| Summary: | 博士 === 國防醫學院 === 生命科學研究所 === 92 === A major objective of therapeutic intervention after ischemic stroke is to promote improved functional outcome. Improved outcome is associated with reduced volume of cerebral infarction and/or the promotion of cerebral plasticity, which is thought to involve dendritic reorganization. The overall goal of this thesis was to examine the role of bone morphogenetic proteins (BMPs) in these processes. In the first 3 studies (Chapters 3 through 5), we used in vivo models of ischemic stroke or neurodegeneration to examine the neuroprotective effects of exogenous BMPs and the effect of traumatic injury on expression patterns of endogenous BMPs. In the subsequent 3 studies (Chapters 6 through 8), in vitro models were used to investigate the role of BMPs in modulating dendritic growth.
We first examined actions of BMPs in adult rat brain in a model of focal stroke. We studied if the neuroprotective effects of intracerebral grafts of fetal kidney tissue, documented previously, were mediated by BMPs. Intracerebral transplantation of fetal kidney tissue, which normally expresses BMPs and GDNF during development, have been reported to reduce ischemic injury in cerebral cortex. In this study, we tested the hypothesis that BMP is involved in this neuroprotective response. Fetal kidney tissue was cut into small pieces and transplanted into cortical areas adjacent to the right middle cerebral artery (MCA) in adult rats. In situ hybridization of brain indicated that these fetal kidney transplants contained high levels of BMP-7 mRNA 3 days after grafting. Immunohistochemical analysis of grafted brain showed co-localization of BMP-7 and PAX-2 immunoreactivity in the graft, suggesting that these transplants contained BMP protein. Some animals were grafted with fetal kidney tissue after intraventricular administration (ICV) of the BMP antagonist noggin (1 µg) or after vehicle, followed by MCA ligation for 60 min. Animals receiving fetal kidney tissue transplantation developed significantly less body asymmetry, as compared to stroke animals that either did not receive transplantation or received fetal kidney grafts and noggin pretreatment. Analysis of these brains after triphenyltetrazolium chloride staining showed that fetal kidney tissue transplantation reduced the volume of infarction in the cerebral cortex. Noggin pretreatment reduced the protection induced by fetal kidney grafting, although noggin itself did not cause increase in cerebral infarction. Eight hours after ischemia, brain homogenates were obtained from grafted and control animals to assay caspase-3 enzymatic activity. This analysis demonstrated that fetal kidney grafts significantly reduced ischemia-induced caspase-3 activity. Reduction of caspase-3 activity could also be antagonized by noggin pretreatment. In conclusion, our data suggested that fetal kidney transplantation reduces ischemia/reperfusion -induced cortical infarction and behavioral deficits in adult rats, which are, at least partially, mediated through the effect of BMPs from the transplants.
In the preceding studies, BMPs or kidney grafts were administered intracerebrally prior to stroke. We also wanted to determine if BMPs would be neuroprotective if given after stroke, a more relevant clinical model . In addition, we wanted to determine if parenteral administration of BMPs could be effective. Adult Sprague-Dawley rats were anesthetized with chloral hydrate. The middle cerebral artery (MCA) was transiently occluded by a filament, inserted through the right internal carotid artery. The filament was removed after 60-min ischemia to allow reperfusion. Some animals were killed 24 hours after MCA occlusion to examine BMP-7 mRNA expression. Other animals received a single dose of intravenous BMP-7 or vehicle at 24 hours after MCA occlusion and were used for subsequent behavioral studies and BMP-7 immunostaining.
BMP-7 mRNA was upregulated 24 hours after MCAo in non-treated animals. BMP-7-immunoreactivity was dose-dependently increased on the ischemic side hippocampus/dentate on the 6th day after MCAo in animals receiving intravenous injection of BMP-7. Animals receiving BMP-7 also showed a decrease in body asymmetry and an increase in locomotor activity from day 7 on after MCAo. Our data thus indicate that BMP-7, given parenterally after stroke, can pass through the blood brain barrier on the ischemic side and induce behavioral recovery in stroke animals at longer testing times.
Given the studies summarized above showing significant activity of BMPs in the adult mammalian CNS, we next studied the regional distribution of endogenous BMP ligand proteins, receptors and antagonists during aging and after lesion of the midbrain dopamine pathways produced by 6-hydroxydopamine. Our objective was to test the hypothesis that these molecules may be part of an endogenous neuroprotective system in the adult brain. We found that there was little change in the levels of these molecules as a function of age. Interestingly, levels of BMP 7 and noggin, a BMP antagonist, were uniquely elevated in substantia nigra. Moreover, after lesions of the midbrain dopamine system by 6-hydroxydopamine, there was a marked reduction in levels of all BMP ligands, receptors and antagonists bilaterally in both substantia nigra and hippocampus. There were also differential changes in BMP ligands, receptors and antagonists in cortex and striatum after such lesions. Taken together, our results indicate significant expression of BMP-related molecules in the adult and aging brain, and suggest a dynamic and differential regulation of these molecules after perturbations.
Finally, as dendritic changes have been postulated to underlie much injury-induced neuroplasticity in the adult brain, we studied action of BMPs on dendritic outgrowth, using sympathetic neurons derived from the superior cervical ganglia (SCG) as a model system. It has been previously reported that BMPs induce dendritic growth in cultured sympathetic neurons. As a first step towards determining if these in vitro observations are relevant to regulation of dendritic growth in vivo, we examined the distribution of BMPs in SCG and target tissues as a function of age and performed functional tests in cultured neurons to determine if BMPs derived from either ganglionic glial cells or target tissues would support dendritic growth. The first set of studies addressing these issues (Chapter 6) focused on the role of BMPs derived from ganglionic glial cells in modulating dendritic growth in sympathetic neurons. In situ hybridization and immunocytochemical studies indicated that the spatiotemporal expression of BMPs 5, 6 and 7 in rat SCG is consistent with their proposed role in dendritogenesis. In vitro, both SCG glia and neurons were found to express BMP mRNA and protein when grown in the presence or absence of the other cell type. However, addition of ganglionic glia to cultured sympathetic neurons caused a marked increase in BMP proteins coincident with a significant decrease in follistatin and noggin. Functional assays indicated that glial-induced dendritic growth is significantly reduced by BMP-7 antibodies and completely inhibited by exogenous noggin and follistatin. These data suggest that glia influence the rapid perinatal expansion of the dendritic arbor in sympathetic neurons by increasing BMP activity via modulation of the balance between BMPs and their antagonists and suggest that BMPs derived locally from surrounding glial cells may be involved in dendritic-based neuronal plasticity following injury in the CNS.
In the next set of studies (Chapter 7), we tested the hypothesis that target-derived BMPs may also be important in regulating dendritic growth in sympathetic neurons. Previous studies have demonstrated a direct correlation between target size and the extent of dendritic arborization in sympathetic neurons, and as shown earlier in Chapter 4, the kidney, which is a sympathetic target, expresses BMPs. To test whether renal-derived BMPs could promote dendritic growth in sympathetic neurons, we used Madin-Darby canine kidney (MDCK) cells as a model. Conditioned medium from MDCK cells induced significant dendritic growth in cultured sympathetic neurons and either BMP-7 antibodies or noggin blocked this effect. That MDCK cells produced BMP-7 was confirmed by immunoprecipitation. In vivo, the thoracic and lumbar sympathetic ganglia innervate the kidney and in vitro experiments demonstrated that neurons derived from these ganglia respond to the dendrite-promoting activity of BMP-7. RT-PCR analyses indicated that these ganglia expressed mRNA for BMPs 2, 5 and 7 during initial stages of dendritic growth but these levels were significantly reduced in the adult animal. In contrast, the kidney expresses mRNA for BMPs 2, 4, 5, 6 and 7, and with the exception of BMP-4, this expression is maintained in the adult. Western blot analyses confirmed renal expression of these BMP proteins from E21 into adulthood. These data suggest that target-derived BMPs may influence dendritic growth during development and into maturity, and suggest that the neuroprotective effects of fetal kidney grafts in ischemic stroke models may be mediated in part by effects on dendritic growth in surviving neurons.
The goal of the last set of studies (Chapter 8) was to examine the relative importance of target-derived BMPs in dendritic growth and to begin to characterize the mechanism(s) underlying retrograde effects of BMPs on dendritic growth. Using cultured SCG sympathetic neurons, we found that BMPs not only trigger dendritic growth, but also are necessary to maintain dendritic arborization. Western blot experiments indicate that BMPs are expressed in SCG from E21 to postnatal day (PND) 21, but are significantly downregulated in the adult. In contrast, as observed earlier in the kidney, expression of mRNA for BMPs, as assessed by RT-PCR, is maintained in SCG target tissues such as the eye and salivary gland from E21 into adulthood. These data suggest that target tissues may regulate dendritic growth in sympathetic neurons via retrograde BMP signaling. To test this hypothesis, we examined dendritic growth in sympathetic neurons cultured in compartmented Campenot chambers. Selective application of BMP-7 to the distal axons of sympathetic neurons induced significant dendritic growth, which was inhibited by the addition of function-blocking BMP-7 antibody to compartments containing distal axons, but not by addition of BMP-7 antibody to compartments containing cell bodies. BMPs 2, 4, 5 and 6 were also observed to induce dendritic growth when added to distal axons. Preliminary investigations of the mechanism(s) of retrograde BMP signaling using sympathetic neurons cultured in Campenot chambers indicate that I125-labelled BMP-7 is transported from distal axons to cell bodies, and this transport is blocked by excess unlabeled BMP-7 but not by excess unlabeled TGF-b. Induction of dendritic growth by BMPs applied to distal axons is blocked in neurons expressing dominant negative dynamin, which inhibits endocytosis, but not binding of BMPs to surface receptors. These data suggest that target-derived BMPs influence dendritic growth following internalization at distal axons and transport back to the cell body. These data also have implications regarding clinical use of BMPs in treatment of ischemic stroke, suggesting that delivery of BMPs to target fields of involved neurons may be as effective as application directly to the site of ischemic injury.
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