Inflammatory mechanisms in focal cerebral ischaemia

Abstract

Stroke is a complex pathophysiological process and the role of inflammation in its initiation and resolution has been much debated. Inflammation is now emerging as a contributor in the development of ischaemic brain damage. The use of anti -inflammatory strategies to reduce damage and improve functional outcome of stroke patients may be valuable in the treatment for a condition that currently has no effective treatment. The exact contribution of the inflammatory response and the involvement of the various components of the immune system are still under investigationIn this thesis, focal cerebral ischaemia was induced in three animal models in an attempt to investigate the contribution of the inflammatory response. The rat monofilament model of middle cerebral artery (MCA) occlusion, considered by some to be a pro- inflammatory model, was set up for the first time in Edinburgh and validated as suitable model for further investigation. Permanent and transient models were established to allow the evaluation of possible reperfusion injury. Both monofilament models were compared with the Endothelin -1 model already established and routinely in use in the laboratory. Analysis of the volume of damage and distribution of the lesion revealed no differences between the three models. However, this observation did not in itself rule out the possibility of different pathophysiological mechanisms in the three models that ultimately resulted in apparently similar sized lesions.FK506, a potent neuroprotectant widely used experimentally, exhibited different neuroprotective efficacies. In all models, FK506 significantly reduced the overall volume of both damage and oedema. The majority of the neuroprotection was observed in the cortex although striatal protection was seen in the transient rat monofilament model. The neuroprotection observed in the transient monofilament model was approximately twice that seen in the permanent model and similar to that in the Endothelin -1 model suggesting distinct pathways that lead to cell death. Data for FK506 administration in the mouse monofilament model demonstrated neuroprotection for the first time in this species was included as an interesting comparison with the rat data.In keeping with the investigation of inflammation in cerebral ischaemia, it was proposed that FK506 neuroprotection was in part mediated through modulation of the inflammatory response. The response of the microglia, the resident immune cells of the central nervous system was examined following FK506 administration. Although the drug appeared to have a noticeable effect the activation state of the microglia, the response was not consistent and difficult to quantify by histological methods. Microglial cultures were established to investigate the effect of FK506 in a less complex system. Ramified microglial cultures were established but the analysis of microglia in vitro by morphology also proved difficult and another method of assessing activation of the cells was pursued. The microglia are known to secrete noxious stimuli when activated amongst which are the pro- inflammatory cytokines. IL-1P, IL -6 and TNFa gene expression was investigated to assess microglial activation. Lipopolysaccharide treated animals and treated microglial preparations were used initially to refine the use of multiplex polymerase chain reaction (MPCR) analysis of gene products. This was extended to tissue from both monofilament models. IL -1(3, IL -6 and TNFa were detected in the cortex and striatum when measured at 3 and 24 hrs post occlusion and showed an earlier cytokine response where reperfusion occurred. It is suggested that the early cytokine response is associated with the endogenous inflammatory cells. Western analysis experiments were performed to verify the presence of the corresponding cytokine proteins with little success. The recent availability of improved cytokine antibodies enabled the examination of cytokines (IL -1f3 and TNFa) in ischaemic by enzyme linked immunosorbant assay (ELISA). No TNFa response was detected despite the presence of mRNA. IL -lß was detected at 3 and 24 hrs post -insult with greater expression at 24 hrs. It may be speculated that this increased expression at the later time is related to the peripheral inflammatory cell infiltration. There was no difference in expression levels between models and FK506 had no affect on the cytokine expression.In summary, the re- introduction of blood to ischaemic tissue appears to alter the response of the individual cells although this does not change their ultimate fate. In instances where reperfusion is established, the tissue appears to be more amenable to neuroprotection by FK506. It is suggested that this is associated with the blockade of the endogenous inflammatory mechanisms that respond acutely to an ischaemic insult.