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.