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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 4, 2005 as doi:10.1096/fj.04-3367fje. |
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Department of Pharmacology and Toxicology, University of Otago School of Medical Sciences, Dunedin, New Zealand
1 Correspondence: Department of Pharmacology and Toxicology, University of Otago, PO Box 913, Dunedin, New Zealand. Email: steve.kerr{at}stonebow.otago.ac.nz
SPECIFIC AIMS
Clomethiazole (CMZ) a GABAA receptor agonist has been shown to be neuroprotective in models of both focal and global ischemia. In the present study we used a modified ‘Levine’ rat-pup model (unilateral carotid artery ligation, coupled with global hypoxia), to evaluate the lasting (90 days) effects of CMZ on HI-induced neuronal damage. We also assessed neuronal activity from hippocampal region CA1 and the activity of key inflammatory mediators, namely nitric oxide synthase (NOS) and arginase, ipsilateral and contralateral to the occlusion.
PRINCIPAL FINDINGS
1. Clomethiazole provides lasting gross histological protection against hypoxia-ischemia-induced neuronal damage
Histological measurements after cerebral HI were carried out at 3 days (TTC staining) and 90 days (H&E staining) to evaluate the extent of neurodegeneration. Assessment after HI + saline treatment revealed extensive damage to both cortical and subcortical regions ipsilateral to the occlusion at both 3 and 90 days postinsult. No gross histological damage was evident in the contralateral hemisphere. Treatment with CMZ (414 mg/kg/day via subcutaneous minipump, for 5 days) was carried out to assess the neuroprotective properties in a model of HI-induced brain damage. CMZ treatment resulted in a significant decrease in infarct size at both 3 and 90 days post-HI compared with HI + saline treatment, extending across both cortical and subcortical regions.
2. Clomethiazole provides lasting preservation of contralateral CA1 neuronal activity after HI
Hippocampi ipsilateral to the occlusion were almost completely degenerated and viable slices could not be obtained. Therefore, only electrophysiological recordings from contralateral hippocampi were carried out. Assessment of evoked potentials across a range of stimulus intensities revealed lasting impairment in neuronal activity 3, 14, and 90 days after HI + saline treatment compared with nonintervention age-matched controls (Fig. 1
). Treatment with CMZ provided lasting (90 days) protection of neuronal function. Assessment of local circuit inhibition 3 and 14 days after HI + saline treatment revealed a biphasic pattern of responding, with increased inhibition at the shorter interstimulus intervals (ISIs) and increased facilitation at the longer ISIs compared with age-matched nonintervention controls. CMZ treatment in general resulted in enhanced inhibition across a range of ISIs relative to HI + saline treatment. Paired-pulse analysis failed to reveal any significant differences between treatment groups 90 days post-HI.
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3. Clomethiazole modulates both NOS and arginase during HI
Assessment of NOS activity 3 days post-HI revealed a significant increase in iNOS activity ipsilateral to the occlusion in the HI + saline treated animals. In addition, a significant increase in total NOS activity was also evidenced ipsilaterally in these same animals. Treatment with CMZ resulted in a further increase in total NOS activity, while significantly decreasing the level of iNOS activity, relative to HI + saline treated animals. Assessment of iNOS and total NOS activities in the contralateral hemisphere and in the cerebellum, failedto reveal any significant differences between treatment groups at either 3 or 90 days post HI.
HI produced significant increases in arginase activity at both 3 and 90 days in the ipsilateral hemisphere and this was prevented by CMZ. Assessment of arginase activity in the contralateral hemisphere and cerebellum, failed to reveal any significances between any of the treatment groups.
CONCLUSIONS AND SIGNIFICANCE
In this study we employed histological, electrophysiological, and biochemical techniques to evaluate the acute and lasting neuroprotective properties of CMZ in a modified ‘Levine’ rat-pup model of HI. Assessment of neuronal damage 3 and 90 days post-HI revealed extensive infarction ipsilateral to the carotid occlusion. CMZ was found to be neuroprotective in this model of HI-induced neuronal damage, and confirms and extends similar results in prior in vivo and in vitro models of ischemia. Notably, this is the first time that CMZ has been shown to be protective out to 90 days postinsult.
A substantial functional (i.e., electrophysiological) impairment was observed contralateral to the occlusion, even in the absence of any gross anatomical damage, suggesting that a lasting transhemispheric diaschisis of hippocampal function follows HI in the rat. As noted, numerous studies of HI damage have used the contralateral hemisphere as an ‘internal control’. However, remote changes in contralateral CNS regions have been documented in which loss of function within an injured area renders other regions less responsive to stimuli. Our study is the first to document a marked functional diaschisis of hippocampal field potentials extending to 90 days post-HI, with CMZ providing virtually complete preservation of function at all time points.
We found that hippocampal local circuit inhibition is not only intact after HI, but is in fact enhanced contralateral to the lesion at short ISI’s, while significant increases in paired-pulse facilitation were evident at longer ISI’s at both 3 and 14 days post-HI. The enhanced inhibition at short ISI’s suggests that diaschisis after HI may involve compensatory remodeling or neurochemical changes that serve to suppress hippocampal excitability. The further enhancement of inhibition after CMZ suggests that even a relatively brief treatment with this compound facilitates these compensatory processes to provide lasting protection.
Assessment of NOS and arginase after HI revealed significant elevations of iNOS and arginase activity in the left (ligated) hemisphere. Previous reports have shown that increased iNOS activity and/or alteration of the arginase/polyamine pathway are associated with damage to neurons. In the present study, CMZ treatment resulted in a significant decrease in arginase and iNOS activity after HI. A significant increase in total NOS activity was also seen, and is most likely due to increased ecNOS or nNOS activity. Previous studies have shown that an increase in total NOS activity, in the absence of increased iNOS activity, is neuroprotective, and that ecNOS is the isoform that underlies these beneficial effects. Our results reinforce the hypothesis that suppression of arginase and iNOS activities by CMZ effectively increases the conversion of L-arginine to L-citrulline by ecNOS and results in neuroprotection.
Our findings support previous work in this area showing that the GABAA allosteric modulator clomethiazole is neuroprotective. Using a robust model of hypoxia-ischemia, we observed virtually complete anatomical and functional neuroprotection out to 3 months post-HI. Ample evidence supports the view that CMZ is both GABAmimetic and antiinflammatory, and these properties together provide neuroprotection. Our findings reinforce this and suggest that widespread functional diaschisis after stroke or focal damage can be prevented by timely treatment with such agents.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3367fje; doi: 10.1096/fj.04-3367fje
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), and HI + CMZ treated animals (
) 3, 14, and 90 days post-HI (n=5, 5, and 4, respectively). Population spike amplitude (mean mV±SE) vs. stimulus intensity 3, 14, and 90 days post-HI (A, C, and E, respectively). CMZ treatment abolished HI-induced functional impairment 3, 14, and 90 days post-HI. Field EPSP slopes (mean mV/ms±SE) vs. stimulus intensity 3, 14, and 90 days post-HI are shown in panels B, D, and F, respectively. HI-induced impairment was significantly reduced by CMZ treatment at all three assessment intervals and was most pronounced at 90 days. HI+saline vs, control: +P < 0.01, #P < 0.001; HI + CMZ vs. HI + saline: *P < 0.05, **P < 0.01, ***P < 0.001.