Role of Withania Somnifera (Ashwagandha) in Ischemic Stroke Rat Model Produced by Unilateral Internal Carotid Artery Ligation: A Histological Staining with 2,3,5 Triphenyltetrazolium Chloride and a Behavior Analysis

Research Article

Analysis. Austin J Anat. 2017; 4(3): 1074.

Role of Withania Somnifera (Ashwagandha) in Ischemic Stroke Rat Model Produced by Unilateral Internal Carotid Artery Ligation: A Histological Staining with 2,3,5 Triphenyltetrazolium Chloride and a Behavior Analysis

Koirala S¹*, Shah S¹, Rouniar GP², Koirala B² and Khanal L¹

¹Department of Human Anatomy, B.P. Koirala Institute of Health Sciences, Nepal

²Department of Clinical Pharmacology and Therapeutics, B.P. Koirala Institute of Health Sciences, Nepal

*Corresponding author: Koirala S, Department of Human Anatomy, B.P. Koirala Institute of Health Sciences, Nepal; Email: koirala_sarun@yahoo.com, sarun.koirala@bpkihs.edu

Received: November 08, 2017; Accepted: December 15, 2017; Published: December 22, 2017

Abstract

Introduction: A stroke is a medical emergency and can cause permanent neurological damage. The more extensive the area of brain affected, the more functions that are likely to be lost.

Objectives: To access and compare neurological deficits after 60 min of induction of stroke and 14 days after treatment with ashwagandha in different groups and to see infract area in brain using 2,3,5 Triphenyltetrazolium Chloride (TTC) stain.

Materials and Methods: 21 Male wistar albino rats were randomly allocated to form 3 groups. Commercially available Withania somnifera root extract (Vitamin world. Inc. NY, USA) was used as intra peritoneal route for 14 days and neurological deficits was assessed. Brain of coronal section of 2mm thickness, immersed in TTC (Sigma Aldrich, Merck Group Germany) solution and incubated at 37°C for 20 min. Inferential analysis was done by t- test to see the significant level at p < 0.05 with 95% confidence interval and ANOVA, Post Hoc test was applied to compare between different groups, regression and correlation was seen.

Results: There was significant differences in area of infract between Withania somnifera treated group and non-medicated group p = 0.017. Latency of fall off time in Rota rod test was seen significant (p = 0.000) in between the group. Similarly, 14 days after the induction of stroke, significant differences was present in different groups in rota rod fall off latency time (p = 0.043) and Grid walking test (p =0.002).

Conclusion: Ashwagandha showed improved neurosensorimotor activities in stroke model produced after unilateral ligation of internal carotid artery.

Keywords: Neurological deficits behavior tests; Ashwaganda

Abbreviations

TTC: 2,3,5 Triphenyltetrazolium Chloride; ICA: Internal Carotid Artery; IP: Intraperitoneal

Introduction

A stroke, known medically as a cerebrovascular accident, is the rapidly developing loss of brain function due to disturbance in the blood supply to the brain which is due to ischemia, lack of blood flow, caused by blockage, thrombosis, arterial embolism or a hemorrhage leakage of blood [1]. As a result, the affected area of the brain is unable to function, which might result in an inability to move one or more limbs on one side of the body, inability to understand or formulate speech, an inability to see one side of the visual field [2].

Ischemia induces production of oxygen free radicals and other reactive oxygen species which damage a number of cellular and extracellular elements and blood vessel endothelium as free radicals directly initiate elements of the apoptosis cascade by means of redox signaling, many antioxidant neuroprotectants drugs works at the level of the endothelium (National Institute of Neurological Disorders and Stroke [3,4].

The Primary objectives of the study was to use the acute stroke model of rat produced by unilateral ligation of internal carotid artery, to see the neuroprotective effect of ashwagandha by conducting neurological deficits behavior tests. Secondary objectives were to access the neurological deficits after 60 min of induction of stroke, to compare the neurological deficits in different groups, 14 days after the i.p. injection of ashwagandha and to see the brain infraction area after unilateral ligation of ICA by using 2,3,5 Triphenyltetrazolium Chloride (TTC) stain.

Materials and Methods

Type of study: Experimental, Quantitative type of animal study

Sample size was calculated by using Resource Equation Method in which, a value E was measured, which is degree of freedom of Analysis Of Variance (ANOVA).

E = Total number of animals - Total number of groups

In this study to see the role of ashwagandha in rat stroke model and we had three groups (one group as control and two experimental groups); where value of E was assumed to be 18.

Then; the total number of animals or sample size (n) is:

E= n-3

18= n-3

N= 21, total sample size= 21, 7 in each group

By Resource Equation Method total 21 Male wistar albino rats weighing 200–220 gm, 7 in each group were randomly allocated to form 3 groups.

Animal Housing and Caring: Male wistar albino rats weighing 200–220g was obtained from the departmental animal house of BPKIHS. All procedures was approved by the IRC, BPKIHS. Animal handling for this research was done in accordance to the Guidelines given by Ethical Review Board for the Care and Use of Animals (ERBA) of the Nepal Health Research Council (NHRC, and NIH publication no. 80- 23 revised 1996). Rats were housed in standard plastic cages under room temperature (22–24°C) with a 12-h light/dark cycle. Unless otherwise stated standard laboratory food and water was provided throughout the experiments. Animals were allowed to acclimatize to the laboratory conditions for seven days prior to the experimental procedures. All efforts were made to minimize the number of animals used and their suffering.

Ethical consideration: Ethical clearance was obtained from Institutional Review Committee (IRC) and approval from Research Committee, BPKIHS, IRC/ 476/ 015.

Requirements and materials: Normal saline, surgical gloves, adhesive tapes, curved scissors, surgical blade, curved forceps, small scissors, cotton, surgical thread, 4.0 silk sutures, 1, 3ml, 5 ml syringe, 2% lidocaine HCl (Xylocaine), 16 number needle, betadine solution and ointment, magnifying glass, razors, cidex solution.

Surgical procedures and experimental design

Animals were anesthetized with Ketamin 50mg/ kg/ i.p. and turned supine position, and fixed to the surgical table using a adhesive tape

The incisional site was shaved and betadine solution was applied in the skin to make aseptic area for surgical process.

Three different experimental groups were made as below:

• Control group was made with only skin incision in neck region coded as Group A0,

• Unilateral internal carotid artery ligation only coded as Group A1

• Unilateral internal carotid artery ligation and treatment with ashwaganda 10 mg/kg i. pt. for 14 days coded as Group A2

A midline neck incision was made and the soft tissue over the trachea was retracted gently.

The Right common carotid artery was carefully isolated from the vagus nerve and the internal carotid artery was ligated using a 4.0 silk suture to create a acute model of stroke in rat model and the skin was sutured.

After 60 min the silk suture was removed to re-vascularize the brain. The mid line neck incision was then sutured.To relief pain and discomfort in the post-operative period, topical 1% lidocaine gel was applied on the sutured area and 0.5 ml saline i.p. was given for volume replenishment after surgery. Ashwagandha 10mg/kg/ i. p. (Root extract-Vitamin world. Inc. NY, USA) was injected in Group coded as Code: A2 for 14 days.

After 14 days of treated in Group A2,at the end of the study neurological deficits was assessed in Group A1, and A0, values were filled in data collection sheet and the animal was given high dose of sodium pentobarbitol (40 mg/ kg/ i.p) to deeply anesthetized, decapitated and brain was removed carefully.

Neurological deficits test and scoring

The acute neurological deficit of the animals in each group was evaluated after reperfusion after 60 min, by placing the rat in the floor, Grid walking test, Grip test and Rota rod test was done at the end of day 14 and values were filled in data collection sheet.

Staining method and image analysis

Properties of stain: 2,3, Triphenyltetrazolium chloride

Chemical structure of 2,3, 5 TTC is. (T8877, Sigma Aldrich Company) (Figure 1).