Neuroprotective effect of alcoholic extract of Terminalia ... - Wseas.us

Recent Researches in Modern Medicine37 0 C with a thermostatically controlledinfrared lamp. In sham rats the right CCAwas surgically prepared for the insertion ofthe filament, but the filament was notinserted.2.4 Study protocolRats (200-250g) were randomly divided intofive groups of 9 rats in each group, fed withdrug or vehicle for 14 days prior toexperiment. The first group served asnormal and received 1% tween 80 in waterorally. The second group served as shamcontrol, received 1% tween 80 in waterorally. The third group served as ischemiccontrol, received 1% tween 80 in waterorally. The fourth and fifth groups weretreated with TABE 500 and 1000mg/kg/p.o., respectively for 14 days prior toMCAO. After 24 hrs, neurologicalexaminations were performed in all groups.Then six rats in each group were decapitatedto obtain brain tissue samples forbiochemical analysis and remaining threebrains in each group were used forhistopathological studies.2.5 Neurological deficitNeurological deficit in the vehicle- anddrug-pretreated groups were determinedafter 24 hrs of induction. Neurologicalfindings were scored on a 5-point scale [13]as follows: no neurological deficit = 0,failure to extend left paw fully = 1, circlingto left = 2, falling to left = 3, did not walkspontaneously and had decreased levels ofconsciousness = 4. The above procedureswere performed in a blinded manner.2.6 Biochemical estimations24 h after induction of ischemia the animalswere killed by cervical dislocation and theirbrains were dissected out quickly. Theinfarcted area was homogenized in 50 mMphosphate buffer (pH 7.0) containing 0.1mM EDTA to give 5% w/v homogenate.The homogenate was centrifuged at 10,000rpm for 10 min at 0 o C in cold centrifuge andthe resulting supernatant was used forbiochemical estimations.2.6.1 Superoxide dismutaseSOD activity was measured according to themethod of Misra and Fridovich [14], at roomtemperature. 100 µl of supernatant wasadded to 880 µl of 0.05 M carbonate buffer(pH 10.2, containing 0.1 mM EDTA), and20 µl of 30 mM epinephrine (in 0.05%acetic acid) was added to the mixture, andthe optical density values were measured at480 nm for 4 min on a Systronics UV–Visible Spectrophoto-meter; activity wasexpressed as the amount of enzyme thatinhibits the oxidation of epinehrine by 50%which is equal to 1 unit.2.6.2 CatalaseCatalase activity was measured by a slightlymodified version of Aebi, [15] at roomtemperature. 100 µl of supernatant wasadded to 10 ul of 100% ethanol and placedin ice bath for 30 min. The tubes werebrought to room temperature followed bythe addition of 10 µl of Triton X-100. In acuvette containing 200 µl of phosphatebuffer and 50µl of above mixture, 250 µl of0.066M H 2 O 2 in phosphate buffer wasadded. The decrease in optical density wasmeasured at 240 nm for 60 sec in SystronicsUV Spectrophotometer. The molarextinction coefficient of 43.6 Mcm -1 wasused to determine catalase activity which isequal to the moles of H 2 O 2 degraded/mgprotein/min.2.6.3 Reduced GlutathioneReduced glutathione levels were measuredaccording to the method of Ellman [16] atroom temperature. 0.75 ml of supernatantwas mixed with 0.75 ml of 4%sulphosalicylic acid and then centrifuged at1200 rpm for 5 min at 4_C. From this 0.5 mlof supernatant was taken and added to 4.5ml of 0.01 M DTNB, and absorbance wasISBN: 978-960-474-278-3 283