Postischemic Neuronal Death Research Articles

Ultrastructural and MRI study of the substantia nigra evolving exofocal post-ischemic neuronal death in the rat.

To clarify the morphological characteristics of exofocal post-ischemic neuronal death (EPND) in the substantia nigra (SN), we investigated the course of light- and electron-microscopic changes of the SN of rats subjected to occlusion of the left middle cerebral artery (MCA) for 1, 2, 4, 7 and 12 days. To assess cellular edema, sequential magnetic resonance (MR) mapping of the apparent diffusion coefficient (ADC) and the T2 value test was performed. Histological and electron-microscopic examination on day 1 showed dotted chromatin clumps in the nuclei of some neurons and mild swelling of the perivascular endfeet of astrocytes in the ipsilateral SN. On day 2, a few cells of the ipsilateral SN pars reticulata (SNr) revealed key morphological signs of apoptosis--apoptotic body-like condensation and segregation of the chromatin and DNA fragmentation-like nuclear remnants. On day 4, 38% of neurons became swollen (pale neurons) with cytoplasmic microvacuoles, which appeared to originate from rough endoplasmic reticulum (rER), mitochondria and Golgi apparatus. Twenty percent of neurons showed massive proliferation of the cisternae of the rER, some of which were fragmented or had lost their normal parallel arrangement. In addition, MR mapping revealed a transient ADC decrease with a T2 increase (signifying a phase of cellular edema), which coordinated with the phase of ultrastructural cellular swelling. Further, the total number of neurons started to decrease gradually, the perivascular endfeet of astrocytes were markedly swollen, and the neuropil became loose on day 4. On day 7, reactive astrocytes and dark neurons occurred most frequently. These results suggest that the EPND in the SN after occlusion of the MCA in adult rats is due to both apoptosis and necrosis, although necrosis seems to be the dominant mechanism of the EPND. However, the morphologic resemblances of EPND to delayed neuronal death suggest these processes have a common pathomechanism.

Comparison of Calpain and Caspase Activities in the Adult Rat Brain after Transient Forebrain Ischemia

The role of calpain and caspase family proteases in postischemic neuronal death remains controversial. This study compared the timing, location, and relative activity of calpains and caspases in the adult rat brain following 10 min of transient forebrain ischemia. Western blots of cortical, striatal, and hippocampal homogenates demonstrated a α-spectrin cleavage pattern indicative of predominant calpain activity, which peaked between 24 and 48 h after reperfusion. However, immunohistochemical evidence of both caspase 3 activation and caspase-mediated substrate cleavage was detected as early as 1 h and as late as 7 days after reperfusion in circumscribed neuronal populations. Simultaneous or sequential caspase and calpain activation was also observed suggesting the potential for interaction of these protease systems. The complex spatiotemporal pattern of calpain and caspase activity observed in this study provides important insights for the development and evaluation of therapeutic strategies to reduce protease-mediated injury following global brain ischemia.

Activation of mGlu1 but not mGlu5 metabotropic glutamate receptors contributes to postischemic neuronal injury in vitro and in vivo

In order to investigate the involvement of mGlu1 and mGlu5 metabotropic glutamate receptors in the development of postischemic neuronal death, we examined the effects of selective agonists and antagonists in models of cerebral ischemia in vitro and in vivo. In murine cortical cell cultures and rat organotypic hippocampal slices exposed to oxygen and glucose deprivation (OGD), the mGlu1 antagonists 1-aminoindan-1,5-dicarboxylic acid (AIDA; 300 μM), ( S)-(+)-2-(3′-carboxybicyclo[1.1.1]pentyl)-glycine (CBPG; 300 μM), 7-hydroxyiminocyclopropan[ b]chromen-1 a-carboxylic acid ethyl ester (CPCCOEt; 10–30 μM) and (+)-2-methyl-4-carboxyphenylglycine (LY367385; 30–100 μM) reduced neuronal loss when added to the medium during OGD and the subsequent 24-h recovery period. On the contrary, the potent and selective mGlu5 antagonist methyl-6-(phenylethynyl)-pyridine (MPEP; 0.1–1 μM) did not exhibit neuroprotection in any of these in vitro models. Incubation with the nonselective mGlu1 and mGlu5 agonist 3,5-dihydroxyphenylglycine (3,5-DHPG; 300 μM) but not with the mGlu5 agonist ( RS)-2-chloro-5-hydroxyphenylglycine (CHPG; 1 mM) enhanced the severity of OGD-induced neuronal damage. In gerbils subjected to global ischemia, intracerebroventricular administration of AIDA (100 nmol two times) or CBPG (300 nmol, two times) afforded consistent protection against CA1 pyramidal cell death, whereas MPEP (10 pmol i.c.v two times and 10 mg/kg i.p two times) failed to reduce postischemic hippocampal damage. Our results suggest that activation of mGlu1 but not mGlu5 receptor contributes to postischemic neuronal injury.

Comet assay as a novel approach for studying DNA damage in focal cerebral ischemia: differential effects of NMDA receptor antagonists and poly(ADP-ribose) polymerase inhibitors.

The single-cell gel electrophoresis (comet assay) was used to evaluate the possibility of detecting single-strand breaks of brain DNA in the early phase of ischemia. Four hours after occlusion of the middle cerebral artery (MCAO) in rats, the percentage of DNA migrating into the comet tail (indicating the presence of breaks) increased from 11.4 +/- 4.70 to 34.7 +/- 9.2 (means +/- SD) in the caudate and from 9.9 +/- 4.3 to 42.8 +/- 14.1 in the cortex. Interestingly, a subpopulation of cells exhibiting higher resistance to the ischemic insult was present in the caudate putamen, but not in the cortex. Administration of MK801, an N-methyl-d-aspartate (NMDA) glutamate receptor antagonist, (1 mg/kg subcutaneously, 10 minutes before MCAO), reduced the ischemia-induced DNA breaks and the infarct volume, suggesting that excessive stimulation of NMDA receptors contributes to the formation of both DNA damage and infarct volume. In contrast, DPQ, an inhibitor of poly(ADP-ribose) polymerase (PARP) (10 mg/kg intraperitoneally, 2 hours before and 1 hour after MCAO), reduced the infarct volume but not DNA damage, suggesting that the neuroprotective actions of PARP inhibitors occur at a later step of the processes leading to postischemic neuronal death.

Primate neurons show different vulnerability to transient ischemia and response to cathepsin inhibition.

Previously, we reported "calpain-induced leakage of lysosomal enzyme cathepsin" as a mechanism of ischemic neuronal death specific for primates. Cathepsin inhibitors such as CA-074 and E-64c were demonstrated to significantly inhibit hippocampal neuronal death. Pyramidal neurons of the hippocampus, Purkinje cells in the cerebellum, and neurons in the caudate nucleus, outer putamen and cortical III, V layers, are known to be vulnerable to ischemia. However, regional differences of the vulnerability and response to neuroprotectants, have not been studied in detail. Here, the monkey brains undergoing transient ischemia were studied to clarify such regional differences by the microscopic counting of surviving neurons. The dead neurons were characterized by eosinophilic coagulation necrosis without apoptotic bodies. The control postischemic brain without treatment showed surviving neurons in caudate nucleus (55.8%), outer putamen (44.4%), cortical III layer (37.8%), CA4 (35.3%), cortical V layer (34.1%), cerebellum (28.2%), CA3 (24.3%), CA2 (16.2%), and CA1 (2.0%). Only the CA1 showed an almost total neuronal loss. In contrast, a single postictal injection of CA-074 or E-64c led to significant inhibition of postischemic neuronal death in all brain regions studied. Overall, more surviving neurons were seen after E-64c treatment than with CA-074: cerebellum, 91.6% vs 85.6%; CA4, 88.6% vs 77.3%; caudate nucleus, 86.1% vs 89.8%; CA2, 83.6% vs 53.0%; outer putamen, 81.3% vs 87.7%; CA1, 80.1% vs 47.4%; CA3, 79.6% vs 60.3%; cortical layer III, 75.5% vs 67.7%; and cortical layer V, 75.0% vs 65.9%, for E-64c and CA-074, respectively. Cathepsin plays a critical role in ischemic neuronal death, and its inhibitors may protect neurons throughout the brain.

The novel and systemically active metabotropic glutamate 1 (mGlu1) receptor antagonist 3-MATIDA reduces post-ischemic neuronal death

We examined the pharmacological properties of 3-methyl-aminothiophene dicarboxylic acid (3-MATIDA) by measuring second messenger responses in baby hamster kidney cells stably transfected with mGlu1a, mGlu2, mGlu4a or mGlu5a receptors and ionotropic glutamate receptor agonist-induced depolarizations in mouse cortical wedges. 3-MATIDA was a potent (IC 50=6.3 μM, 95% confidence limits 3–15) and relatively selective mGlu1 receptor antagonist. When tested on mGlu2, mGlu4 or mGlu5 receptors its IC 50 was >300 μM. When tested in cortical wedges, however, 3-MATIDA was also able to antagonize AMPA or NMDA responses with an IC 50 of 250 μM. When present in the incubation medium of cultured murine cortical cells, 3-MATIDA (1–100 μM) significantly reduced the death of neurons induced by 60 min of oxygen and glucose deprivation (OGD), even when added up to 60 min after OGD. A similar neuroprotective activity was observed when 3-MATIDA was present at 10–100 μM in the medium of rat organotypic hippocampal slice cultures exposed to 30 min OGD. Systemic administration of 3-MATIDA (3–10 mg/kg, immediately and 1 h after the onset of ischemia) reduced the volume of brain infarcts following permanent middle cerebral artery occlusion in rats. Our results show that 3-MATIDA is a potent and possibly selective mGlu 1 receptor antagonist that may be considered as a novel prototype neuroprotective agent.

Vitamin B6 protects primate retinal neurons from ischemic injury

Vitamin B6 derivatives protect the retinal neurons from excitotoxic injury in vitro. However, their in vivo role in a process involving excitotoxicity, such as ischemia, remains unknown. We studied potential protective effects of pyridoxal 5′-phosphate (PLP) and pyridoxal hydrochloride (pyridoxal) on the retinal neurons in a monkey model of transient global ischemia. Daily intravenous injections (15 mg/kg) of pyridoxal and PLP were performed for consecutive 10 days. On the sixth day, whole brain complete ischemia was produced by clipping the innominate and the left subclavian arteries for 20 min. The monkeys were sacrificed 5 days after ischemia and their retinas were processed for histological analysis. The ischemia induced a marked cellular injury in the retina as shown by the loss of ganglion cells and the reduction of thickness of the ganglion cell, inner plexiform, and inner nuclear layers. PLP significantly prevented the ganglion cell loss and the reduction of thickness of the ganglion cell layer. Pyridoxal significantly prevented the ganglion cell loss as well as the reduction of thickness of ganglion cell, inner plexiform and inner nuclear layers. These results suggest that PLP and pyridoxal counteract the postischemic neuronal death in the adult primate retina, offering a potential for a novel pharmacotherapy of retinal ischemic injury.

PET imaging of ischemic neuronal death in the hippocampus of living monkeys.

The aim of the present study was to visualize postischemic hippocampal neuronal death in the living monkey brain, using a high-resolution positron emission tomography (PET) and novel radioligands. In preceding papers, we reported on postischemic hippocampal neuronal death in a model of Japanese monkeys (Macaca fuscata) undergoing a 20-min complete whole-brain ischemia. Using the same model here, we investigated the in vivo bindings of two radiotracers, [11C]Ro15-4513 (a type II benzodiazepine receptor ligand) and [11C](+)3-MPB (a muscarinic cholinergic receptor ligand), in the hippocampus on day 7 after ischemia, as compared to the normal hippocampus. A significant decrease in the in vivo binding of [11C]Ro154513 and [11C(+)3-MPB was observed in the postischemic monkey hippocampus on day 7 after ischemia compared to controls. Light and electron microscopic analyses of postischemic CA1 neurons showed typical features of coagulation necrosis, as associated with a marked reduction of postsynaptic densities and presynaptic vesicles. These results suggest that semiquantification of hippocampal neuronal death is possible in the living primate brain using PET, and that the same procedures can be applied for evaluating neuronal cell loss in patients with ischemic injuries and/or dementia.

Modeling of poly(ADP-ribose)polymerase (PARP) inhibitors. Docking of ligands and quantitative structure-activity relationship analysis.

Poly(ADP-ribose)polymerase-1 (PARP-1) is a nuclear enzyme that has recently emerged as an important player in the mechanisms leading to postischemic neuronal death, and PARP inhibitors have been proposed as potential neuroprotective agents. With the aim of clarifying the structural basis responsible for PARP inhibition, we carried out a computational study on 46 inhibitors available through the literature. Our computational approach is composed of three parts. In the first one, representative PARP inhibitors have been docked into the crystallographic structure of the catalytic domain of PARP by using the Autodock 2.4 program. The docking studies thus carried out have provided an alignment scheme that has been instrumental for superimposing all the remaining inhibitors. Upon the basis of this alignment scheme, a quantitative structure-activity relationship (QSAR) analysis has been carried out after electrostatic and steric interaction energies have been computed with the RECEPTOR program. The QSAR analysis yielded a predictive model able to explain much of the variance of the 46-compound data set. The inspection of the QSAR coefficients revealed that the major driving force for potent inhibition is given by the extension of the contact surface between enzyme and inhibitors while electrostatic energy and hydrogen bonding capability play a minor role. Finally, the projection of the QSAR coefficients back onto the X-ray structure of the catalytic domain of PARP provides insights into the role played by specific amino acid residues. This information will be useful to address the design of new selective and potent PARP inhibitors.

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.

Effect of nordihydroguaiaretic acid on behavioral impairment and neuronal cell death after forebrain ischemia

The purpose of this study was to evaluate the neuroprotective effect of nordihydroguaiaretic acid (NDGA), an antioxidant and/or 5-lipoxygenase inhibitor, on ischemia–reperfusion injury behavioral pharmacologically and histologically in vivo. First, the antioxidant activity of NDGA was evaluated in vitro by measuring the production of thiobarbituric acid reactive substances (TBARS) in rat brain homogenate. Second, the effect of NDGA on learning and memory impairment induced by rat four-vessel occlusion transient ischemia was investigated with the Morris water-maze task. Third, the effect of NDGA on pyramidal cell loss in the hippocampus after transient ischemia was examined. NDGA inhibited the production of TBARS with an IC 50 of 0.1 μM, and significantly attenuated postischemic learning and memory impairment at 10 mg/kg. Furthermore, consecutive 4-day administration of NDGA at 10 mg/kg significantly reduced the postischemic neuronal death. NDGA was found to be potent and effective as an anti-ischemia–reperfusion injury agent in terms of behavioral pharmacology and histology. The present results suggest that NDGA has beneficial effects on behavioral deficits and histological injury caused by ischemia–reperfusion.

Characteristic changes in T 2-value, apparent diffusion coefficient, and ultrastructure of substantia nigra evolving exofocal postischemic neuronal death in rats

To correlate the magnetic resonance (MR) imaging characteristics of exofocal postischemic neuronal death (EPND) in the substantia nigra (SN) with associated histologic changes, we occluded the left middle cerebral artery of rats for 1, 4, 7, or 12 days. Day 1 (post-occlusion) T 2-weighted images revealed high signal intensity indicative of infarction in the ipsilateral caudate nucleus, putamen, and cortex but not the SN. Diffusion-weighted images (DWIs) on day 1 similarly failed to reveal any changes in the SN. However, on day 4, DWIs revealed high signal intensity in the ipsilateral SN, in which the apparent diffusion coefficient (ADC) transiently decreased ( P<0.05) while the T 2-value increased ( P<0.05). These measures returned to and remained at control levels on days 7 and 12. Histologic examination on day 4 revealed dark-staining neurons, markedly swollen perivascular astrocytic end-feet, many swollen neurons with cytoplasmic microvacuoles that mainly originated in the rough endoplasmic reticulum, and strongly roughed neuropils. Reactive astrocytes and dark neurons most frequently appeared on days 7 and 12. The severity of cellular swelling paralleled the change in the ADC. These results demonstrate that a transient high-intensity signal on DWIs, indicative of a decrease in the ADC, is predictive of EPND in the SN.

Molecular mechanisms of ischemic neuronal injury

Brain ischemia triggers a complex cascade of molecular events that unfolds over hours to days. Identified mechanisms of postischemic neuronal injury include altered Ca2+ homeostasis, free radical formation, mitochondrial dysfunction, protease activation, altered gene expression, and inflammation. Although many of these events are well characterized, our understanding of how they are integrated into the causal pathways of postischemic neuronal death remains incomplete. The primary goal of this review is to provide an overview of molecular injury mechanisms currently believed to be involved in postischemic neuronal death specifically highlighting their time course and potential interactions. [Neumar RW. Molecular mechanisms of ischemic neuronal injury. Ann Emerg Med. November 2000;36:483-506.]

1-Aminoindan-1,5-dicarboxylic acid and (S)-(+)-2-(3'-carboxybicyclo[1.1.1] pentyl)-glycine, two mGlu1 receptor-preferring antagonists, reduce neuronal death in in vitro and in vivo models of cerebral ischaemia.

Metabotropic glutamate (mGlu) receptors have been implicated in a number of physiological and pathological responses to glutamate, but the exact role of group I mGlu receptors in causing postischaemic injury is not yet clear. In this study, we examined whether the recently-characterized and relatively selective mGlu1 receptor antagonists 1-aminoindan-1,5-dicarboxylic acid (AIDA) and (S)-(+)-2-(3'-carboxybicyclo[1.1.1]pentyl)-glycine (CBPG) could reduce neuronal death in vitro, following oxygen-glucose deprivation (OGD) in murine cortical cell and rat organotypic hippocampal cultures, and in vivo, after global ischaemia in gerbils. When present in the incubation medium during the OGD insult and the subsequent 24 h recovery period, AIDA and CBPG significantly reduced neuronal death in vitro. The extent of protection was similar to that observed with the nonselective mGlu receptor antagonist (+)-alpha-methyl-4-carboxyphenylglycine [(+)MCPG] and with typical ionotropic glutamate (iGlu) receptor antagonists. Neuroprotection was also observed when AIDA or CBPG were added only after the OGD insult was terminated. Neuronal injury was not attenuated by the inactive isomer (-)MCPG, but was significantly enhanced by the nonselective mGlu receptor agonist (1S,3R)-1-aminocyclopentane-1, 3-dicarboxylic acid [(1S,3R)-ACPD] and the group I mGlu receptor agonist 3,5-dihydroxyphenylglycine (3,5-DHPG). The antagonists (+)MCPG, AIDA and CBPG were also neuroprotective in vivo, because i. c.v. administration reduced CA1 pyramidal cell degeneration examined 7 days following transient carotid occlusion in gerbils. Our results point to a role of mGlu1 receptors in the pathological mechanisms responsible for postischaemic neuronal death and propose a new target for neuroprotection.

Neuroprotective effect of mild hypothermia cannot be explained in terms of a reduction of glutamate release during ischemia

An exogenous glutamate injection into the hypothermic hippocampal CA1 during 5-min ischemia produced the same extent of extracellular glutamate levels as observed in the normothermic CA1 during 5-min ischemia; however, neuronal death was not induced in the hypothermic CA1. Glutamate is released excessively into the extracellular space during ischemia, and is thought to induce brain injury by its neurotoxicity. It has been reported that the massive glutamate release is reduced by mild hypothermia, and it has been proposed that the reduction of ischemia-induced glutamate release exerts the neuroprotective effect on postischemic neuronal death. In the present study, to determine whether the neuroprotective effect of mild hypothermia on postischemic hippocampal CA1 neuronal death is due to the reduction of ischemia-induced glutamate release, gerbils were subjected to 5-min ischemia under hypothermic condition at 31°C and were simultaneously injected exogenously with l-glutamate, so that the hypothermic CA1 around a microdialysis probe was exposed to the same extracellular glutamate levels as seen during normothermic ischemia, and the histological outcome was examined. An injection with 1 mM l-glutamate into the hypothermic CA1 during 5-min ischemia produced a similar extent of increased glutamate (17-fold increase) to that observed in the normothermic CA1 during 5-min ischemia (16-fold increase). However, neuronal death was not induced in the hypothermic CA1. This result indicates that the neuroprotective effect of mild hypothermia cannot be explained in terms of a reduction of glutamate release during ischemia.

Experimental protocol for studying delayed effects of in vitro ischemia on neurotransmitter release from brain slices

The mechanisms by which ischemic injury leads to delayed neuronal death are not completely understood. Notably, no data are so far available on the modifications in neurosecretory responses evoked by a period of ischemia. Superfused brain slices represent a useful preparation in studying the effects of in vitro ischemia on neurotransmitter release. Using this experimental model we describe a protocol which allows to study not only the immediate effects of an ischemic insult, but also, more interestingly, its delayed (1 h) effects on the release of different neurotransmitters. A first pulse ( S 1) of 50 mM KCl was applied at the 60th min of perfusion and a second one was applied at the 210th min ( S 2). In vitro ischemia was performed from the 120th to the 150th min, during the inclusive period between the two depolarizing stimuli. The delayed effects of the ischemic treatment on slice response to KCl were calculated as S 2/ S 1 ratio. This protocol allows to study neurotransmitter release mechanisms associated with postischemic neuronal death. Moreover it will be useful in the evaluation of the neuroprotective potential of new drugs. Themes: Disorders of the nervous system Topics: Ischemia

Enhanced neuronal death from focal ischemia in AMPA-receptor transgenic mice

Excitatory amino acid (EAA) receptors play an important role in neuronal cell death in acute cerebral ischemia. Blocking the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) subtype of EAA receptor has been shown to reduce cell death in global cerebral ischemia. However their role in focal stroke, although suggestive, has remained more contentious. To clarify this issue, we generated transgenic mice overexpressing the AMPA receptor (AMPAR) subunit GluR2-flip which would increase AMPAR-mediated currents. Excitatory neurons in these transgenic mice are thus predicted to be more susceptible than wild-type neurons to EAA (glutamate)-induced excitotoxic damage. Consistent with this prediction, cultured neurons from transgenic mice had a lower LD 50 for exposure to glutamate (10 −3–10 −5 M for 5 min) compared to wild-type neurons. Moreover, transgenic mice subjected to permanent focal ischemia of the middle cerebral artery (MCA) using the intralumenal filament model sustained larger infarctions compared to wild-type controls. Hence we have developed a genetic mouse model that demonstrates the crucial role of AMPAR containing GluR2-flip in the pathogenesis of focal hypoxic-ischemic neuronal cell death. This model will be a valuable tool in elucidating molecular mechanisms of glutamate excitotoxicity and evaluating the efficacy of glutamate receptor antagonists in attenuating post-ischemic neuronal cell death.

Thromboxane Synthetase Inhibitor Ameliorates Delayed Neuronal Death in the CA1 Subfield of the Hippocampus after Transient Global Ischemia in Gerbils

Thromboxane A2 accumulates in the hippocampus after global ischemia and may play a key role in postischemic hypoperfusion. Thromboxane synthetase inhibitor (OKY-046) inhibits the accumulation of thromboxane A2 and promotes prostacycline production. Therefore, we set out to determine whether the inhibition of thromboxane synthesis would ameriolate postischemic neuronal death. Three groups of six Mongolian gerbils were subjected to different treatments: untreated control, untreated ischemia, and treated ischemia. Immediately after forebrain ischemia, OKY-046 (10 mg/kg) was injected intraperitoneally into the treated group. After 7 days of survival, the histopathology of the brain was examined. Pyramidal cell density in the CA1 sector in the treated group was 147 +/- 70 nuclei/mm (mean +/- SD), which was significantly (p < 0.05) higher than than in the untreated group (33 +/- 10 (nuclei/mm). The findings were 231 +/- 7 nuclei/mm for the control group. No significant difference was seen in the profile of temporal muscle temperature before and after ischemia between the groups. Ultrastructurally, the vessels in the CAI sector showed lumen patency in the treated group, whereas occluded vessels with an extended perivascular space were observed in the untreated group. Thromboxane synthetase inhibitor thus partly ameliorates the selective vulnerability of the hippocampus after forebrain ischemia, suggesting that thromboxane A2 is involved in the development of delayed neuronal death, independently of any thermal effect.

Brain μ‐Calpain Autolysis During Global Cerebral Ischemia

Proteolytic degradation of numerous calpain substrates, including cytoskeletal and regulatory proteins, has been observed during brain ischemia and reperfusion. In addition, calpain inhibitors have been shown to decrease degradation of these proteins and decrease postischemic neuronal death. Although these observations support the inference of a role for mu-calpain in the pathophysiology of ischemic neuronal injury, the evidence is indirect. A direct indicator of mu-calpain proteolytic activity is autolysis of its 80-kDa catalytic subunit, and therefore we examined the mu-calpain catalytic subunit for evidence of autolysis during cerebral ischemia. Rabbit brain homogenates obtained after 0, 5, 10, and 20 min of cardiac arrest were electrophoresed and immunoblotted with a monoclonal antibody specific to the mu-calpain catalytic subunit. In nonischemic brain homogenates the antibody identified an 80-kDa band, which migrated identically with purified mu-calpain, and faint 78- and 76-kDa bands, which represent autolyzed forms of the 80-kDa subunit. The average density of the 80-kDa band decreased by 25 +/- 4 (p = 0.008) and 28 +/- 9% (p = 0.004) after 10 and 20 min of cardiac arrest, respectively, whereas the average density of the 78-kDa band increased by 111 +/- 50% (p = 0.02) after 20 min of cardiac arrest. No significant change in the density of the 76-kDa band was detected. These results provide direct evidence for autolysis of brain mu-calpain during cerebral ischemia. Further work is needed to characterize the extent, duration, and localization of mu-calpain activity during brain ischemia and reperfusion as well as its role in the causal pathway of postischemic neuronal injury.

The glycine antagonist and free radical scavenger 7-C1-thio-kynurenate reduces CA1 ischemic damage in the gerbil

We examined whether 7-C1-thio-kynurenate, a potent antagonist at the glycine site of the N-methyl- d-aspartate receptor which also inhibits lipid peroxidation, protected CA1 pyramidal cells following transient forebrain ischemia. Global ischemia was produced in anesthetized gerbils by 5 min bilateral carotid artery occlusion; hippocampal injury was assessed seven days later. 7-C1-thio-kynurenate (100 mg/kg, i.p. × 5) dramatically attenuated ischemia-induced CA1 cell loss (from95 ± 1 to7 ± 3%): the protection was associated with a delayed and marked reduction in the animals' temperature. However, when the gerbils were maintained normothermic for at least 360 min, 7-C1-thio-kynurenate still provided partial (54 ± 11%) but significant protection. No protection was observed when a reduction in temperature with a time course similar to that caused by 7-C1-thio-kynurenate was experimentally induced in saline-treated ischemie animals. In situ hybridization revealed that expression of NMDA-R1, a subunit of the N-methyl- d-aspartate receptor, was selectively reduced in CA1 seven days following global ischemia. In ischemic gerbils treated with 7-C1-thio-kynurenate, protected CA1 cells were still able to express normal amounts of NMDA-R1 messenger RNA. Our results demonstrate that 7-C1-thio-kynurenate, a glutamate receptor blocker possessing radical scavenger properties, is effective in reducing CA1 hippocampal damage following global ischemia in the gerbil. Since there is growing evidence that a positive feedback interaction between activation of glutamate receptors and free radical formation may be responsible for the generation of ischemic brain damage, drugs capable of interfering with both pathogenic mechanisms may be useful in preventing post-ischemic neuronal death.