Stroke is the 3rd leading cause of death and the leading cause of major disability worldwide. Currently, there are no neuroprotective drugs approved for the acute treatment of ischaemic stroke. The vast majority of stroke therapeutics failed in clinical trials due to toxic side effects and/or a clinically irrelevant therapeutic window. This thesis is focused on exploiting the delayed neurodegeneration that occurs in the compromised penumbra, as these cells may be capable of being saved by therapeutic intervention in a clinically obtainable window. In order to investigate the ischaemic cascade and be able to draw conclusions that are applicable to humans, the international gold standard animal model for cerebral ischaemia, the filament insertion middle cerebral artery occlusion (MCAO) model, was established at the University of Otago. This model was validated under new laboratory conditions and employed adult male Sprague Dawley rats. After testing multiple occlusion lengths, it was concluded that a 2hr ischaemic period was sufficient to produce a consistent infarct of optimal size. It has been well documented that neuroinflammation contributes to much of the delayed progression of neural injury post-stroke. Therefore, the catechin (-)-epigallocatechin gallate (EGCG), which is an anti-inflammatory, anti-oxidant and free-radical scavenging agent was investigated in the MCAO model of stroke. 50mg/kg i.p. of EGCG or saline was administered immediately post-MCAO and animals were sacrificed at 72hr post-filament insertion. The results confirmed that treatment with EGCG was neuroprotective and non-toxic. However, EGCG also induced an over 50% increase in the risk of haemorrhagic conversions. The anti-platelet effects of EGCG and lack of toxicity suggests that the catechin may prove to be an efficacious prophylactic for stroke. The contrary findings for EGCG treatment led to the re-evaluation of the neuroinflammatory pathway for alternate mechanisms to target therapeutic interventions.
The temporal profile of the primary inducible enzymes nitric oxide synthase (NOS), cyclooxygenase (COX) and arginase (and their isoforms) were quantified 0, 3 and 7 days post-stroke. In both hemispheres, total NOS activity exhibited a significant and sustained up-regulation to 7 days post-occlusion. In the ipsilateral hemisphere at least half of the total increase was accounted for by inducible NOS (iNOS) expression. Arginase, which competes with NOS for L-arginine, demonstrated a delayed but significant increase in activity by day 7 in the infarcted hemisphere, thereby correlating well with the downward slope of NOS activity (illustrating the switch in the conversion pathway). COX activity was observably increased in the ipsilateral hemisphere, but the up-regulation did not reach significance by day 7. Alternately, the contralateral hemisphere displayed a significant decrease in activity by day 3. These results give conclusive evidence that the contralateral hemisphere is NOT an appropriate internal control and imply that NOS and COX inhibitors may prove to be efficacious for a much longer therapeutic window than current treatments. However, the delayed induction of COX activity may also indicate that this enzyme has a finite therapeutic window, as it may also stimulate remodelling of surviving neural networks. The prolonged up-regulation of inflammatory mediators implies that there may be an induction of an autoimmune component to the response. Therefore, the thymus (T) lymphocyte activation was quantified up to 14 days post-stroke. Cluster of differentiation (CD) 3⁺ T lymphocytes (equally contributed to by CD4⁺ and CD8⁺ T cells) exhibited a significant and sustained up-regulation in the infarcted region from day 3 up to at least day 14 post-ischaemia. Quantitative analysis of all cells present post-stroke determined that immune cells make up an average of 73% of all cells present in the 'peak' ischaemic areas. The CD4⁺ T helper cell response was delineated by double immunohistochemical labelling. Interferon-γ positively labelled with CD4⁺ T cells at days 3, 7 and 14 post-insult detailing a Th1-driven pro-inflammatory response. This evidence indicates that the autoimmune response is critical post-ischaemia and that it may be highly susceptible to modification by anti-inflammatory therapeutic intervention.
The primary downstream effect of the pro-inflammatory/immune cascade is apoptosis. The main organelle responsible for the 'go, no go' response to apoptotic factors is the mitochondria. In order to distinguish whether mitochondrial dysfunction was initiated shortly after ischaemia induction or if it was delayed, like the inflammatory/immune response, to a clinically relevant window, the temporal profile of mitochondrial complex inactivation was studied. It was found that mitochondrial membrane viability was impaired by day 3, followed by a significant decrease in respiratory complex activation and an increase in tissue injury by oxidative stress by 7 days post-ischaemia. These results indicate that targeting the early decrease in membrane viability or mitochondrial permeability transition pore opening combined with anti-apoptotic therapeutics, may attenuate the proceeding mitochondrial impairment in oxidative phosphorylation, reactive oxygen species generation and subsequent cell death cascades. The current investigations into the temporal profile and quantitative contributions of the inflammatory, immune and apoptotic mechanisms post-stroke highlight potential strategies for modulation by acute stroke therapeutics. Furthermore, the general knowledge amassed from these studies dictates the necessity of a new approach to therapeutic intervention. The acknowledgement of so many contributing systems suggests that in addition to a thrombolytic, a combination therapy involving multiple neuroprotectants should be employed to account for the multifaceted nature of the sequelae of ischaemic stroke.
| Identifer | oai:union.ndltd.org:ADTP/217579 |
| Date | January 2006 |
| Creators | Rahman, Rosanna, n/a |
| Publisher | University of Otago. Department of Pharmacology & Toxicology |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://policy01.otago.ac.nz/policies/FMPro?-db=policies.fm&-format=viewpolicy.html&-lay=viewpolicy&-sortfield=Title&Type=Academic&-recid=33025&-find), Copyright Rosanna Rahman |
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