Biomedicine & Pharmacotherapy 110 (2019) 47–58

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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-

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

⁎
Corresponding author.
E-mail address: [email protected] (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. Biomedicine & Pharmacotherapy 110 (2019) 47–58

related kinase/mitogen-activated protein kinase (ERK/MAPK). Upre- 2.3. Drug preparation

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

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

Fig. 2. Diagrammatic Scheme of experimental procedure.

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2.8. Biochemical assessment a fluorescent microscope.

2.8.1. Determination of hippocampal soluble Aβ (1-42), IRS-S307, GSK- 2.9.3. Quantification

3β, and TNF-α Quantitative assessment of neuronal loss and Aβ plaque load was
Soluble Aβ (1–42), IRS-S307, GSK-3β and TNF-α level in the hip- done in H & E and Congo red staining respectively. For both assess-
pocampal homogenate of rats were measured using commercial ELISA ments, 10 random regions in the CA1 subdivision of the hippocampus
kits as per the manufacturer’s instructions. were selected under high microscopic field (40 X) per group. In H & E
stained slides, necrotic and/or degenerated neurons were counted while
2.8.2. Determination of acetylcholinesterase level in Congo red-stained slides, percentage area occupied by Aβ stains in
Acetylcholinesterase (AChE) level in the hippocampus was de- each selected regions was evaluated [35,36]. The obtained data were
termined using Ellman’s method [29]. 0.05 ml of supernatant was then statistically analyzed.
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- 2.10. Statistical analysis
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- The results were analyzed using Graph Pad Prism 6.01 (San Diego,
photometers. Results were expressed in nanomoles of acetylthiocholine CA, United States) and values were expressed as mean ± standard
iodide hydrolyzed per min per mg of protein. error mean (SEM). Escape latency period in MWM and total exploration
time in FO1, FO2 and Novel object in NOR was measured using two-
2.8.3. Determination of hippocampal nitrite level way analysis of variance (ANOVA) and paired student t-test respec-
Hippocampal nitrite level was determined by colorimetric assay tively. The time period during probe trial in MWM, Discrimination
using Griess reagent (0.1% N-(1-napththyl) ethylenediamine dihy- index in NOR and results from all other parameters were measured
drochloride, 1% sulphanilamide and 2.5% phosphoric acid) [30]. Equal using one-way analysis of variance (ANOVA) and followed by Tukey’s
volumes of supernatant and Griess reagent were mixed and then in- post-hoc test. P < 0.05 was set to be statistically significant.
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- 3. Results
trophotometers. Nitrite level was analyzed using the determined
sodium nitrite standard curve and expressed in micromoles per mg 3.1. Astaxanthin attenuated Aβ (1–42) induced memory deficit during
protein [31]. Morris Water Maze (MWM) test

2.8.4. Determination of hippocampal antioxidants In the MWM test, animals were trained for 4 days to locate the
2.8.4.1. Measurement of lipid peroxidation. Lipid peroxidation level was hidden platform. On the first day of training, no significant difference
determined by measuring the amount of malondialdehyde (MDA) in the was observed in mean escape latencies among any group. Rats infused
hippocampus [32]. In 0.1 ml of tissue homogenate 0.45 ml of 5% (w/v) with Aβ (1–42) showed poor ability (p < 0.001) in learning the task on
chilled trichloroacetic acid (TCA) and 0.67% thiobarbituric acid (TBA) all 4 days as compared to the control group. On the other hand, the ASX
was mixed and centrifuged for 10 min at 4000 g. Test tubes were then treated group showed significant improvement in learning abilities
incubated in a boiling water bath for 10 min. The pink color obtained during acquisition trials dose-dependently when compared with Aβ
was measured spectrophotometrically at 535 nm using Perkin Elmer (1–42) infused group. On the final day of acquisition trial, ASX treat-
Lambda 20 spectrophotometers. Results obtained were expressed in ment in Aβ (1–42) infused Wistar rats exhibited a significant decrease
nanomoles per mg protein [33]. 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).
2.8.4.2. Measurement of reduced glutathione (GSH) activity. Reduced Probe trial confirmed the impairment of memory in Aβ (1–42) in-
glutathione (GSH) level in the hippocampus was measured using the fused Wistar rats where these rats showed a significant reduction
method described by Ellman [34] and as adopted by [33]. In this (p < 0.001) of time spent in target quadrant. However, treatment with
colorimetric method, equal quantities of tissue homogenate and 10% ASX showed significant improvement in retention time at 0.5 mg/kg
trichloroacetic acid were mixed and centrifuged for 15 min at (p = 0.0220) and 1 mg/kg (p = 0.0019) when compared with Aβ
3000 rpm. Then the samples were mixed with 0.5 ml 5,5- (1–42) infused group (Fig. 3B). Moreover, a significant decrease
dithiobisnitro benzoic acid (DTNB), 2 ml of phosphate buffer (pH 7.4) (p = 0.0067) in platform crossing frequency of Aβ (1–42) infused
and 0.4 ml of double-distilled water. The absorbance of samples was Wistar rats was observed which was substantially reversed by ASX
recorded at 412 nm within 15 min of the addition of DTNB. treatment at 0.5 mg/kg (p = 0.0124) and 1 mg/kg (p = 0.0221) of
doses.
2.9. Histopathological analysis of brain
3.2. Astaxanthin reverses Aβ (1–42) induced impairment in short term
2.9.1. H & E staining recognition memory task performance
After sacrifice, the whole brain was stored overnight in 10% for-
malin and then embedded in paraffin for 4 h. Paraffin blocks were Non-spatial memory and Short-term memory was assessed using
prepared and 5 μL of coronal sections of hippocampal CA1, CA3, and Novel Object Recognition (NOR) test. The non-significant difference
dentate gyrus region were cut using microtome. Sections were mounted was observed during familiarize phase in between all groups (Fig. 4A).
on silane-coated slides, washed in xylene for deparaffinization, rehy- Although during retention phase Aβ (1–42) infused rats showed no
drated in graded ethanol and finally stained with hematoxylin-eosin (H significant difference in discriminating between familiar and novel
& E). Hippocampal neurons morphology were observed and captured at object. Rats treated with ASX spent more time (p < 0.001) in exploring
40 X magnification under light microscopy. Novel object then familiar object when compared with Aβ (1–42) in-
fused group (Fig. 4B).
2.9.2. Congo red staining The spatial memory deficit in rats infused with Aβ (1–42) was also
For Congo red staining, sections were stained with Congo red so- demonstrated through significant reduction (p < 0.001) in the dis-
lution (0.2%) for 1 h and then counter-stained with hematoxylin solu- crimination index when compared with the control group. Whereas,
tion. Plaques were observed and captured at 400 X magnification under ASX treatment, dose-dependently, improved discrimination index at

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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 Glycogen Synthase Kinase-3β or GSK-3β activity is increased during
amyloid-β (1–42) level AD and it leads to hyperphosphorylation of tau protein leading to the
formation of Neurofibrillary tangles (NFT). I.c.v. administration of Aβ
Increased level of Aβ (1–42) oligomers is an indicator for AD de- (1–42) peptides in rats significantly (p < 0.001) raised the level of
velopment which progressively aggregates to form senile plaques. Aβ GSK-3β activity in the hippocampus when compared with the sham
(1–42) level (pg/ml) was significantly increased in Aβ (1–42) infused control group. After 28 days of treatment, ASX dose-dependently re-
group (p < 0.001) when compared to sham control animals. duced the activity of GSK-3β in rats as compared with Aβ (1–42) in-
Administration of ASX for 28 days significantly (p < 0.001) reduced fused group. ASX at 0.5 mg/kg showed 32.36% (p = 0.0014) of re-
Aβ (1–42) level at 0.5 and 1 mg/kg as compared with Aβ (1–42) infused duction while at 1 mg/kg showed 44.87% of reduction in GSK-3β
group (Fig. 5). 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 Increase in pro-inflammatory markers like TNF-α is found in pa-
indicator of central insulin resistance. Aβ (1–42) infusion in rats pro- tients with the AD. TNF-α was significantly raised (p < 0.001) by Aβ
moted hippocampal IRS-1 (s307) phosphorylation significantly (1–42) infusions when compared with control. This effect was sig-
(p < 0.001) as compared to sham control group. However, ASX nificantly (p = 0.0002) reversed by 0.5 mg/kg of ASX treatment for 28
treatment, dose-dependently, significantly ameliorated phosphoryla- days. 1 mg/kg oral dose of ASX also exhibited significant reduction
tion of hippocampal IRS-1 (s307) at 0.5 mg/kg (p = 0.0047) as well as (p < 0.001) of Aβ (1–42) induced an increase in TNF-α level in Wistar
1 mg/kg (p < 0.001) doses when compared with toxic group (Fig. 6). rats (Fig. 8).

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

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

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

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demonstrating that serine phosphorylation of Insulin receptor sub- present study also demonstrated that i.c.v. administration of Aβ (1–42)
strate-1 (IRS-1) lead to insulin resistance in both in vitro and in vivo peptides significantly enhanced AChE activity. On the other hand,
model of the AD which was prevented by administration of exendin treatment with ASX significantly attenuated escalated AChE activity in
(GLP-1 analog), an antidiabetic drug [9]. Insulin resistance in neurons Aβ (1–42) infused rats. This is the first study to demonstrate the effect
disrupts the insulin-mediated PI3k-Akt-mTOR pathway which is known of ASX on AChE in an AD model. However, an earlier study has de-
to be responsible for cell survival and growth [8,49]. This is the very monstrated ASX mediated reduction of AChE in a doxorubicin-induced
first study to demonstrate that Astaxanthin significantly and dose-de- model of cognitive dysfunction [62].
pendently reduced IRS-1 S307 phosphorylation mediated by i.c.v ad- Nitrite estimation was done in the hippocampus as it is a marker for
ministration of Aβ (1–42) peptides. Previous studies have demonstrated NO production. NO is an endogenously produced free radical and is
the profound action of ASX in increasing insulin sensitivity in the per- known for its noxious effects on our body system. During AD nitrite
iphery [23,24,50]. Interestingly, stimulation of IRS–PI3K–Akt pathway level is found to be significantly increased and can lead to the formation
was found to be associated with ASX mediated improved insulin sen- of peroxynitrite (ONOO−), also known as Reactive Nitrogen Species
sitivity in liver and skeletal muscles [23,50]. It was also reported that (RNS) [63]. Formation of RNS causes lipid peroxidation, protein, and
activation of Stress-activated protein kinases (SAPK), also known as, mitochondrial damage, DNA oxidation and eventually leads to neuronal
Jun amino-terminal kinases (JNK), particularly JNK-1, phosphorylated damage [64]. In our study, i.c.v. administration of Aβ (1–42) peptides
IRS-1 at serine 307 leading to the development of insulin resistance in Wistar rats significantly enhanced hippocampal nitrite level. Aβ has
[23]. Also, another study has reported that elevated Aβ oligomers lead earlier shown impairment of hippocampal LTP via NO/cGMP/PKG/
to activation of JNK protein and via JNK/TNF-α pathway which leads CREB pathway [65]. ASX treatment dose-dependently attenuated nitrile
to the phosphorylation of IRS-1 at multiple serine residues [9]. Thus level in the hippocampus of rats after Aβ (1–42) peptides infusion. ASX
probable reason behind ASX mediated reduction in IRS-1 S307 phos- mediated reduction in Aβ level in hippocampus might be the reason
phorylation can be attenuation of Aβ level, oxidative/nitrosative stress behind this effect. Previous studies have also demonstrated ASX
and TNF-α activity in rat’s hippocampus which have been reported in mediated improvement of cognitive functions via the inhibition of NO
our study. production in the hippocampus [66].
Glycogen Synthase Kinase-3β (GSK-3β) is an enzyme which is Enhanced lipid peroxidation and antioxidant enzyme reduction are
widely implicated in Alzheimer’s disease. GSK-3β plays a very sig- some of the extensively used markers for estimation of oxidative stress.
nificant role in the AD and acts as a causative link between Aβ accu- Postmortem analyses of AD brains have earlier revealed increased lipid
mulation and Tau pathology [51]. The activity of GSK-3β is upregulated peroxidation and protein oxidation [67]. Our brain has relatively
during AD and rise in Aβ level is one of the reported mechanisms be- higher oxygen demand and low level of antioxidant enzymes making it
hind that [52]. GSK-3β once activated leads to hyperphosphorylation of at higher risk towards oxidative stress [68]. Accumulation of Aβ can
Tau protein. Hyperphosphorylation of Tau destabilizes the tau protein lead to an extensive increase in Oxidative stress and vice versa creating
and eventually leads to the formation of Neurofibrillary tangles (NFT) a vicious cycle [69]. In our study also, i.c.v. administration of Aβ
[51]. Inhibition of GSK-3β has shown a decrease in tau hyperpho- peptides in Wistar rats leads to the enhanced lipid peroxidation and
sphorylation AD models [53]. Another mechanism behind over acti- reduction in GSH level [70,71]. ASX, known for its potent antioxidant
vation of GSK-3β is insulin resistance in neurons. Insulin resistance effect [18], significantly reversed the toxic effect of Aβ on hippocampal
disrupts insulin signaling mediated PI3k-Akt-mTOR pathway and ear- MDA and GSH level. Previous studies have demonstrated anti-oxidant
lier studies have shown that activation of Akt protein inhibits stimu- activity of ASX in neurons via stimulation of NrF2/HO-1 pathway and
lation of GSK-3β [49]. Even in our study, i.c.v administration of Aβ increase in SOD activity [20,54]. Our study also verified the antioxidant
showed a significant increase in GSK-3β activity. However, Astaxanthin activity of ASX during AD in the hippocampus via a decrease in lipid
dose-dependently inhibited GSK-3β activity in rats that received Aβ peroxidation and increase in GSH activity.
peptide intracerebroventricularly. Both in vitro and in vivo studies have Moreover, histopathological analysis by H & E staining revealed Aβ
also earlier reported the neuroprotective role of ASX via down- mediated neuronal damage in hippocampal sections of Wistar rats
regulating GSK-3β activity [20,54]. These results indicate that ASX can which was characterized by disorganization of neurons, eosinophilic
contribute in suppressing tau hyperphosphorylation mediated NFT stained cytoplasm, nuclei swelling and neuronal shrinking. These
formation during AD via GSK-3β inhibition. findings are in agreement with the previous report which showed that
If Aβ plays a central role in the AD then chronic neuroinflammation Aβ (1–42) induced memory impairment is associated with neuronal
acts as a medium which provides a neurotoxic environment thereby injury in hippocampal areas of rat brain [72,73]. Congo red staining, a
promoting aggravation of AD complications. Chronic release of pro- histological marker for Aβ accumulation confirmed increased level of
inflammatory cytokines (TNF-α & IL-1β) can enhance Aβ via upregu- Aβ in the hippocampus of Aβ (1–42) infused group as shown by pre-
lating Amyloid Precursor Protein (APP) expression and can also lead to vious studies [71,74]. Significant red color stains (marked with yellow
tau hyperphosphorylation [55]. In our study also, Aβ infused hippo- arrows) depicting the accumulation of Aβ can be clearly seen in the
campal tissue showed a significant increase in TNF-α level. Treatment hippocampal section of Wistar rats administered with Aβ (1–42) pep-
with ASX significantly attenuated TNF-α release induced by Aβ (1–42) tides. We for the first time demonstrated histopathological effects of
peptides. These results were consistent with the anti-inflammatory ef- ASX on Aβ (1–42) induced AD model in Wistar rats. ASX treatment
fect of ASX reported in previous studies [20]. Studies have even in- showed significant protection against Aβ mediated neuronal damage
dicated the presence of NFκB binding domain on BACE-1 promoter and plaque formation in the hippocampus. H & E of ASX treated group
region [56,57]. Stimulation of NFκB by TNF-α & IL-1β can upregulate demonstrated mild neuronal toxicity with lesser number of eosinophilic
BACE-1 expression thus, promoting increased production of neurotoxic stained neurons and increased number of healthy neurons with pro-
Aβ [58]. Interestingly, an earlier study has exhibited inhibition of NFκB minent nuclei. Previous studies have reported ASX as a potential can-
in the hippocampus by ASX [59]. Reduction of TNF-α and inhibition of didate to promote neuron plasticity thereby attenuating neurodegen-
NFκB might be the reason behind attenuation of BACE-1 expression eration and also preserving cognitive functions [75]. On the other hand,
thereby leading to a decrease in Aβ production. Congo red staining of hippocampal sections of ASX treated groups ex-
Production of Aβ during AD has shown a cholinergic deficit in the hibited a protective effect against plaque formation dose-dependently.
hippocampus via enhancing Acetylcholinesterase (AChE) activity and This analysis supports the fact that ASX attenuates production and ac-
thus leading to cognitive decline [60]. Acetylcholinesterase can also cumulation of Aβ in the hippocampus of Wistar rats. Also, these positive
lead to the formation of β-amyloid peptide fragments which can ag- histological effects of ASX are in consideration with our behavioral and
gregate to form cytotoxic complexes with the growing fibrils [61]. The biochemical assessments.

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5. Conclusion 1339–1353, https://doi.org/10.1172/JCI57256.

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Jamia Hamdard in collaboration with Sun Pharmaceutical Industries [20] H. Che, Q. Li, T. Zhang, D. Wang, L. Yang, J. Xu, T. Yanagita, C. Xue, Y. Chang,
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Astaxanthin alleviates brain aging in rats by attenuating oxidative stress and in-
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lab, Department of Pharmacognosy and Phytochemistry, Jamia [22] N. Ito, H. Saito, S. Seki, F. Ueda, T. Asada, Effects of composite supplement con-
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Neurobehavioral Pharmacology Laboratory, Department of Medical Alzheimer Dis. 62 (4) (2018) 1767–1775, https://doi.org/10.3233/JAD-170969.
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