Stroke is a clinical condition caused by a sudden disruption in the cerebral blood supply that most often leads to a focal, but occasionally even a global neurological deficit. Ischemic strokes that occur due to a thrombotic or embolic occlusion of a cerebral blood vessel account for about 80% of all stroke cases and is one of the major causes of mortality and adulthood disabilities worldwide. Pathophysiologically, an ischemic stroke is characterised by the presence of two regions; a central core and a peripheral penumbral area, based on their differential cerebral perfusion thresholds. Cerebral ischemia within the ischemic penumbra triggers a complex series of receptor-mediated molecular mechanisms that activate many downstream kinases and transcription factors known to influence neuronal apoptosis in the penumbral region. However, the current understanding regarding how different receptor-mediated mechanisms could activate these signaling molecules and thereby modulate neuronal apoptosis in the penumbra still remains poor. Moreover, recombinant tissue plasminogen activator (rt-PA), the only drug approved for therapy in acute ischemic stroke patients does not inhibit or prevent the activation of these receptor-mediated mechanisms that promote ischemia-induced cell death. Hence, there is an urgent need to explore such receptor targets as avenues for neuroprotection during stroke therapy. Thus, the focus of this research thesis is to look at the roles of three novel receptors namely: adiponectin receptors (ADRs1&2), Ephrin A2 (EphA2) and complement anaphylatoxin receptors (C5aR/CD88) in ischemic stroke.
The vast majority of the known biological actions of adiponectin are mediated by two transmembrane adiponectin receptors (ADRs 1&2). Thus far, the roles of ADR expression in the brain have been demonstrated in the arcuate and the paraventricular nucleus of hypothalamus, where its activation affects food intake. However, their roles in ischemic stroke remained unknown. The results of the adiponectin receptor project demonstrated that primary cortical neurons express both, ADRs 1 & 2. Furthermore, the activation of neuronal ADR1 in the presence of either of its ligands, globular as well as trimeric adiponectin, promoted neuronal cell death by the downstream activation of p38-MAP kinase and AMP-kinase. This neuronal cell death was further exacerbated by addition of adiponectin during an in vitro model of stroke thereby demonstrating a role for neuronal ADR1 in ischemic strokes.
Similarly, EphA2 receptor, an important guidance molecule during neurogenesis was investigated using in vitro and in vivo techniques of experimental ischemic stroke. It was observed that following middle cerebral artery occlusion (MCAO) in vivo, the EphA2 receptor deficient mice (EphA2-/-) had better blood brain barrier integrity as compared to their wild type (WT) counterparts, and this contributed to a better stroke outcome. In addition, a direct neuronal protective effect was also observed, where EphA2-/- cortical neuronal cultures were less vulnerable to apoptotic cell death as compared to WT neurons, following an in vitro ischemic insult. Together, the dual protective effects of EphA2 receptor deletion at the blood-brain barrier and cortical neuronal level provides a valuable insight into the possibility of exploring its therapeutic potential during stroke intervention.
The third project of this research thesis was designed to investigate the possibility of combining neuroprotective strategies in ischemic stroke therapy. For this purpose, we decided to explore the effect of combining hypothermia and innate immune C5a-receptor (CD88) antagonism, following an ischemic insult. Although many studies have shown that hypothermia targets several cellular processes, its effects on innate immune receptor-mediated apoptotic death still remain unclear. Moreover, whether inhibiting the signaling of innate immune receptors like complement anaphylatoxin C5a receptor (CD88) plays a role in this hypothermic neuroprotection still need to be deciphered. In order to explore the involvement of innate immune receptors in hypothermia induced neuroprotection during ischemic strokes, we investigated whether inhibiting the signaling of complement anaphylatoxin C5a receptor (C5aR/CD88) could attenuate stroke induced neuronal apoptosis. Using various types of ischemic insults in different neuronal cells, we confirmed that hypothermia attenuated apoptotic neuronal cell death in vitro and pharmacologically blocking or knocking out CD88 further enhanced this effect. Thus, this project raised a promising therapeutic possibility of adding CD88 antagonists along with hypothermia to improve stroke outcomes.
At a stage where several major human clinical trials for stroke therapy have failed to produce desirable results either due to either a lack of efficacy or serious adverse effects, a renewed vigor for devising a viable treatment option for ischemia-induced cell death is imperative. The various findings from this thesis, suggest that receptor-mediated mechanisms play a major role in the pathological progress and outcome of ischemic strokes and these findings would be beneficial in providing a rationale to target these receptors in an attempt to devise a neuroprotective, multitargeted approach in stroke therapy.