| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online May 7, 2004 as doi:10.1096/fj.03-1203fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Department of Pharmacology and Toxicology, University of Otago, Dunedin, New Zealand
1Correspondence: Department of Pharmacology and Toxicology, University of Otago, P.O. Box 913, Dunedin, New Zealand. E-mail: ian.appleton{at}stonebow.otago.ac.nz
SPECIFIC AIMS
Hypoxia-ischemia (HI) -induced brain dysfunction, as well as other stroke-like syndromes, remains a growing concern with no current pharmacological treatment available. Spermine, a naturally occurring polyamine, has shown considerable promise as a putative neuroprotectant in models of neurodegeneration. To date, no study has used a model of HI-induced brain damage to assess the putative neuroprotective properties of spermine. More important, the mechanisms of this neuroprotective effect have not been determined. Therefore, in this study we used a modified Levine rat pup model (unilateral carotid artery ligation, coupled with global hypoxia) to evaluate the effects of various polyamines on HI-induced brain damage. In addition, we investigated the biochemical (NOS and arginase) and molecular energetics (mitochondrial complexes) in association with these effects.
PRINCIPAL FINDINGS
1. Spermine, but not putrescine or spermidine, provided gross histological neuroprotection
H and E staining 7 days post-HI revealed extensive neuronal damage ipsilateral to carotid ligation, with no apparent anatomical damage contralaterally in HI + saline-treated animals. Treatment with polyamines (putrescine, spermine, and spermidine) at a dose of 10 mg/kg i.p. for 6 days was carried out to assess their neuroprotective effects in a model of HI-induced brain damage. Only spermine significantly decreased the infarct size. Spermines’ neuroprotective effects were shown to extend to gross preservation of the hippocampus as well as the outer cortical layers.
2. Spermine modulates NOS activity induced by HI
Assessment of NOS activity revealed an increase in iNOS activity (absence of calcium) ipsilateral to the occlusion from HI + saline-treated animals compared with nonintervention controls. No change in total NOS activity (addition of calcium) was evidenced ipsilaterally from these same animals. Treatment with spermine resulted in an increase in total NOS activity, with no change in iNOS activity compared with HI + saline controls. Assessment of iNOS and total NOS activity in the contralateral hemisphere failed to reveal any significant differences between treatment groups.
3. Spermine treatment decreases the elevated arginase activity induced by HI
Assessment of arginase activity ipsilateral to the occlusion from HI + saline-treated animals revealed a significant increase in activity. Treatment with spermine decreased the level of activity compared with HI + saline controls. However, the level of activity was still elevated compared with nonintervention controls. Assessment of arginase activity in the contralateral hemisphere failed to reveal any significance between treatment groups.
4. Spermine provided near-full preservation to mitochondrial respiratory complexes and the mitochondrial membrane marker citrate synthase against HI-induced damage
Complexes I, II-III, IV and citrate synthase activities were assessed in tissue homogenates from left and right hemispheres. HI resulted in a decrease in the activity of complex I compared with untreated control. Spermine provided significant preservation of mitochondrial energetics at complex I compared with HI + saline. Assessment of complex I activity in the right hemisphere failed to show any significant differences between treatment groups. HI resulted in a decrease in complex IV kinetics ipsilaterally compared with controls. Spermine treatment provided significant protection to mitochondrial complex IV kinetics compared with HI + saline, but was still marginally down-regulated compared with controls. No change in mitochondrial complex IV kinetics was seen in the contralateral hemisphere between any groups. Mitochondrial complex II-III activity for both hemispheres elicited no change in activity between treatment groups.
The mitochondrial membrane matrix marker citrate synthase was impaired in the ipsilateral hemisphere following HI + saline compared with nonintervention controls. Treatment with spermine preserved citrate synthase activity against HI-induced damage and was not different from nonintervention control levels. Assessment of citrate synthase activity in the contralateral hemisphere failed to reveal differences between any groups.
5. Spermine modulated LDH changes induced by HI
HI + saline treatment deceased LDH activity in the ipsilateral hemisphere compared with nonintervention controls. Treatment with spermine reversed the HI-induced decrease in LDH activity back to control levels. In contrast, LDH activity in the contralateral hemisphere was increased following HI + saline, though not significantly, compared with controls. No change in LDH activity compared with nonintervention controls was evident after HI + spermine treatment.
CONCLUSIONS AND SIGNIFICANCE
The present study used a modified Levine rat pup model of HI to 1) assess the neuroprotective effect of the polyamines putrescine, spermidine, and spermine, and 2) establish some of the mechanisms behind their neuroprotection. The HI-induced damage is consistent with others in the field with pronounced cortical and subcortical cell loss. The findings in the present study demonstrate for the first time that, of the polyamines, only spermine is neuroprotective in a model of HI-induced brain damage as assessed using gross histological measures. This is in keeping with previous work illustrating spermines’ neuroprotective effects in both in vivo and in vitro models of ischemia. More important, we have shown that HI induces modulation of critical biochemical enzymes (namely, NOS and arginase) and subsequent substantial impairment in mitochondrial energetics. Acute treatment with spermine provided significant alteration to the above biochemical enzymes, resulting in robust anatomical neuroprotection and virtually full preservation of mitochondrial energetics.
Assessment of the biochemical enzymes NOS and arginase following HI revealed elevated levels of activity in iNOS and arginase activity in the left hemisphere. Previous reports have shown that an elevated level of iNOS activity is detrimental to tissue survival. Treatment with spermine in the present study resulted in a decrease in arginase activity, with an increase in total NOS activity with no effect on levels of iNOS activity. The subsequent increase in total NOS activity seen in the present study is most likely to be attributed to an increase in ecNOS or nNOS activity. This is in keeping with previous work showing that increased total NOS activity in the absence of an increase in iNOS activity is neuroprotective. Earlier work has shown that of the NOS isoforms, only elevated levels of ecNOS have shown beneficial effects. We therefore hypothesize for the first time that suppressing arginase activity pushes the utilization of L-arginine down the NOS pathway with a resultant increase in ecNOS-derived NO, resulting in neuroprotection.
This study confirms and extends previous observations that NOS activity is a pivotal element in neurodegenerative/neuroprotective processes. In addition, we speculate for the first time that modulation of the enzyme arginase may be a target for future neurodegenerative pathologies.
|
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1203fje; doi: 10.1096/fj.03-1203fje
This article has been cited by other articles:
|
|
 |
|
  V. J. Adlam, J. C. Harrison, C. M. Porteous, A. M. James, R. A. J. Smith, M. P. Murphy, and I. A. Sammut Targeting an antioxidant to mitochondria decreases cardiac ischemia-reperfusion injury FASEB J, July 1, 2005; 19(9): 1088 - 1095. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
