IN VITRO MODEL OF CEREBRAL STROKE: THERAPEUTIC POTENTIAL OF TRANS RESVERATROL AND CURCUMIN

Abstract

Ischemic cerebral stroke, which occurs due to loss of blood supply to the brain, is the second largest cause of morbidity and mortality across the globe. Further, generation of free radicals in brain is significantly enhanced during reperfusion and produces detrimental effects. However, various mechanisms of neuronal injury in stroke have been suggested. Thus, attempts were made with the drugs, which can intervene in these biochemical events as to prevent the cerebral injury. Few initiatives have already been taken using cell lines viz. PC12 cells, SH-SY5Y cells, human neuroblastoma, primary cultures of neuronal cells and mixed cultures of neuronal and glial cells towards developing in vitro models to understand the mechanisms involved in the pathophysiology of cerebral stroke. The present investigations were carried out to develop a rapid, cost effective and more sensitive mechanism based in vitro model of ischemic stroke as first tier of screening of neuroprotective drugs for their antistroke potential. To achieve these objectives, initial experiments were carried out to optimize the various conditions for oxygen glucose deprivation (OGD) and reoxygenation using PC12 cells. Further, for validation of the in vitro model developed during the study, trans resveratrol and curcumin were used for three time schedules i.e. pre-treatment (for 24 h prior to OGD), post-treatment (for 24 h after OGD) and whole-treatment (for 24 h prior to OGD + 6 h during OGD + 24 h after OGD) at different non cytotoxic concentrations. Trans resveratrol and curcumin were selected as model drugs to study the validity of PC 12 cells-OGD in vitro model, since both of them are known to have strong multiple pharmacological properties. Further, extrapolative studies were carried out using trans resveratrol as a test drug in rat middle cerebral artery occlusion (MCAo) model of ischemic stroke. Experiments were carried out to identify the optimum durations of oxygenglucose deprivation (OGD) and reoxygenation. Further, the precise role of glucose as one of the limiting factors during reoxygenation was demonstrated. The OGD of 6 h followed by a reoxygenation of 24 h with 4-6 mg/ml glucose concentrations in culture medium was found to be the optimum conditions to create the cerebral stroke like situations under in vitro environment using PC 12 cells. Using optimized conditions of OGD-reoxygenation and glucose concentration in culture medium during reoxygenation, studies were carried out for numbers of endpoints involved in the process of causation and progression of cerebral stroke. Significant alterations in the oxidative stress indicator markers have been recorded, which shows the responsiveness of the system to the ischemic insult. OGD induced elevated levels of PGE2 were also indicative that PC12 cells growing in the experimental setup have responded in the same fashion as in case of ischemic stroke. Levels of nitric oxide (NO) following OGD-reoxygenation insult were found to be increased. More than three fold increase in the accumulation of Ca following 6 h OGD and 24 h of reoxygenation was observed. The expression profile of genes involved in apoptosis and oxidative stress induced damages has been studied and significant responses to the ischemic insult in cultured PC12 cells have been found. Statistically highly significant decrease in the dopamine DA-D2 receptor levels were detected in PC12 cells under the experimental conditions. In general, a dose dependent protective potential could be observed in both pre and whole-treatment groups of trans resveratrol. A clear cut synergistic response was observed in whole-treatment group. This synergistic response in efficacy might be due to cumulative response of pre and post-treatment in whole-treatment group. In case of intracellular calcium, it seems that either an additional period of pretreatment or higher concentrations of trans resveratrol might increase the efficacy to restore the levels, as following the post and whole-treatment at highest concentration (25uM), the values brought to the basal level. All the treatment schedules and concentrations of trans resveratrol used were significantly effective in reducing PGE2 levels in PC12 cells received 6 h of OGD and 24 h of reoxygenation. These findings confirmed that OGD-reoxygenation insult reduces the dopamine DA-D2 bindings by 51.6% of normoxia control. Unlike rest of the endpoints studied, only pre-treatment of trans resveratrol was found to restore the bindings in a dose dependent manner, whereas no recovery could be seen in case of whole-treatment group. Expression of the genes were found to drop down significantly and reached almost to the basal level following whole and pre-treatment of trans resveratrol. Contrary to the transcriptional (mRNA) changes, expressions at translational (protein) level were not found to be recovered significantly for any of the gene studied. 11 The treatment schedule of curcumin was similar as in the case of trans resveratrol. Except one or two endpoints, in general pre-treatment of curcumin was found to be better than other treatment schedules followed by whole-treatment group, whereas, the post-treatment group was observed minimum restorative under the experimental set up. Though curcumin is well known for its anti inflammatory and antioxidant activities but at the same time, pro-oxidant activity of curcumin at higher doses and long exposure period has also been reported in various experimental models. In case of intracellular calcium, it is clearly seen that the lowermost concentration of curcumin used in pre-treatment was able to restore the values near to normoxia control, while higher doses in the same group can be considered as pro-oxidant. Like trans resveratrol in case of curcumin also, all the treatment schedules and concentrations were found effective to control the OGD-induced alterations in PC12 cells. However, no adverse affect of higher doses could be detected as almost equal magnitude of reduction in PGE2 levels was recorded. The findings for DA-D2 receptor following the curcumin treatment were almost similar to the trans resveratrol. Incidentally, the trend for the OGD-reoxygenation induced alteration in the expression of mRNA of studied genes known to be involved in the cascade of ischemic cerebral stroke was similar following the treatment of both curcumin and trans resveratrol. To the best of my knowledge, following OGD-reoxygenation insult induced changes in mRNA expression of studied genes and restoration by trans resveratrol and curcumin in PC12 cells have never been studied earlier. Similar to the trans resveratrol, recovery at protein level following curcumin treatments was non significant. On the basis of the observations on the anti-stroke potential activity of trans resveratrol and curcumin, it could be concluded that PC12 cells-OGD in vitro model for cerebral stroke is able to mimic most of the endpoints happened during stroke and responded specifically for both the drugs tested i.e. curcumin and trans resveratrol. In case of trans resveratrol, whole-treatment group was found to be better than other treatment groups while pre-treatment of curcumin could be recorded better. In totality, curcumin has shown better protective potential than trans resveratrol in PC 12 cells- OGD in vitro model under the experimental conditions. However, trans resveratrol

was selected for in vivo validation studies in rat MCAo model of cerebral stroke since studies using curcumin in same in vivo model have already been carried out by others. In vivo studies were conducted using rat MCAo model of cerebral stroke to study the extrapolation of the data received through PC12 cell-OGD in vitro model. Trans resveratrol at 20 mg/kg, p.o.(per oral dose) was selected for the study. The data confirmed that the ischemic insult following 24 h and 7 days reperfusion was capable of inducing significant neurobehavioral impairment in MCAo rat model. A self recovery was also reported by 7 days of reperfusion without any drug treatment. The pre-treatment of trans resveratrol of seven days was found to be the best amongst the treatment groups as it significantly restored the parameters studied in time dependent manner i.e. 24 h and 7 days of reperfusion. Oxidative stress indicator markers were found to be altered following MCAo-reperfusion. A decrease in body weight following MCAo, observed in the present study, is consistent with the earlier reports of post ishemic loss in body weight. Interestingly, a significantly lower magnitude of antioxidant activity of trans resveratrol in post-treatment schedules was observed. Further, after multiple doses of post-treatment i.e. for 7 days during reperfusion period trans resveratrol was rather found to be causing adverse or no effects in general for the parameters studied. These findings are also in coordination with the infarction volume observed in the present investigations. These studies would be of immense significance not only in understanding the mechanisms involved in the ischemic cerebral stroke at cellular and molecular levels, but would also be useful in adopting this PC 12 cells-OGD system as a cost effective, reliable, more sensitive with better predictive values for screening of antistroke potential of drug candidate molecules in very rapid way. Further, this system would also be useful to extrapolate the data with rat MCAo model of ischemic stroke and to the clinical situations.