An Experimental
Study Evaluating the dose of oral D-Galactose on aging Induction in wistar
albino male Rats
Sriram B. S1, Ravichandra.V2*,
Rajendra Holla3, Shailaja Moodithaya4, Kishan Prasad5
1Research
Scholar, Department of Pharmacology, K.S Hegde Medical Academy,
Mangaluru -575008, Karnataka, India
2Associate
Professor, Department of Pharmacology, K.S Hegde Medical Academy,
Mangaluru -575008, Karnataka India.
3Professor
and HOD, Department of Pharmacology, K.S Hegde Medical Academy,
Mangaluru -575008, Karnataka India.
4Additional
Professor, Department of Physiology, K.S Hegde Medical Academy,
Mangaluru -575008, Karnataka India.
5Professor,
Department of Pathology, K.S Hegde Medical Academy, Mangaluru -575008,
Karnataka India
*Corresponding Author E-mail:
ravi75chandra@yahoo.co.in
ABSTRACT:
Objective: The present study aimed to evaluate dose required
for oral D-galactose in aging induction on Wistar albino male rats, by
evaluating Cognitive, Biochemical and Histological variables. Methods:
Male Wistar albino rats were randomly separated into six groups (n=6). Group 1
(Control) received distilled water; Group 2 (Standard) received 120mg/kg subcutaneous
(S.C) D -galactose; Group 3,4,5 and 6 received oral D-galactose in dose of 100,
120, 150 and 200mg/kg body weight respectively. Results: Group 2 (120mg/kg)
S.C and Group 3 (100mg/kg) orally administered D-galactose, showed increase in
plasma MDA (Malondialdehyde) levels, appearance of lipofuscin pigment
deposition in hepatocytes as well as increase in duration to find food along
with total errors in Cognitive tests respectively compared to control. There is
also significant decrease in plasma Glutathione levels in Group 3 compared to
standard. Conclusion: Oral D-galactose at dose of 100mg/kg can be
employed as model for aging induction.
KEYWORDS: D-galactose, Radial maze, Heb William maze,
Malondialdehyde (MDA), Glutathione (GSH), Lipofuscin.
INTRODUCTION:
Aging is a chronic biological process characterized by
alterations in physiological and psychological functioning ultimately leading
to death[1]. To screen for antiaging drugs in preclinical studies
D-galactose model of aging (rats/mice) is employed. D-galactose is a reducing
sugar which gets converted to Galactitol, this induces oxidative stress inside
mitochondria by forming Advanced Glycation end (AGE) products[2].
Chronic
systemic D-galactose administration by subcutaneous/intraperitoneal causes
oxidative damage by activating nitric oxide synthase, NF
B signaling pathway which resembles characteristic
signs of Alzheimer’s disease in rodents[3]. Many studies also points
out role of proinflammatory cytokines like IL-6 by inducing inflammation, still
mechanism of action of D-galactose in aging induction is not very clear.[4].
On the other side there is insisting evidence regarding administration of oral
D-galactose produces protective effect in animal model of Alzheimer’s disease
induced by streptozotocin, attributing its role as concentration or
administration route dependent[5]
Considering
the fact that many studies conducted so far has administered D-galactose by
intraperitoneal /subcutaneous routes for induction of aging, oral
administration has not received sufficient attention. The present study
evaluated the effects of oral D-galactose in various doses by comparing it with
subcutaneous (standard) and control. Since chronic oral D-galactose
administration can serve useful and alternate way for inducing aging.
MATERIALS AND METHODS:
Chemicals:
D-galactose (Batch No - 8870910, Sisco
research laboratories private limited, India). All other chemicals and regents
were of analytical grade.
Animals:
Male rats of 3 months old (200-250g) was
used for study, after obtaining permission from Institutional Animal Ethics
Committee (115/1999) KSHEMA. They were kept in standard laboratory environment
with food and water ad libitum. Rats were maintained in natural light
and dark cycle at room temperature of 22 -24oC and strictly followed
guidelines of CPCSEA of government of India.
Experimental design:
Rats are divided into 6 groups, (n=6 rats)
in each group Group 1: Control-1.0ml of distilled water is administered orally.
Group 2 -1.0ml of D-galactose is administered subcutaneously. Group 3 -1.0ml of
D-galactose (100mg/kg) dissolved in distilled water given orally. Group 4 -1.0ml
of D -galactose (120mg/kg) dissolved in distilled water given orally. Group 5 –
1.0ml of D-galactose (150mg/kg) dissolved in distilled water given orally.
Group 6 -1.0ml of D -galactose (200mg/kg) dissolved in distilled water given
orally. The experiment is carried out for 42 days and on 43 rd day rats are
subjected for cognitive analysis using Radial and Heb williammaze. On 54th day
rats are sacrificed by euthanisia, liver is taken and blood is drawn using cardiac
puncture and subjected for histological and biochemical analysis.
Radial maze:
The cognitive parameters like spatial and working
memory are analyzed using.
Radial maze:
It is a locally fabricated wooden radial arm maze;
elevated 50cm above the floor consist of an octagonal central hub 36cm in
diameter with eight radial arms, latency to find food is recorded as measure of
working memory evaluation
Heb William maze:
Heb-Williams Maze: It is an “Incentive based
exteroceptive behavioral test” based on spatial working memory of an animal. In
this model, the animal learns the path to trace the food the incentive with
training, it requires less time and commits less number of errors to reach the
food [6]
Liver lipofuscin:
In each group, rats are sacrificed and
liver cells are subjected for histological analysis using Haematoxylin and
Eosin staining and lipofuscin is analysed in liver.[7]
Blood samples:
Blood is collected using cardiac puncture
and collected in heparinized tube, then subjected for centrifugation and plasma
is separated out and MDA (malondialdehyde) and Glutathione levels are
determined using spectrophotometer.[8]
Statistical analysis:
Statistical analysis was performed using
Graph pad prism version 5.0.One way ANOVA was performed followed by Dunnetts
multiple comparison test as post hoc. p value less than 0.05 was considered
significant.
Radial maze:
Table
no. 1 showing the results of Latency to find food of Radial maze experiment on
post 44, 45, 46, 47th day of D -galactose administration
|
Groups
|
44th day
|
45th day
|
46th day
|
47th day
|
|
NC
|
267.5±3.33
|
175.5±2.25
|
163.3±4.54
|
146.7±2.87
|
|
D-Gal S.C
|
423.2±3.86***
|
396.8±2.40***
|
384.3±3.83***
|
324.2±4.07***
|
|
D-Gal 1
|
491.8±7.91***
|
515.2±2.85***
|
525.7±2.58***
|
547± 2.82***
|
|
D-Gal 2
|
273.8±2.85***
|
255.5±3.72***
|
245.5±2.16***
|
215±2.19***
|
|
D-Gal 3
|
283.8±2.85***
|
285.2±2.56***
|
265.5±2.21***
|
226.3±2.94***
|
|
D-Gal 4
|
277.3±4.17***
|
264.8±2.22***
|
257.2±3.06***
|
234.5±2.34***
|
N=
6 rats in each group, are expressed as Mean ± SD, One way ANOVA followed by
Dunnett’s multiple comparison test is used,***p= 0.001 compared to control,
p< 0.05 is considered as significant.
Table
no 2 showing the results of Total errors of Radial maze experiment on post 44,
45, 46, 47th day of D -galactose administration
|
Groups
|
44th day
|
45th day
|
46th day
|
47th day
|
|
NC
|
9.16±4.49
|
9.33±2.16
|
8.00±3.03
|
4.5±3.27
|
|
D-Gal S.C
|
12.33±5.00
|
7.83±4.16
|
8.66±4.17
|
7.5±3.93
|
|
D-Gal 1
|
17.67±2.58**
|
17.83±3.31**
|
14.00±3.46**
|
12.5±2.42**
|
|
D-Gal 2
|
10.17±2.40
|
10.00±3.28
|
7.66±3.32
|
4.00±3.03
|
|
D-Gal 3
|
10.67±3.07
|
9.5±3.20
|
4.83±3.06
|
4.33±3.55
|
|
D-Gal 4
|
14.5±3.61
|
9.83±2.63
|
8.5±3.27
|
5.83±3.54
|
N=
6 rats in each group, Values are expressed as Mean ± SD, One way ANOVA followed
by Dunnett’s multiple comparison test is used test is used,**p= 0.01 compared
to control , p< 0.05 is considered as significant.
Heb William maze:
Table
no 3 showing the results of Time taken to reach Reward chamber (TRC) of Heb
William maze on post 49, 50, 51 and 52nd day of D -galactose administration
|
Groups
|
49th day
|
50th day
|
51stday
|
52nd day
|
|
Control
|
46.67±3.32
|
35.33±3.07
|
24.17±8.88
|
22.17±11.3
|
|
D-gal S.C 120 mg/kg
|
135.3±2.16***
|
116.5±3.01***
|
87.5±3.01***
|
109.2±2.13***
|
|
D-Gal Oral100mg/kg
|
227.2±2.04***
|
209.5±1.87***
|
196.0±3.63***
|
184.7±2.80***
|
|
D-Gal Oral 120 mg/kg
|
125.3±3.32***
|
114.7±2.50***
|
97.5±3.67***
|
94.83±2.85***
|
|
D-Gal Oral 150 mg/kg
|
109.2±1.72***
|
107.7±2.73***
|
103.3±2.65***
|
108.00±2.60***
|
|
D-Gal Oral 200 mg/kg
|
147.2±3.12***
|
111.0±2.96***
|
107.3±3.67***
|
117.8±2.13***
|
N= 6 rats in each group, Values are
expressed as Mean ± SD, One way ANOVA followed by tukeys multiple comparison
test is used ,p= 0.001 compared to control , p< 0.05 is considered as
significant
Plasma MDA levels
N=
6 rats in each group, Values are expressed as Mean ± SD, One way ANOVA followed
by Dunnett’s multiple comparison test is used, ***p= 0.001 compared to control,
p< 0.05 is considered as significant.
Plasma
GSH levels
N= 6 rats in each group, Values
are expressed as Mean ± SD, One way ANOVA followed by Dunnets multiple comparison
test is used, *p= 0.05 compared to control; **p= 0.01 compared to control;
p< 0.05 is considered as significant.
Histological
analysis:
|
Group 1 Control
|
Group 3 D -galactose oral
(100 mg/kg)
|
|
Group
2 D-galactose S.C (120 mg/kg)
|
Group
4 D-galactose oral (150 mg/kg)
|
|
Group
5 D-galactose oral (150 mg/kg)
|
Group
6 D -galactose oral (200 mg/kg)
|
RESULTS:
There
is significant increase in latency to find food as well as in time taken to
reach reward chamber in all groups compared to control in radial maze and hebwilliam
maze respectively (Table No.1 and 3, p< 0.001 compared to control). With
reference to total number of errors committed it is observed only in Group 3 (D-galactose
100mg/kg orally) (Table No.2, p< 0.01) compared to control.
The
plasma MDA levels are raised in all groups (Figure no.1, p< 0.001) compared
to control. However plasma Glutahione is decreased in Group 2 and Group 3
(Figure no.2, p< 0.05 for group 2, p< 0.01 for group 3) compared to
control.
Histologically,
in hepatocytes there is appearance of Lipofuscin pigment in kupffer cells which
is observed in group 2 and group 3 compared to control and in all other groups
there is no significant changes.
DISCUSSION:
D-galactose
which is a reducing sugar used as an experimental model for inducing aging by
administering via subcutaneous/intraperitoneal route. Till date there is
limited findings regarding its effectiveness via oral route. The present study
aimed at understanding D-galactose efficacy and dose through oral route in
aging induction. There is significant increase in latency to find food and time
taken to reach reward chamber in all groups which could be attributed to the
cognitive decline caused by formation of galactitol via neuroinflammation[9].
This indicates effectiveness of D-galactose in all doses as well as route in
aging induction. Interestingly D-galactose at dose of 100mg/kg (group 3) caused
increase in errors committed compared to control which was not observed in
other groups, this could be dose dependent effect on working memory[10].
Many
studies has confirmed role of oxidative stress in aging through free radicals
formation[8]. The plasma MDA levels were significantly increased in
all groups compared to control which indicates D-galactose potential in raising
oxidative stress thereby contributing to aging. Interestingly the plasma
glutathione which is protective antioxidant was decreased only in group 3 (100mg/kg
orally) and not in other groups compared to control. This could be due to hormetic
effects of D-galactose which is characterized by High dose protection and low
dose determination. [11]
Lipofuscin
is a wear and tear pigment which is specific of aging process and found in
retina, heart, skin and liver of aging animals. [12] In the present
study Lipofuscin pigment was observed in hepatocytes of group 2 (120 mg/kg S.C)
and group 3 (100mg/kg) suggesting aging induction.
CONCLUSION:
D-galactose
given orally at dose of 100mg/kg is effective as subcutaneous/intraperitoneal
model in aging induction and can be employed as alternate route for chronic
administration.
ACKNOWLEDGEMENT:
We
would like to thank Dr. Prakash P.S., Dean, K. S Hegde Medical Academy for
extending support and encouragement for the study.
CONFLICT OF INTEREST:
The Author(s) declare(s) that they have no conflicts
of interest to disclose.
AUTHORS
CONTRIBUTION:
First
author (S.B.S and R.V.) initiated and conducted the research. The second
authors (S.M, R.H and K.P) conducted animal care and prepared the manuscript.
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