Acute Neuronal Death Research Articles

Glutamate and the glutamate receptor system: a target for drug action.

Glutamate is the most important excitatory neurotransmitter in the central nervous system. In the process, glutamate fulfills numerous physiological functions, but also plays an important role in the pathophysiology of different neurological and psychiatric diseases, especially when an imbalance in glutamatergic neurotransmission occurs. Under certain conditions, glutamate has a toxic action resulting from an activation of specific glutamate receptors, which leads to acute or chronic death of nerve cells. Such mechanisms are currently under discussion in acute neuronal death within the context of hypoxia, ischaemia and traumas, as well as in chronic neurodegenerative or neurometabolic diseases, idiopathic parkinsonian syndrome, Alzheimer's dementia and Huntington's disease. It is hoped that glutamate antagonists will lead to novel therapies for these diseases, whereby the further development of glutamate antagonists for blocking disease-specific subtypes of glutamate receptors may be of major importance in the future.

Brevetoxin derivatives act as partial agonists at neurotoxin site 5 on the voltage-gated Na + channel

Brevetoxins (PbTx-1 to PbTx-10) are potent lipid-soluble polyether neurotoxins produced by the marine dinoflagellate Karina brevis, an organism associated with ‘red tide’ blooms in the Gulf of Mexico. Ingestion of shellfish contaminated with K. brevis produces neurotoxic shellfish poisoning (NSP) in humans. NSP symptoms emanate from brevetoxin activation of neurotoxin site 5 on voltage-gated sodium channels (VGSC) [Toxicon 20 (1982) 457]. In primary cultures of rat cerebellar granule neurons (CGN), brevetoxins produce acute neuronal injury and death. The ability of a series of naturally occurring and synthetic brevetoxins to trigger Ca 2+ influx in CGN was explored in the present study. Intracellular Ca 2+ concentration was monitored in fluo-3-loaded CGN using a fluorescent laser imaging plate reader. The naturally occurring derivatives PbTx-1, PbTx-2 and PbTx-3 all produced a rapid and concentration-dependent increase in cytosolic [Ca 2+]. The maximum response to PbTx-1 was approximately two-fold greater than that of either PbTx-2 or PbTx-3. Two synthetic derivatives of PbTx-3, α-naphthoyl-PbTx-3 and β-naphthoyl-PbTx-3, were also tested. Both α- and β-naphthoyl-PbTx-3 stimulated a rapid and concentration-dependent Ca 2+ influx that was, however, less efficacious than that of PbTx-3. These data indicate that, analogous to neurotoxin site 2 ligands, activators of neurotoxin site 5 display a range of efficacies, with PbTx-1 being a full agonist and other derivatives acting as partial agonists.

Selective targeting of synthetic antioxidants to mitochondria: towards a mitochondrial medicine for neurodegenerative diseases?

Mitochondria are the major source of superoxide, and are responsible for activating apoptosis and oxidative damage during acute neuronal cell death and neurodegenerative disorders like Alzheimer and Parkinson diseases. While the molecular mechanisms by which mitochondrial oxidative stress triggers apoptosis are still investigated, attempts to achieve neuroprotection using antioxidant molecules have already been successful in several models of neuronal cell death. To increase the availability of antioxidant drugs at the mitochondrial level within cells, Michael P. Murphy recently proposed to covalently couple antioxidant molecules to a membrane-permeable lipophilic cation serving as carrier. Since mitochondria maintain at rest a potential of −180 mV, the diffusible cationic moiety drives the accumulation of the complex inside the matrix towards a diffusion equilibrium: for a monovalent cationic carrier, a thousand-fold accumulation of the complex is theoretically achievable; for a divalent cation, a million-fold accumulation is expected. Such mitochondria-targeted versions of natural antioxidants have successfully been synthesized and were found to counteract the pro-apoptotic effects of exogenous oxidative insults, while having no effects in models mimicking physiological apoptosis. Based on these observations, we carried out the synthesis of targeted variants of the artificial free radical scavengers 4-hydroxy-2,2,6,6-tetramethylpiperidin- N-oxide (TEMPOL) and Salen–Mn(III) complex of o-vanillin (EUK-134). Our preliminary results indicate that these targeted compounds, while delaying apoptosis after an exogenous oxidative insult, are not more active than their untargeted variants. This questions the general efficiency of the targeting procedure used and/or suggests that the main pro-apoptotic effector targets of exogenous oxidative insults are not located within mitochondria.

Selective release of calpain produced alphalI-spectrin (alpha-fodrin) breakdown products by acute neuronal cell death.

Activation of calpain results in the breakdown of alpha II spectrin (alpha-fodrin), a neuronal cytoskeleton protein, which has previously been detected in various in vitro and in vivo neuronal injury models. In this study, a 150 kDa spectrin breakdown product (SBDP150) was found to be released into the cell-conditioned media from SH-SY5Y cells treated with the calcium channel opener maitotoxin (MTX). SBDP150 release can be readily quantified on immunoblot using an SBDP150-specific polyclonal antibody. Increase of SBDP150 also correlated with cell death in a time-dependent manner. MDL28170, a selective calpain inhibitor, was the only protease inhibitor tested that significantly reduced MTX-induced SBDP150 release. The cell-conditioned media of cerebellar granule neurons challenged with excitotoxins (NMDA and kainate) also exhibited a significant increase of SBDP150 that was attenuated by pretreatment with an NMDA receptor antagonist, R(-)-3-(2-carbopiperazine-4-yl)-propyl-1-phosphonic acid (CPP), and MDL28170. In addition, hypoxic/hypoglycemic challenge of cerebrocortical cultures also resulted in SBDP150 liberation into the media. These results support the theory that an antibody-based detection of SBDP150 in the cell-conditioned media can be utilized to quantify injury to neural cells. Furthermore, SBDP150 may potentially be used as a surrogate biomarker for acute neuronal injury in clinical settings.

Fas (CD95/Apo-1)/Fas ligand expression in neonates with pontosubicular neuron necrosis.

Pontosubicular neuron necrosis (PSN) represents an age-specific response to severe hypoxic-ischemic injury occurring in human neonates but not in older children or adults. Histologically, PSN is characterized by acute neuronal death in the pontine nuclei and the hippocampal subiculum bearing the hallmarks of apoptosis. In animal models of hypoxic-ischemic injury, induction of neuronal apoptosis can be triggered by Fas (CD95/Apo-1), a cell surface receptor of the tumor necrosis factor-alpha superfamily, which transduces apoptotic death signals when cross-linked by its natural ligand. Here, we have investigated the expression of Fas/Fas ligand in human autopsy material consisting of 13 PSN cases and 10 age-matched cases without PSN. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling, immunohistochemistry, and double labeling for Fas/Fas ligand and the astrocyte marker glial fibrillary acid protein, the microglia/macrophage specific marker KiM1P, and the neuronal marker NeuN were performed on formalin-fixed brain specimens. Although mainly neurons of both PSN and controls expressed Fas receptor, expression was significantly increased (p = 0.001) in PSN cases in which predominantly degenerating cells with signs of early apoptosis showed Fas expression. In contrast, Fas ligand expression was found mainly on astrocytes and microglial cells. There was no significant difference between cases with and without PSN. We conclude that in the developing human brain, cells expressing the Fas receptor may be susceptible to undergoing apoptosis in response to hypoxic-ischemic injury.

Traumatically Induced Axotomy Adjacent to the Soma Does Not Result in Acute Neuronal Death

Traumatic axonal injury (TAI), a consequence of traumatic brain injury (TBI), results from progressive pathologic processes initiated at the time of injury. Studies attempting to characterize the pathology associated with TAI have not succeeded in following damaged and/or disconnected axonal segments back to their individual neuronal somata to determine their fate. To address this issue, 71 adult male Sprague Dawley rats were subjected to moderate central fluid percussion injury and killed between 30 min and 7 d after injury. Antibodies to the C terminus of beta-amyloid precursor protein (APP) identified TAI in continuity with individual neuronal somata in the mediodorsal neocortex, the hilus of the dentate gyrus, and the dorsolateral thalamus. These somata were followed with immunocytochemical markers of neuronal injury targeting phosphorylated 200 kDa neurofilaments (RMO-24), altered protein translation (phosphorylated eukaryotic translation initiation factor 2 alpha), and cell death [terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)], with parallel electron microscopic (EM) assessment. Despite the finding of TAI within 20-50 micrometer of the soma, no evidence of cell death, long associated with proximal axotomy, was seen via TUNEL or routine light microscopy/electron microscopy. Rather, there was rapid onset (<6 hr after injury) subcellular change associated with impaired protein synthesis identified by EM, immunocytochemical, and Western blot analyses. When followed 7 d after injury, these abnormalities did not reveal dramatic progression. Rather, some somata showed evidence of potential reorganization and repair. This study demonstrates a novel somatic response to TAI in the perisomatic domain and also provides insight into the multifaceted pathology associated with TBI.

Evaluation of preconditioning treatments to protect near-pure cortical neuronal cultures from in vitro ischemia induced acute and delayed neuronal death

We evaluated the efficacy of cycloheximide, heat stress, NMDA receptor blockade (MK801/AP-5), oxygen–glucose deprivation, hypoxia, hypothermia and TNFα preconditioning to protect cortical neurons from in vitro ischemic insults that result in acute necrotic and delayed apoptotic neuronal death. Preconditioning treatments were performed 22–24 h before in vitro ischemia. In vitro ischemia was carried out in 96-well microtitre strip-plates by washing neuronal cultures with a balanced salt solution containing 25 mM 2-deoxy- d-glucose and incubating in an anaerobic chamber. Glutamate receptor blockers were present during in vitro ischemia to induce delayed neuronal death. Cycloheximide, heat stress, MK801 and oxygen–glucose deprivation preconditioning were neuroprotective in both acute and delayed in vitro ischemic neuronal death models. AP-5 preconditioning and a 12 h post-MK801 preconditioning interval protected neurons from acute ischemic neuronal death only. Hypoxia, TNFα and hypothermic preconditioning provided no neuronal protection in the in vitro ischemia models. This study has confirmed for the first time that several preconditioning treatments can protect neurons from in vitro ischemia induced acute necrotic and delayed apoptotic neuronal death. In addition, a unique feature of this study is the finding that preconditioning could be induced in near-pure primary cortical neuronal cultures, thus confirming that ischemic tolerance is an intrinsic property of neurons and provides a simplified culture system for identifying neuroprotective proteins.

Histological and elemental changes in the rat brain after local irradiation with carbon ion beams.

The left cerebral hemispheres of adult Sprague-Dawley rat brains were irradiated at doses of 30, 50, or 100 Gy with charged carbon particles (290 MeV/nucleon; 5 mm spread-out Bragg peak). The spread-out Bragg peak used here successfully and satisfactorily retained its high-dose localization in the defined region. A histological examination showed that necrotic tissue damage, hemorrhage in the thalamus, and vasodilatations around the necrotic region were induced at 8 weeks after 100 Gy irradiation. The regions with tissue damage correlated well with those expected from the radiation-dose distribution, indicating an advantage of charged carbon particles for irradiating restricted brain regions. An X-ray fluorescent analysis demonstrated a decrease in the concentrations of K and P, and an increase in the concentrations of Cl, Fe, Zn in the damaged region at 8 weeks post-irradiation, though no significant changes were observed before 4 weeks of post-irradiation. This may indicate that even the very high radiation doses used here did not induce acute and immediate neuronal cell death, in contrast with ischemic brain injury where acute neuronal cell death occurred and the elemental concentrations changed within a day after the induction of ischemia.

Establishment of neuronal in vitro models of ischemia in 96-well microtiter strip-plates that result in acute, progressive and delayed neuronal death

Using 96-well microtiter strip-plates we established in vitro ischemia models with acute, progressive and delayed neuronal death onset. In vitro ischemia was induced by washing neuronal cultures with a balanced salt solution with (acute/delayed models) or without (progressive model) 25 mM 2-deoxy- D-glucose and incubating in an anaerobic chamber. Reperfusion was performed by removing cultures from the anaerobic chamber and washing and/or adding Dulbecco’s modified Eagle medium containing N2 supplement. Acute neuronal death resulted in cell swelling during in vitro ischemic incubation with the majority of neurons appearing swollen and necrotic within 3 h post-insult. Progressive neuronal death was characterized by cell shrinkage during and immediately following in vitro ischemia with increasing neuronal degeneration resembling both necrosis and apoptosis over a 24-h period post- in vitro ischemia. Delayed neuronal death was induced by glutamate-receptor blockade during in vitro ischemia. Neurons appeared morphologically normal immediately following and up to 6 h after in vitro ischemia and then started to degenerate over the next 42 h by a process resembling apoptosis. We monitored oxygen consumption during in vitro ischemia and found it to be similar for the three models and have shown that plastic culture wells store oxygen. The establishment of acute, progressive and delayed in vitro models of ischemia using 96-well microtiter strip-plates will provide useful tools to further investigate ischemic neuronal death/survival mechanisms and provide a high-throughput system to evaluate potential neuroprotective agents. Oxygen storage in plastic culture wells is likely to contribute to the extended oxygen- and oxygen–glucose-deprivation times required to induce significant neuronal injury in vitro.

Circulating Insulin-Like Growth Factor I Mediates the Protective Effects of Physical Exercise against Brain Insults of Different Etiology and Anatomy

Physical exercise ameliorates age-related neuronal loss and is currently recommended as a therapeutical aid in several neurodegenerative diseases. However, evidence is still lacking to firmly establish whether exercise constitutes a practical neuroprotective strategy. We now show that exercise provides a remarkable protection against brain insults of different etiology and anatomy. Laboratory rodents were submitted to treadmill running (1 km/d) either before or after neurotoxin insult of the hippocampus (domoic acid) or the brainstem (3-acetylpyridine) or along progression of inherited neurodegeneration affecting the cerebellum (Purkinje cell degeneration). In all cases, animals show recovery of behavioral performance compared with sedentary ones, i.e., intact spatial memory in hippocampal-injured mice, and normal or near to normal motor coordination in brainstem- and cerebellum-damaged animals. Furthermore, exercise blocked neuronal impairment or loss in all types of injuries. Because circulating insulin-like growth factor I (IGF-I), a potent neurotrophic hormone, mediates many of the effects of exercise on the brain, we determined whether neuroprotection by exercise is mediated by IGF-I. Indeed, subcutaneous administration of a blocking anti-IGF-I antibody to exercising animals to inhibit exercise-induced brain uptake of IGF-I abrogates the protective effects of exercise in all types of lesions; antibody-treated animals showed sedentary-like brain damage. These results indicate that exercise prevents and protects from brain damage through increased uptake of circulating IGF-I by the brain. The practice of physical exercise is thus strongly recommended as a preventive measure against neuronal demise. These findings also support the use of IGF-I as a therapeutical aid in brain diseases coursing with either acute or progressive neuronal death.

Protein aggregation after focal brain ischemia and reperfusion.

Two hours of transient focal brain ischemia causes acute neuronal death in the striatal core region and a somewhat more delayed type of neuronal death in neocortex. The objective of the current study was to investigate protein aggregation and neuronal death after focal brain ischemia in rats. Brain ischemia was induced by 2 hours of middle cerebral artery occlusion. Protein aggregation was analyzed by electron microscopy, laser-scanning confocal microscopy, and Western blotting. Two hours of focal brain ischemia induced protein aggregation in ischemic neocortical neurons at 1 hour of reperfusion, and protein aggregation persisted until neuronal death at 24 hours of reperfusion. Protein aggregates were found in the neuronal soma, dendrites, and axons, and they were associated with intracellular membranous structures during the postischemic phase. High-resolution confocal microscopy showed that clumped protein aggregates surrounding nuclei and along dendrites were formed after brain ischemia. On Western blots, ubiquitinated proteins (ubi-proteins) were dramatically increased in neocortical tissues in the postischemic phase. The ubi-proteins were Triton-insoluble, indicating that they might be irreversibly aggregated. The formation of ubi-protein aggregates after ischemia correlated well with the observed decrease in free ubiquitin and neuronal death. The authors concluded that proteins are severely damaged and aggregated in neurons after focal ischemia. The authors propose that protein damage or aggregation may contribute to ischemic neuronal death.

NMDA But Not Non-NMDA Excitotoxicity is Mediated by Poly(ADP-Ribose) Polymerase

Poly(ADP-ribose) polymerase (PARP-1), a nuclear enzyme that facilitates DNA repair, may be instrumental in acute neuronal cell death in a variety of insults including, cerebral ischemia, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced parkinsonism, and CNS trauma. Excitotoxicity is thought to underlie these and other toxic models of neuronal death. Different glutamate agonists may trigger different downstream pathways toward neurotoxicity. We examine the role of PARP-1 in NMDA- and non-NMDA-mediated excitotoxicity. NMDA and non-NMDA agonists were stereotactically delivered into the striatum of mice lacking PARP-1 and control mice in acute (48 hr) and chronic (3 week) toxicity paradigms. Mice lacking PARP-1 are highly resistant to the excitoxicity induced by NMDA but are as equally susceptible to AMPA excitotoxicity as wild-type mice. Restoring PARP-1 protein in mice lacking PARP-1 by viral transfection restored susceptibility to NMDA, supporting the requirement of PARP-1 in NMDA neurotoxicity. Furthermore, Western blot analyses demonstrate that PARP-1 is activated after NMDA delivery but not after AMPA administration. Consistent with the theory that nitric oxide (NO) and peroxynitrite are prominent in NMDA-induced neurotoxicity, PARP-1 was not activated in mice lacking the gene for neuronal NO synthase after NMDA administration. These results suggest a selective role of PARP-1 in glutamate excitoxicity, and strategies of inhibiting PARP-1 in NMDA-mediated neurotoxicity may offer substantial acute and chronic neuroprotection.

Neurotoxic APP C-terminal and β-amyloid domains colocalize in the nuclei of substantia nigra pars reticulata neurons undergoing delayed degeneration

Increased amyloid precursor protein (APP) expression and intracellular accumulation of its toxic fragments have been associated with acute neuronal death processes. However, the role of APP fragments in delayed neurodegeneration remains poorly understood. We have characterized the appearance of APP domains in rat substantia nigra pars reticulata (SNpR) neurons targeted for delayed degeneration following neurotoxic striatal lesion. From 4 to 8 days postlesion (dpl) SNpR neurons ipsilateral to the lesion showed marked cytosolic accumulation of full length APP. Moreover, the nuclei of affected neurons also showed intense immunoreactivity (IR) for APP C-terminal and β-amyloid domains but not for an N-terminal sequence. These data suggested the presence of APP C-terminal fragment. The absence of nuclear IR for a β1–40 specific antibody supports this conclusion. Ultrastructural analysis of nigral sections from 6 dpl rats using a β-amyloid domain antibody showed pronounced accumulation of immunogold-silver reaction product in the nuclei of affected SNpR neurons that was absent in control, contralateral SNpR neurons. These findings suggest that intranuclear APP C-terminal fragment may play a role in genomic events contributing to delayed neuron degeneration in the SNpR.

Atheroemboli to the brain: Size threshold for causing acute neuronal cell death

Objective: The objective of this study was to investigate the dilemma posed by the observations that carotid angioplasty dislodges significant numbers of plaque fragments but is reported to have a low rate of neurologic consequences. We examined the fragments released by ex vivo carotid angioplasty. The smaller and most numerous were separated by size and injected into rats to determine the tolerance of the brain to microemboli. Methods: Ex vivo angioplasty was performed on a total of 20 human carotid plaques removed en bloc. Plaques were placed within polytetrafluoroethylene grafts, and three manipulations were performed: guide wire insertion, 3.5- or 4.0-mm balloon angioplasty, and 5-mm angioplasty with or without a Palmaz stent. After each manipulation, the lumen was flushed, effluent was collected, and fragments were counted under 100× magnification. Using 200-μm and 500-μm micropore mesh, we separated fragments by size into two groups: (1) less than 200 μm and (2) 200 to 500 μm. We then injected rats with saline alone (Group A), with 100 fragments less than 200 μm (Group B), or with 100 fragments 200 to 500 μm (Group C). Animals were euthanized at 1, 3, and 7 days, and brain sections were examined for cell viability and expression of HSP- 72. Results: The total number of fragments dislodged from the plaques varied from 30 to 553. The mean number of fragments released with each manipulation was as follows: guide wire passage, 24; initial balloon angioplasty, 97; second balloon angioplasty, 68; and second angioplasty plus stent, 172. Sixteen of the 20 plaques dislodged fragments that were 1 mm or more in greatest dimension. There was no evidence of brain ischemia in Group A at any time. Group B also showed no injury at 1 or 3 days. However, injection of 200- to 500-μm fragments (Group C) caused a scattered pattern of neuronal cell death. At 7 days, brain sections from both Group B and Group C animals had a scattered pattern of ischemic neurons. There were no classic wedge-shaped infarctions. Discussion: The brain appears to have a surprising tolerance for microembolization in the acute setting. Thus, carotid angioplasty may dislodge plaque fragments, but there may still be a low incidence of stroke. However, even small plaque fragments, less than 200 μm, may cause neuronal ischemia at later time points. Periprocedural microemboli could cause subtle neurologic dysfunction in late follow-up. (J Vasc Surg 2000;32:68-76.)

Improved selective, simple, and contrast staining of acidophilic neurons with vanadium acid fuchsin

Acidophilia is one of the hallmarks of acute neuronal damage and death in brain ischemia, excitotoxic and traumatic lesions and epileptic seizures. We here describe a novel and simple method for visualizing acidophilic neurons on paraffin sections, using vanadium acid fuchsin (VAF) staining and toluidine blue or hematoxylin counterstaining. Paraffin sections of the brain fixed in ethanol–formalin–acetic acid mixture are stained in 0.1% acid fuchsin containing 0.125% of ammonium metavanadate and 1% of glacial acetic acid, differentiated if overstained in 0.01% of borax solution, and counterstained with 0.05–0.025% of toluidine blue in acetate buffer (pH 3.3) or Gill's II hematoxylin. The sections are dehydrated, cleared in xylene and mounted in Canada balsam or any synthetic mounting media for light microscopy. VAF combined with toluidine blue or hematoxylin results in highly selective and reproducible color contrast staining of acidophilic neurons as well as glial nuclei and hyperchromatic neurons. As a progressive method, acid fuchsin staining usually does not require differentiation. The red acidophilic neurons are clearly visible on the background of non-damaged cells, which significantly facilitates the identification, and localization of damaged neurons, even at low magnification under the light microscope. Theme: Disorders of the Nervous System. Topics: Neuronal damage and death.

The role of excitotoxicity in ALS--what is the evidence?

It is well accepted that excitotoxic mechanisms contribute to the pathogenesis of acute neuronal death in stroke, epilepsy, or brain trauma. It is less widely acknowledged that excitotoxic mechanisms play a role in the pathogenesis of chronic neurological disorders, in particular neurodegenerative diseases. However, evidence is accumulating that this mechanism is indeed part of the pathogenesis of late-onset neurodegenerative diseases. One of the clinical examples may be amyotrophic lateral sclerosis, a disease in which antiexcitotoxic strategies have neuroprotective effects in both, an established animal model and in man. In addition, there is accumulating neuropathological, pathobiochemical and pathophysiological evidence which indicates that excitotoxic mechanisms are part of the pathogenesis of the human disease and consequently part of the mechanisms explaining selective vulnerability ("pathoclisis") in the human motor system.

Immunohistochemical nitrotyrosine distribution in neonatal rat cerebrocortical slices during and after hypoxia

The peroxynitrite contributions to hypoxic damage in brain slices that arise from N-methyl- d-aspartate (NMDA) receptor activation were studied by following the temporal-spatial course of nitrotyrosine (NT) formation during six conditions: hypoxia ( pO 2<5 mmHg) with or without 10 μM MK-801 treatment; with exposure to 10, 100 and 1000 μM NMDA; and no treatment (control). In each experiment, twenty 350-μm thick cerebrocortical slices, obtained from the parietal lobes of ten 7-day-old Sprague–Dawley rats, were metabolically recovered and allowed to respire in a well-oxygenated perfusion system. Thirty minutes exposures to hypoxia or NMDA were followed by 2 h of oxygenated reperfusion. MK-801 administration began 15 min prior to hypoxia and was discontinued during reperfusion. Anti-NT serum immunohistochemistry stains in 20-μm frozen sections of slices taken during oxygenated reperfusion, after hypoxia or NMDA exposure, were positive in both neurons and endothelial cells. NT-positive neurons were detected sooner after hypoxia than after NMDA exposure, suggesting that mechanisms of superoxide generation were different in both groups. After hypoxia and even more so after NMDA exposure, more intense NT-positive staining was observed in endothelial cells than in neurons. Cell damage after hypoxia was attenuated by MK-801. MK-801 decreased post-hypoxia counts of NT-stained endothelial cells by 78.5% ( p<0.001) and NT-stained neurons by 54.1% ( p<0.05). Our findings suggest that NMDA receptor activation in hypoxic brain slices is associated with increased post-hypoxic peroxynitrite production that contributes to acute neuronal death and endothelial cell injury. Peroxynitrite injury to endothelial cells, caused either by increased peroxynitrite from within or from increased vulnerability to peroxynitrite from without, might play an important role in hypoxic–ischemic brain injury and NMDA-induced brain injury.

Spreading depression determines acute cellular damage in the hippocampal slice during oxygen/glucose deprivation.

During ischaemia neurons depolarize and release the neurotransmitter L-glutamate, which accumulates extracellularly and binds to postsynaptic receptors. This initiates a sequence of events thought to culminate in immediate and delayed neuronal death. However, there is growing evidence that during ischaemia the development of spreading depression (SD) can be an important determinant of the degree and extent of ischaemic damage. In contrast, SD without metabolic compromise (as occurs in migraine aura) causes no discernible damage to brain tissue. SD is a profound depolarization of neurons and glia that propagates like a wave across brain tissue. Brain cell swelling, an early event of both the excitotoxic process and of SD, can be assessed by imaging associated intrinsic optical signals (IOSs). We demonstrate here that IOS imaging clearly demarcates the ignition site and migration of SD across the submerged hippocampal slice of the rat. If SD is induced by elevating [K+]O, the tissue fully recovers, but in slices that are metabolically compromised at 37.5 degrees C by oxygen/glucose deprivation (OGD) or by ouabain exposure, cellular damage develops only where SD has propagated. Specifically, the evoked CA1 field potential is permanently lost, the cell bodies of involved neurons swell and their dendritic regions increase in opacity. In contrast to OGD, bath application of L-glutamate (6-10 mM) at 37.5 degrees C evokes a non-propagating LT increase in CA1 that reverses without obvious cellular damage. Moreover, application of 2-20 mM glutamate or various glutamate agonists fail to evoke SD in the submerged hippocampal slice. We propose that SD and OGD together (but not alone) constitute a 'one-two punch', causing acute neuronal death in the slice that is not replicated by elevated glutamate. These findings support the proposal that SD generation during stroke promotes and extends acute ischaemic damage.

Evidence for neuronal apoptosis in pontosubicular neuron necrosis

Pontosubicular neuron necrosis (PSN) is characterized by acute neuronal death in the subiculum and the pons occurring in a circumscribed perinatal period. The morphological changes in PSN are quite similar to those described during apoptosis, a form of programmed cell death. Morphological re-evaluation of the lesions by light and electron microscopy revealed the typical changes of apoptosis with condensed basophilic nuclei and the formation of apoptotic bodies. By analysing DNA fragmentation in situ with a recently established technique, we were able to show that neuronal death in PSN is apoptotic. The demonstration of DNA fragmentation by the in situ tailing technique was reliable in the human autopsy material used in this study and was only slightly affected by autolysis or formalin fixation. The subiculum and the pons are shown to be susceptible to apoptosis at different times during development. PSN thus provides a model in which the process of nerve cell apoptosis in the developing human central nervous system can be studied.

Delayed neuronal injury induced by sub-lethal NMDA exposure in the hippocampal slice

Stroke produces neuronal death by two general processes which differ in their temporal course. Acute neuronal death occurs within minutes, while delayed neuronal death evolves within 24 h. To better examine mechanisms of delayed death, we developed a new in vitro model of delayed neuronal injury using extended electrophysiological recordings in paired hippocampal slices. We exposed one hippocampal slice of each pair to 10 μM N-methyl- d-aspartate (NMDA) until the orthodromic CA1 PS disappeared. Thereafter, NMDA-treated slices regained near full recovery of PS amplitude within one hour. However, 10 h later, NMDA-treated slices demonstrated a rapid decline in PS amplitude of 82% ± 15. CA1 orthodromic evoked PS was lost completely at an average 12.4 ± 1.6 h after NMDA exposure. This sudden loss of response contrasted with paired, untreated slices, where CA1 PS could be elicited for 22.6 ± 4.0 h ( P < 0.05). Treatment with 10 mM MgCl 2 begun after NMDA exposure and continued for 35 min, prevented delayed loss of CA1 orthodromic PS, which then could be elicited for 20.3 ± 2.1 h. These results indicate that delayed injury can be evaluated using the hippocampal slice. They also suggest that activation of NMDA receptors can induce delayed neuronal injury in CA1 neurons, and that magnesium treatment after NMDA can prevent this injury.