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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 29, 2004 as doi:10.1096/fj.04-2806fje. |
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Department of Pharmacology and Toxicology, University of Otago, Dunedin, New Zealand
1 Correspondence: Department of Pharmacology and Toxicology, University of Otago, PO Box 913, Dunedin, New Zealand. E-mail: ian.appleton{at}stonebow.otago.ac.nz
SPECIFIC AIMS
Cerebral hypoxia-ischemia (HI) can result in brain damage that has been likened to ischemic stroke, for which there are no current pharmacological treatments available. (–)-Epigallocatechin gallate (EGCG) is a potent polyphenolic antioxidant and has been shown to be neuroprotective in a gerbil model of global ischemia. However, the neuroprotective effects of EGCG have not been investigated in an animal model of HI and mechanisms of action underlying EGCG’s neuroprotective actions in vivo have not been fully elucidated. In this study we examined 1) the neuroprotective effects of EGCG in a modified "Levine" model of HI using Wistar rat pups; 2) the role key inflammatory enzymes nitric oxide synthase (NOS) and arginase play in the neuroprotective properties of EGCG; and 3) the effects of HI and EGCG on mitochondrial energetics (mitochondrial complexes) and integrity (citrate synthase).
PRINCIPAL FINDINGS
1. EGCG provided gross histological neuroprotection against HI-induced brain damage
Unilateral common carotid artery occlusion followed by global hypoxia caused extensive neuronal damage in the ipsilateral hemisphere of the brain, particularly in the hippocampus and cerebral cortex, as evidenced by hematoxylin and eosin staining. The mean volume of infarction was 99.6 ± 15.6 mm3. There was no apparent neuronal damage in the contralateral hemisphere. EGCG (50 mg/kg) i.p. treatment for 4 days beginning 1 day prior to HI significantly (P<0.05) reduced the mean volume of infarction (38.0±16.4 mm3). Hippocampus and cortical structures were protected by EGCG administration.
2. EGCG had no effect on arginase activity
Arginase activity was assessed in nonintervention controls, HI + saline controls, and HI + EGCG animals. HI did not significantly alter arginase activity in the ipsilateral hemisphere from nonintervention controls. Treatment with EGCG did not significantly modify arginase activity compared with HI + saline controls.
3. EGCG inhibits total NOS and inducible NOS (iNOS) activity but not nitrite levels
Hypoxia-ischemia significantly (P<0.05) induced total NOS activity (calcium-dependent) and iNOS activity (calcium-independent) in the ipsilateral hemisphere compared with nonintervention controls. EGCG administration significantly (P<0.05) inhibited the HI-induced increase of total NOS and iNOS activity back to the level of nonintervention controls. HI increased nitrite levels in the ipsilateral hemisphere, but this did not reach significance. Treatment with EGCG maintained nitrite levels similar to nonintervention controls.
4. EGCG differentially modulates NOS isoform protein expression
Protein expression of iNOS, endothelial NOS (eNOS), and neuronal NOS (nNOS) was assessed using Western blot analysis. HI significantly (P<0.05) induced iNOS protein expression in the ipsilateral hemisphere compared with nonintervention controls. Treatment with EGCG maintained iNOS protein expression levels close to nonintervention control levels. HI had no effect on eNOS and nNOS protein expression, whereas EGCG administration significantly (P<0.05) increased eNOS and nNOS protein expression compared with HI + saline controls.
5. EGCG protected mitochondrial respiratory complex activity and the mitochondrial membrane marker citrate synthase against HI-induced damage
Ipislateral and contralateral hemispheres were assayed for complexes I–V and citrate synthase activity. HI significantly (P<0.01) inhibited the activity of complex I compared with nonintervention controls. EGCG administration significantly (P<0.05) preserved complex I activity compared with HI + saline controls. Complex II–III activity was significantly (P<0.01) reduced after HI compared with nonintervention controls. Treatment with EGCG significantly (P<0.01) protected complex II–III activity against HI-induced impairment. HI caused a significant (P<0.001) decrease in complex IV kinetics vs. nonintervention controls. This decrease was inhibited significantly (P<0.001) by EGCG. Complex V activity was reduced significantly (P<0.05) by HI compared with nonintervention controls. EGCG significantly (P<0.05) preserved complex V activity compared with HI + saline controls. There were no changes in the activity of any complexes in any of the treatment groups in the contralateral hemisphere.
HI + saline controls had significantly (P<0.001) reduced citrate synthase activity (a mitochondrial membrane matrix marker) compared with nonintervention controls. Treatment with EGCG significantly (P<0.001) inhibited the impairment of citrate synthase activity induced by HI. Citrate synthase activity was not altered in the contralateral hemisphere in any of the treatment groups.
6. EGCG did not significantly preserve aconitase activity
Aconitase activity was assessed in the ipsilateral and contralateral hemispheres as a marker for oxidative stress. HI induced a significant (P<0.05) reduction in aconitase activity compared with nonintervention controls. Aconitase activity returned closely to control levels in HI + EGCG animals but did not reach significance. There were no differences in aconitase activity between any of the treatment groups in the contralateral hemisphere.
CONCLUSIONS AND SIGNIFICANCE
The present study investigated, for the first time, whether exogenous administration of EGCG was neuroprotective in an animal model of HI-induced brain damage. It was demonstrated that HI caused extensive neuronal damage ipsilateral to the common carotid artery occlusion producing a large volume of infarction, particularly in hippocampus and cerebral cortex brain structures. This study revealed that EGCG was neuroprotective against HI-induced brain damage by reducing the volume of infarction compared with HI + saline controls and hippocampal and cortical structures remained intact. Studies have identified the neuroprotective effect of EGCG in a model of global ischemia and the neuroprotective effect of green tea extract (in which the major constituent is EGCG) in models of focal and global ischemia. Therefore, EGCG is an effective neuroprotectant against ischemia-induced brain injury. However, the mechanisms of action underlying the neuroprotection have not been fully elucidated in vivo.
Nitric oxide has been implicated as a key neurotransmitter in the mechanisms of HI-induced neuronal damage. Earlier studies have revealed that iNOS and nNOS-derived NO is detrimental to cell survival whereas eNOS-derived NO produces beneficial effects (for example, increasing cerebral blood flow and scavenging reactive oxygen species). Here, we have shown that total NOS and iNOS activity was increased with HI, but this was prevented by administration of EGCG. This indicates that the increase in total NOS activity was due to the increase in iNOS activity, suggesting that iNOS-derived NO after HI is damaging to the brain. To further assess the involvement of the various isoforms of NOS, Western blot analysis was performed. Protein expression during HI, with or without EGCG, mirrored the iNOS activity data, suggesting that part of EGCG’s neuroprotective effect is due to inhibition of iNOS. HI had no effect on the protein expression of nNOS or eNOS. However, both of these isoforms were increased with administration of EGCG. Thus, the potentially damaging effects of NO from an increase in nNOS may have been counteracted by the induction of eNOS, which produces beneficial effects. Therefore, up-regulation of eNOS by EGCG may have played a major part in producing neuroprotection. eNOS has been identified as a novel target for stroke protection and agents are being investigated that can selectively induce its activity and expression. We propose that the neuroprotective actions of EGCG with respect to NOS modulation are mediated in part by induction of eNOS and inhibition of iNOS.
Mitochondria are major targets of ischemic damage implicated in the impairment of ATP production leading to apoptosis of the cell. In the present study, mitochondrial respiratory complexes and membrane integrity were all compromised by HI. Previous reports have shown that complexes I and IV are particularly susceptible to hypoxic damage, and a major cause of mitochondrial injury may be due to the actions of reactive oxygen and nitrogen species such as superoxide and peroxynitrite. However, EGCG provided significant preservation of complexes I–V activity as well as citrate synthase activity, meaning mitochondrial function was protected and neurodegeneration did not occur.
This study confirms and extends the neuroprotective effects of EGCG. We provide evidence that iNOS inhibition and eNOS induction play a pivotal role in neuroprotection provided by EGCG; mitochondrial function was protected from HI-induced damage (Fig. 1
). We therefore speculate that EGCG offers a novel means of therapeutic intervention in addition to its antioxidant role in prevention of brain damage associated with HI and other neurodegenerative disorders.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2806fje;
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