Extracellular signal-regulated kinase and c-Jun N-terminal protein kinase in ischemic tolerance.

The alterations and involvement of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal protein kinase (JNK) activation were examined in the hippocampal CA1 region in a rat model of global brain ischemic tolerance. Western blotting study showed that ERK activation (diphosphorylation) level was decreased (3.75-, 0.56-, and 0.23-fold vs sham control) and JNK activation level was increased (3.82-, 4.63-, and 5.30-fold vs sham control) 3 days after more severe ischemic insults (6 min, 8 min, and 10 min of ischemia, respectively). These alterations were significantly prevented by pretreatment with preconditioning ischemia, which also provided neuronal protection against ischemic injury. Inhibition of ERK activation after preconditioning ischemia by PD98059, a specific ERK kinase inhibitor, significantly prevented the inhibitory effects of preconditioning ischemia on both JNK activation and ischemic injury. The results suggest that ERK activation after preconditioning ischemia may result in the prevention of JNK activation and thus be involved in the protective responses in ischemic tolerance in hippocampal CA1 region.     

SNAP-25 in hippocampal CA1 region is involved in memory consolidation

As a synaptosomal protein, SNAP-25 plays a role in a number of neuronal functions including axonal growth, dendrite formation, fusion of synaptic vesicles with membrane and the expression of long-term potentiation (LTP) in the hippocampus. Using a learning/memory behavior screening, we identified SNAP-25 as one of the differentially expressed genes in the hippocampus upon behavioral training. The inhibition of SNAP-25 with intracerebroventricular antisense oligonucleotide caused a deficit in long- but not short-term memory for step-down inhibitory avoidance. Intra-CA1 infusion of the SNAP-25 antisense oligonucleotide impaired long-term contextual fear memory and spatial memory and interfered with the LTP of synaptic transmission in the CA1 region. The inhibitory effect on LTP was not mediated by a pre-synaptic mechanism because paired pulse facilitation of synaptic transmission was not affected after administration of the antisense oligonucleotide. Together, the results suggest that SNAP-25 in the CA1 region is involved in memory consolidation.     

Orthodromic activation of hippocampal CA1 region of the rat

(1) The posterior alveus (PA), the anterior alveus (AA) and the Schaffer collaterals (SCH) evoked field potential components which were organized as parasagittal strips of various widths. Spatially continuous and interactive lamellae are suggested. (2) By correlation with unit activities, the early postsynaptic components evoked by PA, AA and SCH were inferred to be extracellular excitatory postsynaptic potentials (EPSPs) and the late, long-duration components, the inhibitory postsynaptic potentials (IPSPs). The hypothesis that interneurons as well as pyramidal cells generate the field is proposed and discussed. (3) One- and two-dimensional profiles of deep evoked potentials and current source-sink analysis revealed excitatory synapses in stratum oriens for the PA and AA inputs and in stratum radiatum for the SCH input. The late dipole field evoked by PA and AA possessed current sources in strata radiatum and pyramidale, the sites of the inhibitory synapses. The late dipole field evoked by SCH had another component possibly generated by recurrent activity, afterpotentials or relayed activity through CA3.     

Human c-Jun N-terminal kinase expression and activation in the nervous system

Differential expression and localization of c-Jun N-terminal kinases (JNKs) in the human brain may reflect transduction of a variety of extracellular stimuli to selective cellular responses. Of the three JNKs, JNK1 and 2 are widely distributed in tissues and JNK3 is predominantly restricted to brain where it is expressed in neurons. Although there is considerable molecular conservation among all three JNKs, we distinguished expression of each by in situ hybridization, immunoblot analysis with a panel of antibodies, and stress-activation using c-Jun as substrate. In the human central nervous system (CNS), there are at least 10 isoforms: JNK3alpha1 and JNK1alpha1 were the major JNK isoforms expressed; JNK2 was not detected. On immunoblots of brain homogenates, antibody selectivity identified JNK3alpha1 as a 45-kDa protein, JNK1alpha1, a slightly lower band at 44 kDa, and a 50-kDa band of unknown specificity. Recombinant human JNK3alpha1, transfected either into CHO, COS-1, or Neuro2A (N2A) cells, was strongly expressed as a 45-kDa protein in each. Transfected JNK3alpha1, and endogenous JNK1, each immunoprecipitated from N2A cells, phosphorylated recombinant forms of human c-Jun. Kinase activity of each JNK was modestly stimulated in N2A cells by anisomycin but not by ceramide, UV irradiation, or heat shock. Endogenous JNK activation, especially at a low level, may reflect a chronic and cumulative stress process that contributes to hyperphosphorylation of cytoskeletal proteins such as those found in Alzheimer's disease (AD), and ultimately, induction of apoptosis.     

GluR6-containing KA receptor mediates the activation of p38 MAP kinase in rat hippocampal CA1 region during brain ischemia injury

Our previous study showed that kainate (KA) receptor subunit GluR6 played an important role in ischemia-induced MLK3 and JNK activation and neuronal degeneration through the GluR6-PSD95-MLK3 signaling module. However, whether the KA receptors subunit GluR6 is involved in the activation of p38 MAP kinase during the transient brain ischemia/reperfusion (I/R) in the rat hippocampal CA1 subfield is still unknown. In this present study, we first evaluated the time-course of phospho-p38 MAP kinase at various time-points after 15 min of ischemia and then observed the effects of antagonist of KA receptor subunit GluR6, GluR6 antisence oligodeoxynucleotides on the phosphorylation of p38 MAP kinase induced by I/R. Results showed that inhibiting KA receptor GluR6 or suppressing the expression of KA receptor GluR6 could down-regulate the elevation of phospho-p38 MAP kinase induced by I/R. These drugs also reduced the phosphorylation of MLK3, MKK3/MKK6, MKK4, and MAPKAPK2. Additionally, our results indicated administration of three drugs, including p38 MAP kinase inhibitor before brain ischemia significantly decreased the number of TUNEL-positive cells detected at 3 days of reperfusion and increased the number of the surviving CA1 pyramidal cells at 5 days of reperfusion after 15 min of ischemia. Taken together, we suggest that GluR6-contained KA receptors can mediate p38 MAP kinase activation through a kinase cascade, including MLK3, MKK3/MKK6, and MKK4 and then induce increased phosphorylation of MAPKAPK-2 during ischemia injury and ultimately result in neuronal cell death in the rat hippocampal CA1 region.     

挤压伤至股骨干骨折对大鼠海马CA1区形态学变化

目的观察挤压伤(CS)至股骨干骨折后缺血/再灌注(I/R)引起的SD大鼠海马CA1区形态学的变化特征。方法建立大鼠CS模型,SD大鼠双侧后肢挤压6 h,再灌注0、6、12、24 h,4%多聚甲醛灌注,用刀片把脑组织从正中矢状位分为左右两半,自上丘至视交叉节段取脑组织,一半用于高尔基染色,一半制备成石蜡切片,分别用于HE染色和Nissl染色。结果挤压伤至股骨干骨折I/R后开始出现大鼠海马神经元疏松紊乱、肿胀变性,形态结构受损,凋亡数目增加,神经元大量丢失,海马CA1区树突棘密度减少,挤压再灌注12 h最为显著,24 h上述形态有所改善,但仍低于正常水平。结论 I/R引起SD大鼠海马CA1区神经元细胞受损,受损程度在解压后12 h达到高峰。     

Role of c-Jun N-terminal kinase in early brain injury after subarachnoid hemorrhage

The c-Jun N-terminal kinase (JNK) is induced by cerebral ischemia and injurious blood components acutely after subarachnoid hemorrhage (SAH). We hypothesized that inhibition of JNK will prevent damage to the neurovascular unit in the early brain injury period after SAH. Ninety-nine male SD rats (300-350 g) were randomly assigned to sham, SAH, and SAH treated with JNK inhibitor groups. SAH was induced by endovascular perforation. The JNK inhibitor SP600125 was administered intraperitoneally at 1 hr before and 6 hr after SAH. At 24 hr after SAH, we observed increased phosphorylation of JNK and c-Jun. Signs of neurovascular damage were observed in the hemorrhagic brains; these included the increases of aquaporin (AQP)-1 expression and brain water content as well as enhanced matrix metalloproteinase (MMP)-9 activity, vascular collagen IV loss, increased VEGF tissue level, and Evans blue extravasation. The appearances of cleaved caspase-3 expression, TUNEL-positive cells, and apoptotic morphology in cerebral tissues were associated with neurological deficit after SAH. JNK inhibition prevented c-Jun phosphorylation and suppressed AQP1, MMP-9, VEGF, and caspase-3 activation, with concomitant diminution of neuronal injury, blood-brain barrier preservation, reduced brain swelling, and improved neurological deficit in rats after SAH. This study demonstrates a multitude of beneficial effects of JNK inhibition, including protection of the neurovascular unit in early brain injury after SAH.     

Antioxidant enzymes are differently changed in experimental ischemic hippocampal CA1 region following repeated restraint stress

Restraint stress induces physiological changes in the brain. In the present study, we observed the effects of repeated stress on ischemic damage associated with oxidative stress in gerbils. Animals were placed into restrainers for 5h (between 09:30 h and 14:30 h) for 21 consecutive days prior to 5 min of transient cerebral ischemia. Experimental groups were divided into 4 groups; 1) sham-operated control-group (sham-group), 2) ischemia-operated control-group (ischemia-group), 3) sham-operated stress-group (stressed-sham-group), and 4) ischemia-operated stress-group (stressed-ischemia-group). Serum corticosterone level in the ischemia-group was highest (330% vs the sham-group) at 12h post-ischemia, and serum corticosterone levels in the stressed-ischemia-group were significantly lower than the ischemia-group. Locomotor activity in the ischemia-group was significantly increased (300% vs the sham-group) at 1 day post-ischemia; however, locomotor activity in the stressed-ischemia-group was less increased compared to the ischemia-group. A few NeuN (neuron-specific soluble nuclear antigen)-positive ((+)) cells were found in the stratum pyramidale (SP) of the hippocampal CA1 region (CA1) 4 days post-ischemia in the ischemia-group; however, in the stressed-ischemia-group at 4 days post-ischemia, 83.8% of NeuN(+) neurons were found. In addition, we found a few Fluro-Jade B (a marker for neuronal degeneration)(+) and TUNEL(+) cells in the stressed-ischemia-group at 4 days post-ischemia. In gliosis, glial fibrillary acidic protein(+) astrocytes in the stressed-ischemia-groups was similar to the ischemia-groups; however, ionized calcium-binding adapter molecule 1(+) microglia in the stressed-ischemia-groups were much less activated than the ischemia-groups. Among antioxidants, Cu,Zn-superoxide dismutase (SOD1) immunoreactivity in the SP was higher in the stressed-ischemia-groups than the ischemia-groups. Catalase immunoreactivity in the SP of the stressed-ischemia-groups was similar to the ischemia-groups. However, Mn-superoxide dismutase and glutathione peroxidase immunoreactivity were lower than the ischemia-groups. In brief, our results indicate that repeated restraint stress significantly attenuates ischemic neuronal damage and locomotor activity following ischemia. In addition, SOD1 among antioxidants significantly increases in the stressed-ischemia-groups.     

Neuroprotection against focal ischemic brain injury by inhibition of c-Jun N-terminal kinase and attenuation of the mitochondrial apoptosis-signaling pathway.

c-Jun N-terminal kinase (JNK) is an important stress-responsive kinase that is activated by various forms of brain insults. In this study, we have examined the role of JNK activation in neuronal cell death in a murine model of focal ischemia and reperfusion; furthermore, we investigated the mechanism of JNK in apoptosis signaling, focusing on the mitochondrial-signaling pathway. We show here that JNK activity was induced in the brain 0.5 to 24 h after ischemia. Systemic administration of SP600125, a small molecule JNK-specific inhibitor, diminished JNK activity after ischemia and dose-dependently reduced infarct volume. c-Jun N-terminal kinase inhibition also attenuated ischemia-induced expression of Bim, Hrk/DP5, and Fas, but not the expression of Bcl-2 or FasL. In strong support of a role for JNK in promoting the mitochondrial apoptosis-signaling pathway, JNK inhibition prevented ischemia-induced mitochondrial translocation of Bax and Bim, release of cytochrome c and Smac, and activation of caspase-9 and caspase-3. The potential mechanism by which JNK promoted Bax translocation after ischemia was further studied using coimmunoprecipitation, and the results revealed that JNK activation caused serine phosphorylation of 14-3-3, a cytoplasmic sequestration protein of Bax, leading to Bax disassociation from 14-3-3 and subsequent translocation to mitochondria. These results confirm the role of JNK as a critical cell death mediator in ischemic brain injury, and suggest that one of the mechanisms by which JNK triggers the mitochondrial apoptosis-signaling pathway is via promoting Bax and Bim translocation.     

TAK1抑制剂5Z-7-oxozeaenol对大鼠脑外伤海马CA1区的神经保护作用研究

目的探讨转化生长因子β激活激酶(TAK1)选择性抑制剂5Z-7-oxozeaenol在创伤性颅脑损伤中对伤侧海马的保护作用及可能机制;方法 72只雄性Sprague-Dawley大鼠随机分为3组:假手术组,外伤+二甲基亚砜组,外伤+5Z-7-oxozeaenol组,按照改良的Feeney法制作大鼠颅脑外伤模型,外伤后10 min通过左侧侧脑室给药,24 h后取标本,Western blot检测TAK1和磷酸化TAK1的表达,并用凝胶迁移实验检测其下游核因子κB和活化蛋白-1的活性,采用原位末端脱氧核苷酸转移酶介导的生物素脱氧尿嘧啶核苷酸缺口末端标记法(TUNEL)检测海马CA1区神经元的凋亡情况,胶质纤维酸性蛋白(GFAP)免疫组化染色检测星形胶质细胞的表达;结果与溶剂组相比,治疗组伤侧海马TAK1和磷酸化TAK1的表达量、核因子κB和活化蛋白-1的DNA结合活性显著降低,同时海马CA1区TUNEL和GFAP阳性细胞明显减少(P<0.05)。结论 5Z-7-oxozeaenol通过抑制TAK1信号通路降低海马组织中NF-κB和AP-1的活性起到抗凋亡作用。     

Herpes simplex virus type 1-induced encephalitis has an apoptotic component associated with activation of c-Jun N-terminal kinase

Herpes simplex virus type 1 (HSV-1) triggered apoptosis in hippocampal cultures, as determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and immunohistochemistry with antibody specific for the large fragment of activated caspase 3. The levels of phosphorylated (activated) c-Jun N-terminal kinase (JNK) were also increased in HSV-1-infected hippocampal cultures as were the levels of activated c-Jun, its target. JNK activation was involved in HSV-1-induced apoptosis as evidenced by apoptosis inhibition with the JNK inhibitor SP600125. HSV-2 activated the mitogen-activated protein kinase/extracellular regulated protein kinase (MEK/ERK) survival pathway and did not trigger apoptosis in hippocampal cultures. The MEK specific inhibitor U0126 inhibited ERK activation and caused a significant increase in the percent TUNEL(+) cells in HSV-2-infected cultures, indicating that the failure of HSV-2 to trigger apoptosis is due to its ability to activate the MEK/ERK survival pathway. JNK was also activated in brain tissues from patients with HSV-associated acute focal encephalitis (HSE) that were positive for HSV-1 antigen. JNK activation correlated with apoptosis, as determined by immunohistochemistry with antibody to activated caspase 3 or cleaved poly (ADP-ribose) polymerase (PARP). The data suggest that HSE has an apoptotic component that may contribute to disease pathogenesis.     

c-Jun N-terminal kinase mediates lactacystin-induced dopamine neuron degeneration

Parkinson disease is characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta. It has been proposed that dysfunction of the ubiquitin proteasome system plays an important role in the pathogenesis of Parkinson disease, but the mechanisms underlying ubiquitin proteasome system-related neuron degeneration are unknown. Here, we demonstrate that the proteasome inhibitor lactacystin induces phosphorylation of c-Jun N-terminal kinase (JNK) and c-Jun, the release of cytochrome c, activation of both caspase-9 and caspase-3, and sequential apoptosis of dopaminergic neurons in vitro. Most of these effects can be attenuated by the JNK inhibitor SP600125. Furthermore, infusion of lactacystin in rats in vivo also leads to phosphorylation of JNK before nigral neuron loss; chronic administration of SP600125 also blocks this loss. These results indicate that JNK is involved in proteasome inhibition-induced dopaminergic neuron degeneration through caspase-3-mediated apoptotic pathways, suggesting that this kinase may be a therapeutic target for the prevention of substantia nigra pars compacta degeneration in Parkinson disease patients.     

亚低温对大鼠全脑缺血再灌注损伤后海马CA1区c-Jun蛋白表达的影响

目的研究亚低温对大鼠全脑缺血再灌注损伤后海马CA1区神经元的保护作用,并探讨其可能的机制。方法采用四血管阻断法建立大鼠全脑缺血模型。SD大鼠,随机分为假手术组(SH组)、常温组(IR组)和亚低温组(HIR组)。各组在全脑缺血15min后分别再灌注6h、12h、1d、3d,采用苏木素-伊红(HE)染色观察各时间点海马CA1区细胞形态学变化和TUNEL法检测海马CA1区神经元凋亡,免疫印迹检测c-Jun蛋白表达。结果(1)HE染色结果 IR组和HIR组于全脑缺血再灌注后6h,HE染色未见明显改变,IR组缺血再灌注1d时CA1区出现严重改变,3d时损伤最严重,出现细胞数目减少,细胞胞体缩小、胞核固缩深染,损伤严重,排列紊乱,核膜不清,核仁消失。而HIR组海马存活的锥体细胞数较之IR组12h、1d、3d时间点均明显增加(P<0.05)。(2)TUNEL标记IR组于缺血再灌注后6h在海马CA1区阳性细胞开始增多,缺血再灌注1 d时阳性细胞数最多。而HIR组各时间点阳性细胞数均较IR组明显减少(P<0.01)。(3)免疫印迹结果全脑缺血再灌注后6h c-Jun蛋白在IR组海马CA1区表达开始增加,12h达高峰,持续到3d;HIR组在各时间点的表达均弱于IR组(P<0.01)。结论亚低温通过减少海马CA1区c-Jun的表达,抑制海马CA1区神经元的凋亡,可能是亚低温脑保护作用的机制之一。     

Effect of ischemic preconditioning on antioxidant status in the gerbil hippocampal CA1 region after transient forebrain ischemia

Ischemic preconditioning (IPC) is a condition of sublethal transient global ischemia and exhibits neuro-protective effects against subsequent lethal ischemic insult. We, in this study, examined the neuroprotective effects of IPC and its effects on immunoreactive changes of antioxidant enzymes including superoxide dismutase (SOD) 1 and SOD2, catalase (CAT) and glutathione peroxidase (GPX) in the gerbil hippocampal CA1 region after transient forebrain ischemia. Pyramidal neurons of the stratum pyramidale (SP) in the hippocampal CA1 region of animals died 5 days after lethal transient ischemia without IPC (8.6%(ratio of remanent neurons) of the sham-operated group);however, IPC prevented the pyramidal neurons from subsequent lethal ischemic injury (92.3%(ratio of remanent neurons) of the sham-operated group). SOD1, SOD2, CAT and GPX immunoreactivities in the sham-operated animals were easily detected in pyramidal neurons in the stratum pyramidale (SP) of the hippocampal CA1 region, while all of these immunoreac-tivities were rarely detected in the stratum pyramidale at 5 days after lethal transient ischemia without IPC. Meanwhile, their immunoreactivities in the sham-operated animals with IPC were similar to (SOD1, SOD2 and CAT) or higher (GPX) than those in the sham-operated animals without IPC. Furthermore, their immunoreactivities in the stratum pyramidale of the ischemia-operated animals with IPC were steadily maintained after lethal ischemia/reperfusion. Results of western blot analysis for SOD1, SOD2, CAT and GPX were similar to immunohistochemical data. In conclusion, IPC maintained or increased the expression of antioxidant enzymes in the stratum pyramidale of the hippocampal CA1 region after subsequent lethal transient forebrain ischemia and IPC exhibited neuroprotective effects in the hippocampal CA1 region against transient forebrain ischemia.     

缺血预处理后海马CA1区反应性星形胶质细胞增生与迟发性神经元缺血耐受性的关系

目的 探讨缺血预处理后海马CA1区反应性星形胶质细胞增生与迟发性神经元缺血耐受性的关系。方法 实验动物被随机分为手术组、缺血组、预缺血组、预缺血后再缺血组。阴断沙土鼠双侧颈总动脉造成前脑缺血模型。采用细胞特异性抗原胶质纤维酸性蛋白（GFAP）免疫组化法标记星形胶质细胞。结果 预缺血后1－7天,海马CA1区GFAP阳性的星形胶质细胞数轻度增加,至28天时增生非常显著（P＜0．01）。预缺血后1－7天再缺血,海马CA1区存活正常神经元数逐渐下降,预缺血后28天再缺血又显著增加（P＜0．01）。结论 缺血预处理后,神经元可出现迟发性缺血耐受,反应性星形胶质细胞增生可能起了重要作用。     

Domoic acid-induced hippocampal CA1 hyperexcitability independent of region CA3 activity.

Domoic acid (DOM) is a potent agonist of AMPA and kainic acid (KA) receptors in the CNS and is known to produce seizures acutely, and lasting excitotoxic damage in several brain regions. While the excitotoxic effects of DOM are well documented, its seizurogenic properties are less clear. In this study, we assessed the acute effects of DOM and KA in region CA1 of intact rat hippocampal slices (CA3-on) and in slices lacking region CA3 (CA3-off). Orthodromic Schaffer collateral-evoked CA1 field potentials (population spikes and somal EPSP's) were monitored during DOM and KA (10-500 nM) administration. In CA3-off slices both KA and DOM produced immediate increases in CA1 population spike amplitude. With prolonged exposure, lasting dose-dependent reductions in spike amplitude and EPSP slope were observed, possibly due to depolarising conduction block following excessive AMPA/KA receptor activation; DOM was several-fold more potent than KA in this regard. Population spike threshold did not vary with DOM, but in CA3-on slices a dose-dependent steepening of the I/O curve and increase in maximum spike amplitude was seen. CA1 hyperexcitability, as evidenced by the appearance of prominent second and third population spikes, was equivalently increased across a range of DOM concentrations in both CA3-on and CA3-off slices and, in general, DOM-induced CA1 hyperexcitability was not enhanced by the presence of CA3 for any of the other variables assessed in this study. These findings show that DOM directly promotes neuronal hyperactivity in region CA1, presumably due to tonic AMPA and/or KA-receptor mediated depolarization, and further suggests that DOM-induced hyperactivity in the recurrently networked, AMPA/KA-receptor rich region CA3 does not contribute to the onset and spread of limbic seizures during relatively mild DOM intoxication.     

Nicotine reverses consolidated long-term potentiation in the hippocampal CA1 region

Long-term potentiation (LTP) has a memory-like consolidation period during which it becomes progressively stabilized. However, it is unknown how the consolidation is achieved. The present study demonstrates that nicotine reverses stabilized LTP in the hippocampal CA1 region, providing the first evidence that consolidated LTP can be reversed. The nicotine-induced reversal appeared to work by reversing cellular processes involved in stabilizing LTP, as LTP was readily induced again after reversal. The effect of nicotine was mediated, in large part, via desensitization of alpha7 nicotinic acetylcholine receptors (nAChRs), as an alpha7 nAChR-selective antagonist mimicked the nicotine effect. A non-selective N-methyl-d-aspartate receptor (NMDAR) antagonist completely abolished the nicotine-induced reversal, whereas an NR2B-containing NMDAR-selective antagonist had no effect. Furthermore, both the protein phosphatase 1/protein phosphatase 2A inhibitor okadaic acid and the protein phosphatase 2B (calcineurin) inhibitor cyclosporin A blocked the nicotine-induced reversal. Taken together, our results suggest that the reversal of stabilized LTP depends on the activation of NR2A-containing NMDARs and dephosphorylation. Thus, the consolidation of LTP appears to be the interruption of signaling leading to NR2A-containing NMDAR-dependent activation of protein phosphatases, which can be circumvented by nicotine-induced signaling. LTP induced in chronic nicotine-treated hippocampi contained a component that is immune to reversal, and thus acute nicotine was no longer effective to reverse consolidated LTP. These results demonstrate the differential effects of acute and chronic nicotine exposure on the cellular processes that are potentially involved in learning and memory.     

Indomethacin ameliorates ischemic neuronal damage in the gerbil hippocampal CA1 sector

The purpose of our experiment was to examine whether the cyclooxygenase inhibitor indomethacin ameliorates neuronal injury in the gerbil hippocampal CA1 sector following 5 minutes of forebrain ischemia. Thirty minutes before bilateral carotid artery occlusion, Mongolian gerbils were injected intraperitoneally with 1 (n = 10), 2 (n = 10), 5 (n = 12), or 10 (n = 7) mg/kg of indomethacin. Seven days after occlusion, the gerbils were perfusion-fixed and neuronal density in the hippocampal CA1 sector was assessed. The mean +/- SEM neuronal density in nine unoperated normal gerbils was 307 +/- 9/mm, in 10 untreated ischemic gerbils 55 +/- 21/mm, and in seven vehicle-treated ischemic gerbils 15 +/- 9/mm. The mean +/- SEM neuronal density in ischemic gerbils treated with 1, 2, 5, or 10 mg/kg indomethacin was 132 +/- 28/mm, 154 +/- 29/mm, 176 +/- 30/mm, and 136 +/- 39/mm, respectively. Indomethacin at any dose significantly ameliorated ischemic neuronal damage in the gerbil hippocampal CA1 sector.