Uncoupling protein-2 prevents neuronal death and diminishes brain dysfunction after stroke and brain trauma.

Whereas uncoupling protein 1 (UCP-1) is clearly involved in thermogenesis, the role of UCP-2 is less clear. Using hybridization, cloning techniques and cDNA array analysis to identify inducible neuroprotective genes, we found that neuronal survival correlates with increased expression of Ucp2. In mice overexpressing human UCP-2, brain damage was diminished after experimental stroke and traumatic brain injury, and neurological recovery was enhanced. In cultured cortical neurons, UCP-2 reduced cell death and inhibited caspase-3 activation induced by oxygen and glucose deprivation. Mild mitochondrial uncoupling by 2,4-dinitrophenol (DNP) reduced neuronal death, and UCP-2 activity was enhanced by palmitic acid in isolated mitochondria. Also in isolated mitochondria, UCP-2 shifted the release of reactive oxygen species from the mitochondrial matrix to the extramitochondrial space. We propose that UCP-2 is an inducible protein that is neuroprotective by activating cellular redox signaling or by inducing mild mitochondrial uncoupling that prevents the release of apoptogenic proteins.

Combining neuroprotective treatment of embryonic nigral donor tissue with mild hypothermia of the graft recipient.

Around 80-95% of the immature dopaminergic neurons die when embryonic ventral mesencephalic tissue is transplanted. Cell death occurs both during the preparation of donor tissue and after graft implantation, but the effect of combining successful neuroprotective treatments before and after transplantation has not been extensively investigated. We therefore treated embryonic rat mesencephalic tissue with a combination of the lipid peroxidation inhibitor tirilazad mesylate (3 microM) and the caspase inhibitor Ac.YVAD.cmk (500 microM) and transplanted the tissue into hemiparkinsonian rats kept hypothermic (32-33 degrees C) or normothermic (37 degrees C) during, and 90 min following, graft surgery. Suspension cell number did not differ between untreated or tirilazad/YVAD-treated preparations prior to transplantation. When graft survival was evaluated 6 weeks after implantation, both tirilazad/YVAD pretreatment and mild hypothermia increased the survival of transplanted dopaminergic neurons. Approximately 50-57% of the embryonic dopaminergic neurons survived the dissociation and grafting procedure in rats rendered hypothermic, but there was no significant additive effect on graft survival with a combined treatment. All groups of rats exhibited behavioral recovery in the amphetamine-induced rotation test. There was a significantly enhanced functional capacity of grafts placed in hypothermic as compared to normothermic rats. However, tirilazad/YVAD pretreated implants did not afford greater behavioral improvement than control-treated grafts. Our results suggest that neuroprotective treatments administered prior to and immediately after neural graft implantation may under certain conditions rescue, at least in part, the same subset of dopaminergic neurons. The study also emphasizes the importance of the immediate time after grafting for transplant survival, with relevance both for primary mesencephalic implants and stem cell grafts.

Cerebral ischemia upregulates vascular endothelin ET(B) receptors in rat.

BACKGROUND AND PURPOSE: Elevated levels of endothelin-1 (ET-1) have been reported in cerebral ischemia. A role for ET may prove more important if the vascular receptors were changed. We addressed whether there is any change in ET receptor expression in cerebral ischemia. METHODS: The right middle cerebral artery (MCA) was occluded in male Wistar rats for 2 hours with the intraluminal filament method. The basilar artery and both MCAs were removed after 46 hours of recirculation. The contractile responses to ET-1, a combined ET(A) and ET(B) receptor agonist, and sarafotoxin 6c (S6c), a selective ET(B) receptor agonist, were examined in vitro, and ET receptor mRNA was quantified by real-time polymerase chain reaction. RESULTS: S6c, which had no contractile effect per se on fresh or sham-operated rat cerebral arteries, induced a marked contraction in the occluded MCA (E(max) [maximum contraction, calculated as percentage of the contractile capacity of 63.5 mmol/L K+]=68+/-68%; P<0.0001), while there was no difference in the responses to ET-1 after cerebral ischemia. Real-time polymerase chain reaction revealed a significant upregulation of both the ET(A) and ET(B) receptors (both P<0.05) in the occluded MCA compared with the nonoccluded MCA from the same rats. CONCLUSIONS: Focal cerebral ischemia in rat induces increased transcription of both ET(A) and ET(B) receptors, which results in the appearance of a contractile response to the ET(B) receptor agonist S6c. These results suggest a role for ET receptors in the pathogenesis of a vascular component after cerebral ischemia.

Comprehensive regional and temporal gene expression profiling of the rat brain during the first 24 h after experimental stroke identifies dynamic ischemia-induced gene expression patterns, and reveals a biphasic activation of genes in surviving tissue.

In order to identify biological processes relevant for cell death and survival in the brain following stroke, the postischemic brain transcriptome was studied by a large-scale cDNA array analysis of three peri-infarct brain regions at eight time points during the first 24 h of reperfusion following middle cerebral artery occlusion in the rat. K-means cluster analysis revealed two distinct biphasic gene expression patterns that contained 44 genes (including 18 immediate early genes), involved in cell signaling and plasticity (i.e. MAP2K7, Sprouty2, Irs-2, Homer1, GPRC5B, Grasp). The first gene induction phase occurred at 0-3 h of reperfusion, and the second at 9-15 h, and was validated by in situ hybridization. Four gene clusters displayed a progressive increase in expression over time and included 50 genes linked to cell motility, lipid synthesis and trafficking (i.e. ApoD, NPC1, G3P-dehydrogenase1, and Choline kinase) or cell death-regulating genes such as mitochondrial CLIC. We conclude that a biphasic transcriptional up-regulation of the brain-derived neurotrophic factor (BDNF)-G-protein coupled receptor (GPCR)-mitogen-activated protein (MAP) kinase signaling pathways occurs in surviving tissue, concomitant with a progressive and persistent activation of cell proliferation signifying tissue regeneration, which provide the means for cell survival and postischemic brain plasticity.

Npas4, a novel helix-loop-helix PAS domain protein, is regulated in response to cerebral ischemia.

Basic helix-loop-helix PAS domain proteins form a growing family of transcription factors. These proteins are involved in the process of adaptation to cellular stresses and environmental factors such as a change in oxygen concentration. We describe the identification and characterization of a recently cloned PAS domain protein termed Npas4 in ischemic rat brain. Using gene expression profiling following middle cerebral artery occlusion, we showed that the Npas4 mRNA is differentially expressed in ischemic tissue. The full-length gene was cloned from rat brain and its spatial and temporal expression characterized with in situ hybridization and Northern blotting. The Npas4 mRNA is specifically expressed in the brain and is highly up-regulated in ischemic tissues following both focal and global cerebral ischemic insults. Immunohistochemistry revealed a strong expression in the limbic system and thalamus, as well as in layers 3 and 5 in the cortex of the unchallenged brain. When overexpressed in HEK 293 cells, Npas4 appears as a protein of approximately 100 kDa. In brain samples, however, in addition to the 100 kDa band a specific 200 kDa immunoreactive band was also detected. Ischemic challenge lead to a decrease in the 200 kDa form and a simultaneous increase in the 100 kDa immunoreactivity. This could indicate a novel regulatory mechanism for activation and/or deactivation of this protein in response to ischemic brain injury.

Postischemic exercise attenuates whereas enriched environment has certain enhancing effects on lesion-induced subventricular zone activation in the adult rat.

Experimental stroke increases cell proliferation and neurogenesis in the subventricular zone (SVZ) and in the dentate gyrus subgranular zone (SGZ) in the adult mammalian brain. This study examined the effects of postischemic voluntary exercise (running wheel) and environmental enrichment on the SVZ and SGZ 1 week after focal cortical ischemia in adult spontaneously hypertensive rats. Immunohistochemical labeling was performed for incorporation of specific cell markers such as Ki67 and 5-bromodeoxyuridine (proliferating and newborn cells), terminal deoxynucleotidyl transferase-mediated dUTP in situ nick-end labeling (apoptotic cells), Sox-2 and glial fibrillary acidic protein (neural stem and progenitor cells), polysialylated neural cell adhesion molecule and doublecortin (neuroblasts). Postischemic exercise and environmental enrichment differentially modulated SVZ cell genesis but lacked effects on the SGZ. Lesion-induced proliferation of neural stem/progenitor cells and neuronal precursors was attenuated in stroke runners without any effects on apoptosis or neuronal migration in the forebrain. Running activity did not affect the SVZ in intact rats. In contrast to postischemic wheel running, postischemic environmental enrichment did not have attenuating effects on the ipsilateral SVZ and increased proliferating putative neural stem cells and neuronal precursors contralaterally. A significant functional improvement, assessed using a rotating pole, was observed only in the postischemically enriched group and was likely due to other types of plasticity than neuronal replacement at this early time point. It may be concluded that in contrast to enriched environment, exercise during the first postischemic week might be detrimental for regenerative processes initiated in the SVZ after stroke.

Does low-energy ultrasound, known to enhance thrombolysis, affect the size of ischemic brain damage?

OBJECTIVE: In view of the potentially beneficial effects of ultrasound on fibrinolysis, we aimed to study possible drawbacks of low-energy ultrasound on nonperfused and perfused brain tissue. METHODS: A model of transient focal ischemia in anesthetized rats was used. Rats were randomly assigned to 1 of 3 groups: the first exposed to ultrasound, the second a control of the ultrasound group, and the third a method control group. In each group, the right middle cerebral artery was occluded for 1.5 hours, during which time the rats in the ultrasound group were exposed to 1 hour of pulsed ultrasound (1 MHz; spatial-average temporal-average intensity, 0.1 W/cm2 at a duty cycle of 10%). The occlusion period was followed by a 24-hour recirculation period, after which the brains were excised and evaluated. RESULTS: Ultrasound did not affect the volume of ischemic damage in nonperfused brain tissue or add ischemic damage to perfused brain tissue. CONCLUSIONS: Under these experimental conditions, ultrasound does not cause additional ischemic damage to the rat brain during middle cerebral artery occlusion.

Mineralocorticoid receptor expression and increased survival following neuronal injury.

Glucocorticoids, acting via the mineralocorticoid receptor, are required for granule neuronal survival in the rat dentate gyrus. Whether this mineralocorticoid receptor-mediated neuroprotective effect has more general applicability is unknown. Here we report increased mineralocorticoid receptor expression in rat hippocampal and cortical neurons exposed in vitro to low levels of staurosporine and in rat hippocampal pyramidal neurons exposed in vivo to hypothermic transient global ischaemia. In both the cell culture system and the in vivo system increased mineralocorticoid receptor expression is associated with increased neuronal survival, and this increase is reversed by mineralocorticoid receptor antagonism. Modulation of mineralocorticoid receptor gene expression may therefore be an important target for reduction of brain injury in conditions caused by cerebral ischaemia including brain damage following cardiac arrest and stroke.

Cyclosporin A prevents calpain activation despite increased intracellular calcium concentrations, as well as translocation of apoptosis-inducing factor, cytochrome c and caspase-3 activation in neurons exposed to transient hypoglycemia.

Blockade of mitochondrial permeability transition protects against hypoglycemic brain damage. To study the mechanisms downstream from mitochondria that may cause neuronal death, we investigated the effects of cyclosporin A on subcellular localization of apoptosis-inducing factor and cytochrome c, activation of the cysteine proteases calpain and caspase-3, as well as its effect on brain extracellular calcium concentrations. Redistribution of cytochrome c occurred at 30 min of iso-electricity, whereas translocation of apoptosis-inducing factor to nuclei occurred at 30 min of recovery following 30 min of iso-electricity. Active caspase-3 and calpain-induced fodrin breakdown products were barely detectable in the dentate gyrus and CA1 region of the hippocampus of rat brain exposed to 30 or 60 min of insulin-induced hypoglycemia. However, 30 min or 3 h after recovery of blood glucose levels, fodrin breakdown products and active caspase-3 markedly increased, concomitant with a twofold increase in caspase-3-like enzymatic activity. When rats were treated with neuroprotective doses of cyclosporin A, but not with FK 506, the redistribution of apoptosis-inducing factor and cytochrome c was reduced and fodrin breakdown products and active caspase-3 immuno-reactivity was diminished whereas the extracellular calcium concentration was unaffected. We conclude that hypoglycemia leads to mitochondrial permeability transition which, upon recovery of energy metabolism, mediates the activation of caspase-3 and calpains, promoting cell death.