Pre ischemic administration of l name and citicoline exerts synergistic neuroprotection in rat model of transient focal cerebral ischemia

Abstract

Current investigation was carried out to determine the suitable therapeutic time window for L NAME a non selective NOS inhibitor citicoline a membrane stabilizer and their combination to ascertain the possible synergistic neuroprotective effects in rat model of transient focal cerebral ischemia Transient focal cerebral ischemia was induced in rats by middle cerebral artery occlusion reperfusion surgical procedure wherein four hypen zero nylon filament was inserted into the external carotid artery and proceeded further into the internal carotid artery until a resistance was felt The nylon filament was held in position by ligating it along with the internal carotid artery with suture thread The rat was sutured externally and maintained in cage under incandescent lamp for two hours After two hours of ischemia the external suture of the animal was removed and the nylon filament was pulled out slowly allowing restoration of blood flow in the brain for seventy h Ischemia was considered to be initiated from the time of insertion of the filament into the internal carotid artery and reperfusion was considered when the filament was totally pulled out from the middle cerebral artery L NAME and Citicoline were administered at three different time phases of ischemia I e thirty min before the induction of ischemia pre one h after the onset of ischemia during and two h after the onset of reperfusion post After seventy h of reperfusion the animals were assessed for neurological and behavioural anxiety cognition and motor activity functions The results of neurological behavioural biochemical and molecular investigations revealed that pre ischemic administration as the suitable therapeutic time window of L NAME and Citicoline Hence in the combination study both L NAME and Citicoline were administered at the pre ischemic phase to determine the synergistic neuroprotection Following behavioural assessments the animals were sacrificed and the ipsilateral brains were collected for the determination of neurochemicals Glutamate and Aspartate and biochemicals glutamine synthetase activity ATP NAD nitrate or nitrite sodium potassium ATPase super oxide dismutase and reduced glutathione For infarct measurement histopathology and GFAP expression whole brains were collected Collected whole brains were stored in the deep freezer for brain infarct measurement and in ten percentage neutral buffered formalin for histopathology and GFAP expression respectively Neurological deficit was assessed following Bedersons scoring method Anxiety was determined following elevated plus maze cognition in Y maze and motor activity was assessed using rota rod test and beam walk test Neurochemicals glutamate and aspartate were determined in HPTLC and biochemicals were determined following standard protocols ATP and NAD was determined using HPLC Brain infarction percentage was evaluated using TTC staining Histopathology of the brain sections were performed using H and E staining Expression of Glial Fibrillary Acidic Protein was determined using immunohistochemistr Irrespective of the treatment phase both L NAME and Citicoline produced significant neuroprotection when compared to vehicle treated ischemic reperfusion group However in both the cases pre ischemic administration exerted best therapeutic outcome The neurochemicals glutamate and aspartate were decreased along with decrease in glutamine synthetase nitrate or nitrite in the treatment groups in the order of Combination gt Citicoline gt L NAME An increase in the ATP NAD sodium potassium ATPase super oxide dismutase and reduced glutathione was observed in the treatment groups in the order of Combination gt Citicoline gt L NAME In the TTC staining percentage brain infarction was significantly reduced in all the treatment groups when compared to IR group Neuronal damage was found to be reduced in the treatment groups when compared to IR Expression of GFAP was also reduced significantly in the treatment groups than the IR group Brain infarction neuronal damage and GFAP expression was reduced in the treatment groups in the order of Combination gt Citicoline gt L NAME It is evident from the observations of the present study that combination of L NAME and citicoline exhibits synergistic effect and affords better neuroprotection than the individual administration of L NAME and Citicoline Results also demonstrate that the suitable therapeutic time window of L NAME and Citicoline is before the onset of ischemia Hence it is concluded that L NAME Citicoline and their combination may be used as a prophylactic agent in the patients with known risk of stroke such as hypertension cardiac abnormalities such as patent foramen ovale and atrial septal aneurysm carotid artery stenosis and transient ischemic attacks