Neuroprotective Effects of Glial Cell line-Derived Neurotrophic Factor、Bone Morphogenetic Proteins and Hyperbaric Oxygenation against Stroke

Autor: Cheng-Fu Chang, 張成富
Rok vydání: 2001
Druh dokumentu: 學位論文 ; thesis
Popis: 90
This dissertation research focused on focal cerebral ischemia, using the middle cerebral artery occlusion model in rats and mice. Studies were conducted primarily to examine influences of TGF-β family trophic factors on the sequalae of pathophysiological events. One study examined changes in the receptor for GDNF to test the hypothesis of an endogenous GDNF neuroprotective system.(Time Course Study of GFRa-1 Expression in an Animal Model of Stroke. Sarabi A, Chang CF, Wang Y, Hoffer BJ, and Morales M. Exp Neurol. 2001;170:283-289.) Previous studies have shown that intracerebral administration of glial cell line-derived neurotrophic factor (GDNF) reduces ischemia-mediated cerebral infarction. The biological effects of GDNF are mediated by GDNF-family receptor alpha-1 (GFRa-1) and c-Ret. In this study, we examined the levels of expression of GFRa-1 and c-Ret in a rat model of stroke. Adult Sprague-Dawley rats were anesthetized with chloral hydrate. The right middle cerebral artery was ligated at its distal branch for 90 min. Animals were sacrificed at 0, 6, 12, and 24 h after reperfusion and levels of expression of GFRa-1 and c-Ret mRNA were determined by in situ hybridization histochemistry. We found that GFRa-1 mRNA was up-regulated in CA3, dentate gyrus (DG), cortex, and striatum. The peak of up-regulation in DG was 6 h after reperfusion. GFRa-1 mRNA levels in CA3 were gradually up-regulated over the 24-h reperfusion period. In cortex, GFRa-1 mRNA was up-regulated at all time points; however, the peak of up-regulation was observed at 0 and 24 h after reperfusion. In striatum, an initial up-regulation of GFRa-1 was found at 0 h after ischemia. In striatum, up-regulation of c-Ret mRNA was detected as early as 0 h after reperfusion. A gradual increase was found at 6, 12, and 24 h after reperfusion. In conclusion, our results indicate that there are both regional and temporal differences in up-regulation of GFRa-1 and c-Ret after ischemia. Since GDNF is neuroprotective, up-regulation of GFRa-1 and c-Ret could enhance the responsiveness to GDNF and reduce neuronal damage. The selective up-regulation of GFRa-1 and c-Ret in different brain areas suggests that there may be regional differences in GDNF-induced neuroprotection in stroke. These changes in receptors for GDNF supports the hypothesis of an endogenous GDNF neuroprotective mechanism and extends earlier data showing a parallel upregulation of GDNF itself in stroke. A second study investigated another member of the TGF-β family, termed Bone Morphogenic Protein-6 (BMP-6) in this same rat model of focal ischemia. (Bone Morphogenetic Protein-6 Reduces Ischemia-Induced Brain Damage in Rats. Wang Y, Chang CF, Morales M, Chou J, Chen H-L, Chiang Y-H, Lin S-Z, Cadet J L, Deng X, Wang, J-Y, Chen, S-Y, Kaplan PL, and Hoffer,BJ. Stroke. 2001;32:2170-2178.) Bone morphogenetic protein-6 (BMP-6) and its receptors are expressed in adult and fetal brain.Receptors for BMP-6 are upregulated in adult brain after injury, leading to the suggestion that BMP-6 is involved in the physiological response to neuronal injury. The purpose of my study was to determine whether there was a neuroprotective effect of BMP-6 in vivo and in vitro. Lactate dehydrogenase and microtubule-associated protein-2 (MAP-2) activities were used to determine the protective effect of BMP-6 against H2O2 in primary cortical cultures. The neuroprotective effects of BMP-6 were also studied in chloral hydrate—anesthetized rats. BMP-6 or vehicle was injected into right cerebral cortex before transient right middle cerebral artery (MCA) ligation. Animals were killed for triphenyl-tetrazolium chloride staining, caspase-3 immunoreactivity and enzymatic assays, and TUNEL assay. A subgroup of animals were used for locomotor behavioral assays. Application of H2O2 increased lactate dehydrogenase activity and decreased the density of MAP-2 neurons in culture. Both responses were attenuated by BMP-6 pretreatment. Complementary in vivo studies showed that pretreatment with BMP-6 increased motor performance and generated less cerebral infarction induced by MCA ligation/reperfusion in rats. Pretreatment with BMP-6 did not alter cerebral blood flow or physiological parameters. There was decreased ischemia-induced caspase-3 immunoreactivity, caspase-3 enzymatic activity, and density of TUNEL-positive cells in ischemic cortex in BMP6-treated animals. BMP-6 thus reduces ischemia/reperfusion injury, perhaps by attenuating molecular events underlying apoptosis. Because GDNF and BMP-6 utilize entirely different receptors and intracellular second messengers, these experiments also suggest a “cocktail” of trophic factors may provide optimal neuroprotection in stroke. A third study examined another approach to provide neuroprotection for cerebral ischemia, utilizing hyperbaric oxygen. (Hyperbaric oxygen therapy for treatment of postischemic stroke in adult rats. Chang CF, Niu KC, Hoffer BJ, Wang Y, and Borlongan CV. Exp Neurol. 2000;166:298-306.) The hypothesized efficacy of hyperbaric oxygen (HBO) therapy for treatment of stroke was tested in this study. Adult rats were subjected to occlusion of the middle cerebral artery and subsequently exposed to HBO (3 atm, 2 x 90 min at a 24-h intervals. Animals terminated shortly after the second HBO treatment) or hyperbaric pressure (HBP; 3 atm, 2 x 90 min at a 24-h interval; animals terminated shortly after the second treatment) immediately after the ischemia or after a 60-min delay generally displayed recovery from motor deficits at 2.5 and 24 h of reperfusion. There was also a reduction in cerebral infarction at 24 h of reperfusion compared to ischemic animals subjected to normal atmospheric pressure. While both HBO and HBP treatments promoted beneficial effects, HBO produced more consistent protection than HBP. Treatment with HBO immediately or 60 min after reperfusion produced equally significant attenuations of both cerebral infarction and motor deficits. In contrast, protective effects of HBP treatment against ischemia were noted only when administered immediately after ischemia; there was a significantly reduced infarction volume, but only a trend toward decreased behavioral deficits. The present results demonstrate that HBO and, to some extent HBP, reduce ischemic brain damage and behavioral dysfunctions. Thus, we have validated the use of HBO in clinical situations after acute stroke. The last study in this thesis examined the mechanisms of methamphetamine facilitation of ischemic cerebral injury. (Methamphetamine potentiates ischemia/reperfusion insults after transient middle cerebral artery ligation.Wang Y, Hayashi T, Chang CF, Chiang YH, Tsao LI, Su TP, Borlongan C, and Lin SZ. Stroke. 2001;32:775-82.) Previous studies have indicated that both methamphetamine (MA) and ischemia/reperfusion injuries involve reactive oxygen species formation and activation of apoptotic mechanisms. That MA could have a synergistic or additive effect with stroke-induced brain damage is possible. The purpose of the present study was to investigate whether administration of MA in vivo would potentiate ischemic brain injury. Adult CD-1 mice were pretreated with MA or saline. Each animal was later anesthetized with chloral hydrate and placed in a stereotaxic frame. A subset of animals received intracerebral administration of GDNF. The right middle cerebral artery and bilateral carotids were transiently occluded for 45 minutes. Regional cerebral blood flow was measured by laser Doppler. Animals were sacrificed for triphenyltetrazolium chloride staining and p53 mRNA Northern blot assay after 24 hours of reperfusion. Cortical and striatal GDNF levels were assayed by ELISA. We found that pretreatment with MA increased ischemia-induced cerebral infarction. Ischemia or MA alone enhanced p53 (a pro-apoptotic molecule) mRNA expression. Moreover, MA potentiated expression of p53 mRNA in the ischemic mouse brain. MA pretreatment decreased GDNF levels in ischemic striatum. Intracerebral administration of GDNF before ischemia reduced MA-facilitated infarction. Our data indicate that MA exacerbates ischemic insults in brain, perhaps through the inhibition of GDNF-mediated pathways and suggest that MA may antagonize endogenous neuroprotective pathways as part of its mechanism of action. In addition, MA may also upregulate pro-apoptotic mechanisms, contributing to the ultimate extent of infarction. Taken together, these studies strengthen the hypothesis that apoptotic mechanisms are important in determining the extent of infarction after focal cerebral ischemia. They further provide a preclinical basis for exploring new potential therapies based on trophic proteins, particularly in the TGF-β family.
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