Post-ischemic Neurodegeneration Research Articles

Mechanistic Role of Calpains in Postischemic Neurodegeneration

The calpain family of proteases is causally linked to postischemic neurodegeneration. However, the precise mechanisms by which calpains contribute to postischemic neuronal death have not been fully elucidated. This review outlines the key features of the calpain system, and the evidence for its causal role in postischemic neuronal pathology. Furthermore, the consequences of specific calpain substrate cleavage at various subcellular locations are explored. Calpain substrates within synapses, plasma membrane, endoplasmic reticulum, lysosomes, mitochondria, and the nucleus, as well as the overall effect of postischemic calpain activity on calcium regulation and cell death signaling are considered. Finally, potential pathways for calpain-mediated neurodegeneration are outlined in an effort to guide future studies aimed at understanding the downstream pathology of postischemic calpain activity and identifying optimal therapeutic strategies.

Abstract 2837: Delayed Calpain Inhibition Is Neuroprotective After Transient Global Brain Ischemia

Introduction/Hypothesis: The delay in neurodegeneration after transient global brain ischemia offers a potentially broad therapeutic window for inhibiting molecular injury mechanisms. One such mechanism, calpain-mediated proteolysis, peaks between 24 and 72 hours after transient forebrain ischemia (TFI) in rats. This study tests the hypothesis that calpain inhibition is neuroprotective when initiated 22 hours after TFI. Methods: Male Long Evans rats (400 – 450 g) were anesthetized (halothane), mechanically ventilated, and instrumented for temperature and hemodynamic monitoring. Each underwent 10 minutes of TFI induced by reversible bilateral carotid occlusion and hypovolemic hypotension (MAP 30 mm Hg). Twenty-two hours after TFI, rats were block randomized (n=6/group) to receive either calpain inhibitor (CEP-3453 (Cephalon), 60 mg/kg IV bolus and 30 mg/kg IV infusion for 50 hours) or vehicle (normal saline IV bolus and infusion for 50 hours). Sham operated rats served as controls. Rats were euthanized 72 hours after injury and brains were processed for immunohistochemistry. Hippocampal CA1 sector calpain activity was analyzed by immunofluorescence using primary antibody that specifically detects calpain-cleaved alpha-spectrin (Ab38). Neurodegeneration was quantified by counting normal appearing Hoechst-stained neuronal nuclei in the hippocampal CA1 pyramidal layer. Ab38 immunofluorescence intensity and normal nuclei counts were compared between vehicle and CEP-3453 treatment groups using a two-tailed Student’s t-test (alpha error 0.05). Results: Baseline and post-injury hemodynamic parameters and temperature were not significantly different between vehicle and CEP-3453 treatment groups. Relative to sham operated controls, mean CA1 sector Ab38 immunofluorescence increased by 263 ± 281% in vehicle treated rats and 68 ± 147% in CEP-3453 treated rats (p=0.17). Normal CA1 pyramidal layer nuclei averaged 10 ± 12% of control in vehicle treated rats and 50 ± 42% of control in CEP-3453 treated rats (p=0.047). Conclusion: These results suggest a causal role for calpains in delayed post-ischemic neurodegeneration, and demonstrate a broad therapeutic window for calpain inhibition in this model of transient global brain ischemia.

Somatostatin Receptor 2 Is Activated in Cortical Neurons and Contributes to Neurodegeneration after Focal Ischemia

Somatostatin receptor 2 (SSTR2) mediates neuromodulatory signals of somatostatin and cortistatin in the cerebral cortex. Recently, SSTR2 has been shown to enhance conserved death ligand- and mitochondria-mediated apoptotic pathways in non-neuronal cells. Whether somatostatin receptors are activated in cerebrocortical neurons and contribute to neurodegeneration after experimental focal ischemia was unknown until now. Here we examined internalization of SSTR2 in a rat model of middle cerebral artery occlusion (MCAO) by confocal microscopy. At 3 and 6 hr after MCAO, SSTR2 was internalized excessively in cerebrocortical neurons adjacent to the infarct, which was prevented by intracerebroventricular application of the SSTR2-selective antagonist BIM-23627. SSTR2 internalization was associated with a transient depletion of somatostatin from axonal terminals and increased expression of SSTR2 mRNA. The initial loss of somatostatin was followed by an increase in somatostatin mRNA levels, whereas cortistatin mRNA expression was decreased. In SSTR2-deficient mice with lacZ under the control of the SSTR2 promoter, MCAO-induced upregulation of SSTR2 gene expression was less pronounced than in wild types. SSTR2-deficient mice exhibited a 40% reduction of infarct size after permanent distal MCAO and a 63% reduction after transient proximal MCAO. In summary, we provide direct evidence for activation of SSTR2 by an endogenous ligand after focal ischemia. Activation of functional SSTR2 receptors contributes to increased SSTR2 gene expression and postischemic neurodegeneration.

Poly(ADP-ribose) polymerase inhibitors attenuate necrotic but not apoptotic neuronal death in experimental models of cerebral ischemia.

An excessive activation of poly(ADP-ribose) polymerase (PARP) has been proposed to play a key role in post-ischemic neuronal death. We examined the neuroprotective effects of the PARP inhibitors benzamide, 6(5H)-phenanthridinone, and 3,4-dihydro-5-[4-1(1-piperidinyl)buthoxy]-1(2H)-isoquinolinone in three rodent models of cerebral ischemia. Increasing concentrations of the three PARP inhibitors attenuated neuronal injury induced by 60 min oxygen-glucose deprivation (OGD) in mixed cortical cell cultures, but were unable to reduce CA1 pyramidal cell loss in organotypic hippocampal slices exposed to 30 min OGD or in gerbils following 5 min bilateral carotid occlusion. We then examined the necrotic and apoptotic features of OGD-induced neurodegeneration in cortical cells and hippocampal slices using biochemical and morphological approaches. Cortical cells exposed to OGD released lactate dehydrogenase into the medium and displayed ultrastructural features of necrotic cell death, whereas no caspase-3 activation nor morphological characteristics of apoptosis were observed at any time point after OGD. In contrast, a marked increase in caspase-3 activity was observed in organotypic hippocampal slices after OGD, together with fluorescence and electron microscope evidence of apoptotic neuronal death in the CA1 subregion. Moreover, the caspase inhibitor Z-VAD-FMK reduced OGD-induced CA1 pyramidal cell loss. These findings suggest that PARP overactivation may be an important mechanism leading to post-ischemic neurodegeneration of the necrotic but not of the apoptotic type.

Apoptosis and necrosis occur in separate neuronal populations in hippocampus and cerebellum after ischemia and are associated with differential alterations in metabotropic glutamate receptor signaling pathways.

It was evaluated whether postischemic neurodegeneration is apoptosis and occurs with alterations in phosphoinositide-linked metabotropic glutamate receptors (mGluRs) and their associated signaling pathways. A dog model of transient global incomplete cerebral ischemia was used. The CA1 pyramidal cells and cerebellar Purkinje cells underwent progressive delayed degeneration. By in situ end-labeling of DNA, death of CA1 and Purkinje cells was greater at 7 days than 1 day after ischemia, whereas death of granule neurons in dentate gyrus and cerebellar cortex was greater at 1 than at 7 days. Ultrastructurally, degenerating CA1 pyramidal neurons and cerebellar Purkinje cells were necrotic; in contrast, degenerating granule neurons were apoptotic. In agarose gels of regional DNA extracts, random DNA fragmentation coexisted with internucleosomal fragmentation. By immunoblotting of regional homogenates, mGluR1alpha, mGluR5, phospholipase Cbeta (PLCbeta), and Galphaq/11 protein levels in hippocampus at 1 and 7 days after ischemia were similar to control levels, but in cerebellar cortex, mGluR1alpha and mGluR5 were decreased but PLCbeta was increased. By immunocytochemistry, mGluR and PLCbeta immunoreactivity dissipated in CA1 and cerebellar Purkinje cell/ molecular layers, whereas immunoreactivities for these proteins were enhanced in granule neurons. It was concluded that neuronal death after global ischemia exists as two distinct, temporally overlapping forms in hippocampus and cerebellum: necrosis of pyramidal neurons and Purkinje cells and apoptosis of granule neurons. Neuronal necrosis is associated with a loss of phosphoinositide-linked mGluR transduction proteins, whereas neuronal apoptosis occurs with increased mGluR signaling.

CD95 Ligand (Fas-L/APO-1L) and Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Mediate Ischemia-Induced Apoptosis in Neurons

Programmed cell death plays an important role in the neuronal degeneration after cerebral ischemia, but the underlying mechanisms are not fully understood. Here we examined, in vivo and in vitro, whether ischemia-induced neuronal death involves death-inducing ligand/receptor systems such as CD95 and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). After reversible middle cerebral artery occlusion in adult rats, both CD95 ligand and TRAIL were expressed in the apoptotic areas of the postischemic brain. Further recombinant CD95 ligand and TRAIL proteins induced apoptosis in primary neurons and neuron-like cells in vitro. The immunosuppressant FK506, which most effectively protects against ischemic neurodegeneration, prevented postischemic expression of these death-inducing ligands both in vivo and in vitro. FK506 also abolished phosphorylation, but not expression, of the c-Jun transcription factor involved in the transcriptional control of CD95 ligand. Most importantly, in lpr mice expressing dysfunctional CD95, reversible middle cerebral artery occlusion resulted in infarct volumes significantly smaller than those found in wild-type animals. These results suggest an involvement of CD95 ligand and TRAIL in the pathophysiology of postischemic neurodegeneration and offer alternative strategies for the treatment of cardiovascular brain disease.

Post-ischemic changes of [ 3H]glycine binding in the gerbil brain after cerebral ischemia

Sequential changes of [ 3H]glycine binding in the gerbil were investigated in selectively vulnerable areas 1 h to 7 days after 10 min of cerebral ischemia. A significant reduction in [ 3H]glycine binding was found in the hippocampus and thalamus from as early as 1 h after ischemia. In contrast, the striatum and frontal cortex showed a significant decline in [ 3H]glycine binding from 5 h after recirculation. Thereafter, a severe reduction in [ 3H]glycine binding was observed in all regions 7 days after ischemia. MAP2 (microtubule-associated protein 2) immunoreactivity was unaffected in the hippocampus, frontal cortex and thalamus up to 48 h after ischemia. Thereafter, a severe loss of MAP2-immunoreactive neurons was found in these regions, especially in the hippocampal CA1 sector. However, the striatum showed a severe loss of MAP2 immunoreactivity from 24 h after ischemia. These results demonstrate that transient cerebral ischemia causes severe reduction in [ 3H]glycine binding throughout the brain, and this reduction precedes the neuronal damage in selectively vulnerable areas. These findings suggest that a neurotransmitter, glycine, may play a key role in the pathogenesis of post-ischemic neurodegeneration in selectively vulnerable areas.

An immunohistochemical observation of manganese superoxide dismutase in rat substantia nigra after occlusion of middle cerebral artery

We investigated immunohistochemically the localization and changes of manganese superoxide dismutase (Mn-SOD) in substantia nigra after 1 h of middle cerebral artery (MCA) occlusion in the rat with reperfusion periods of 1, 3, 7, and 14 days. In normal rats, Mn-SOD immunoreactivity was observed intensely in many large neurons in substantia nigra pars reticulata (SNr) and weakly in neurons in the pars compacta (SNc). The immunoreactivity of the neurons in both SNr and SNc was reduced after 1 day and almost lost 3 days after MCA occlusion, although these neurons showed completely normal morphology. The immunoreactivity recovered only in the large neurons in SNr from the 7th day and well preserved after 2 weeks, when SNr showed obvious atrophy. The results indicate that oxidative stress may be involved in the pathogenesis of the secondary postischemic neurodegeneration in SN.

Excitotoxins and free radicals: accomplices in post-ischemic and other neurodegeneration

Excitotoxins and free radicals: accomplices in post-ischemic and other neurodegeneration