Effect of dihydroergotoxine mesylate (Hydergine®) on delayed neuronal death in the gerbil hippocampus

The CA 1 neurons in the gerbil hippocampus exhibiting necrosis with delayed onset following 5 min ischemia were reduced markedly by the systemic administration of dihydroergotoxine mesylate (Hydergine; HYG). Immediately after 5 min of forebrain ischemia, the animals were injected intraperitoneally with HYG. Seven days after ischemia, perfusion-fixed brains were processed by conventional histology. The number of neurons per millimeter in the CA 1 pyramidal cell layer were calculated and they were labelled neuronal density. In the control group, the neuronal density was 66.03 +/- 7.37 (mean +/- SEM), in the vehicle group, it was 11.25 +/- 4.93. The neuronal density in the HYG group was 69.19 +/- 6.49. The difference in the neuronal density between the HYG group and the control group was not statistically significant. These data indicate that HYG protects on the CA 1 neurons, and this suggest that the suppression of adrenoceptors by this drugs may be the main mechanism of action. This morphologic outcome may explain the functional amelioration of mental impairment by HYG.     

Effect of L-arginine and NG-nitro-L-arginine on delayed neuronal death in the gerbil hippocampus

Abstract

To assess the role of nitric oxide (NO) in cerebral ischemia, we investigated the effect of L-arginine, a substrate of NO synthase (NOS), and N^G-nitro-L-arginine (L-NNA), a NOS inhibitor_l on neuronal death in the CA1 hippocampal region. Seventy-two Mongolian gerbils were used in the study. Both carotid arteries were occluded for 4 min to induce forebrain ischemia. Temporal muscle temperature was strictly maintained at 37.5±0.3°C during the ischemia. L-arginine (70 and 100 mg kg-¹) or L-NNA (11 10 and 100 mg kg-¹) was administered intraperitoneally 4 times: 30 min before_l 3h, 6h and 24h after induction of ischemia. Four days after ischemic insult_l the animals were perfusion-fixed, and the neuronal densities in the media, middle and lateral CA1 subfield were estimated. A verage neuronal cell density of the control group was 2-3 mm in each subfield. L-arginine at doses of 10 and 100 mg kg-¹ did not prevent neuronal death. L-NNA at doses of 1 and 10 mg kg-¹ did not protect neuronal cells from ischemia either. However_l in ischemia gerbils treated with 100 mg kg-¹ L-NNAI the average neuronal cell density in the lateral CA 1 subfield was 54.4± 19.1. L-NNA (100 mg kg-¹) significantly (p< O. 05) reduced the occurrence of neuronal death in the lateral CA1 subfield. The present results suggest that NO plays an important role in the development of neuronal injury after global ischemia. [Neural Res 1997; 19: 426-430]     

Hyperbaric oxygenation prevents delayed neuronal death following transient ischaemia in the gerbil hippocampus

The mechanism of the neuroprotective effect of hyperbaric oxygenation remains unclear although its clinical benefits have been well recognized for human ischaemic neuronal disease. The preventive effect of hyperbaric oxygenation against delayed neuronal death was investigated in the gerbil following transient forebrain ischaemia. Delayed neuronal death in the gerbil was produced by clips on both the common carotid arteries (10 min). Morphological examination was carried out after several protocols of hyperbaric oxygenation, modified from the protocols for human ischaemic neuronal disease. Neurons in the hippocampal CA₁ were well preserved in the gerbils treated with hyperbaric oxygenation, more so than in the gerbils with no hyperbaric oxygenation. Moreover, more neurons were preserved in the CA₁ treated with hyperbaric oxygenation within 6 h of the ischaemia, than when the hyperbaric oxygenation was started 24 h after the ischaemia. The induction of heat shock proteins (HSP72 and HSP27) became weaker in the gerbils with hyperbaric oxygenation than in those without hyperbaric oxygenation, as seen immunohistochemically. We also observed an increase in dense bodies, that were shown to be lysosomes and myelinoid structures in the cytoplasm of the neurons ultrastructurally, in the hippocampus with hyperbaric oxygenation. However, no oxygen toxicity to the neurons was detected, up to at least two atmospheres absolute. This experimental system was useful to investigate the preventive mechanism of hyperbaric oxygenation against delayed neuronal death in the gerbil, and to determine the clinical indications and the most effective protocol for hyperbaric oxygenation for ischaemic neuronal damage in the human brain.     

Delayed neuronal death and delayed neuronal recovery in the human brain following global ischemia

Summary The understanding of delayed hippocampal death as a therapeutic window for post-ischemic treatment of the brain has led to numerous investigations focusing upon underlying cellular mechanisms and pharmacological potentials in gerbils and rats. Nevertheless, studies on the occurrence of delayed neuronal death in the human brain have been singular and dealt with only small files of patients. To complement these limited data, in the present study 26 adult patients with a history of a single cardiac arrest were included. Following successful resuscitation, individual survival ranged from less than 1 h to 186 days ( = 11 days). The severity of the resultant ischemic injury in hippocampus CA1, among Purkinje cells, or in frontal neocortex, respectively, was quantified by direct counting of necrotic neurons. Additionally, hippocampal specimens were immunostained for neuron-specific enolase. The data obtained demonstrate the occurrence of delayed neuronal death in human hippocampus and, in a minor form, in cerebellar Purkinje cells. This is in contrasts to the immediate manifestation of ischemic neuronal necrosis in the neocortex. Unlike previous findings in experimental animals and in humans, the delay of CA1 cell death could be defined as lasting about 7 days following cardiac arrest. Moreover, the immunohistochemical results indicate delayed neuronal recovery in CA1, which in the time course reciprocally corresponds to delayed manifestation of hippocampal neuronal death. Interpretation of the results must consider the lack of information about the exact individual duration of cardiac arrest and resuscitation, as well as missing data concerning pre-ischemic physiological variables.     

Involvement of calmodulin in neuronal cell death

A large body of evidence indicates that disturbances of Ca(2+) homeostasis may be a causative factor in the neurotoxicity following cerebral ischemia. However, the mechanisms by which Ca(2+) overload leads to neuronal cell death have not been fully elucidated. Calmodulin, a major intracellular Ca(2+)-binding protein found mainly in the central nervous system, mediates many physiological functions in response to changes in the intracellular Ca(2+) concentration, whereas Ca(2+) overload in neurons after excitotoxic insult may induce excessive activation of calmodulin signaling pathways, leading to neuronal cell death. To determine the role of calmodulin in the induction of neuronal cell death, we generated primary rat cortical neurons that express a mutant calmodulin with a defect in Ca(2+)-binding affinity. Neurons expressing the mutant had low responses of calmodulin-dependent signaling to membrane depolarization by high KCl and became resistant to glutamate-triggered excitotoxic neuronal cell death compared with the vector or wild-type calmodulin-transfected cells, indicating that blocking calmodulin function is protective against excitotoxic insult. These results suggest that calmodulin plays a crucial role in the processes of Ca(2+)-induced neuronal cell death and the possibility that the blockage of calmodulin attenuates brain injury after cerebral ischemia.     

Histochemical study of Ca2+-ATPase activity in ischemic CA1 pyramidal neurons in the gerbil hippocampus

Although cytosolic Ca²⁺ accumulation plays a pivotal role in delayed neuronal death, there have been no investigations on the role of the cellular Ca²⁺ export system in this novel phenomenon. To clarify the function of the Ca²⁺-pump in delayed neuronal death, the plasma membrane Ca²⁺-ATPase activity of CA1 pyramidal neurons was investigated ultracytochemically in normal and ischemic gerbil hippocampus. To correlate enzyme activity with delayed neuronal death, histochemical detection was performed at various recirculation times after 5 min of ischemia produced by occlusion of the bilateral carotid arteries. At 10 min after ischemia, CA1 pyramidal neurons showed weak Ca²⁺-ATPase activity. Although enzyme activity had almost fully recovered 2 h after ischemia, it was reduced again 6 h after ischemia. Thereafter, Ca²⁺-ATPase activity on the plasma membrance of CA1 pyramidal neurons decreased progressively, losing its localization on day 3. On day 4 following ischemia, reaction products were diffusely scattered throughout the whole cell body. Our results indicate that, after once having recovered from ischemic damage, severe disturbance of the membrane Ca²⁺ export system proceeds from the early stage of delayed neuronal death and disturbs the re-export of accumulated cytosolic Ca²⁺, which might contribute to delayed neuronal death. Occult disruption of Ca²⁺ homeostasis seems to occur from an extremely early stage of delayed neuronal death in CA1 pyramidal cells.     

Temporal alterations in voltage gated Ca2+ channel immunoreactivities in the gerbil hippocampus following ischemic insults

In the present study, temporal changes of voltage-gated Ca²⁺ channel (VGCC) immunoreactivities were evaluated in the gerbil hippocampus following ischemia. P/Q-type VGCC immunoreactivity was elevated in the hippocampus in the 3 h post-ischemic group. In the 30 min post-ischemic group, N-type VGCC immunoreactivity began to increase only in the CA1 region. L-type (1C) VGCC immunoreactivity was significantly increased in the 12 h post-ischemic group. L-type (1D) VGCC immunoreactivity began to increase in the CA1 region in the 30 min post-ischemic group and peaked in the 12 h post-ischemic group. These findings suggest that the altered VGCC immunoreactivities following ischemia may play an important role in the ischemic neuronal injury.     

一氧化氮合酶抑制剂对缺血性海马迟发性神经元死亡的保护作用

目的：研究一氧化氮在缺血性海马迟发性神经元死亡中的作用,观察非选择性一氧化氮合酶抑制剂Ｎ＾Ｇ－ｎｉｔｒｏ－Ｌ－ａｒｇｉｎｉｎｅ对缺血性海马ＤＮＤ的影响。方法：实验分为假手术组,生理盐水治疗组,Ｌ－ＮＮＡ治疗组。采用大鼠４血管关闭方法制作了全脑缺血再灌流模型,以假手术组为对照,检测了脑缺血１０ｍｉｎ再灌流７２ｈ海马区ＮＯＳ活性的变化并观察计量了海马ＣＡ１区组织病理改变;     

Fine structural nature of delayed neuronal death following ischemia in the gerbil hippocampus

Summary An unusual, delayed neuronal death (DND) has been noticed in the hippocampus of the Mongolian gerbil following brief ischemia (Kirino 1982). On day 1 following 5–10min of ischemia, light microscopy showed the CA1 pyramidal cells unchanged. On day 2, the cells showed massive growth of membranous cytoplasmic organelles instead of overt cellular disintegration. These neurons were destroyed extensively by day 4 after ischemic insult. Following longer ischemia (20–30min), however, the changes in the CA1 pyramidal cells appeared faster and resembled the wellcharacterized ischemic cell change (ICC). To further clarify the differences between ICC and DND, gerbils were submitted to transient 5–30min ischemia. They were perfusion-fixed following a given survival period and then processed for electron microscopy. Following transient ischemia, specimens showed slow cell changes with growth of cisterns of the endoplasmic reticulum (ER). In some CA1 neurons, the cytoplasm was shrunken and darkly stained, but they also displayed accumulation of ER cisterns. Occasionally, the CA1 cells demonstrated highly shrunken dark perikarya, no different than in ICC. These results indicate that DND seems to be the typical disease process of the CA1 sector and that a severer insult makes the change faster and more similar to ICC. ICC seems to occur when the CA1 pyramidal cells are damaged so severely that they cannot react with proliferous activity.     

Intraventricular administration of the neurotrophic factor midkine ameliorates hippocampal delayed neuronal death following transient forebrain ischemia in gerbils

Midkine (MK) is a growth factor with neurotrophic activities, and is expressed during the early stages of experimental cerebral infarction in rats in the zone surrounding the infarct. To evaluate in vivo activity of MK in preventing neuronal death, MK produced in yeast (Pichia pastoris) was administered into the brain ventricle immediately before occlusion of the bilateral common carotid artery of Mongolian gerbils. MK administration at the dose of 0.5-2 microg immediately before occlusion was found to ameliorate delayed neuronal death in the hippocampal CA1 region caused by transient ischemia 7 days after the insult. The hippocampal neurons of the MK-administered gerbils tended to degenerate 14 and 21 days after the insult, but their numbers remained higher than those in saline-administered controls; however, the hippocampal neurons were degenerated 28 days after the insult. MK administration at 2 h after occlusion did not ameliorate the neuronal death. These findings suggested that the therapeutic time window was narrow. The two to four times repeated administration of 2 microg MK immediately before and at 1, 2, or 3 weeks after the occlusion were not significantly different for the hippocampal neuronal death at 28 days after the insult compared with a single injection, but were significantly effective compared with vehicle administration alone. These findings suggested that the therapeutic time window was relatively narrow. The potent neuroprotective activity of MK observed in vivo suggested that MK might be useful as a therapeutic reagent for prevention of neuronal death in neurodegenerative diseases.     

Expression of Bax and Bcl-2 protein in the gerbil hippocampus following transient forebrain ischemia and its modification by phencyclidine

Abstract

To determine the effect of phencyclidine (a noncompetitive NMDA receptor antagonist) on expression of Bax and Bcl-2 (apoptosis-regulating proteins) in gerbil hippocampus after transient forebrain ischemia, brain sections were immunohistochemically evaluated 48, 72, 96 hand 7 days following ischemia. In ischemic control animals, the expression of Bax in CA 7 neurons was increased with time and peaked at 72 h, then disappeared at 96 h. In the phencyclidine (5 mg kg^-1 , intraperitoneally)-treated animals, the intensity of Bax expression at 72 h was weaker than that of ischemic control animals. Furthermore, at 96 h, Bax expression was still observed in CA1 neurons. No expression of Bcl-2 in the CA1 neurons was detected in either control or phencyclidine-treated animals. From these results, it is possible that NMDA receptor antagonists exert their preventive effect against delayed neuronal death through inhibition of Bax protein expression, although the precise relationship between the function of Bax protein and delayed neuronal death is still unclear. [Neural Res 1997; 19: 629-633]     

大鼠脑缺血后海马CA1区胶质纤维酸性蛋白表达与迟发性神经元死亡的关系

目的观察大鼠大脑缺血再灌注后海马CA1区胶质纤维酸性蛋白(GFAP)的表达与迟发性神经元死亡的关系。方法采用大鼠大脑中动脉阻塞再灌注模型(MCAO),将大鼠随机分为MCAO后3d、7d、30d组及假手术组,应用免疫荧光与TUNEL染色法分别观察脑缺血再灌注后不同时间点缺血侧海马CA1区GFAP表达情况和迟发性神经元死亡(DND)的变化。结果(1)3d组海马DND阳性(DND 组)的MCAO大鼠、海马DND阴性(DND-组)的MCAO大鼠与假手术组大鼠比较,缺血侧海马CA1区GFAP染色的平均光密度无显著性差异(P>0.05),但GFAP阳性细胞的形态发生变化;(2)7d组大鼠缺血侧海马CA1区GFAP阳性细胞大量活化增殖,表现为胞体变大,突起增多;DND( )、DND(-)组海马CA1区GFAP染色的平均光密度较假手术组增高(P<0.01),且DND(-)组的GFAP平均光密度较DND( )组明显增高(P<0.01);(3)30d组大鼠缺血侧海马CA1区GFAP表达呈瘢痕样改变,DND( )、DND(-)组与假手术组比较其GFAP染色的平均光密度明显增高(P<0.05),且DND( )组的GFAP平均光密度较DND(-)组明显增高(P<0.05)。结论大鼠MCAO后星形胶质细胞反应性变化的差异可能与海马CA1区迟发性神经元死亡的发生有关。     

小檗碱对大鼠海马CA_1区迟发性神经元坏死的影响

本文采用Ｐｕｌｓｉｎｅｌｌｉ－Ｂｒｉｅｒｌｅｙ４血管结扎致ＳＤ大鼠全脑缺血（１０ｍｉｎ）再灌流模型，分别观察了早期不同再灌流时间（１２、２４、４８ｈ）点上，大鼠海马ＣＡ１区神经元的超微结构以及再灌７ｄ时光镜结构变化，同时观察了小檗碱对ＣＡ１区迟发性神经元坏死的影响。结果显示脑缺血再灌流早期，ＣＡ１区神经元超微结构发生明显改变，７ｄ时光镜下绝大部分细胞脱失；而用药组大鼠海马ＣＡ１区神经元在相应时间点上超微结构变化相对较轻，７ｄ时仍有绝大多数（８２％）细胞存活，细胞密度为１７２±１２．２个／ｍｍ，显著高于缺血对照组２７±７．６个／ｍｍ，Ｐ＜０．００１。提示小檗碱对大鼠短暂脑缺血再灌流造成的海马ＣＡ１区迟发性神经元坏死具有显著的对抗作用。     

Quin2 protects against neuronal cell death due to Ca overload

Quin2-acetoxymethylester (quin2/AM) (50 μM), administered directly to the motoneuronal pool of the frog spinal cord, could be loaded into the motoneuron as well as the other cells in the lumbar region. Depolarizing responses of the ventral root to l-glutamate in the quin2-loaded side persisted even after prolonged exposure to A23187 (2.0 μM), while the responses in the unloaded side were markedly reduced. Histologically confirmed neuronal cell loss from the motoneuronal pool induced by A23187 (2.0 μM) or by a high concentration of l-glutamate (10 mM) was prevented by pretreatment with quin2/AM. A23187- and l-glutamate-induced histological and functional damage in neuronal cells and the protective effects of quin2 on them provide further evidence for cell death due to Ca²⁺ overloading.     

Late expression of Na+/H+ exchanger 1 (NHE1) and neuroprotective effects of NHE inhibitor in the gerbil hippocampal CA1 region induced by transient ischemia

Although acidosis may be involved in neuronal death, the participation of Na⁺/H⁺ exchanger (NHE) in delayed neuronal death in the hippocampal CA1 region induced by transient forebrain ischemia has not been well established. In the present study, we investigated the chronological alterations of NHE1 in the hippocampal CA1 region using a gerbil model after ischemia/reperfusion. In the sham-operated group, NHE1 immunoreactivity was weakly detected in the CA1 region. Two and 3 days after ischemia/reperfusion, NHE1 immunoreactivity was observed in glial components, not in neurons, in the CA1 region. Four days after ischemia/reperfusion, NHE1 immunoreactivity was markedly increased in CA1 pyramidal neurons as well as glial cells. These glial cells were identified as astrocytes based on double immunofluorescence staining. Western blot analysis also showed that NHE protein level in the CA1 region began to increase 2 days after ischemia/reperfusion. The treatment of 10 mg/kg 5-(N-ethyl-N-isopropyl) amiloride, a NHE inhibitor, significantly reduced the ischemia-induced hyperactivity 1day after ischemia/reperfusion. In addition, NHE inhibitor potently protected CA1 pyramidal neurons from ischemic damage, and NHE inhibitor attenuated the activation of astrocytes and microglia in the ischemic CA1 region. In addition, NHE inhibitor treatment blocked Na⁺/Ca²⁺ exchanger 1 immunoreactivity in the CA1 region after transient forebrain ischemia. These results suggest that NHE1 may play a role in the delayed death, and the treatment with NHE inhibitor protects neurons from ischemic damage.     

NGF delays rather than prevents the cholinergic terminal damage and delayed neuronal death in the hippocampus after ischemia

Cerebral ischemia induces damage of cholinergic terminals in the hippocampus, which preceded the delayed neuronal death (DND) of the CA1 pyramidal cells. We investigated the effects of nerve growth factor (NGF) on the cholinergic terminal damage after ischemia. Continuous NGF infusion (0.5 μg/7 days) into the lateral ventricle before and after 5 min ischemia prevented a decrease in choline acetyltransferase (ChAT)-immunoreactivity and disturbance of acetylcholine (ACh) release on the 4th day after ischemia, but not on day 7, i.e., NGF infusion caused delay in the progress of the cholinergic terminal damage. These findings show that the cholinergic terminal damage may result from deficiency of endogenous NGF in an ischemic brain. In addition, we investigated whether NGF would prevent the DND after ischemia. NGF infusion also caused delay in the progress of the DND until day 14. Our results suggested that the neuroprotective effect of NGF on the DND may be secondarily yielded by maintenance of communication between cholinergic terminal and the target CA1 cell, and that prevention of cholinergic terminal damage may be useful for the treatment of cerebrovascular disease.     

Effect of cyclooxygenase and lipoxygenase inhibitors on delayed neuronal death in the gerbil hippocampus

The purpose of our study was to examine whether cyclooxygenase and lipoxygenase inhibitors ameliorate delayed neuronal death in the hippocampal CA1 sector in Mongolian gerbils after 5 minutes of forebrain ischemia. Gerbils were injected intraperitoneally with cyclooxygenase inhibitors piroxicam and flurbiprofen or with lipoxygenase inhibitors AA-861 and BW-755C. Seven days after ischemic insult, the animals were perfusion-fixed, and the neuronal density in the hippocampal CA1 sector was estimated. The average neuronal density in unoperated normal gerbils was 247 +/- 9/mm (mean +/- SEM). In ischemic gerbils with vehicle administration, the average neuronal densities were 13 +/- 2, 14 +/- 2, 13 +/- 2, and 13 +/- 1 for piroxicam, flurbiprofen, AA-861, and BW-755C, respectively. The average neuronal densities in ischemic gerbils treated with 1.5 and 10 mg/kg piroxicam and 1.5 and 10 mg/kg flurbiprofen were 13 +/- 2, 194 +/- 9, 19 +/- 5, and 143 +/- 12, respectively. In ischemic gerbils treated with 15 and 100 mg/kg AA-861 and 30 mg/kg BW-755C, the average neuronal densities were 12 +/- 1, 13 +/- 1, and 14 +/- 2, respectively. At their higher doses, both piroxicam and flurbiprofen significantly (p less than 0.01) ameliorated delayed neuronal death in the hippocampal CA1 sector. Our results suggest that cyclooxygenase products play an important role in the development of delayed neuronal injury after cerebral ischemia.     

Veratridine delays apoptotic neuronal death induced by NGF deprivation through a Na+-dependent mechanism in cultured rat sympathetic neurons

Superior-cervical ganglion (SCG) cells dissociated from newborn rats depend on nerve growth factor (NGF) for survival. Membrane depolarization with elevated K+ is known to prevent neuronal death following NGF deprivation and/or to promote survival via a Ca2+-dependent mechanism. Here we have exploited the possibility of whether or not a Na+-dependent pathway for neuronal survival is present in these cells. Veratridine (ec50=40 nM), a voltage-dependent Na+ channel activator, significantly delayed the onset of apoptotic cell death in NGF-deprived SCG neurons that had been cultured for 7 days in the presence of NGF. This effect was blocked completely by Na+ channel blockers including tetrodotoxin (TTX, 1 μM), benzamil (25 μM) and flunarizine (1 μM), but was not attenuated by nimodipine (1 μM), an L-type Ca²⁺ channel blocker. The saving effect of veratridine on cultured neurons was observed even in low Ca²⁺ media (0–1.0 mM), but was completely abolished in a low Na⁺ medium (38 mM). Sodium-binding benzofuran isophthalate was employed as a fluorescent probe for monitoring the level of cytoplasmic free Na⁺, which revealed a sustained increase in its level (12.9 mM, 307% of that of control) in response to veratridine (0.75 μM). The TTX or flunarizine completely blocked veratridine-induced Na⁺ influx in these cultured neurons. Moreover, no appreciable increase in intracellular Ca²⁺ was detected under these conditions. Though Na⁺ channels were effectual in SCG neurons which were freshly isolated from newborn rats, the Na⁺-dependent saving effect of veratridine was not observed in these young neurons. These lines of evidence suggest that the death-suppressing effect of veratridine on cultured SCG neurons depends on the Na⁺ influx via voltage-dependent Na⁺ channels, and suggests the presence of Na⁺-dependent regulatory mechanism(s) in neuronal survival.