ml of supernatant was
mixed with 0.1 ml 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB, Ellman's
reagent), 0.1 ml of acetylthiocholine iodide and 3 ml of sodium phos-
phate buffer (pH 8). Change in absorbance was measured at the 30 s
interval for 2 min at 412 nm using Perkin Elmer Lambda 20 spectro-
photometers. Results were expressed in nanomoles of acetylthiocholine
iodide hydrolyzed per min per mg of protein.
2.8.3. Determination of hippocampal nitrite level
Hippocampal nitrite level was determined by colorimetric assay
using Griess reagent (0.1% N-(1-napththyl) ethylenediamine dihy-
drochloride, 1% sulphanilamide and 2.5% phosphoric acid) [30]. Equal
volumes of supernatant and Griess reagent were mixed and then in-
cubated for 10 min at room temperature in dark. Supernatant’s absor-
bance was measured at 540 nm with a Perkin Elmer Lambda 20 spec-
trophotometers. Nitrite level was analyzed using the determined
sodium nitrite standard curve and expressed in micromoles per mg
protein [31].
2.8.4. Determination of hippocampal antioxidants
2.8.4.1. Measurement of lipid peroxidation. Lipid peroxidation level was
determined by measuring the amount of malondialdehyde (MDA) in the
hippocampus [32]. In 0.1 ml of tissue homogenate 0.45 ml of 5% (w/v)
chilled trichloroacetic acid (TCA) and 0.67% thiobarbituric acid (TBA)
was mixed and centrifuged for 10 min at 4000 g. Test tubes were then
incubated in a boiling water bath for 10 min. The pink color obtained
was measured spectrophotometrically at 535 nm using Perkin Elmer
Lambda 20 spectrophotometers. Results obtained were expressed in
nanomoles per mg protein [33].
2.8.4.2. Measurement of reduced glutathione (GSH) activity. Reduced
glutathione (GSH) level in the hippocampus was measured using the
method described by Ellman [34] and as adopted by [33]. In this
colorimetric method, equal quantities of tissue homogenate and 10%
trichloroacetic acid were mixed and centrifuged for 15 min at
3000 rpm. Then the samples were mixed with 0.5 ml 5,5-
dithiobisnitro benzoic acid (DTNB), 2 ml of phosphate buffer (pH 7.4)
and 0.4 ml of double-distilled water. The absorbance of samples was
recorded at 412 nm within 15 min of the addition of DTNB.
2.9. Histopathological analysis of brain
2.9.1. H & E staining
After sacrifice, the whole brain was stored overnight in 10% for-
malin and then embedded in paraffin for 4 h. Paraffin blocks were
prepared and 5 μL of coronal sections of hippocampal CA1, CA3, and
dentate gyrus region were cut using microtome. Sections were mounted
on silane-coated slides, washed in xylene for deparaffinization, rehy-
drated in graded ethanol and finally stained with hematoxylin-eosin (H
& E). Hippocampal neurons morphology were observed and captured at
40 X magnification under light microscopy.
2.9.2. Congo red staining
For Congo red staining, sections were stained with Congo red so-
lution (0.2%) for 1 h and then counter-stained with hematoxylin solu-
tion. Plaques were observed and captured at 400 X magnification under
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Biomedicine & Pharmacotherapy 110 (2019) 47–58
a fluorescent microscope.
2.9.3. Quantification
Quantitative assessment of neuronal loss and Aβ plaque load was
done in H & E and Congo red staining respectively. For both assess-
ments, 10 random regions in the CA1 subdivision of the hippocampus
were selected under high microscopic field (40 X) per group. In H & E
stained slides, necrotic and/or degenerated neurons were counted while
in Congo red-stained slides, percentage area occupied by Aβ stains in
each selected regions was evaluated [35,36]. The obtained data were
then statistically analyzed.
2.10. Statistical analysis
The results were analyzed using Graph Pad Prism 6.01 (San Diego,
CA, United States) and values were expressed as mean ± standard
error mean (SEM). Escape latency period in MWM and total exploration
time in FO1, FO2 and Novel object in NOR was measured using two-
way analysis of variance (ANOVA) and paired student t-test respec-
tively. The time period during probe trial in MWM, Discrimination
index in NOR and results from all other parameters were measured
using one-way analysis of variance (ANOVA) and followed by Tukey’s
post-hoc test. P < 0.05 was set to be statistically significant.
3. Results
3.1. Astaxanthin attenuated Aβ (1–42) induced memory deficit during
Morris Water Maze (MWM) test
In the MWM test, animals were trained for 4 days to locate the
hidden platform. On the first day of training, no significant difference
was observed in mean escape latencies among any group. Rats infused
with Aβ (1–42) showed poor ability (p < 0.001) in learning the task on
all 4 days as compared to the control group. On the other hand, the ASX
treated group showed significant improvement in learning abilities
during acquisition trials dose-dependently when compared with Aβ
(1–42) infused group. On the final day of acquisition trial, ASX treat-
ment in Aβ (1–42) infused Wistar rats exhibited a significant decrease
in mean escape latency period at 0.5 mg/kg (p = 0.0027) and 1 mg/kg
(p < 0.001) when compared with Aβ (1–42) infused group (Fig. 3A).
Probe trial confirmed the impairment of memory in Aβ (1–42) in-
fused Wistar rats where these rats showed a significant reduction
(p < 0.001) of time spent in target quadrant. However, treatment with
ASX showed significant improvement in retention time at 0.5 mg/kg
(p = 0.0220) and 1 mg/kg (p = 0.0019) when compared with Aβ
(1–42) infused group (Fig. 3B). Moreover, a significant decrease
(p = 0.0067) in platform crossing frequency of Aβ (1–42) infused
Wistar rats was observed which was substantially reversed by ASX
treatment at 0.5 mg/kg (p = 0.0124) and 1 mg/kg (p = 0.0221) of
doses.
3.2. Astaxanthin reverses Aβ (1–42) induced impairment in short term
recognition memory task performance
Non-spatial memory and Short-term memory was assessed using
Novel Object Recognition (NOR) test. The non-significant difference
was observed during familiarize phase in between all groups (Fig. 4A).
Although during retention phase Aβ (1–42) infused rats showed no
significant difference in discriminating between familiar and novel
object. Rats treated with ASX spent more time (p < 0.001) in exploring
Novel object then familiar object when compared with Aβ (1–42) in-
fused group (Fig. 4B).
The spatial memory deficit in rats infused with Aβ (1–42) was also
demonstrated through significant reduction (p < 0.001) in the dis-
crimination index when compared with the control group. Whereas,
ASX treatment, dose-dependently, improved discrimination index at
S.O. Rahman et al. Biomedicine & Pharmacotherapy 110 (2019) 47–58

Fig. 3. Effect of Astaxanthin on Morris water maze performance of rats. (A) Escape latencies in the water maze during four consecutive days test, (B) % time spent in
target quadrant during probe trial, (C) Platform crossing frequency during probe trial. The values are expressed as mean ± SEM (n = 8). ##P < 0.01 and #P < 0.001
vs. sham control, *P < 0.05, **P < 0.01 and ***p < 0.001 vs. Aβ (1–42) infused group.

0.5 mg/kg (p = 0.0070) and 1 mg/kg (p = 0.0005) as compared with 3.5. Astaxanthin significantly attenuated Aβ (1–42) induced hippocampal
Aβ (1–42) infused group (Fig. 4C). GSK-3β stimulation

3.3. Astaxanthin dose-dependently ameliorated hippocampal soluble
amyloid-β (1–42) level
Increased level of Aβ (1–42) oligomers is an indicator for AD de-
velopment which progressively aggregates to form senile plaques. Aβ
(1–42) level (pg/ml) was significantly increased in Aβ (1–42) infused
group (p < 0.001) when compared to sham control animals.
Administration of ASX for 28 days significantly (p < 0.001) reduced
Aβ (1–42) level at 0.5 and 1 mg/kg as compared with Aβ (1–42) infused
group (Fig. 5).
Glycogen Synthase Kinase-3β or GSK-3β activity is increased during
AD and it leads to hyperphosphorylation of tau protein leading to the
formation of Neurofibrillary tangles (NFT). I.c.v. administration of Aβ
(1–42) peptides in rats significantly (p < 0.001) raised the level of
GSK-3β activity in the hippocampus when compared with the sham
control group. After 28 days of treatment, ASX dose-dependently re-
duced the activity of GSK-3β in rats as compared with Aβ (1–42) in-
fused group. ASX at 0.5 mg/kg showed 32.36% (p = 0.0014) of re-
duction while at 1 mg/kg showed 44.87% of reduction in GSK-3β
activity against Aβ (1–42) peptides only infused rats (Fig. 7).

3.4. Astaxanthin dose-dependently prevented Aβ (1–42) mediated 3.6. Astaxanthin treatment prevented Aβ (1–42) induced rise in
hippocampal IRS-1 (s307) phosphorylation hippocampal TNF-α level

Phosphorylation of Insulin Receptor Substrate-1 at serine 307 is an
indicator of central insulin resistance. Aβ (1–42) infusion in rats pro-
moted hippocampal IRS-1 (s307) phosphorylation significantly
(p < 0.001) as compared to sham control group. However, ASX
treatment, dose-dependently, significantly ameliorated phosphoryla-
tion of hippocampal IRS-1 (s307) at 0.5 mg/kg (p = 0.0047) as well as
1 mg/kg (p < 0.001) doses when compared with toxic group (Fig. 6).
Increase in pro-inflammatory markers like TNF-α is found in pa-
tients with the AD. TNF-α was significantly raised (p < 0.001) by Aβ
(1–42) infusions when compared with control. This effect was sig-
nificantly (p = 0.0002) reversed by 0.5 mg/kg of ASX treatment for 28
days. 1 mg/kg oral dose of ASX also exhibited significant reduction
(p < 0.001) of Aβ (1–42) induced an increase in TNF-α level in Wistar
rats (Fig. 8).

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S.O. Rahman et al. Biomedicine & Pharmacotherapy 110 (2019) 47–58

Fig. 4. Effect of Astaxanthin on Novel Object Recognition task in Aβ (1–42) infused rats. (A) Total exploring time during familiarization phase where FO1 and FO2
are two similar objects, (B) total exploring time during retention phase where F is familiar object while N is Novel Object and (C) Discrimination index obtained from
retention phase. The values are expressed as mean ± SEM (n = 8). #P < 0.001 vs. sham control, *P < 0.05, **p < 0.01 and ***p < 0.001 vs. Aβ (1–42) infused
group.

Fig. 5. Hippocampal soluble Aβ (1–42) levels after 28 days of treatment with

Fig. 6. Hippocampal IRS-S307 level after 28 days of treatment with
Astaxanthin in Aβ (1–42) peptides infused rats. The values are expressed as
Astaxanthin in Aβ (1–42) peptides infused rats. The values are expressed as
mean ± SEM. ###P < 0.001 vs. sham control and ***p < 0.001 vs. Aβ (1–42)
mean ± SEM. ###P < 0.001 vs. sham control, **p < 0.01 and ***p < 0.001
infused group.
vs. Aβ (1–42) infused group.

3.7. Astaxanthin ameliorated hippocampal acetylcholinesterase (AChE)

control group. Oral administration of ASX with the dose of 1 mg/kg
activity
exhibited a very significant decrease (p = 0.0002) in AChE activity as
compared to Aβ (1–42) infused group. However, at 0.5 mg/kg, ASX also
According to the cholinergic hypothesis, the activity of AChE sig-
showed a decrease in AChE activity but with lesser significance
nificantly increases during AD which leads to degradation of
(p = 0.0107) when compared with Aβ (1–42) infused rats (Table 1).
Acetylcholine (ACh). Infusion of Aβ (1–42) peptide in rats showed a
significant increase (p < 0.001) in AChE activity as compared to the

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doses significantly decreased (p < 0.001) the level of MDA in Aβ

(1–42) infused group (Table 1).
Glutathione is an antioxidant enzyme and reduced glutathione level
is a marker for oxidative stress. Aβ peptide infusion leads to the sub-
stantial reduction (p < 0.001) in GSH when compared with sham
control group. This reduction in GSH activity was counteracted by ASX
treatment for 28 days. At both 0.5 (p = 0.0010) and 1 (p = 0.0004)
mg/kg ASX showed significant reversal of reduction in GSH activity
after i.c.v. administration of Aβ (1–42) peptides in Wistar rats (Table 1).

3.10. Astaxanthin treatment showed a significant reduction in neuronal

Fig. 7. Hippocampal GSK-3β level after 28 days of treatment with Astaxanthin
in Aβ (1–42) peptides infused rats. The values are expressed as mean ± SEM.
###
P < 0.001 vs. sham control, **p < 0.01 and ***p < 0.001 vs. Aβ (1–42)
infused group.
Fig. 8. Hippocampal TNF-α level after 28 days of treatment with Astaxanthin in
Aβ (1–42) peptides infused rats. The values are expressed as mean ± SEM.
###
P < 0.001 vs. sham control and ***p < 0.001 vs. Aβ (1–42) infused group.
3.8. Astaxanthin ameliorated Aβ (1–42) induced elevation in hippocampal
nitrite level
During the AD, brain nitrite expression is increased which leads to
nitrosative stress. Hippocampal nitrite level was significantly increased
(p = 0.005) in Aβ (1–42) infused group when compared with the
control group. Administration of 0.5 (p = 0.0151) and 1 mg/kg
(p = 0.0012) of ASX for 28 days significantly reduced (p < 0.05 and
p < 0.01 respectively) hippocampal nitrite level as compared with Aβ
(1–42) infused group (Table 1).
3.9. Astaxanthin reversed Aβ (1–42) mediated increase in MDA while a
decrease in GSH activity in the hippocampus
Increase in lipid peroxidation (Malondialdehyde level) and a de-
crease in antioxidant enzymes (Glutathione level) are an integral part of
AD-affected brains. Even in our study i.c.v. infusion of Aβ (1–42)
peptides lead to significant escalation (p < 0.001) in MDA level as
compared with sham control group. Administration of ASX at both
damage and amyloidosis in the histopathological analysis
Fig exhibits toxic effects of Aβ (1–42) peptide infusion in the hip-
pocampus of Wistar rats. H&E staining displayed intact neurons with
prominent nucleoli while Congo red staining displayed no Aβ plaque
accumulation in the hippocampus of rat from the sham control group.
A significant increase in (p = 0.001)protruding eosinophilic cyto-
plasm, pyknotic nuclei, swelling of neurons, pyramidal cells shrinkage
and dispersed vacuolization can be observed from H& E staining of rat’s
hippocampus after i.c.v. administration of Aβ (1–42) peptides. Oral
treatment with ASX showed mild neuronal toxicity (p = 0.0111 for
0.5 mg/kg and p = 0.002 for 1 mg/kg of dose) with lesser number of
eosinophilic stained neurons. Reduced pyknotic nuclei and vacuoliza-
tion can also be observed in ASX treated groups (Fig. 9).
On the other hand, significant accumulation of amyloid beta pla-
ques can be observed after hippocampal Congo red staining of Aβ
(1–42) peptide infused rats. These plaques, which appeared red in
color, were shown widely distributed in the hippocampus regions.
Extensive vacuolization and neuronal injury can also be observed in this
slide. In hippocampal sections of rat treated with ASX for 28 days
showed a reduction in Congo red staining at 0.5 mg/kg (p = 0.0047)
and 1 mg/kg (p = 0.002) when compared with hippocampal Congo red
staining of Aβ (1–42) infused group (Fig. 10).
4. Discussions
For Decades now, numerous studies have upgraded our knowledge
regarding Alzheimer’s disease (AD) and its related abnormalities. In
spite of that, complications of the AD have made the development of its
therapeutic intervention quite a challenging task [37]. In the past
decade, many clinical trials for therapies against AD have been com-
menced but they were completed with very high failure rate either due
to serious adverse effect or no significant therapeutic efficacy [38,39].
Thus it is imperative to keep exploring different approaches that can be
used to target the AD complications which could be translated into
successful clinical trials. Our study was based on the hypothesis that
Central insulin resistance is associated with AD condition. Although it is
still not clear that whether it’s a cause or consequence of AD. We in-
vestigated the effect of astaxanthin (ASX) on amyloid beta (Aβ) induced

Table 1
Effect of Astaxanthin on hippocampal AChE, Nitrite, MDA and GSH level in Aβ (1–42) peptides infused rats.
Groups AChE Activity (nmol/min/mg protein) Nitrite (μmol MDA (nmol/ mg protein) GSH (μmol/ mg protein)
/mg protein)

Control 104 ± 10.9 83.16 ± 10.8 1.110 ± 0.1064 4.139 ± 0.3764

Aβ (1-42) 313 ± 24.4### 218.2 ± 13.69### 5.689 ± 0.8591### 1.107 ± 0.0636###
Aβ (1-42) + AS X 0.5 mg 232 ± 9.48* 162.1 ± 14.96* 2.890 ± 0.1938*** 2.784 ± 0.1843***
Aβ (1-42) + AS X 1 mg 199 ± 18.6*** 145.2 ± 8.122** 2.422 ± 0.3433*** 2.930 ± 0.2302***
ASX per se 108 ± 9.65 87 ± 7.848 1.207 ± 0.1043 3.910 ± 0.3235

The values are expressed as mean ± SEM.

###
P < 0.001 vs. sham control.
* p < 0.05.
** p < 0.01.
*** p < 0.001 vs. Aβ (1–42) infused group.

53
S.O. Rahman et al. Biomedicine & Pharmacotherapy 110 (2019) 47–58

Fig. 9. Histology of CA 1 region of hippocampus stained with H & E after 35 days of experimental protocol (40X). (A) Sham control group represents healthy neurons
with prominent nuclei, (B) Aβ (1–42) infused group representing neuronal damage, eosinophilic stained cytoplasm, vacuolization and neuronal shrinkage, (C)
Astaxanthin (0.5 mg/kg) & (D) Astaxanthin (1 mg/kg) treated groups representing mild neuronal injury with lesser number of eosinophilic stained neurons, (E)
Astaxanthin per se group representing intact neurons with prominent nuclei, (F) Quantitative assessment of number of degenerated neurons in hippocampal sections
of rats brain. The values are expressed as mean ± SEM. ###p < 0.001 vs control, *p < 0.05 and ***p < 0.001 vs. Aβ (1–42) infused group.

AD in Wistar rat. We also demonstrated the role of ASX on hippocampal
insulin resistance by measuring its effect on Insulin Receptor Substrate-
1 (IRS-1) serine phosphorylation.
Intracerebroventricular administration of Aβ (1–42) peptides into
rat hippocampus showed a significant rise in Aβ level in both bio-
chemical and histopathological analysis. Hence, it can be considered as
a reliable model for an AD in rats. Aβ (1–42) infusion in rats also ex-
hibited a significant impairment of cognitive abilities in behavioral
studies and also enhanced GSK-3β activity, TNF-α activity, oxidative
stress markers, nitrile level, Acetylcholinesterase activity, and neuro-
degeneration when compared with sham control animals. These results
were in accordance with earlier studies reported [26,40]. Interestingly,
the rise in Aβ level also demonstrated a significant increase in IRS-1
phosphorylation at Serine 307 (S307). An in vitro study has already
reported that Aβ (1–42) peptides administration can phosphorylate IRS-
1 at S307 leading to insulin resistance [9,41]. This indicates that Aβ
infusion in rats demonstrated a significant insulin resistance in the
hippocampus of Wistar rats.
Different techniques are now available for behavioral assessment of
cognitive disability and memory impairment. We performed Morris
Water Maze (MWM) test and Novel Object Recognition (NOR) test to
study spatial and non-spatial memory and learning the ability of rats
respectively. In the present study, rats infused with Aβ (1–42) peptides
exhibited a significant increase in escape latency during acquisition
trial and a decrease in time spent in target quadrant during probe trial.
ASX dose-dependently showed a significant reversal of poor learning
and consolidation of memory in the treatment group when compared
with Aβ (1–42) infused group rats in both acquisition and probe trials.

54
S.O. Rahman et al. Biomedicine & Pharmacotherapy 110 (2019) 47–58

Fig. 10. Histology of CA1 region of hippocampus stained with Congo red after 35 days of experimental protocol (40X). (A) Sham control group represents healthy
neurons with no Aβ accumulation detected, (B) Aβ (1–42) infused group representing extensive Aβ accumulation and neurodegeneration, (C) Astaxanthin (0.5 mg/
kg) treated group representing decreased Aβ accumulation with mild neurodegeneration, (D) Astaxanthin (1 mg/kg) treated group exhibiting significant reduction in
Aβ accumulation with lesser number of eosinophilic stained neurons, (E) Astaxanthin per se group representing intact neurons and no Aβ accumulation, (F)
Quantitative assessment of percentage area occupied by Aβ stain in hippocampal sections of rats brain. The values are expressed as mean ± SEM. **p < 0.01 and
***p < 0.001 vs. Aβ (1–42) infused group.

In addition, rats in our Aβ (1–42) infused group was unable to differ-
entiate between a familiar and novel object which was significantly
restored by ASX treatment dose-dependently. Our results are consistent
with previous findings reported by Madhu et al., where betulinic acid
was used as a treatment in the rat model of AD [42].
Aβ accumulation or plaque formation is well known as an integral
part of the AD which leads to neurodegeneration particularly in the
hippocampus. Senile plaques can be formed either due to excessive
production of Aβ by β- and γ-secretases or reduced degradation of Aβ
by Aβ-degrading proteases such as an Insulin-degrading enzyme (IDE)
[43]. Also, Aβ (1–42) oligomers are more toxic and prone to aggregate
than Aβ (1–40) [44]. In our study oral administration of ASX for 28
days significantly attenuated Aβ (1–42) level in the hippocampus of
Wistar rats when compared with Aβ (1–42) infused group. Previous
studies have demonstrated the neuroprotective role of ASX against Aβ
oligomers both in vitro and in vivo [20,45]. The probable reason for the
anti-amyloidogenic action of ASX can be due to its potent anti-oxidant
effects mediated by NFATc4 activation and reduction of mitochondrial
ROS production or maybe via stimulation of Nrf2/HO-1 pathway or
significant reduction in γ-secretase expression [19]. Thus the reduction
in γ-secretase expression leads to the attenuation of Aβ production and
formation of Aβ plaques in the hippocampus. Also, no such study was
found which evaluated the role of ASX on Aβ-degrading proteases.
Whether ASX modifies Aβ degradation or not needs further evaluation.
Suzanne de la Monte, in 2005, published the very first study which
reported that AD might be a neuroendocrine disease affected by neu-
ronal insulin resistance and thus she coined the term ‘’type 3 diabetes’’
[46]. Since then, various studies have indicated the connection of AD
with neuronal insulin resistance [47,48]. Another team of scientists
leads by Theresa R Bomfim also published their study in 2012

55
S.O. Rahman et al.
demonstrating that serine phosphorylation of Insulin receptor sub-
strate-1 (IRS-1) lead to insulin resistance in both in vitro and in vivo
model of the AD which was prevented by administration of exendin
(GLP-1 analog), an antidiabetic drug [9]. Insulin resistance in neurons
disrupts the insulin-mediated PI3k-Akt-mTOR pathway which is known
to be responsible for cell survival and growth [8,49]. This is the very
first study to demonstrate that Astaxanthin significantly and dose-de-
pendently reduced IRS-1 S307 phosphorylation mediated by i.c.v ad-
ministration of Aβ (1–42) peptides. Previous studies have demonstrated
the profound action of ASX in increasing insulin sensitivity in the per-
iphery [23,24,50]. Interestingly, stimulation of IRS–PI3K–Akt pathway
was found to be associated with ASX mediated improved insulin sen-
sitivity in liver and skeletal muscles [23,50]. It was also reported that
activation of Stress-activated protein kinases (SAPK), also known as,
Jun amino-terminal kinases (JNK), particularly JNK-1, phosphorylated
IRS-1 at serine 307 leading to the development of insulin resistance
[23]. Also, another study has reported that elevated Aβ oligomers lead
to activation of JNK protein and via JNK/TNF-α pathway which leads
to the phosphorylation of IRS-1 at multiple serine residues [9]. Thus
probable reason behind ASX mediated reduction in IRS-1 S307 phos-
phorylation can be attenuation of Aβ level, oxidative/nitrosative stress
and TNF-α activity in rat’s hippocampus which have been reported in
our study.
Glycogen Synthase Kinase-3β (GSK-3β) is an enzyme which is
widely implicated in Alzheimer’s disease. GSK-3β plays a very sig-
nificant role in the AD and acts as a causative link between Aβ accu-
mulation and Tau pathology [51]. The activity of GSK-3β is upregulated
during AD and rise in Aβ level is one of the reported mechanisms be-
hind that [52]. GSK-3β once activated leads to hyperphosphorylation of
Tau protein. Hyperphosphorylation of Tau destabilizes the tau protein
and eventually leads to the formation of Neurofibrillary tangles (NFT)
[51]. Inhibition of GSK-3β has shown a decrease in tau hyperpho-
sphorylation AD models [53]. Another mechanism behind over acti-
vation of GSK-3β is insulin resistance in neurons. Insulin resistance
disrupts insulin signaling mediated PI3k-Akt-mTOR pathway and ear-
lier studies have shown that activation of Akt protein inhibits stimu-
lation of GSK-3β [49]. Even in our study, i.c.v administration of Aβ
showed a significant increase in GSK-3β activity. However, Astaxanthin
dose-dependently inhibited GSK-3β activity in rats that received Aβ
peptide intracerebroventricularly. Both in vitro and in vivo studies have
also earlier reported the neuroprotective role of ASX via down-
regulating GSK-3β activity [20,54]. These results indicate that ASX can
contribute in suppressing tau hyperphosphorylation mediated NFT
formation during AD via GSK-3β inhibition.
If Aβ plays a central role in the AD then chronic neuroinflammation
acts as a medium which provides a neurotoxic environment thereby
promoting aggravation of AD complications. Chronic release of pro-
inflammatory cytokines (TNF-α & IL-1β) can enhance Aβ via upregu-
lating Amyloid Precursor Protein (APP) expression and can also lead to
tau hyperphosphorylation [55]. In our study also, Aβ infused hippo-
campal tissue showed a significant increase in TNF-α level. Treatment
with ASX significantly attenuated TNF-α release induced by Aβ (1–42)
peptides. These results were consistent with the anti-inflammatory ef-
fect of ASX reported in previous studies [20]. Studies have even in-
dicated the presence of NFκB binding domain on BACE-1 promoter
region [56,57]. Stimulation of NFκB by TNF-α & IL-1β can upregulate
BACE-1 expression thus, promoting increased production of neurotoxic
Aβ [58]. Interestingly, an earlier study has exhibited inhibition of NFκB
in the hippocampus by ASX [59]. Reduction of TNF-α and inhibition of
NFκB might be the reason behind attenuation of BACE-1 expression
thereby leading to a decrease in Aβ production.
Production of Aβ during AD has shown a cholinergic deficit in the
hippocampus via enhancing Acetylcholinesterase (AChE) activity and
thus leading to cognitive decline [60]. Acetylcholinesterase can also
lead to the formation of β-amyloid peptide fragments which can ag-
gregate to form cytotoxic complexes with the growing fibrils [61]. The
56
Biomedicine & Pharmacotherapy 110 (2019) 47–58
present study also demonstrated that i.c.v. administration of Aβ (1–42)
peptides significantly enhanced AChE activity. On the other hand,
treatment with ASX significantly attenuated escalated AChE activity in
Aβ (1–42) infused rats. This is the first study to demonstrate the effect
of ASX on AChE in an AD model. However, an earlier study has de-
monstrated ASX mediated reduction of AChE in a doxorubicin-induced
model of cognitive dysfunction [62].
Nitrite estimation was done in the hippocampus as it is a marker for
NO production. NO is an endogenously produced free radical and is
known for its noxious effects on our body system. During AD nitrite
level is found to be significantly increased and can lead to the formation
of peroxynitrite (ONOO−), also known as Reactive Nitrogen Species
(RNS) [63]. Formation of RNS causes lipid peroxidation, protein, and
mitochondrial damage, DNA oxidation and eventually leads to neuronal
damage [64]. In our study, i.c.v. administration of Aβ (1–42) peptides
in Wistar rats significantly enhanced hippocampal nitrite level. Aβ has
earlier shown impairment of hippocampal LTP via NO/cGMP/PKG/
CREB pathway [65]. ASX treatment dose-dependently attenuated nitrile
level in the hippocampus of rats after Aβ (1–42) peptides infusion. ASX
mediated reduction in Aβ level in hippocampus might be the reason
behind this effect. Previous studies have also demonstrated ASX
mediated improvement of cognitive functions via the inhibition of NO
production in the hippocampus [66].
Enhanced lipid peroxidation and antioxidant enzyme reduction are
some of the extensively used markers for estimation of oxidative stress.
Postmortem analyses of AD brains have earlier revealed increased lipid
peroxidation and protein oxidation [67]. Our brain has relatively
higher oxygen demand and low level of antioxidant enzymes making it
at higher risk towards oxidative stress [68]. Accumulation of Aβ can
lead to an extensive increase in Oxidative stress and vice versa creating
a vicious cycle [69]. In our study also, i.c.v. administration of Aβ
peptides in Wistar rats leads to the enhanced lipid peroxidation and
reduction in GSH level [70,71]. ASX, known for its potent antioxidant
effect [18], significantly reversed the toxic effect of Aβ on hippocampal
MDA and GSH level. Previous studies have demonstrated anti-oxidant
activity of ASX in neurons via stimulation of NrF2/HO-1 pathway and
increase in SOD activity [20,54]. Our study also verified the antioxidant
activity of ASX during AD in the hippocampus via a decrease in lipid
peroxidation and increase in GSH activity.
Moreover, histopathological analysis by H & E staining revealed Aβ
mediated neuronal damage in hippocampal sections of Wistar rats
which was characterized by disorganization of neurons, eosinophilic
stained cytoplasm, nuclei swelling and neuronal shrinking. These
findings are in agreement with the previous report which showed that
Aβ (1–42) induced memory impairment is associated with neuronal
injury in hippocampal areas of rat brain [72,73]. Congo red staining, a
histological marker for Aβ accumulation confirmed increased level of
Aβ in the hippocampus of Aβ (1–42) infused group as shown by pre-
vious studies [71,74]. Significant red color stains (marked with yellow
arrows) depicting the accumulation of Aβ can be clearly seen in the
hippocampal section of Wistar rats administered with Aβ (1–42) pep-
tides. We for the first time demonstrated histopathological effects of
ASX on Aβ (1–42) induced AD model in Wistar rats. ASX treatment
showed significant protection against Aβ mediated neuronal damage
and plaque formation in the hippocampus. H & E of ASX treated group
demonstrated mild neuronal toxicity with lesser number of eosinophilic
stained neurons and increased number of healthy neurons with pro-
minent nuclei. Previous studies have reported ASX as a potential can-
didate to promote neuron plasticity thereby attenuating neurodegen-
eration and also preserving cognitive functions [75]. On the other hand,
Congo red staining of hippocampal sections of ASX treated groups ex-
hibited a protective effect against plaque formation dose-dependently.
This analysis supports the fact that ASX attenuates production and ac-
cumulation of Aβ in the hippocampus of Wistar rats. Also, these positive
histological effects of ASX are in consideration with our behavioral and
biochemical assessments.
S.O. Rahman et al.
5. Conclusion
We conclude that our study was in accordance with available evi-
dence that correlates neuronal insulin resistance with Alzheimer’s dis-
ease [76]. Our study demonstrated increased IRS-1 (S307) phosphor-
ylation which as per the previous studies meant enhanced hippocampal
insulin resistance after i.c.v. administration of Aβ (1–42) peptides in
Wistar rats. The behavioral, biochemical and histopathological in-
vestigation revealed that oral treatment with Astaxanthin for 28 days
not only attenuated AD-related complications but also reversed Aβ in-
duced insulin resistance in hippocampal neurons.
These findings suggest the potential therapeutic efficacy of ASX
against neuronal insulin resistance and AD complications. ASX, known
for its pleiotropic actions (anti-oxidative, anti-inflammatory, anti-dia-
betic and also anti-amyloidogenic), can be considered as a potential
candidate for the treatment of AD.
However, the authors would also like to highlight few limitations in
this study that could be considered in further investigations of ASX on
hippocampal insulin sensitivity by evaluating its role on IRS-PI3K-Akt
pathway, GLUT-4 transporter, and JNK-1 activation; as well as on
neurodegeneration by evaluating its role on cell proliferation (BrdU
labelling), hippocampal volume and gliosis (GFAP labelling).
Funding
This project was funded by the Pharmaceutical Medicine Program of
Jamia Hamdard in collaboration with Sun Pharmaceutical Industries
Ltd.
Acknowledgment
We express our gratitude to Pharmaceutical biotechnology research
lab, Department of Pharmacognosy and Phytochemistry, Jamia
Hamdard for providing us with astaxanthin and also to
Neurobehavioral Pharmacology Laboratory, Department of Medical
Elementology and Toxicology, Jamia Hamdard for providing various
facilities used during the course of the study.
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