MATERIAL AND METHODS
Adult Wistar rats were procured from Institutional Advanced Small Animal Facility PGIMER, Chandigarh, India. The necessary approvals were collected before experimental studies. All the experimental animals were accommodated in the groups of 2/3 under standard polypropylene cages under controlled hygienic environmental conditions and had proper access to food and waterad libitum at temperature 23 ± 4ºC under a 12-hour light-dark cycle throughout the experiment.
Experimental Design and Statistical Analysis:
TBI Model Standardization by Marmarou’s weight-drop method: TBI instrument was designed with essential modifications to stabilize the actual energy impact for model standardization which included 6 subgroups of Wistar rats (150-450gm). The standardization was done by calculating the difference in the impact of injury concerning the adjustments of height and the weight falls. The impact of freefalling brass weights was correlated with by physical formula of Impact force and the reading of the impactometer. The rats were anesthetized withketamine and xylazine (i.e. 100mg/kg and 10mg/kg) before inducing the diffuse type injury by Marmarou’s weight drop method on Day0. The injury progression and neurobehavioural tests were assessed on Day0, Day1, Day3, Day7, and Day14. The treatment experiments were continued with the selected group having a lower seizure threshold and less mortality after TBI.
Epileptogenesis confirmation for Post-traumatic epilepsy: After following the TBI procedure of Marmarou’s weight-drop method, rats were administrated with a sub-convulsive dose of pentylenetetrazole, 35mg/kg after 2 weeks of surgery i.e. on Day14. The rats were assessed for 2 hours for seizure detection in a plexiglass chamber(Racine et al., 1972). The effect of Post-traumatic complications was assessed for molecular changes in the rat hippocampus and final results were correlated with histological changes.
Evaluation of neuroprotective effects of Flufenamic Acid, Fasudil Hydrochloride and Valproic Acid in TBI induced Epileptogenesis model: Four treatment groups have been completed with the administration of Fasudil Hydrochloride (FH), Flufenamic Acid (FFA), Valproic Acid (VPA), and Valproic Acid + Flufenamic Acid (FFA). FH was dissolved and diluted in normal saline before use and was administered by i.p. route of 10 mg/kg. FFA was dissolved in CMC and was administered by the oral route as 20mg/kg after standardization to injured rats. VPA was dissolved and diluted in normal saline before use and was administered by i.p. route as 350 mg/kg animals. Forth treatment group has been designed with the combination of a half dose of VPA and the half dose of more effective treatment between FH and FFA. All the drugs were administered to TBI-induced rats after 6 hours of the injury and once every 24 hours for 14 Days. An equal volume of vehicle was administered in control and sham animals i.e. normal saline. No drugs have been given to TBI and EPLT group. All the groups were examined for seizure scoring with sub convulsive dose of PTZ i.e. 35mg/kg after Day14 for 2 hours except control and sham groups. At the end of the animal experiment, the rats were sacrificed under ether anesthesia by decapitation procedure. The brains were washed by saline perfusion method before collection and stored in appropriate storage conditions till molecular experiments.