Neuropathy‐induced apoptosis: Protective effect of physostigmine

Traumatic, infectious, metabolic, and chemical noxa to the nervous system are the etiology of a crippling disease generally termed neuropathy. Motor disorders, altered sensibility, and pain are the pathognomonic traits. Cellular alterations induced by this chronic pathology include mitochondrial dysfunctions that lead to the activation of the apoptotic cascade. Energy imbalance can compromise the maintenance of mitochondrial membrane potential, furthering the release of cytochrome C and the subsequent cleavage and activation of caspases. Chronic constriction injury (CCI) of the rat sciatic nerve is a neuropathy model able to induce a strong mitochondrial impairment with a consequent apoptotic induction. In this model, the acetylcholinesterase inhibitor physostigmine is administered at 0.125 mg/kg i.p. (twice per day) starting from the operation and for 15 days after. The cholinergic activation reduces cytosolic levels of cytochrome C, suggesting an improved stability of the mitochondrial membrane, and the expression level of the active caspase 3 fragments (19, 16 kDa) is reduced significantly with respect to saline treatment. Accordingly, physostigmine impairs caspase 3 protease activity. In fact, the target of the activated caspase 3, the 89‐kDa PARP fragment, is significantly less expressed in the ligated nerve of physostigmine‐treated rats, reaching levels that are comparable to those in the contralateral unligated nerve. Finally, this natural acetylcholinesterase inhibitor reduces DNA fragmentation both in the proximal and in the distal parts of the nerve. This protection correlates with the induction of XIAP. Therefore, apoptosis, central to tissue degeneration, is prevented by repeated physostigmine treatment of CCI animals. © 2009 Wiley‐Liss, Inc.     

Neuroprotective effects of acetyl-L-carnitine on neuropathic pain and apoptosis: a role for the nicotinic receptor

Several pathologies related to nervous tissue alterations are characterized by a chronic pain syndrome defined by persistent or paroxysmal pain independent or dependent on a stimulus. Pathophysiological mechanisms related to neuropathic disease are associated with mitochondrial dysfunctions that lead to an activation of the apoptotic cascade. In a model of peripheral neuropathy obtained by the loose ligation of the rat sciatic nerve, acetyl-L-Carnitine (ALCAR; 100 mg/kg intraperitoneally [i.p.] twice daily for 14 days) was able to reduce hyperalgesia and apoptosis. In the present study, different mechanisms for the analgesic and the antineuropathic effect of ALCAR are described. The muscarinic blocker atropine (5 mg/kg i.p.) injected simultaneously with ALCAR did not antagonize the ALCAR antihyperalgesic effect on the paw-pressure test but significantly reduced the analgesic effect of ALCAR. Conversely, the antineuropathic effect of ALCAR was prevented by cotreatment with the nicotinic antagonist mecamylamine (2 mg/kg i.p. twice daily for 14 days). A pharmacological silencing of the nicotinic receptors significantly reduced the X-linked inhibitor of apoptosis protein-related protective effect of ALCAR on the apoptosis induced by ligation of the sciatic nerve. Taken together, these data highlight the relevance of nicotinic modulation in neuropathy treatment.     

Reovirus-induced neuronal apoptosis is mediated by caspase 3 and is associated with the activation of death receptors

Reovirus infection of the central nervous system (CNS) is an important experimental system for understanding the pathogenesis of neurotropic viral infection. Infection of neonatal mice with T3 reoviruses causes lethal encephalitis in which injury results from virus-induced apoptosis. We now show that this apoptosis in vivo is associated with activation of caspase 3, and use neuroblastoma and primary neuronal cultures to identify the cellular pathways involved. Reovirus-induced apoptosis in neuronal cultures is initiated by activation of the tumor necrosis factor (TNF) receptor superfamily death receptors and is inhibited by treatment with soluble death receptors (DRs). The DR-associated initiator caspase, caspase 8, is activated following infection, this activation is inhibited by a cell-permeable peptide inhibitor (IETD-CHO). In contrast to our previous findings in non-neuronal cell lines, reovirus-induced neuronal apoptosis is not accompanied by significant release of cytochrome c from the mitochondria or with caspase 9 activation following infection. This suggests that in neuronal cells, unlike their non-neuronal counterparts, the mitochondria-mediated apoptotic pathway associated with cytochrome c release and caspase 9 activation does not play a significant role in augmenting reovirus-induced apoptosis. Consistent with these results, peptide caspase inhibitors show a hierarchy of efficacy in inhibiting reovirus-induced apoptosis, with inhibitors of caspase 3 > caspase 8 > caspase 9. These studies provide a comprehensive profile of the pattern of virus-induced apoptotic pathway activation in neuronal culture.     

Caspase-dependent and -independent death of camptothecin-treated embryonic cortical neurons.

This study investigates the mechanisms underlying death of cultured embryonic cortical neurons exposed to the DNA-damaging agent camptothecin and in particular the interdependence of the roles of cyclin-dependent kinases (Cdks), caspases, and mitochondrial function. Camptothecin evokes rapid neuronal death that exhibits nuclear features of apoptosis. This death is accompanied by loss of cytochrome c and mitochondrial transmembrane potential as well as by induction of caspase-3-like activity and caspase-2 processing. The Cdk inhibitor flavopiridol provides long-term rescue from death and prevents loss of cytochrome c and mitochondrial transmembrane potential as well as caspase activation and processing. General caspase inhibitors rescue neurons from this rapid apoptotic death but do not prevent them from undergoing delayed death in which nuclear features of apoptosis are absent. Moreover, the caspase inhibitors do not affect early cytochrome c release and delay but do not prevent the loss of transmembrane potential. Agents that directly disrupt mitochondrial function without inducing cytochrome c release lead to a caspase-independent death. These observations favor a model in which (1) DNA damage leads to Cdk activation, which lies upstream of release of cytochrome c and caspase activation; (2) cytochrome c release is caspase-independent and may occur upstream of caspase activation; (3) early apoptotic death requires caspases; and (4) delayed nonapoptotic death that occurs in the presence of caspase inhibitors is a consequence of prolonged loss of mitochondrial function. These findings shed light on the mechanisms by which DNA damage kills neurons and raise questions regarding the general utility of caspase inhibitors as neurotherapeutic agents.     

A population of PC12 cells that is initiating apoptosis can be rescued by nerve growth factor.

Programmed cell death, or apoptosis, occurs asynchronously in neuronal cells. To overcome this asynchrony, rat pheochromocytoma (PC12) cells were separated at different stages of apoptosis on the basis of cell density. Live cells that exhibited no apoptotic features floated to the top of density gradients. The most dense cells showed extensive loss of cytochrome c from mitochondria, caspase activation, chromatin condensation, and DNA fragmentation. These cells were committed to apoptosis and could not be rescued by reculturing in with nerve growth factor (NGF). Cells of intermediate density displayed no DNA fragmentation, but had begun to show cytochrome c loss, caspase activation, and chromatin condensation. This population displayed upregulation of the prodeath factor, c-Jun, and downregulation of prosurvival kinase, Akt. Importantly, apoptosis was reversible by NGF in this population. These studies suggest that increased cell density correlates with an initial step in the apoptosis mechanism that precedes irreversible commitment to suicide.     

Diabetic neuropathy in the rat: 1. Alcar augments the reduced levels and axoplasmic transport of substance P

This study examined the sciatic nerve axonal transport of substance P-like immunoreactivity (SPLI) and its basal content in stomach, sciatic nerve and lumbar spinal cord of 8- and 12-week alloxan-diabetic rats, respectively. One group of diabetic rats received acetyl-l-carnitine (ALCAR) throughout the experimental period. Alloxan treatment caused hyperglycemia and reduced body growth. Axonal transport of SPLI was studied by measurement of 24-hour accumulation at a ligature on the sciatic nerve. There was a marked reduction (from 50% to 100% according to the nerve segment examined) of anterograde and retrograde accumulation of SPLI in the constricted nerve of 8-week diabetic rats. In the sciatic nerve of ALCAR-treated diabetic rats, the accumulation of SPLI was comparable to control values. In the sciatic nerve, lumbar spinal cord and stomach of 12-week diabetic rats, there is a significant reduction of SPLI content. ALCAR treatment prevented SPLI loss in these tissues. Sciatic nerves showed the typical sorbitol increase and myo-inositol loss that were significantly counteracted by ALCAR. This study suggests that ALCAR treatment prevents diabetes-induced sensory neuropathy by improving altered metabolic pathways such as polyol activity and myo-inositol synthesis, and by preventing the reduction of synthesis and axonal transport of substance P. © 1995 Wiley-Liss, Inc.     

Differential roles of NMDA and non-NMDA receptor activation in induction and maintenance of thermal hyperalgesia in rats with painful peripheral mononeuropathy

Central activation of excitatory amino acid receptors has been implicated in neuropathic pain following nerve injury. In a rat model of painful peripheral mononeuropathy, we compared the effects of non-competitive NMDA receptor antagonists (MK 801 and HA966) and a non-NMDA receptor antagonist (CNQX) on induction and maintenance of thermal hyperalgesia induced by chronic constrictive injury (CCI) of the rat common sciatic nerve. Thermal hyperalgesia to radiant heat was assessed by using a foot-withdrawal test and NMDA/non-NMDA receptor antagonists were administered intrathecally onto the lumbar spinal cord before and after nerve injury. Four daily single treatments with 20 nmol HA966 or CNQX beginning 15 min prior to nerve ligation (pre-injury treatment), reliably reduced thermal hyperalgesia in CCI rats on days 3, 5, 7 and 10 after nerve ligation. Thermal hyperalgesia was also reduced in CCI rats receiving a single post-injury treatment with HA966 (20 or 80 nmol) or MK 801 (5 or 20 nmol) on day 3 after nerve ligation when thermal hyperalgesia was well developed. In contrast, a single post-injury CNQX (20 or 80 nmol) treatment failed to reduce thermal hyperalgesia or to potentiate effects of HA966 or MK 801 (5 or 20 nmol) on thermal hyperalgesia in CCI rats. Moreover, multiple post-injury CNQX treatments utilizing the same dose regime as employed for the pre-injury treatment attenuated thermal hyperalgesia but only when the treatment began 1 or 24 h (but not 72 h) after nerve ligation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cell death in the superficial dorsal horn in a model of neuropathic pain

The aims of this study were to investigate the occurrence of apoptotic cell death in the dorsal horn of the adult rat spinal cord following chronic constriction injury (CCI) to the sciatic nerve and to correlate this with behavioural responses. Six groups of six rats were used as follows: 1) CCI, 2) CCI, 3) MK801 + CCI, 4) axotomy, 5) sham, and 6) naive. Group 1 animals were behaviourally tested for thermal hyperalgesia 8 days following surgery and sacrificed and the spinal cords removed and frozen. The rest of the groups underwent the same procedure 14 days following surgery. The lumbar region of the spinal cord was cryosectioned and the incidence of apoptotic cells investigated using the TUNEL technique plus Hoechst double labelling. By 8 days post-CCI, hyperalgesia had developed in the ipsilateral paw, which was still present 14 days after the injury compared to the contralateral paw and naive and sham animals. Preemptive MK-801 prevented the onset of hyperalgesia. Significant numbers of apoptotic cells were present in the ipsilateral dorsal horn of the spinal cord 8 and 14 days following CCI compared to the contralateral side and to naive and sham animals. Preemptive treatment with MK-801 reduced the extent of apoptosis resulting from CCI to the level seen in control animals. This study demonstrates that cells undergo apoptosis as a result of CCI simultaneous with the occurrence of hyperalgesia. Furthermore, MK-801 prevents the onset of hyperalgesia and reduces the extent of apoptotic cell death, suggesting, perhaps, that apoptosis contributes to the initiation/maintenance of hyperalgesia.     

Clonidine reduces hypersensitivity and alters the balance of pro- and anti-inflammatory leukocytes after local injection at the site of inflammatory neuritis

Perineural alpha2-adrenoceptor activation relieves hypersensitivity induced by peripheral nerve injury or sciatic inflammatory neuritis. This effect is associated with a reduction in pro-inflammatory cytokines, as well as a reduction in local leukocyte number and their capacity to produce pro-inflammatory cytokines. Curiously, clonidine's antinociceptive effect appears with a 2-3-day delay after injection. Previous observations have shown that alpha-adrenoceptor activation induces apoptosis in leukocytes, which would reduce leukocyte number. Additionally, macrophage scavenging of apoptotic cells results in a shift to an anti-inflammatory phenotype, with expression of transforming growth factor (TGF)-beta1. We therefore examined the effects of perineural clonidine 24 h and 3 days after its injection on apoptosis, TGF-beta1 expression and lymphocyte and macrophage phenotype in acute sciatic inflammatory neuritis. Perineural clonidine reduced ipsilateral neuritis-induced hypersensitivity in a delayed manner (3 days after treatment), along with a reduction at this time in lymphocyte number and an increase in caspase-3 and TGF-beta1 expressing cells and macrophages co-expressing TGF-beta1 in the sciatic nerve. One day after injection clonidine treatment was associated with a reduction in lymphocytes and pro-inflammatory Th-1 cells as well as increased numbers of caspase-3 and TGF-beta1 expressing cells and macrophages co-expressing TGF-beta1 in sciatic nerve. Clonidine's effects were prevented by co-administration of an alpha2-adrenoceptor antagonist. These data suggest that alpha2-adrenoceptor activation in sciatic inflammatory neuritis increases local apoptosis and anti-inflammatory products early after treatment. This early effect likely underlies the delayed anti-inflammatory and anti-hypersensitivity effects of perineural clonidine in this setting.     

Activation of caspase-9 and -3 during H2O2-induced apoptosis of PC12 cells independent of ceramide formation

The treatment of PC12 cells with H2O2 (100-500 microM) resulted in typical apoptotic changes including fragmentation and condensation of nuclei, and DNA fragmentation observed as DNA ladder. H2O2-induced apoptosis was associated with activation of caspase-3 as assessed by cleavage of specific fluorogenic substrate peptide and processing of procaspase-3 and poly(ADP-ribose) polymerase. However, formation of ceramide, which often locates upstream of caspase-3, was not observed. The inhibitory peptide relatively specific for caspase-3, z-DEVD-FMK and non-selective caspase inhibitor z-VAD-FMK inhibited activation of caspase-3 and apoptotic cell death. However, the relatively specific inhibitors, Ac-YVKD for caspase-1 and Ac-IETD for caspase-8/6, did not affect the occurrence of apoptotic cell death. As an upstream activation of caspase-3, induction of cytochrome c release followed by processing of procaspase-9 was observed by Western blotting, although the formation of intracellular ceramide was not observed. On the other hand, in PC12 cells overexpressing Bcl-2, the number of apoptotic cells was markedly decreased and activation of both caspases-9 and -3 was prevented. These results suggest that cytochrome c and caspase-9 initiate the activation of executor caspase-3 in H2O2-treated PC12 cells, and that Bcl-2 inhibits H2O2-induced release of cytochrome c from mitochondria and then proteolytic processing of procaspase-9.     

C2-ceramide mediates cerebellar granule cells apoptosis by activation of caspases-2, -9, and -3

Ceramide is able to induce the apoptotic death of cerebellar granule cells (CGC) in culture. However, previous reports did not agree on whether ceramide-induced apoptosis of CGC requires caspase activation. Here we have shown that addition of C2-ceramide is able to produce extensive death of cultured CGC, which is associated with chromatin condensation, ladder-like DNA fragmentation, and activation of caspases. Our results show that C2-ceramide activates caspases-3, -9, and -2 but not caspases-1 and -8. Caspase-9 activation was associated with cytochrome c release from mitochondria toward the cytosol and was followed by activation of caspases-2 and -3. PARP proteolysis was also observed after caspase-3 and -2 activation. The involvement of caspases-9, -3, and -2 in ceramide-mediated apoptotic death of CGC was further supported by the use of specific inhibitors.     

Ameroid rings for gradual chronic constriction of the sciatic nerve in rats: contribution of different nerves to neuropathic pain

Mononeuropathy was induced by placing an ameroid ring around the sciatic nerve and was compared with chronic constriction injury (CCI) of the sciatic nerve [Pain 33 (1988) 87] in rats. Mechanical allodynia was assessed and the role of sciatic and saphenous afferents (Adelta and C) in thermal hyperalgesia investigated. A shorter duration of mechanical allodynia in ameroid rats as compared to CCI rats was observed. Thermal hyperalgesia was observed in the saphenous innervated skin of the hindpaw for Adelta and C nociceptors in ameroid and for Adelta nociceptors only in CCI rats, respectively. The sciatic innervated skin showed a thermal hypoalgesia with a fast onset for Adelta afferents and a slower onset for C afferents in CCI and ameroid rats. The duration of both thermal hypo- and hyperalgesia was longer in ameroid rats. We conclude that ameroid rings are a useful tool for the investigation of long-duration hyperalgesic effects of nerve injury, as the effects were more stable and seen for a longer time (>8 weeks) as compared to the CCI model. The uninjured saphenous afferents, in particular C fibers, mediate thermal hyperalgesia after chronic constriction of the sciatic nerve using an ameroid ring.     

七叶皂甙钠对坐骨神经结扎术后大鼠热痛觉及脊髓神经细胞凋亡的影响

目的探讨七叶皂甙钠对坐骨神经结扎术后大鼠热痛觉及脊髓神经细胞凋亡的影响。方法90只SD大鼠随机分为正常对照组、坐骨神经慢性压迫损伤(CCI)模型组(CCI组)和药物治疗组(药物组),每组30只大鼠。采用坐骨神经中段结扎法制作大鼠CCI模型。药物组大鼠术后给予腹腔注射0.1%七叶皂甙钠5 mg/(kg.d)治疗,CCI组给予等量生理盐水替代。各组于术前1 d取5只大鼠测定热痛觉,于术后8h、1 d、3 d、7 d、14 d及21 d时分别取5只大鼠测定热痛觉后处死。分别应用TUNEL法及免疫组化染色法检测大鼠相应损伤段脊髓组织中凋亡神经细胞数及肿瘤坏死因子-α(TNF-α)的表达。结果各组大鼠术前1 d时热痛觉的比较差异无统计学意义。CCI组及药物组大鼠术后热痛觉均较正常对照组更敏感(均P<0.05);药物组大鼠术后热痛觉较CCI组迟钝(均P<0.05)。CCI组及药物组大鼠术后各时间点脊髓组织中凋亡细胞数均明显多于正常对照组(均P<0.05);药物组大鼠术后各时间点脊髓组织中凋亡细胞数均明显少于CCI组(均P<0.05)。CCI组大鼠术后8 h～14 d,药物组术后8 h～7 d脊髓组织TNF-α的表达均明显高于正常对照组(均P<0.05);两组TNF-α的表达均于术后8 h起逐渐增高,至术后3 d时达最高,其后逐渐减少(均P<0.05);并分别于术后21 d及14 d时降至正常对照组水平。结论结扎大鼠坐骨神经可产生神经病理性疼痛,并可诱发脊髓神经细胞凋亡。七叶皂甙钠能缓解坐骨神经结扎术后大鼠热痛觉增敏,这可能与其降低TNF-α在脊髓后角的表达及抑制神经细胞的凋亡有关。     

Differential activation of PKC delta in the substantia nigra of rats following striatal or nigral 6-hydroxydopamine lesions

Parkinsonian neurodegeneration is associated with heightened levels of oxidative stress and the activation of apoptotic pathways. In an in vitro cellular model, we reported that 6-hydroxydopamine (6-OHDA) induces apoptotic cell death via the induction of mitochondrial dysfunction, the activation of caspase 3 and the consequent proteolytic activation of the redox-sensitive kinase, protein kinase C (PKC)δ, in PC12 cells. Here we have investigated the involvement of PKCδ in 6-OHDA-induced cell death in vivo . The nigrostriatal pathway of rats was lesioned by unilateral infusion of 6-OHDA into either the striatum or substantia nigra pars compacta (SNpc). Infusion into the SNpc resulted in rapid loss of tyrosine hydroxylase (TH)-positive cells (87% decrease after 4 days), consistent with a necrotic-like mode of cell death. In contrast, striatal infusion initiated a slower, progressive decline in TH immunoreactivity (25% decrease in the SNpc after 4 days); cell appearance was characteristic of apoptosis. This is consistent with a transient increase in active caspase 3 immunoreactivity at 4 days post-infusion, and a concomitant proteolytic activation of PKCδ in the SNpc of striatal-lesioned rats. Cleavage of PKCδ did not occur in the striatum or cerebellum of lesioned animals, or in the SNpc of sham-operated controls. No increase in caspase 3 immunoreactivity or proteolytic activation of PKCδ was detected in nigral-lesioned rats. These results suggest that after 6-OHDA infusion into the striatum, retrograde neurotoxicity induces caspase 3-dependent PKCδ proteolytic activation in the cell bodies of the SNpc, implicating this kinase in the neurodegenerative process.     

Caspase inhibition therapy abolishes brain trauma-induced increases in Abeta peptide: implications for clinical outcome

The detrimental effects of traumatic brain injury (TBI) on brain tissue integrity involve progressive axonal damage, necrotic cell loss, and both acute and delayed apoptotic neuronal death due to activation of caspases. Post-injury accumulation of amyloid precursor protein (APP) and its toxic metabolite amyloid-beta peptide (Abeta) has been implicated in apoptosis as well as in increasing the risk for developing Alzheimer's disease (AD) after TBI. Activated caspases proteolyze APP and are associated with increased Abeta production after neuronal injury. Conversely, Abeta and related APP/Abeta fragments stimulate caspase activation, creating a potential vicious cycle of secondary injury after TBI. Blockade of caspase activation after brain injury suppresses apoptosis and improves neurological outcome, but it is not known whether such intervention also prevents increases in Abeta levels in vivo. The present study examined the effect of caspase inhibition on post-injury levels of soluble Abeta, APP, activated caspase-3, and caspase-cleaved APP in the hippocampus of nontransgenic mice expressing human Abeta, subjected to controlled cortical injury (CCI). CCI produced brain tissue damage with cell loss and elevated levels of activated caspase-3, Abeta(1-42) and Abeta(1-40), APP, and caspase-cleaved APP fragments in hippocampal neurons and axons. Post-CCI intervention with intracerebroventricular injection of 100 nM Boc-Asp(OMe)-CH(2)F (BAF, a pan-caspase inhibitor) significantly reduced caspase-3 activation and improved histological outcome, suppressed increases in Abeta and caspase-cleaved APP, but showed no significant effect on overall APP levels in the hippocampus after CCI. These data demonstrate that after TBI, caspase inhibition can suppress elevations in Abeta. The extent to which Abeta suppression contributes to improved outcome following inhibition of caspases after TBI is unclear, but such intervention may be a valuable therapeutic strategy for preventing the long-term evolution of Abeta-mediated pathology in TBI patients who are at risk for developing AD later in life.     

Autophagy is activated by apoptotic signalling in sympathetic neurons: an alternative mechanism of death execution

Autophagy is a mechanism whereby cells digest themselves from within and so may be used in lieu of apoptosis to execute cell death. Little is known about its role in neurons. In newly isolated sympathetic neurons, two independent apoptotic stimuli, NGF-deprivation or cytosine arabinoside added in the presence of NGF, caused a 30-fold increase in autophagic particle numbers, many autophagosomes appearing before any signs of DNA-fragmentation. The anti-autophagic drug 3-methyladenine also delayed apoptosis, its neuroprotection correlating with inhibition of cytochrome c release from mitochondria and prevention of caspase activation. In contrast, autophagic activity remained elevated in neurons treated with the pan-caspase inhibitor Boc-Asp(OMe)fmk, which inhibited morphological apoptosis but did not inhibit cytochrome c release nor prevent cell death. We propose that the same apoptotic signals that cause mitochondrial dysfunction also activate autophagy. Once activated, autophagy may mediate caspase-independent neuronal death.     

AMPA-induced dark cell degeneration of cerebellar Purkinje neurons involves activation of caspases and apparent mitochondrial dysfunction

Cerebellar Purkinje neurons (PNs) are selectively vulnerable to AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepriopionic acid)-induced delayed neurotoxicity known as dark cell degeneration (DCD) that is expressed as cytoplasmic and nuclear condensation, neuron shrinkage, and failure of physiology. The present study was initiated to determine whether AMPA-receptor-induced DCD in PNs is associated with Bax translocation to the mitochondria, cytochrome C release from the mitochondria, changes in mitochondrial potential, and activation of representative initiator and executor caspases that include caspase-9, caspase-3, and caspase-7. AMPA consistently and rapidly hyperpolarized mitochondria as reflected by an increase in MitoTracker Red CMS Ros fluorescence. Increases in Bax immunoreactivity were quantitatively and temporally variable and Bax failed to localize to mitochondria. Additionally, we observed a marked increase in immunoreactivity of cytochrome C although its release from mitochondria was not apparent. Mitochondrial membrane hyperpolarization and increases in cytochrome C immunoreactivity preceded caspase activation. Immunohistochemical analyses revealed the active form of caspases-3 and -9 were markedly and significantly increased in PNs following 30 microM AMPA, and caspase-9 activation preceded caspase-3. Increases in active caspase-7 immunoreactivity were less frequently encountered in PNs. Thus DCD shares some characteristics of apoptotic programmed cell death, but lacks typical mitochondrial pathophysiology associated with classic apoptosis. These findings suggest that AMPA-induced DCD is a form of active PCD that lies on a spectrum between classical apoptosis and passive necrosis.