A Middle Cerebral Artery Occlusion Modelling Study of Combinatorial Treatment (Acute Phase) and Post-Ischemic Exercise (Chronic Phase) in Rats

Abstract

Stroke is the third leading cause of death and disability both locally and internationally. Despite the enormous availability of scientific literatures on brain ischemia, currently, only intravenous recombinant tissue-type plasminogen activator (rt-PA, Alteplase) thrombolytic treatment and antiplatelet therapy are approved pharmacological managements which induce the degradation of the arterial thrombus and restoration of vessel patency and blood flow to improve clinical prognosis. In the present study, experimental brain ischemia was modeled with intraluminal suture technique induced middle cerebral artery occlusion in rats (MCAo). Stroke can be temporally classified into varying phases where pharmacological intervention plays an important mediating role in the early phase and physiotherapy in addition to medication are proven clinical approach for managing brain ischemia later. Studies showed that in early phases of MCAo, cell death advances via a continuum between apoptotic and non-apoptotic modes that hinged on cellular energy level. In the current acute phase study, concomitant administration of both z-VAD-fmk and 3-AB yielded better outcome with bigger infarct volume reduction in comparison with single inhibitor administration following MCAo. In addition, combinatorial treatment remained effective even when administered at 24hr post MCAo. Although there was no general correlation between intracellular ATP level and infarct size, only treatment with PARP inhibitors had showed that intracellular ATP level was inversely related to the size of infarct. Although physiotherapy has been clinically proven, its molecular basis has not been well elucidated. The present chronic phase study demonstrated that eight weeks of moderate forced treadmill post-ischemic exercise were able to reduce secondary damages, and achieved better clinical outcome following brain ischemia via multi-modal neuroprotective and angiogenic (in turn supporting neuroregeneration) mechanisms. The elevated level of activated caspase-3 seen in the MCAo rats was reduced subsequent to post-ischemic exercise when TGF-ß1 was further increased and smad7 was reduced concomitantly in the ipsilateral cortices of the MCAo-runner rats suggesting an anti-caspase-dependent-apoptosis property of TGF-ß1¿s role in neuroprotection. Paradoxically, up-regulated positive TUNEL staining within the ipsilateral cortices of the MCAo-runner rats may be taken as an indication as the body¿s effort to modulate imminent cell death via apoptotic pathways which will be less prone to inflammatory events. In addition to neuroprotection, neuroregeneration after ischemic insults is pivotal for a more favorable prognosis. For neuroregeneration to happen, increase in angiogenic and erythropoietic activities are needed to form new and effective oxygen delivery vascular structures. Interestingly, distinct Pecam-1 immunoreactivities were seen with marginal PDGFB increased in sham-runner rats. However, opposite observations could be observed in the ipsilateral cortices of the MCAo-runner rats with up-regulated PDGFB expressions that were not reciprocated by strong Pecam-1 immunoreactivities. Thus suggested that brain ischemia may produce or remove another unidentified factor that may form either inhibitive or necessary condition for the angiogenic property of PDGFB to work. With changes to the angiogenic/erythropoietic factors and vasculatures, antioxidants¿ profiles were also dissimilar in the rats possibly reflecting the differential conditions. In its essence, the present study has shown that combinatorial administration of both z-VAD-fmk and 3-AB was better than if the inhibitors were given individually in the acute phase stroke. The current data from the chronic phase of brain ischemia experiments also showed that post-ischemic exercises could induce endogenous factors that may confer neuroprotection and possibly neuroregeneration.