Subsequent to an hypoxic-ischaemic (HI)-insult a multi-faceted complex cascade of events occurs that ultimately results in cellular and neurological impairments within cortical and sub-cortical central nervous system (CNS) regions. In the present studies a modified �Levine� rat-pup model of HI (left carotid artery ligation + 1 hour global hypoxia on post-natal day (PND) 26) was employed to assess the neuroprotective properties of clomethiazole (CMZ; a γ-aminobutyric acid (GABA)A receptor agonist). In this study, histological and electrophysiological paradigms were used to assess the long-term neuroprotective properties of CMZ (414mg/kg/day via mini-pumps). Key enzymes involved in inflammation, namely nitric oxide synthase (NOS) and arginase, were also examined to assess other potential CMZ mechanisms. Assessments were carried out 3- and 90-days post-HI, with extensive ipsilateral CNS lesions evident at a gross histological level, at both the early and long-term stages, with CMZ significantly decreasing the lesion size at 3- and 90-days (P<0.01; P<0.05). Evoked field potential analyses were used to assess hippocampal CA1 neuronal activity ex vivo. Electrophysiological measurements contralateral to the occlusion revealed impaired neuronal function following HI relative to short- and long-term controls (P<0.001, 3- and 14-days; P<0.01, 90-days), with CMZ providing near complete protection (P<0.001 at 3- and 14-days; P<0.01 at 90-days). Both inducible NOS (iNOS) and arginase activities were significantly increased at 3-days (P<0.01), with arginase activity remaining elevated at 90-days post-HI (P<0.05) ipsilaterally. CMZ suppressed the HI-induced increase in iNOS and arginase activities (P<0.001; P<0.05). These data provide evidence of long-term functional neuroprotection afforded by CMZ in a model of HI-induced neurodegeneration. In addition, under conditions of HI, functional deficits were not restricted to the ipsilateral hemisphere and were due, at least in part, to changes in the activity of NOS and arginase.
Underlying mitochondrial dysfunction is eminently present in many neuropathological conditions. The full extent of mitochondrial dysfunction in cortical, hippocampal and cerebellar tissues was assessed following HI. Assessment of mitochondrial FAD-linked respiration at both 1- and 3-days post-HI revealed a significant decrease in activity from ipsilateral cortical and hippocampal regions (P<0.001). In addition, significant changes in respiratory function were also evident in contralateral regions and cerebellum, 3-days post-HI (P<0.05). Assessment of the mitochondrial electron transport chain (complexes I-V) and mitochondrial markers of integrity (citrate synthase) and oxidative stress (aconitase) confirmed ipsilateral mitochondrial impairment following HI. Complexes I, II-III, V and citrate synthase were also impaired, in contralateral regions and cerebellum, 3-days post-HI. CMZ treatment provided significant protection to all mitochondrial aspects of neuronal tissue assessed. This study provides evidence of the full extent of mitochondrial damage following an HI-insult and may contribute, in part, to the impairment seen contralaterally. In addition, protection afforded by CMZ extends to preservation of mitochondrial function and integrity.
Cerebral ischaemia-induced angiogenesis has been shown within and around infarcted regions and may contribute to a more favourable neurological outcome. The level of angiogenesis was examined using platelet endothelial cell adhesion molecule-1 (PECAM-1 / CD31). CD31 immunolabelling 7-days post-HI revealed a significant increase in angiogenesis compared with non-intervention controls (P<0.001). Treatment with CMZ decreased the level of angiogenesis compared to HI + saline (P<0.001) back to non-intervention control levels. Conversely, N[omega]-nitro-L-arginine methyl ester (L-NAME) treatment (5mg/kg/day) exacerbated the ischaemic lesion (P<0.001) and resulted in a marked decrease in angiogenesis compared to non-intervention controls (P<0.001). The extent of cerebral infarction in these studies is dependent on the level of NOS activity with CMZ increasing total NOS levels compared to HI + saline, while L-NAME halted the HI-induce increase in total NOS activity (P<0.001). These results show for the first time, that angiogenesis may be used as an assessment of neurodegeneration / neuroprotection in pathologies of cerebral ischaemia and are directly correlated with changes in NOS activity.
These studies have therefore shown that following HI, damage also occurs contralateral to the occlusion, and is not restricted to the ipsilateral hemisphere. In addition, the neuroprotective effects of CMZ have been shown to extend out to 90-days post-HI, whereby significant protection to CA1 neuronal activity was seen. These studies also provide in vivo evidence that CMZ may also afford neuroprotection via anti-inflammatory pathways, as evidenced by a decrease in iNOS and arginase activities. Furthermore, these studies have also show evidence that angiogenesis (CD31) can be used as a diagnostic tool to assess neuroprotection / neurodegeneration.
| Identifer | oai:union.ndltd.org:ADTP/217565 |
| Date | January 2005 |
| Creators | Clarkson, Andrew N., 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 Andrew N. Clarkson |
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