Kainic acid dose affects delayed cell death mechanism after status epilepticus

Kainic acid (KA)-induced status epilepticus (SE) produces hippocampal neuronal death, which varies from necrosis to apoptosis or programmed cell death (PCD). We examined whether the type of neuronal death was dependent on KA dose. Adult rats were induced SE by intraperitoneal injection of KA at 9 mg/kg (K9) or 12 mg/kg (K12). Hippocampal neuronal death was assessed by TUNEL staining, electron microscopy, and Western blotting of caspase-3 on days 1, 3 and 7 after SE induction. K12 rats showed higher a mortality rate and shorter latency to the onset of SE when compared with K9 rats. In both groups, acidophilic and pyknotic neurons were evident in CA1 at 24h after SE and neuronal loss developed from day 3. The degenerated neurons became TUNEL-positive on days 3 and 7 in K9 rats but not in K12 rats. Caspase-3 activation was detected on days 3 and 7 in K9 rats but was undetectable in K12 rats. Ultrastructural study revealed shrunken neurons exhibiting pyknotic nuclei containing small and dispersed chromatin clumps 24h after SE in CA1. No cells exhibited apoptosis. On days 3 and 7, the degenerated neurons were necrotic with high electron density and small chromatin clumps. There were no ultrastructural differences between the K9 and K12 groups. These results revealed that differences in KA dose affected the delayed cell death (3 and 7 days after SE); however, no effect was seen on the early cell death (24h after SE). Moderate-dose KA induced necrosis, while low-dose KA induced PCD.     

Mu-calpain mediates hippocampal neuron death in rats after lithium-pilocarpine-induced status epilepticus

Status epilepticus (SE) is a severe clinical manifestation of epilepsy which causes brain damage. The pathological process and underlying mechanisms involved in the programmed cell death (PCD) are still not fully clear. In the current study, rats were induced SE by lithium–pilocarpine administration. Our data showed hippocampal neurons death appeared at 6 h after SE and sustained for 7 days. By blotting the activation of μ-calpain and its specific cleavage of nonerythroid -spectrin (SpII) (145 kDa) was evident at 1 and 3 days after SE, which coincided with Bid activation, apoptosis inducing factor (AIF) translocation and cytochrome c release from mitochondria, whereas, activated caspase-3 and caspase-3-specific fragments of SpII (120 kDa) predominantly appeared at 5 and 7 days after SE. Moreover, MDL-28170, a calpain inhibitor, partially rescued the neuron death and attenuated the expression of activated μ-calpain, cleavage of Bid (15 kDa), AIF translocation and cytochrome c release. Taken together, our study indicated that μ-calpain mediated hippocampal neuron PCD is prior to caspase-3 activation. It functioned via translocation of Bid, AIF and cytochrome c release.     

Strain differences in seizure-induced cell death following pilocarpine-induced status epilepticus

Mouse strains differ from one another in their susceptibility to seizure-induced excitotoxic cell death. Previously, we have demonstrated that mature inbred strains of mice show remarkable genetic differences in susceptibility to the neuropathological consequences of seizures in the kainate model of status epilepticus. At present, while the cellular mechanisms underlying strain-dependent differences in susceptibility remain unclear, some of this variation is assumed to have a genetic basis. However, it remains unclear whether strain differences in susceptibility to seizure-induced cell death observed following kainate administration are observed following systemic administration of other chemoconvulsants. In rodents, the cholinomimetic convulsant pilocarpine is widely used to induce status epilepticus (SE), followed by hippocampal damage and spontaneous recurrent seizures, resembling temporal lobe epilepsy. This model has initially been described in rats, but is increasingly used in mice. We characterized neuronal pathologies after pilocarpine-induced status epilepticus (SE) in eight inbred strains of mice focusing on the hippocampus. A ramping-up dose protocol for pilocarpine was used and behavior was monitored for 4-5 h. While we did not observe any significant differences in seizure latency or duration to pilocarpine among the inbred strains, we did observe a significant difference in susceptibility to the neuropathological consequences of pilocarpine-induced SE. Of the eight genetically diverse mouse strains screened for pilocarpine-induced status, BALB/cJ and BALB/cByJ were the only two strains that were resistant to the neuropathological consequences of seizure-induced cell death. Additional studies of these murine strains may be useful for investigating genetic influences on pilocarpine-induced status epilepticus.     

The role of MMP-9 in integrin-mediated hippocampal cell death after pilocarpine-induced status epilepticus

Recent studies demonstrate that matrix metalloproteinase-9 (MMP-9) is closely involved in the pathogenesis of epilepsy. This study investigated the role of MMP-9 in hippocampal cell death after pilocarpine-induced status epilepticus (SE). We showed that MMP-9 expression and activity significantly increased and β1-integrin levels decreased on day 3 after SE. β1-integrin degradation was also observed in hippocampal ex vivo extracts incubated with recombinant active MMP-9. Treatment with a selective MMP-9 inhibitor attenuated MMP-9 up-regulation, β1-integrin degradation, the reduction of ILK activity and Akt phosphorylation, and subsequent hippocampal damage after SE. However, co-treatment with anti-β1-integrin antibody almost completely blocked the protective effects of the MMP-9 inhibitor on both integrin-mediated survival signaling and hippocampal cell death. Our study demonstrates that MMP-9 induces apoptotic hippocampal cell death by interrupting integrin-mediated survival signaling after SE and suggests that MMP-9 may be a promising target for a neuroprotective approach to preventing seizure-induced hippocampal damage.     

Hippocampal programmed cell death after status epilepticus: evidence for NMDA-receptor and ceramide-mediated mechanisms

PURPOSE: Status epilepticus (SE) can result in acute neuronal injury with subsequent long-term age-dependent behavioral and histologic sequelae. To investigate potential mechanisms that may underlie SE-related neuronal injury, we studied the occurrence of programmed cell death (PCD) in the hippocampus in the kainic acid (KA) model. METHODS: In adult rats, KA-induced SE resulted in DNA fragmentation documented at 30 h after KA injection. Ceramide, a known mediator of PCD in multiple neural and nonneural tissues, increased at 2-3 h after KA intraperitoneal injection, and then decreased to control levels before increasing again from 12 to 30 h after injection. MK801 pretreatment prevented KA-induced increases in ceramide levels and DNA fragmentation, whether there was reduction in seizure severity or not (achieved with 5 mg/kg and 1 mg/kg of MK801, respectively). RESULTS: Both ceramide increases and DNA fragmentation were observed after KA-induced SE in adult and in P35 rats. Ceramide did not increase after KA-induced SE in P7 pups, which also did not manifest any DNA fragmentation. Intrahippocampal injection of the active ceramide analogue C2-ceramide produced widespread DNA fragmentation, whereas the inactive ceramide analogue C2-dihydroceramide did not. CONCLUSIONS: Our data support the hypotheses that (a) N-methyl-d-aspartate-receptor activation results in ceramide increases and in DNA fragmentation; (b) ceramide is a mediator of PCD after SE; and (c) there are age-related differences in PCD and in the ceramide response after SE. Differences in the ceramide response could, potentially, be responsible for observed age-related differences in the response to SE.     

不同潜伏期致痫大鼠海马神经元线粒体损伤及Fas、Bax、caspase-3的表达

目的观察不同潜伏期致癫痫状态大鼠海马CA3区神经元线粒体超微结构损伤及Fas、Bax、cas-pase-3的表达变化。方法分别采用海人酸腹腔注射(A组)和尾静脉注射(B组)诱发不同潜伏期的大鼠癫痫持续状态(SE)。于SE终止后不同时点取海马,电镜观察线粒体的超微结构,半定量RT-PCR检测Fas、Bax的mRNA水平,免疫组化检测caspase-3蛋白的表达。结果A组潜伏期为97±11min,线粒体肿胀,神经元呈凋亡征;B组潜伏期为48±13min,线粒体肿胀且伴膜的崩解,神经元呈坏死表现。对照组及B组未检测到Fas及Bax mRNA;A组Fas及Bax的mRNA表达均于SE后6h出现,24h增加,48h达高峰,并持续至72h。两组动物均在SE后6h出现caspase-3表达增高(P<0.001),24h达顶峰(P<0.001);A组高表达持续至72h,B组在48h点急剧降低。结论不同潜伏期的SE导致了不同程度的线粒体损伤,进而决定了神经元死亡的分子机制。     

人脑出血后海马神经元损伤与Caspase-3的关系

目的　研究人脑出血后海马神经元损伤与凋亡促进因子Caspase- 3之间的关系。方法　选取10例因脑出血而死亡的尸检脑标本,应用HE染色,Caspase- 3免疫组织化学染色和Caspase- 3m RNA原位杂交,观察脑出血时海马CA1 区神经元损伤变化与Caspase- 3表达之间的关系。结果　海马CA1 区,出血5 h可检测Caspase- 3免疫组化染色的阳性细胞(10 .4个/高倍视野) ,2 2～4 8h时达高峰(2 1.7～2 4 .3个/高倍视野)。Caspase- 3m RNA原位杂交阳性表达始于5 h(6 .75个/高倍视野) ,2 4 h达高峰(14 .6 0～18.30个/高倍视野) ;二者均在72 h后呈明显下降趋势。出血2 4 h,可检测到TUNEL阳性细胞,持续至72 h。Caspase- 3m RNA与Caspase- 3呈正相关,相关系数为0 .717(P<0 .0 5 )。4～16 h受损神经元形态基本正常,2 4～4 8h细胞凋亡特征明显,72 h后,几乎所有神经元形态呈现严重病理变化。结论　人脑出血后海马神经元发生一系列形态学变化,其演变规律与凋亡促进因子Caspase- 3有密切相关性。     

Immunohistochemical investigation of caspase-1 and effect of caspase-1 inhibitor in delayed neuronal death after transient cerebral ischemia

The localization of caspase-1 protein, interleukin-1beta (IL-1beta)-converting enzyme, was immunohistochemically examined in the hippocampal CA-1 subfield by a transient occlusion of bilateral common carotid arteries in Mongolian gerbils. Immunoreactivities for caspase-1 were found in microglias, astrocytes, endothelial cells of capillaries and some non-pyramidal neurons. Immunopositive microglias increased in number from 3 days until 7 days from the transient ischemia, and astrocytes also increased in number from 3 days until 28 days. At the electron microscopic level, caspase-1 immunoreaction endproducts were associated with Golgi apparatus in glial cells, endothelial cells of blood vessels and non-pyramidal neurons. The delayed neuronal death of CA-1 pyramidal cells was significantly protected by the treatment of specific caspase-1 inhibitor (Ac-WEHD-CHO) or broad caspase family inhibitor (z-VAD-FMK). Cell death was protected in a dose dependent manner by the former by 43-57%, and by the latter by 66-91% when injected at 1 and 10 microg, respectively. On the other hand, the protective effect of specific caspase-3 inhibitor (Ac-DMQD-CHO) was less significant at higher dose (10 microg) by 33% (P<0.05), and not detectable at lower dose (1 microg) by 13% (P=0.27). Furthermore, a significant decrease of microglias and astrocytes was found in the CA-1 as well as the reduction of IL-1beta and caspase-1 immunoreactivities by the treatment of Ac-WEHD-CHO. Extravasation of serum albumin was also extremely reduced by this treatment. These findings suggest that the inhibition of caspase-1 activity ameliorates the ischemic injury by inhibiting the activity of IL-1beta.     

细胞周期调控对大鼠局灶性脑缺血后迟发性神经元死亡的影响

目的探讨细胞周期调控对脑神经细胞凋亡的影响。方法采用光化学方法制作大鼠局灶性缺血模型,术后立即腹腔注射细胞周期抑制剂Olomoucine进行干预。HE染色来测定皮层梗死灶的体积;应用免疫组织化学法观察缺血后第3天假手术组、缺血对照组和干预组大鼠损伤侧皮层病灶周围凋亡神经细胞的表达;聚丙烯酰胺凝胶电泳(Western blot)和半定量逆转录-聚合酶链式反应(RT-PCR)观察Caspase-3蛋白及mRNA的表达。结果 HE染色显示对照组梗死灶明显大于干预组;免疫组织化学可见病灶周围凋亡神经细胞的阳性表达,干预组较假手术组显著增高,但明显低于对照组(P<0．05);Western blot和RT-PcR显示Caspase-3蛋白和mRNA的表达,干预组较假手术组增高(P<0．05),而较对照组明显降低(P<0．05)。结论脑缺血损伤后细胞周期调控参与了迟发性神经元的死亡过程。     

Caspase-3 is activated following axotomy of neonatal facial motoneurons and caspase-3 gene deletion delays axotomy-induced cell death in rodents

In this report, we examined the possible functions of the cell death protease, caspase-3, in the axotomy-induced apoptosis of facial motoneurons in newborn rodents. Using in situ hybridization and Western blot, we found higher levels of caspase-3 mRNA and pro-caspase-3 protein expression in motoneurons of neonatal and 2-week-old rats than adult rats. Following facial motoneuron axotomy, caspase-3 mRNA and protein expression increased in motoneurons of both neonatal and adult rats. However, using an antibody directed to the activated form of the caspase-3 protease, we found that catalytically active caspase-3 was present only in axotomized neonatal motoneurons. As motoneurons in neonatal but not adult rodents are susceptible to axotomy-induced apoptosis, we hypothesized that caspase-3 may play a role in their demise. To determine the necessity of caspase-3 activation in axotomy-induced apoptosis, we counted the number of surviving motoneurons at 4 and 7 days following axotomy in wild type mice and caspase-3 gene-deleted mice. There were nearly three times more surviving motoneurons in caspase-3 gene-deleted mice than in wild type mice at both 4 days (mean 1074 vs. 464, P<0.005) and 7 days (mean 469 vs. 190, P<0.005) following injury, indicating a slower rate of death. Examination of the dying motoneurons using TUNEL staining (for fragmented DNA) and bisbenzimide staining (for nuclear morphology) revealed incomplete nuclear condensation in caspase-3-deficient motoneurons. These results demonstrate that caspase-3 activation plays important roles in the rapid demise of axotomized neonatal motoneurons.     

Resistance of neurofilaments to degradation, and lack of neuronal death and mossy fiber sprouting after kainic acid-induced status epilepticus in the developing rat hippocampus

Neurofilament (NF) proteins, the major constituent of intermediate filaments in neurons, have an important role in cellular stability and plasticity. We have now studied the short-term (hours) and long-term (up to 1 week) effects of kainic acid (KA)-induced status epilepticus (SE) on the reactivity of NF proteins, and mossy fiber (MF) sprouting and neuronal death up to 4 weeks in 9-day-old rats. In Western blotting, the expression of the phosphorylation-independent epitopes of NF-L, NF-M, and NF-H rapidly but transiently increased after the treatment, whereas the phosphorylated NF-M remained elevated for 7 days. However, the treatment did not change the immunoreactivity of NF proteins, and no neuronal death or mossy fiber sprouting was detected at any time point. Our findings indicate seizure-induced reactivity of NF proteins but their resistance to degradation, which could be of importance in neuronal survival and may also prevent MF sprouting in the developing hippocampus.     

Early release of cytochrome C and activation of caspase-3 in hyperglycemic rats subjected to transient forebrain ischemia

The mechanisms underlying the aggravating effect of hyperglycemia on brain damage are still elusive. The present study was designed to test our hypothesis that hyperglycemia-mediated damage is caused by mitochondrial dysfunction with mitochondrial release of cytochrome c (cyt c) to the cytoplasm, which leads to activation of caspase-3, the executioner of cell death. We induced 15 min of forebrain ischemia, followed by 0.5, 1, and 3 h of recirculation in sham, normoglycemic and hyperglycemic rats. Release of cyt c was observed in the neocortex and CA3 in hyperglycemic rats after only 0.5 h of reperfusion, when no obvious neuronal damage was observed. The release of cyt c persisted after 1 and 3 h of reperfusion. Activation of caspase-3 was observed after 1 and 3 h of recovery in hyperglycemic animals. No cyt c release or caspase-3 activation was observed in sham-operated controls while a mild increase of cyt c was observed in normoglycemic ischemic animals after 1 and 3 h of reperfusion. The findings that there is caspase activation and cyt c relocation support a notion that the biochemical changes that constitute programmed cell death occur after ischemia and contribute, at least in part, to hyperglycemia-aggravated ischemic neuronal death.     

Activation of cell death pathway after a brief period of global ischemia in diabetic and non-diabetic animals

Mitochondria play a critical role in the pathogenesis of cerebral ischemia. Acute hyperglycemia has been shown to activate the mitochondria-initiated cell death pathway after an intermediate period of ischemia. The objective of the present study was to determine if diabetic hyperglycemia induced by streptozotocin activates the cell death pathway after a brief period of global ischemia. Five minutes of global ischemia was induced in nondiabetic and diabetic rats. Brain samples were collected after 30 min, 6 h, 1, 3, and 7 days of recirculation as well as from sham-operated controls. Histopathological examination in the hippocampal CA1, CA3, hilus, and dentate gyrus regions, as well as in the cortical and thalamic areas, showed that neuronal death in diabetic animals increased compared to nondiabetic ischemic controls. Neuronal damage maturation occurred after 7 days of recovery in nondiabetic rats, while it was shortened to 3 days of recovery in diabetic animals. Western blot analyses revealed that release of cytochrome c markedly increased after 1 and 3 days of reperfusion in diabetic rats. Caspase-3 activation was evident in the nuclear fraction of the cortex of diabetic rats after 3 days recovery and it was preceded by activation of caspase-9, but not activation of caspase-8. Electron microscopy demonstrated that chromatin condensation and mitochondrial swelling were features of the diabetes-mediated ischemic neuronal damage. However, no apoptotic bodies were observed in any sections examined. These results suggest that a brief period of global ischemia in diabetic animals activates a neuronal cell death pathway involving cytochrome c release, caspase-9 activation, and caspase-3 cleavage, all of which are most likely initiated by early mitochondria damage.     

Intravenous human umbilical cord blood transplantation for stroke: impact on infarct volume and caspase-3-dependent cell death in spontaneously hypertensive rats

Transplantation of human umbilical cord blood cells (HUCBC) produces reliable behavioral and morphological improvements in animal models of stroke. However, the mechanisms of action still have not been fully elucidated. The aim of the present study is the evaluation of potential neuroprotective effects produced by HUCBC in terms of reduced infarct volume and caspase-3-dependent cell death. Permanent middle cerebral artery occlusion was induced in 90 spontaneously hypertensive rats. The animals were randomly assigned to the control group (n = 49) or the verum group (n = 41). The cell suspension (8 × 10⁶ HUCBC per kilogram bodyweight) or vehicle solution was intravenously administered 24 h after stroke onset. Fifty subjects (n = 25/25) were sacrificed after 25, 48, 72 and 96 h, and brain specimens were removed for immunohistochemistry for MAP2, cleaved caspase-3 (casp3) and GFAP. Another 42 animals (n = 26/16) were sacrificed after 0, 6, 24, 36 and 48 h and their brains processed for quantitative PCR for casp3 and survivin. The infarct volume remained stable over the entire experimental period. However, cleaved casp3 activity increased significantly in the infarct border zone within the same time frame. Numerous cleaved casp3-positive cells were colocalized with the astrocytic marker GFAP, whereas cleavage of neuronal casp3 was observed rarely. Neither the infarct volume nor casp3 activity was significantly affected by cell transplantation. Delayed systemic transplantation of HUCBC failed to produce neuroprotective effects in a permanent stroke model using premorbid subjects.     

Discovery of a novel compound: insight into mechanisms for acrylamide-induced axonopathy and colchicine-induced apoptotic neuronal cell death

The exposure of humans and experimental animals to certain industrial toxins such as acrylamide is known to cause nerve damage classified as axonopathy, but the mechanisms involved are poorly understood. Here we show that acrylamide induces morphological changes and tyrosine phosphorylation of focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2), a member of the FAK subfamily, in human differentiating neuroblastoma SH-SY5Y cells. Furthermore, we identified a novel molecule designated 'compound-1' that inhibits the morphological and biochemical events. Daily oral administrations of the compound also effectively alleviated behavioral deficits in animals elicited by acrylamide in inclined plane testing, landing foot spread testing and rota-rod performance testing. The compound also effectively inhibited the biological and biochemical responses caused by another axonopathy inducer, colchicine, including tyrosine phosphorylation of Pyk2, formation of an 85-kDa poly(ADP-ribose)polymerase (PARP) fragment and apoptosis-associated induction of the NAPOR gene as well as neuronal cell death. Our findings not only provide insight into FAK and Pyk2 functions in neuronal cells, but may also be important in the development of therapeutic agents for peripheral neuropathy and neurodegeneration.     

慢性糖尿病大鼠局灶性脑缺血再灌注损伤后海马区Caspase-3和β-淀粉样蛋白的表达意义

目的 观察天冬酰胺内肽酶-3(Caspase-3)和β-淀粉样蛋白(Aβ)在慢性糖尿病(cDM)大鼠脑缺血再灌注海马神经元损伤中的表达意义.方法 采用链脲佐菌素(STZ)诱导和线栓法制备慢性糖尿病(cDM)和大脑中动脉闭塞模型(MCAO),分别在各时间点(1d、2d、3d、4d、5d、7d),应用HE染色和免疫组化方法比较慢性糖尿病脑缺血再灌注组(简称cDM组)与缺血再灌注组海马神经元缺失、Caspase-3和Aβ免疫组化阳性细胞数变化.结果 cDM组(慢性糖尿病脑缺血再灌注组)海马区神经元缺失,于2d开始出现,4d达高峰,7d减少,而且cDM组Caspase-3和Aβ表达上调,均在2d开始出现,4d达高峰,7d减少,在各时间点分别明显高于缺血再灌注组,差异具有统计学意义(P＜0.05).结论慢性糖尿病大鼠脑缺血再灌注损伤后海马CA1区神经元发生严重缺失,Caspase-3和Aβ明显上调提示Caspase-3介导的细胞凋亡机制和Aβ神经毒性作用可能是慢性糖尿病加重脑缺血再灌注海马神经元损伤机制之一.     

Arachidonic acid peroxides induce apoptotic Neuro-2A cell death in association with intracellular Ca(2+) rise and mitochondrial damage independently of caspase-3 activation

The present study aimed at understanding the effects of arachidonic acid peroxides on neuronal cell death using the mouse neuroblastoma cell line, Neuro-2A cells. Arachidonic acid peroxides were produced by ultraviolet (UV) radiation. UV-radiated arachidonic acid significantly reduced Neuro-2A cell viability at concentrations of more than 0.1 muM, with being more potential than non-radiated arachidonic acid. Nuclei of Neuro-2A cells killed with UV-radiated arachidonic acid were reactive to Hoechst 33342, a marker of apoptosis, and the effect was much greater than that achieved with non-radiated arachidonic acid. UV-radiated arachidonic acid persistently increased intracellular Ca(2+) concentrations and dissipated mitochondrial membrane potential in Neuro-2A cells. UV-radiated arachidonic acid-induced Neuro-2A cell death, whereas it was not affected by a pancaspase inhibitor or a caspase-3 inhibitor, was significantly inhibited by an inhibitor of caspase-1, -8, or -9. The results of the present study suggest that arachidonic acid peroxides induce apoptotic neuronal cell death in association with intracellular Ca(2+) rise and mitochondrial damage, in part via a caspase-dependent pathway regardless of caspase-3.     

Hypoxic-ischemic injury in neonatal brain: involvement of a novel neuronal molecule in neuronal cell death and potential target for neuroprotection

Perinatal hypoxia-ischemia (HI) is the most common cause of various neurological disabilities in children with high societal cost. Hypoxic-ischemic brain damage is an evolving process and ample evidence suggests distinct difference between the immature and mature brain in the pathology and consequences of brain injury. Therefore, it is of utmost importance to better understand the mechanisms underlying the hypoxic-ischemic injury in neonatal brain to devise effective therapeutic strategies. Nonetheless, the mechanism(s) involved in this pathology in the developing brain remain inadequately understood. Effective neuroprotective strategies will include either inhibition of the death effector pathways or induction of their regulatory and survival promoting cellular proteins. Neuronal pentraxins (NPs) define a family of novel proteins "long pentraxins" that are exclusively expressed in the central neurons, and are homologous to the C-reactive and acute-phase proteins in the immune system. NPs have been shown to be involved in the excitatory synaptic remodeling. We found that the neuronal protein 'neuronal pentraxin 1' (NP1) is induced in neonatal rat brain following HI, and NP1 induction preceded the time of actual tissue loss in brain. In demonstrating this we also found that NP1 gene silencing is neuroprotective against hypoxia-induced neuronal death. This is the first evidence for a pathophysiological function of NP1 in central neurons. Our results suggest that NP1 is part of a death program triggered by HI. Most importantly, our findings of specific interactions of NP1 with the excitatory glutamate receptors AMPA GluR1 subunit and their co-localization suggest a role for this novel neuronal protein NP1 in the excitotoxic cascade. Blockade of AMPA-induced neuronal death following inhibition of NP1 expression further implicates a regulatory interaction between NP1 and AMPA glutamate receptors. Subsequent experiments using NP1 loss-of-function strategies, we have demonstrated specific requirements of NP1 induction in HI-induced neuronal death. Together our findings clearly identify a novel role for NP1 in the coupling between HI and cerebral cell death. Thus, NP1 could be a new molecular target in the central neurons for preventing hypoxic-ischemic neuronal death in developing brain. These very novel results could lead to more effective neuroprotective strategies against hypoxic-ischemic brain injury in neonates.