Sulthiame but not levetiracetam exerts neurotoxic effect in the developing rat brain

Antiepileptic drugs (AEDs) used to treat seizures in pregnant women, infants, and young children can cause cognitive impairment. One mechanism implicated in the development of neurocognitive deficits is a pathologic enhancement of physiologically occurring apoptotic neuronal death in the developing brain. We investigated whether the newer antiepileptic drug levetiracetam (LEV) and the older antiepileptic drug sulthiame (SUL) have neurotoxic properties in the developing rat brain. SUL significantly enhanced neuronal death in the brains of rat pups ages 0 to 7 days at doses of 100 mg/kg and above, whereas LEV did not show this neurotoxic effect. Dosages of both drugs used in the context of this study comply with an effective anticonvulsant dose range applied in rodent seizure models. Thus, LEV is an AED which lacks neurotoxicity in the developing rat brain and should be considered in the treatment of epilepsy in pregnant women, infants, and toddlers once general safety issues have been properly addressed.     

Caffeine-induced neuronal death in neonatal rat brain and cortical cell cultures

We examined the potential neurotoxicity of caffeine and. Intraperitoneal administration of caffeine (50 mg/kg, 3 times a day) produced neuronal death in various brain areas of neonatal rats 24 h later. Caffeine at doses > 300 microM was also neurotoxic in murine cortical cell cultures. Caffeine-induced neuronal death was accompanied by cell body shrinkage and attenuated by anti-apoptotic drugs including cycloheximide, high potassium, and growth factors. Two necrotic pathways, excitotoxicity and oxidative stress, did not mediate caffeine neurotoxicity. The pro-apoptotic protease caspase-3 was activated to mediate neuronal death following exposure to caffeine. The present findings suggest that caffeine may cause caspase-3-dependent neuronal cell apoptosis in neonatal rat as well as.     

Otilonium and pinaverium trigger mitochondrial-mediated apoptosis in rat embryo cortical neurons in vitro

In the frame of a repositioning programme with cholinergic medicines in clinical use searching for neuroprotective properties, we surprisingly found that spasmolytic antimuscarinics otilonium and pinaverium exhibited neurotoxic effects in neuronal cultures. We decided to characterize such unexpected action in primary cultures of rat embryo cortical neurons. Neurotoxicity was time- and concentration-dependent, exhibiting approximate EC₅₀ values of 5 μM for both drugs. Seven antimuscarinic drugs endowed with a quaternary ammonium, and another 10 drugs with different cholinergic activities, carrying in their molecule a ternary ammonium did not exhibit neurotoxicity. Both drugs caused a concentration-dependent blockade of whole-cell inward currents through voltage-activated calcium channels (VACCs). Consistent with this, they also blocked the K⁺-elicited [Ca²⁺]_c transients. Neither antioxidant catalase, glutathione, n-acetylcysteine, nor melatonin protected against neurotoxicity of otilonium or pinaverium. However cyclosporine A, a blocker of the mitochondrial permeability transition pore, prevented the neurotoxic effects of otilonium and pinaverium monitored as the fraction of cells undergoing apoptosis. Furthermore, the caspase-9 and caspase-3 inhibitor Ac-LEHD-CHO mitigated the apoptotic neuronal death of both drugs by around 50%. Data are compatible with the hypothesis that otilonium and pinaverium elicit neuronal death by activating the intrinsic mitochondrial-mediated signaling pathway of apoptosis. This may have its origin in the mitigation of Ca²⁺ entry and the uncoupling of the Ca²⁺-dependent generation of mitochondrial bioenergetics, thus causing the opening of the mitochondrial mPTP to elicit apoptotic neuronal death.     

Propofol and sodium thiopental protect against MK-801-induced neuronal necrosis in the posterior cingulate/retrosplenial cortex

N-Methyl-D-aspartate (NMDA) antagonists act by an anti-excitotoxic action to provide neuroprotection against acute brain injury, but these agents can also cause toxic effects. In low doses they induce reversible neuronal injury, but in higher doses they cause irreversible degeneration of cerebrocortical neurons. GABAmimetic drugs protect against the reversible neurotoxic changes in rat brain. Here we show that two GABAmimetic anesthetic agents--propofol and sodium thiopental--protect against the irreversible neurodegenerative reaction induced by the powerful NMDA antagonist, MK-801.     

Protective Function of Nicotinamide Against Ketamine-induced Apoptotic Neurodegeneration in the Infant Rat Brain

During development, anesthetics activate neuroapoptosis and produce damage in the central nervous system that leads to several types of neurological disorders. A single dose of ketamine (40 mg/kg) during synaptogenesis in a 7-day-old rat brain activated the apoptotic cascade and caused extensive neuronal cell death in the forebrain. In this study, we investigated the protective effect of nicotinamide against ketamine-induced apoptotic neurodegeneration. After 4 h, neuronal cell death induced by ketamine was associated with the induction of Bax, release of cytochrome c into the cytosol, and activation of caspase-3. One single dose of 1 mg/g nicotinamide was administered to a developing rat and was found to inhibit ketamine-induced neuroapoptosis by downregulating Bax, inhibiting cytochrome c release from mitochondria into cytosol, and inhibiting the expression of activated caspase-3. TUNEL and immunohistochemical analyses showed that ketamine-induced cell death occurred through apoptosis and that it was inhibited by nicotinamide. Fluoro-Jade-B staining demonstrated an increased number of dead cells in the cortex and thalamus after ketamine treatment; treatment with nicotinamide reduced the number of dead cells in these brain regions. Our findings suggest that nicotinamide attenuated ketamine-induced neuronal cell loss in the developing rat brain and is a promising therapeutic and neuroprotective agent for the treatment of neurodevelopmental disorders.     

Beta-bungarotoxin is a potent inducer of apoptosis in cultured rat neurons by receptor-mediated internalization

The neurotoxic phospholipase A(2), beta-bungarotoxin (beta-BuTx), is a component of the snake venom from the Taiwanese banded krait Bungarus multicinctus. beta-BuTx affects presynaptic nerve terminal function of the neuromuscular junction and induces widespread neuronal cell death throughout the mammalian and avian CNS. To analyse the initial events of beta-BuTx-mediated cell death, the toxin was applied to cultured rat hippocampal neurons where it induced neuronal cell death in a concentration-dependent manner (EC(50) approximately equal to 5 x 10(-13) M) within 24 h. Fluorescence labelled beta-BuTx was completely incorporated by neurons within < 10 min. Binding and uptake of beta-BuTx, as well as induction of cell death, were efficiently antagonized by preincubation with dendrotoxin I, a blocker of voltage-gated potassium channels devoid of phospholipase activity. Binding of beta-BuTx was selective for neurofilament-positive cells. As evident from intense annexin-V and TUNEL stainings, application of beta-BuTx induced apoptotic cell death exclusively in neurons, leaving astrocytes unaffected. No evidence was obtained for any contribution of either caspases or calpains to beta-BuTx-induced apoptosis, consistent with the inability of the inhibitors Z-Asp-DCB and calpeptin, respectively, to protect neurons from beta-BuTx-induced cell death. These observations indicate that induction of cell death by beta-BuTx comprises several successive phases: (i) binding to neuronal potassium channels is the initial event, followed by (ii) internalization and (iii) induction of apoptotic cell death via a caspase-independent pathway.     

Anticancer agents are potent neurotoxins in vitro and in vivo

Neurotoxicity of anticancer agents complicates treatment of children with cancer. We investigated neurotoxic effects of common cytotoxic drugs in neuronal cultures and in the developing rat brain. When neurons were exposed to cisplatin (5-100 microM), cyclophosphamide (5-100 microM), methotrexate (5-100 microM), vinblastin (0.1-1 microM), or thiotepa (5-100 microM), a concentration-dependent neurotoxic effect was observed. Neurotoxicity was potentiated by nontoxic glutamate concentrations. The N-methyl-D-aspartate receptor antagonist MK 801 (10 microM), the AMPA receptor antagonists GYKI 52466 (10 microM) and NBQX (10 microM), and the pancaspase inhibitor Ac-DEVD-CHO (1 nM) ameliorated neurotoxicity of cytotoxic drugs. To investigate neurotoxicity in vivo, we administered to 7-day-old rats the following: cisplatin (5-15 mg/kg i.p.), cyclophosphamide (200-600 mg/kg i.p.), thiotepa (15-45 mg/kg), or ifosfamide (100-500 mg/kg) and their brains were analyzed at 4 to 24 hours. Cytotoxic drugs produced widespread lesions within cortex, thalamus, hippocampal dentate gyrus, and caudate nucleus in a dose-dependent fashion. Early histological analysis demonstrated dendritic swelling and relative preservation of axonal terminals, which are morphological features indicating excitotoxicity. After longer survival periods, degenerating neurons displayed morphological features consistent with active cell death. These results demonstrate that anticancer drugs are potent neurotoxins in vitro and in vivo; they activate excitotoxic mechanisms but also trigger active neuronal death.     

Microglial cells prevent nitric oxide-induced neuronal apoptosis in vitro

Apoptotic neuronal death is known to occur in the developing brain and in the mature brain of patients with ischemic and degenerative disorders. Although microglial cells are known to become activated in specific conditions, it has not been elucidated whether they enhance or prevent neuronal apoptosis. The present study was intended to observe how microglial cells are involved in neuronal death. When rat primary cortical neurons were incubated with a nitric oxide (NO) donor sodium nitroprusside (SNP; 300 μM) for 10 min, neuronal death occurred 12–16 hr later. The NO-induced neuronal death was inhibited by cycloheximide, and the SNP-treated neurons were characterized by nuclear fragmentation and intact cell membrane under electron microscopy. Agarose gel electrophoresis demonstrated DNA fragmentation of the SNP-treated neurons. Thus, the NO-induced neuronal death appeared to be apoptosis. When neurons were cocultured with rat primary microglial cells, the SNP treatment failed to induce the neuronal death. Because microglia-conditioned medium also prevented apoptotic neuronal death, microglial cells were considered to secrete antiapoptotic factors. The microglia-conditioned medium rescued neurons even when they were added to neuronal cultures after the SNP treatment, implying that the factors acted on neurons in a manner other than scavenging NO. Interleukin-3, interleukin-6, macrophage colony-stimulating factor, and basic fibroblast growth factor, which are known to be secreted by microglial cells, were not effective in preventing NO-induced neuronal death. Among microglia-derived substances, tumor necrosis factor α and plasminogen, which are heat-labile proteins, inhibited neuronal apoptosis. The neuroprotective action of the microglia-conditioned medium, however, still remained, even after it was heated. These findings suggest that microglial cells protect neurons against NO-induced lethal damage by secreting heat-labile and heat-stable neuroprotective factors in vitro. J. Neurosci. Res. 53:415–425, 1998. © 1998 Wiley-Liss, Inc.     

IL-8 induces expression of matrix metalloproteinases, cell cycle and pro-apoptotic proteins, and cell death in cultured neurons

Neuronal cell loss is a critical feature of age-related neurodegenerative diseases such as Alzheimer's disease (AD). In the AD brain, a marked increase in pro-inflammatory cytokines and chemokines, including IL-8, has been documented. The objective of this study was to determine the effect of IL-8 on cell viability and expression of neurotoxic, apoptotic, and cell cycle proteins in cultured neurons. Incubation of cultured neurons with IL-8 for 24 h resulted in neuronal cell death. RT-PCR analysis of primary rat neuronal cultures treated with IL-8 for 24 h showed induction of genes for matrix metalloproteinases (MMP-2 and MMP-9), proinflammatory proteases with neurotoxic properties. Gelatin zymography demonstrated IL-8 induced MMP-2 and MMP-9 activity. Western blot analysis showed that IL-8 also increased levels of the pro-apoptotic protein Bim (Bcl-2-interacting mediator of cell death). In addition, message levels of the cell cycle protein cyclin D1, an early marker for G1/S transition and a protein implicated as a regulator of neuronal apoptosis, were elevated after IL-8 exposure. These results suggest that IL-8 could be an important mediator of neuronal death in AD both via its effects on release of neurotoxins such as MMPs as well as by induction of cell cycle and pro-apoptotic proteins.     

Developmental exposure to methylmercury elicits early cell death in the cerebral cortex and long-term memory deficits in the rat

Experiments were performed to assess the neurotoxic effects induced by prenatal acute treatment with methylmercury on cortical neurons. To this purpose, primary neuronal cultures were obtained from cerebral cortex of neonatal rats born to dams treated with methylmercury (4 and 8 mg/kg by gavage) on gestational day 15, the developmental stage critical for cortical neuron proliferation. Prenatal exposure to methylmercury 8 mg/kg significantly reduced cell viability and caused either apoptotic or necrotic neuronal death. Moreover, this exposure level resulted in abnormal neurite outgrowth and retraction or collapse of some neurites, caused by a dissolution of microtubules. The severe and early cortical neuron damage induced by methylmercury 8 mg/kg treatment correlated with long term memory impairment, since adult rats (90 days of age) born to dams treated with this dose level showed a significant deficit in the retention performance when subjected to a passive avoidance task. Prenatal exposure to methylmercury 4 mg/kg significantly increased the neuronal vulnerability to a neurotoxic insult. This was determined by measuring the increment of chromatin condensation induced by glutamate, at a concentration (30 μM) able to induce an excitotoxic damage. This exposure level eliciting apoptotic death did not result in cognitive dysfunctions. In conclusion, the methylmercury-induced disruption of glutamate pathway during critical windows of brain development may interfere with cell fate and proliferation resulting in a more or less severe cortical lesions associated or not with loss of function later in life, depending on the exposure levels. Therefore, the early biochemical effects and long-term behavioral changes elicited by high methylmercury levels suggest that the developing brain is impaired in its ability to recover following toxic insult, and the initial effects on cortical neurons may lead to permanent cognitive dysfunctions.     

Disseminated corticolimbic neuronal degeneration induced in rat brain by MK-801: potential relevance to Alzheimer's disease

Blockade of N-methyl-D-aspartate (NMDA) glutamate receptors by MK-801 induces neuronal degeneration in the posterior cingulate/retrosplenial cortex and other corticolimbic regions although damage in the latter has not been adequately characterized. This disseminated corticolimbic damage is of interest since NMDA hypofunction, the mechanism that triggers this neurodegenerative syndrome, has been postulated to play a role in the pathophysiology of Alzheimer's disease (AD). Several histological methods, including electron microscopy, were used to evaluate the neurotoxic changes in various corticolimbic regions of rat brain following MK-801 or a combination of MK-801 plus pilocarpine. We found that MK-801 triggers neuronal degeneration in a widespread pattern similar to that induced by phencyclidine and that females showed more damage than males. The neurotoxic reaction involved additional brain regions when muscarinic receptors were hyperactivated by administering pilocarpine with MK-801. Ultrastructural evaluation revealed that a major feature of the neurotoxic action involves degeneration of dendritic spines which entails loss of synaptic complexes. The ultrastructural appearance of degenerating neurons was generally inconsistent with an apoptotic mechanism, although evidence equivocally consistent with apoptosis was observed in some instances. The cell death process evolved relatively slowly and was still ongoing 7 days posttreatment. Relevance of these results to AD is discussed.     

Serum amyloid P component induces neuronal apoptosis and beta-amyloid immunoreactivity

Previously we have reported serum amyloid P component (SAP) induced cell death in cerebro-cortical cultures of rat brain. In this paper we studied the types of target cells and the molecular mechanism of SAP-induced cell death. Immuno-electron and light microscopy revealed that SAP penetrates the plasma membrane and translocates selectively into the nuclei of neurons. Neuronal cells with SAP immunoreactivity exhibit the morphological hallmarks of apoptosis in vitro. The apoptotic mechanism of cell death is also supported by the increased Bax/Bcl-2 ratio. In addition to neurotoxic effects, we detected elevated beta-amyloid (Abeta) immunoreactivity following SAP treatment. This study supports the thesis that SAP plays an important role in the pathomechanism of neurodegenerative diseases, including Alzheimer's disease by inducing neuronal apoptosis.     

Endogenous Zinc Mediates Apoptotic Programmed Cell Death in the Developing Brain

Endogenous zinc can mediate the apoptotic programmed cell death (PCD) in the developing brain. Intensive accumulation of labile zinc occurs in almost all neurons undergoing PCD in the developing rat brain. Based on the greater frequency of neurons with intensive zinc accumulation compared to apoptotic neurons, it is inferred that cytosolic zinc accumulation precedes apoptotic PCD. To determine the role of intracellular labile zinc in developmental apoptosis, we subcutaneously injected the membrane-permeant zinc chelator, N,N,N′,N-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) into postnatal rats for 7 days after birth. TPEN chelated intraneuronal zinc without modulating the expression of the zinc-regulating proteins, ZnT-1, ZnT-3, and synaptophysin. The frequency of apoptotic neurons significantly decreased in TPEN-treated rat brains compared with that in normal postnatal rats. Activating cleavages of caspase-9 and -3, and mitochondrial pro-apoptotic Bax expression were reduced, whereas expression of anti-apoptotic Bcl-2 was increased. Thus, intracerebral zinc chelation may arrest PCD in the developing brain by interfering with the caspase-dependent apoptotic pathway. The present study demonstrates that intracellular zinc acts as a key mediator of developmental apoptosis and therefore provides the first in vivo evidence that endogenous labile zinc causes neuronal apoptosis.     

3,4-methylenedioxyamphetamine upregulates p75 neurotrophin receptor protein expression in the rat brain

The p75 neurotrophin receptor,which is a member of the tumor necrosis factor receptor superfamily,facilitates apoptosis during development and following central nervous system injury.Previous studies have shown that programmed cell death is likely involved in the neurotoxic effects of 3,4-methylenedioxy-N-methylamphetamine (MDMA),because MDMA induces apoptosis of immortalized neurons through regulation of proteins belonging to the Bcl-2 family.In the present study,intraperitoneal injection of different doses of MDMA (20,50,and 100 mg/kg) induced significant behavioral changes,such as increased excitability,increased activity,and irritability in rats.Moreover,changes exhibited dose-dependent adaptation.Following MDMA injection in rat brain tissue,the number of apoptotic cells dose-dependently increased and p75 neurotrophin receptor expression significantly increased in the prefrontal cortex,cerebellum,and hippocampus.These findings confirmed that MDMA induced neuronal apoptosis,and results suggested that this effect was related by upregulated protein expression of the p75 neurotrophin receptor.     

Oxidized proteins in astrocytes generated in a hyperbaric atmosphere induce neuronal apoptosis

In the present study, we investigated the influence of the oxidative damage to astrocytes on neuronal cell survival using cultures of rat cerebral astrocytes and neurons. The exposure of astrocytes to hyperbaric oxygen induced a time-dependent apoptotic cell death, as observed by DNA ladder assessment. When astrocytes damaged by oxidative stress were cocultured with normal neurons from the cerebrum of a newborn rat, neuronal cell death was markedly induced, although normal astrocytes not subjected to hyperoxia cocultured with normal neurons showed no neuronal cell apoptosis. It was found that either the supernatant from the homogenate of astrocytes cultured in hyperbaric oxygen atmosphere or a protein mixture extracted from the supernatant induced neuronal cell death. The level of protein carbonyls, an index of protein oxidation analysis, in cultured astrocytes increased significantly with oxidative stress, and vitamin E inhibited the increase in the level of such oxidized proteins in astrocytes. Furthermore, a two-dimensional (2D) electrophoresis of a protein mixture extracted from the supernatant showed several changes in proteins. These results imply that reactive oxygen species (ROS) induced by oxidative stress attack astrocytes to induce oxidatively denatured proteins in the cells that act as a neurotoxic factor, and that vitamin E protects neurons by inhibiting astrocyte apoptosis caused by oxidative stress.     

Bilirubin induces apoptosis via activation of NMDA receptors in developing rat brain neurons

Increased amounts of bilirubin, the end product of heme degradation, are known to be detrimental to the central nervous system, especially in preterm newborns. In an attempt to delineate the cellular mechanisms by which unconjugated bilirubin exerts its toxic effects on neuronal cells in the developing brain, bilirubin (0.25-5 microM) was added to the extracellular medium of 6-day-old primary cultured neurons from the embryonic rat forebrain, and cell alterations were studied over the ensuing 96 h. Bilirubin decreased cell viability dose dependently with an ED(50) around 1 microM. At the dose of 0.5 microM, it triggered delayed cell death that affected 24% of the neurons. Nuclear incorporation of the fluorescent dye DAPI (4,6-diamidino-2-phenylindole) depicted the presence of apoptosis (16%). Apoptosis features were confirmed by DNA fragmentation reflected by a progressive loss of [(3)H]thymidine and sequential changes in macromolecular synthesis, as shown by the time course of [(3)H]leucine incorporation, as well as by the beneficial effects of cycloheximide and caspase inhibitors. In parallel, treatments with glutamate receptor antagonists showed that MK-801, but not NBQX, protected neurons against bilirubin neurotoxicity, suggesting a role for NMDA receptors in bilirubin effects. Coupled with previous work about glutamate toxicity in the same culture model, these data support the hypothesis that low levels of free bilirubin may promote programmed neuronal death corresponding to an apoptotic process which involves caspase activation and requires the participation of NMDA receptors, along with bilirubin-induced inhibition of protein kinase C activity.     

Excitotoxic neuronal death in the immature brain is an apoptosis-necrosis morphological continuum

Glutamate-induced excitotoxicity is a clinically relevant degenerative process that causes selective neuronal death by mechanisms that remain unclear. Cell death is usually classified as apoptotic or necrotic based on biochemical and morphological criteria. Excitotoxic lesions in the adult rat striatum result in neuronal death associated with apoptotic DNA laddering despite a necrotic appearance of neurons ultrastructurally. This suggests that apoptosis and necrosis may not be mutually exclusive modes of cell death. Here, we characterized normal developmental cell death in the newborn rat brain with respect to DNA fragmentation patterns and ultrastructural morphology to establish a standard for apoptosis in the nervous system, and we concluded that it is essentially indistinguishable from apoptosis described in other tissues. We then investigated whether brain maturity could influence the morphology of neuronal death in vivo in the excitotoxically lesioned newborn rat forebrain. Kainic acid induced DNA laddering and death of neurons exhibiting a variety of morphologies, ranging from necrosis to apoptosis. In neurons that were dying by apoptosis, morphologic changes were characterized by a highly ordered sequence of organelle abnormalities, with swelling of endoplasmic reticulum and Golgi vesiculation preceding most nuclear changes and mitochondrial disruption. We concluded that brain maturity influences the morphologic phenotype of neurodegeneration and that excitotoxic neuronal death in the immature brain is not a uniform event but, rather, a continuum of apoptotic, necrotic, and overlapping morphologies. This excitotoxic paradigm might prove useful for analyzing the mechanisms that govern cell death under physiological and pathological conditions. J. Comp. Neurol. 378:70–87, 1997. © 1997 Wiley-Liss, Inc.     

Influence of corticotrophin releasing factor on neuronal cell death in vitro and in vivo

Several studies have demonstrated that antagonists of the corticotrophin releasing factor (CRF) receptor markedly inhibit experimentally induced excitotoxic, ischaemic and traumatic brain injury in the rat, and that CRF expression is elevated in response to experimentally induced stroke or traumatic brain injury. CRF is also induced by the pro-inflammatory cytokine interleukin 1 (IL-1), which participates in various forms of neurodegeneration. The aim of this study was to test the hypothesis that CRF is toxic directly in vivo or in vitro. In primary cultures of rat cortical neurons, exposure to CRF (10 pM-100 nM) for 24 h failed to cause cell death directly, or to modify the neurotoxic effects of N-methyl-D-aspartate (NMDA). Similarly, infusion of CRF (0.3-5 microg) into specific brain regions of the rat did not induce cell death and did not significantly alter the neuronal damage produced by infusion of excitatory amino acids. These data demonstrate that CRF is not directly neurotoxic, and suggest that either CRF mediates neuronal damage by indirect actions (e.g. on the vasculature) and/or that CRF is not the endogenous ligand which contributes to neurodegeneration through activation of CRF receptors.     

Distinguishing excitotoxic from apoptotic neurodegeneration in the developing rat brain.

Much confusion has arisen recently over the question of whether excitotoxic neuronal degeneration can be considered an apoptotic phenomenon. Here, we addressed this question by using ultrastructural methods and DNA fragmentation analysis to compare a prototypic apoptotic in vivo central nervous system cell death process (physiologic cell death in the developing rat brain) with several central nervous system cell death processes in the in vivo infant rat brain that are generally considered excitotoxic (degeneration of hypothalamic neurons after subcutaneous administration of glutamate and acute neurodegeneration induced by hypoxia/ischemia or by concussive head trauma). We found by ultrastructural analysis that glutamate induces neurodegenerative changes in the hypothalamus that are identical to acute changes induced in the infant rat brain by either hypoxia/ischemia or head trauma, and that these changes are fundamentally different both in type and sequence from those associated with physiologic cell death (apoptosis). In addition, we show by ultrastructural analysis that concussive head trauma induces both excitotoxic and apoptotic neurodegeneration, the excitotoxic degeneration being very acute and localized to the impact site, and the apoptotic degeneration being delayed and occurring in regions distant from the impact site. Thus, in the head trauma model, excitotoxic and apoptotic degeneration can be distinguished not only by ultrastructural criteria but by their temporal and spatial patterns of expression. Whereas ultrastructural analysis provided an unambiguous means of distinguishing between excitotoxic and apoptotic neurodegeneration in each example analysed in this study, DNA fragmentation analysis (TUNEL staining or gel electrophoresis) was of no value because these tests were positive for both processes.