Poly(ADP-ribose) polymerase during reperfusion after transient forebrain ischemia: its role in brain edema and cell death

The activation of poly(ADP-ribose) polymerase (PARP) in the reperfused brain after ischemia has been assumed but never has been directly presented. Our studies indicate a different dynamic of PARP activity alteration in hippocampus during reperfusion after 3 and 10 min of transient forebrain ischemia in gerbils. The phasic stimulation of PARP activity was observed during reperfusion 15 min, 120 min, and 4 d after 3 min of ischemia with subsequent lowering of its activity close to control value on the seventh day of reperfusion. After 10 min of ischemic insult, PARP activity significantly increased from the third to the seventh day of reperfusion. The protein level of PARP was not significantly changed during reperfusion after 3 and 10 min of ischemia, with one exception: On the third day after 10 min of ischemia, PARP protein level was 28% lower compared to control; however, no enhancement of 85-kDa protein immunoreactivity was observed. These data indicate the lack of PARP cleavage in hippocampus of gerbils subjected to ischemia-reperfusion injury. The inhibitor of PARP, 3-aminobenzamide (3-AB) in a dose of 30 mg/kg b.w. (body weight) injected intravenously directly after 3 min of ischemia protects >60% of neuronal cells against death in the CA1 layer of hippocampus but has no effect after 10 min of ischemic episode. 3-AB decreased forebrain edema significantly after 3 and 10 min of ischemia. Our data indicate that PARP inhibitor(s) might offer a potent therapeutic strategy for short global ischemia. The combination of PARP inhibitor with potent antioxidant might enhance its ameliorating effect.     

Inhibition of poly(ADP-ribose) polymerase activity protects hippocampal cells against morphological and ultrastructural alteration evoked by ischemia-reperfusion injury

Poly(ADP-ribose) polymerase 1 (PARP-1 EC 2.4.2.30) is a nuclear enzyme that plays an important role in cell survival and death. PARP is involved in DNA repair machinery, however, massive DNA damage leads to over-activation of PARP-1 and to depletion of its substrate bNAD+ which causes cell death. Our previous study indicated that the PARP activity was significantly activated during ischemia-reperfusion injury. In this study we investigated the effect of PARP inhibitor, 3-aminobenzamide (3-AB) on intracellular organelles alteration. Gerbils were submitted to 3 and 10 min transient global ischemia followed by recirculation and survival for 1 till 7 days. The histological and electron microscopic examination indicated a pronounced protective effect of 3-AB on the swelling of astrocytes and neurons 1 day after 3 and 10 min ischemic insult. It decreased also the swelling of pericytes. 3-AB decreases evoked by ischemia swelling of mitochondria and Golgi apparatus. The significant ameliorating effect of 3-AB was also observed on the 7th day of reperfusion after 3 min ischemia and was also visible on the 1st day after 10 min ischemia. However, 7 days after prolonged 10 min ischemia almost all neurons in the CA1 hippocampal layer died and 3-AB was not able to protect these cells. In spite of that, 3-AB markedly decreased immunostaining of glial fibrillary acidic protein (GFAP), which was enhanced in the stratum: oriens, radiatum and lacunosum-moleculare at the 7th day after 10 min ischemia. These data indicated that inhibition of PARP may have a protective effect on neuronal cells affected by ischemia-reperfusion injury.     

3-Aminobenzamide reduces brain infarction and neutrophil infiltration after transient focal cerebral ischemia in mice

Poly(ADP-ribose) polymerase (PARP) was shown to be detrimental in cerebral ischemia but the mechanisms whereby PARP is deleterious have yet to be determined. They may include a role in neutrophil infiltration known to aggravate ischemic damage. In this context, we investigated the effect of 3-aminobenzamide (3-AB), a PARP inhibitor, on brain damage and neutrophil infiltration after transient focal cerebral ischemia in mice. Ischemia was induced in male Swiss mice, anaesthetized with chloral hydrate (400 mg/kg, i.p.), by a 15-min-occlusion of the left middle cerebral artery using an intraluminal suture. Treatments with 3-AB were first administered intraperitoneally 15 min before reperfusion and endpoints measured at 24 h. Among the range of dosages studied (20-320 mg/kg), 40 mg/kg gave the maximal neuroprotection with a 30% decrease in the infarct volume and tended to improve the neurological score evaluated by a grip test. The same dosage was, however, devoid of effect when injection was delayed 2 or 6 h after reperfusion. Myeloperoxidase (MPO) activity used as an index of neutrophil infiltration showed that infiltration peaked 48 h after reperfusion in our model. At this time point, 3-AB (40 mg/kg given 15 min before reperfusion) markedly reduced the neutrophil infiltration, as evidenced by a 72%-decrease in MPO activity, and was still neuroprotective. Our results confirm that 3-AB reduces brain damage. Moreover, for the first time, a quantitative study shows that 3-AB decreases neutrophil infiltration elicited by cerebral ischemia.     

Long-term neuroprotective effect of inhibiting poly(ADP-ribose) polymerase in rats with middle cerebral artery occlusion using a behavioral assessment

Poly(ADP-ribose) polymerase (PARP) can initiate an energy-consuming and inefficient repair cycle following cerebral ischemia/reperfusion by transferring ADP ribose units to nuclear proteins eventually leading to cellular dysfunction and neuronal death. 3-Aminobenzamide (3-AB) is a selective inhibitor of PARP that can significantly reduce brain damage after focal ischemia in rats and displays a low toxicity in vivo. The goals of this study were to determine if inhibiting PARP with 3-AB has a long-term neuroprotective effect and if functional outcome improves in rats following focal ischemia and treatment with 3-AB. Focal ischemia was induced by a 2-h occlusion of the middle cerebral artery (MCA), using an intraluminal filament. Motor functions were evaluated from 5 to 28 days after reperfusion in four groups of rats: stroke without treatment; stroke treated with 3-AB at doses of 15 mg/kg, stroke treated with 3-AB at doses of 55 mg/kg; and the non-ischemic control rats. Functional behaviors were tested by a series of motor function tasks (foot placing, parallel bar crossing, rope and ladder climbing), as well as a neurological examination. Infarct volume of stroke brain in the same rat was determined by Nissl staining 28 days after surgery. Comparison of the untreated stroke group (n=11) and the treated stroke groups indicates that impairment of motor function was significantly (P<0.001) reduced by administration of 3-AB at doses of 15 mg/kg (n=9) or 55 mg/kg (n=10). Neurological outcome was also improved significantly (P<0.001). Infarct volume was significantly (P<0.01) reduced in both treated groups. Long-term neuroprotection following ischemia/reperfusion injury to the brain can be obtained by administration of a PARP inhibitor. The motor tests employed in this study can be used as sensitive, objective and reproducible measurements of functional impairment in rats following an ischemic stroke.     

Response of Hippocampal Neurons and Glial Cells to Alternating Magnetic Field in Gerbils Submitted to Global Cerebral Ischemia

The purpose of this study was to determine whether exposure to an extremely low-frequency magnetic field (ELF-MF, 50 Hz) affects the outcome of postischemic damage in the hippocampus of Mongolian gerbils. After 10-min bilateral carotid occlusion, the gerbils were continuously exposed to ELF-MF (average magnetic induction at the center of the cage was 0.5 mT) for 7 days. The impact of ELF-MF was estimated immediately (the 7th day after reperfusion) and 7 days after cessation of exposure (the 14th day after reperfusion) compared with ischemic gerbils without ELF-MF exposure. Applying stereological methods, histological evaluation of changes in the hippocampus was done for determining its volume, volume densities of degenerating neurons and astrocytes, as well as the number of microglial cells per unit area. ELF-MF per se did not induce any morphological changes, while 10-min global cerebral ischemia led to neuronal death, especially in CA1 region of the hippocampus, as expected. Ischemic gerbils exposed to ELF-MF had significantly a lower degree of cell loss in the examined structure and greater responses of astrocytes and microglial cells than postischemic gerbils without exposure on the seventh day after reperfusion (immediate effect of ELF-MF). Similar response was observed on the 14th day after reperfusion (delayed effect of ELF-MF); however, differences in measured parameters were low and insignificant. Applied ELF-MF has possible neuroprotective function in the hippocampus, as the most sensitive brain structure in the model of global cerebral ischemia, through reduction of neuronal death and activation of astrocytes and microglial cells.     

Calpain inhibitor entrapped in liposome rescues ischemic neuronal damage

Transient forebrain ischemia induces activation of calpain and proteolysis of a neuronal cytoskeleton, fodrin, in gerbil hippocampus. This phenomenon precedes delayed neuronal death in hippocampal CA1 neurons. We examined effects of a calpain inhibitor on delayed neuronal death after transient forebrain ischemia. In gerbils, a selective calpain inhibitor entrapped in liposome was given transvenously and 30 min later, 5-min forebrain ischemia was produced by occlusion of both common carotid arteries. On day 7, CA1 neuronal damage was examined in the hippocampal slices stained with cresyl violet. Calpain-induced proteolysis of fodrin was also examined by immunohistochemistry and immunoblot. Additionally, to assure entrapment of the inhibitor by CA1 neurons, the inhibitor-liposome complex was labeled with FITC and given to gerbils. Fluorescence in the hippocampal slices was examined by confocal laser scanning microscope. Selective CA1 neuronal damage induced by forebrain ischemia was prevented by administration of the inhibitor in a dose-dependent manner. Calpain-induced proteolysis of fodrin was also extinguished by the calpain inhibitor in a dose-dependent manner. Bright fluorescence of the FITC-labeled inhibitor was observed in the CA1 neurons. The data show an important role of calpain in the development of the ischemic delayed neuronal death. Calpain seems to produce neuronal damage by degrading neuronal cytoskeleton. Our data also show a palliative effect of the calpain inhibitor on the neurotoxic damage, which offers a new and potent treatment of transient forebrain cerebral ischemia.     

Activation of mitogen-activated protein kinases after transient forebrain ischemia in gerbil hippocampus.

We investigated the expression, activation, and distribution of c-Jun N-terminal kinases (JNKs), p38 mitogen-activated protein kinases (p38s) and extracellular signal-regulated kinases (ERKs) using Western blotting and immunohistochemistry in gerbil hippocampus after transient forebrain ischemia to clarify the role of these kinases in delayed neuronal death (DND) in the CA1 subfield. Immunoblot analysis demonstrated that activities of JNK, p38, and ERK in whole hippocampus were increased after 5 min of global ischemia. We used an immunohistochemical study to elucidate the temporal and spatial expression of these kinases after transient global ischemia. The immunohistochemical study showed that active JNK and p38 immunoreactivities were enhanced at 15 min of reperfusion and then gradually reduced and disappeared in the hippocampal CA1 region. On the other hand, in CA3 neurons, active JNK and p38 immunoreactivities were enhanced at 15 min of reperfusion and peaked at 6 hr of reperfusion and then gradually reduced but was continuously detected 72 hr after ischemia. Active ERK immunoreactivity was observed transiently in CA3 fibers and dentate gyrus. Pretreatment with SB203580, a p38 inhibitor, but not with PD98059, an ERK kinase 1/2 inhibitor, reduced ischemic cell death in the CA1 region after transient global ischemia by inhibiting the activity of p38. These findings indicate that the p38 pathway may play an important role in DND during brain ischemia in gerbil. Components of the pathway are important target molecules for clarifying the mechanism of neuronal death.     

Ischemic tolerance and extracellular amino acid concentrations in gerbil hippocampus measured by intracerebral microdialysis

Preconditioning of the brain with sublethal ischemia induces tolerance to subsequent longer periods of ischemia. To elucidate the role of excitatory and inhibitory amino acids in the induction of ischemic tolerance, we measured the extracellular concentrations of the amino acids in the gerbil hippocampus with intracerebral microdialysis. Mongolian gerbils were subjected to 3 min of forebrain ischemia 4 days after preconditioning with 2 min of ischemia or sham operation. Microdialysis probes were implanted into the hippocampus before the second ischemia and the amino acid concentrations in the dialysates were measured with HPLC. During and immediately after 3 min of ischemia without preconditioning, the concentrations of glutamate, glycine, γ-aminobutyric acid, and taurine, but not glutamine, increased significantly. The increased amino acid levels rapidly returned to baseline after reperfusion. Preconditioning of the brain did not alter the amount of any amino acid released during and after the second ischemia. The excitotoxic index also unchanged in the preconditioned hippocampus. Thus, the results clearly show that ischemic tolerance is not induced through the alteration of the amounts of excitatory and inhibitory amino acids released during subsequent ischemia.     

A forebrain ischemic preconditioning model established in C57Black/Crj6 mice

Although many kinds of rat and gerbil cerebral ischemic preconditioning models are available, only a focal ischemic preconditioning model in mice has been reported. As most genetic alterations have been performed in mice, it is urgent to develop mouse ischemic preconditioning models for investigating the molecular mechanisms of ischemic preconditioning in transgenic mice. In the present study, we developed a forebrain ischemic preconditioning model in C57Black/Crj6 (C57BL/6) mice. Forebrain ischemia was induced in C57BL/6 mice (8-10 weeks old) by bilateral common carotid artery occlusion (BCCAO) for 18 min. The conditioning ischemic insult lasting for 6 min was carried out 48 h before the 18-min BCCAO. On the seventh day after BCCAO, neuronal damage was visualized by microtubule-associated protein-2 immunohistochemistry and quantified by cresyl violet staining. Terminal deoxytransferase-mediated dUTP-nick end labeling (TUNEL) was performed 72 h after reperfusion to detect DNA fragmentation. Ischemia for 18 min resulted in injury to the striatum, cortex and hippocampus. In comparison to the hippocampus, striatal neuronal injury was more severe and reproducible. Although the conditioning ischemia itself caused neither noticeable striatal neuronal damage nor DNA fragmentation, it significantly reduced striatal neuronal damage and DNA fragmentation caused by the subsequent 18-min ischemia. These results indicate that striatal neuronal injury after transient BCCAO can be strongly reduced by a sublethal ischemic episode in C57BL/6 mice. As many kinds of gene-altered C57BL/6 mice are available, this preconditioning model may be useful for investigating the molecular mechanisms of ischemic preconditioning in transgenic mice.     

Postischemic inhibition of GABA reuptake by tiagabine slows neuronal death in the gerbil hippocampus

The neuroprotective effects of enhancing neuronal inhibition with a γ-aminobutyric acid (GABA) uptake inhibitor were studied in gerbil hippocampus following transient ischemia. We used in vivo microdialysis to determine a suitable dosing regimen for tiagabine (NNC 328) to elevate extracellular levels of GABA within the hippocampus. In anesthetized (normothermic) gerbils, tiagabine (45 mg/kg, i. p.) selectively elevated extracellular GABA levels 450% in area CA1 of the hippocampus. In gerbils subjected to cerebral ischemia via 5-min bilateral carotid occlusion, extracellular GABA levels increased 13-fold in area CA1, returning to baseline within 30–45 min. When tiagabine was injected 10 min following onset of reperfusion, GABA levels remained elevated (200–470%) for 90 min. In addition, tiagabine significantly reduced the ischemic-induced elevation of glutamate levels in area CA1 during the postischemic period when GABA levels were elevated. There was no effect of postischemic tiagabine on aspartate or six other amino acids. Using the same dosing regimen, we evaluated the degree of neuroprotection in the hippocampus of gerbils 4 and 21 days after ischemia. Tiagabine decreased body temperature a maximum of 2.7°C beginning 30 min into reperfusion and lasting 90 min. In untreated gerbils sacrificed 4 and 21 days after ischemia, there was severe necrosis (99%) of the pyramidal cell layer in area CA1. Whereas tiagabine significantly protected the CA1 pyramidal cell layer in ischemic gerbils at 4 days (overt necrosis confined to about 17% of area CA1), the protection diminished significantly 21 days postischemia. When normothermia was maintained both during and after ischemia in a separate group of tiagabine-treated animals, approximately 77% of the CA1 pyramidal cell layer was necrotic at 4 days. Based on these findings, we suggest that (1) tiagabine slows the development of hippocampal degeneration following ischemia, and (2) that mild, postischemic hypothermia is responsible, in large part, for the neuroprotective actions of this drug. We conclude that the histological outcome after administration of cerebral neuroprotectants should be assessed following long-term survival. © 1995 Wiley-Liss, Inc.     

Temperature modulation of ischemic neuronal death and inhibition of calcium/calmodulin-dependent protein kinase II in gerbils

We used brief bilateral carotid artery occlusion in gerbils to examine the effects of temperature on ischemia-induced inhibition of calcium/calmodulin-dependent protein kinase II activity and neuronal death. In normothermic (36 degrees C) gerbils, ischemia induced a severe loss of hippocampal CA1 pyramidal neurons measured 7 days after ischemia (28.4 neurons/mm, n = 10; control density in 10 naive gerbils 262.1 neurons/mm) and a significant decrease in forebrain calcium/calmodulin-dependent protein kinase II autophosphorylation measured 2 hours after ischemia (12.9 fmol/min, n = 6; control phosphorylation in six naive gerbils 23.5 fmol/min). The effect of temperature on these indicators of ischemic damage was examined by adjusting intracerebral temperature before and during the ischemic insult. Hyperthermic (39 degrees C) gerbils showed almost complete loss of neurons in the CA1 region (3.0 neurons/mm, n = 11) and extension of neuronal death into the CA2, CA3, and CA4 regions. In addition, hyperthermia exacerbated ischemia-induced inhibition of calcium/calmodulin-dependent protein kinase II activity (4.2 fmol/min, n = 6). Hypothermia (32 degrees C) protected against ischemia-induced CA1 pyramidal cell damage (257.0 neurons/mm, n = 20) and inhibition of calcium/calmodulin-dependent protein kinase II activity (26.0 fmol/min, n = 6). Our results are consistent with the hypothesis that loss of calcium/calmodulin-dependent protein kinase II activity may be a critical event in the development of ischemia-induced cell death.     

Eicosanoid production in the caudate nucleus and dorsal hippocampus after forebrain ischemia: a microdialysis study.

Thromboxane (Tx)B2 and 6-keto-prostaglandin (6-keto-PG) F1 alpha formation in the hippocampus and caudate nucleus were evaluated by microdialysis during and following forebrain ischemia. Spontaneously hypertensive rats were subjected to bilateral carotid artery occlusion with simultaneous hypotension for 8, 14, or 20 min. Dialysate was collected during the ischemic interval and during the reperfusion period. TxB2 and 6-keto-PGF1 alpha levels were measured by radioimmunoassay. In both structures, TxB2 production increased significantly during the reperfusion period in all three ischemic groups. By contrast, increased 6-keto-PGF1 alpha elaboration was observed after only the longest ischemic duration. While TxB2 levels gradually decreased during the 3-h reperfusion period in all groups, the levels in the group subjected to 8 min of ischemia returned to control values most rapidly. A relationship between the duration of ischemia and TxB2 production was therefore evident. 6-Keto-PGF1 alpha levels increased in only the group subjected to 20 min of ischemia and, by contrast to the pattern of TxB2 change, 6-keto-PGF1 alpha levels remained elevated throughout the reperfusion period. During reperfusion, the ratio of TxB2 to 6-keto-PGF1 alpha increased substantially versus the preischemic period in both structures. The data demonstrate that eicosanoid elaboration following cerebral ischemia can be evaluated by the microdialysis technique. In addition, they indicate that the thresholds (duration of ischemia) for the postischemic production and the temporal profiles of TxB2 and 6-keto-PGF1 alpha in the caudate and hippocampus differ. They also demonstrate that there is regional heterogeneity in the patterns of eicosanoid elaboration after forebrain ischemia.     

Protective effect of pitavastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor, on ischemia-induced neuronal damage

We investigated the neuroprotective effects of a novel 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor (pitavastatin) on ischemic neuronal damage in gerbils using immunohistochemistry. The animals were allowed to survive for 14 days after 5 min of ischemia induced by bilateral occlusion of the common carotid arteries. Five days after ischemia, severe neuronal cell loss was observed in the hippocampal CA1 sector. Prophylactic treatment with pitavastatin dose-dependently prevented the hippocampal CA1 neuronal cell loss 5 days after ischemia. Immunohistochemical study did not show the change of nNOS and iNOS expression in the hippocampus except for, in a few regions, up to 1 day after ischemia. Thereafter, the expression of iNOS was observed in the hippocampal CA1 sector 5 and 14 days after ischemia. In contrast, the expression of nNOS and eNOS gradually decreased in the hippocampal CA1 sector up to 14 days after ischemia. Prophylactic treatment with pitavastatin also prevented the expression of iNOS and the decrease of eNOS expression and the number of nNOS-positive cells in the hippocampal CA1 sector 5 days after ischemia. However, prophylactic treatment with pitavastatin at a dose of 10 mg kg(-1) did not change the immunoreactivity of iNOS and nNOS in the hippocampus at an early phase after ischemia. In contrast, this drug prevented the reduction of eNOS immunoreactivity in the hippocampal CA1 neurons at an early phase after ischemia. These findings demonstrate that the HMG-CoA reductase inhibitor pitavastatin can protect hippocampal CA1 neurons after transient forebrain ischemia through up-regulation of eNOS expression in this region. Thus pharmacological modulation of eNOS expression may offer a novel therapeutic strategy for cerebral ischemic stroke.     

3-硝基丙酸诱导沙士鼠脑缺血耐受时海马促红细胞生成素的表达

目的:探讨促红细胞生成素(erythropoietin,Epo)在小剂量3-硝基丙酸(3-nitropropionic acid,3-NPA)预处理诱导脑缺血耐受鼠脑海马的表达变化,揭示3-NPA预处理的机制。方法:将沙土鼠腹腔注射5mg/kg3-NPA后3天,阻断沙土鼠双侧预总动脉3.5min造成前脑缺血模型,通过尼氏染色和免疫组化观察海马锥体细胞损伤和Epo的表达变化。结果:对照组海马CA_1区已失去正常结构,锥体细胞大部分丧失,存活神经元计数显著低于3-NPA预处理组。对照组脑缺血再灌注6h,海马可检测到脑源性Epo的表达,再灌注12h,脑源性Epo表达达到较高水平,以后随时间延长逐渐下调。3-NPA预处理组海马Epo的表达在脑缺血再灌注后6h和12h高于对照组,1d、3d、7d与对照组比较无显著性差异。结论:3-NPA预处理可以诱导脑缺血耐受,在3-NPA预处理后脑缺血再灌注早期Epo的表达增强,提示Epo可能是3-NPA预处理形成的机制之一。     

Selective proteasomal dysfunction in the hippocampal CA1 region after transient forebrain ischemia.

Delayed neuronal death in the hippocampal CA1 region after transient forebrain ischemia may share its underlying mechanism with neurodegeneration and other modes of neuronal death. The precise mechanism, however, remains unknown. In the postischemic hippocampus, conjugated ubiquitin accumulates and free ubiquitin is depleted, suggesting impaired proteasome function. The authors measured regional proteasome activity after transient forebrain ischemia in male Mongolian gerbils. At 30 minutes after ischemia, proteasome activity was 40% of normal in the frontal cortex and hippocampus. After 2 hours of reperfusion, it had returned to normal levels in the frontal cortex, CA3 region, and dentate gyrus, but remained low for up to 48 hours in the CA1 region. Thus, the 26S proteasome was globally impaired in the forebrain during transient ischemia and failed to recover only in the CA1 region after reperfusion. The authors also measured 20S and 26S proteasome activities directly after decapitation ischemia (at 5 and 20 minutes) by fractionating the extracts with glycerol gradient centrifugation. Without adenosine triphosphate (ATP), only 20S proteasome activity was detected in extracts from both the hippocampus and frontal cortex. When the extracts were incubated with ATP in an ATP-regenerating system, 26S proteasome activity recovered almost fully in the frontal cortex but only partially in the hippocampus. Thus, after transient forebrain ischemia, ATP-dependent reassociation of the 20S catalytic and PA700 regulatory subunits to form the active 26S proteasome is severely and specifically impaired in the hippocampus. The irreversible loss of proteasome function underlies the delayed neuronal death induced by transient forebrain ischemia in the hippocampal CA1 region.     

Protective effect of pitavastatin,a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor,on ischemia-induced neuronal damage

Abstract

We investigated the neuroprotective effects of a novel 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor (pitavastatin) on ischemic neuronal damage in gerbils using immunohistochemistry. The animals were allowed to survive for 14 days after 5 min of ischemia induced by bilateral occlusion of the common carotid arteries. Five days after ischemia, severe neuronal cell loss was observed in the hippocampal CA1 sector. Prophylactic treatment with pitavastatin dose-dependently prevented the hippocampal CA1 neuronal cell loss 5 days after ischemia. Immunohistochemical study did not show the change of nNOS and iNOS expression in the hippocampus except for, in a few regions, up to 1 day after ischemia. Thereafter, the expression of iNOS was observed in the hippocampal CA1 sector 5 and 14 days after ischemia. In contrast, the expression of nNOS and eNOS gradually decreased in the hippocampal CA1 sector up to 14 days after ischemia. Prophylactic treatment with pitavastatin also prevented the expression of iNOS and the decrease of eNOS expression and the number of nNOS-positive cells in the hippocampal CA1 sector 5 days after ischemia. However, prophylactic treatment with pitavastatin at a dose of 10 mg kg^-1 did not change the immunoreactivity of iNOS and nNOS in the hippocampus at an early phase after ischemia. In contrast, this drug prevented the reduction of eNOS immunoreactivity in the hippocampal CA1 neurons at an early phase after ischemia. These findings demonstrate that the HMG-CoA reductase inhibitor pitavastatin can protect hippocampal CA1 neurons after transient forebrain ischemia through up-regulation of eNOS expression in this region. Thus pharmacological modulation of eNOS expression may offer a novel therapeutic strategy for cerebral ischemic stroke.     

Hydroxyl radical production and lipid peroxidation paralles selective post-ischemic vulnerability in gerbil brain

The salicylate trapping method was used to investigate the changes in hydroxyl radical (·OH) levels in the selectively vulnerable hippocampus compared to the cerebral cortex of gerbils subjected to a 10 min period of near complete forebrain ischemia. Salicylate-derived 2,5-dihydroxybenzoic acid (2,5-DHBA) was measured in sham-operated animals and at 1, 5, and 15 min of reperfusion. A basal level of 2,5-DHBA was also seen in non-ischemic gerbil brain, both in the hippocampus and cortex. The hippocampal basal level was 160% higher than in the cortex (P < .01). Treatment with the cytochrome P450 inhibitor SKF-525A (50 mg/kg s.c. 30 min before measurement) did not affect this basal level in either hippocampus or cortex, which argues against a contribution of metabolic salicylate hydroxylation as its source. In contrast, pretreatment with the arachidonic acid cyclo-oxygenase inhibitor ibuprofen (20 mg/kg s.c.) decreased (?68.8%) the level of salicylate hydroxylation in the hippocampus, but not the cortex. In animals subjected to 10 min of forebrain ischemia, a selective increase in 2,5-DHBA was observed in the hippocampus at 1 min of reprerfusion which subsided by 5 min. No increase in salicylate hydroxylation was apparent in the cortex within the same time frame. The increase in ·OH in the hippocampus at 1 min of reperfusion was accompanied by a significant decrease (?15%; P < .03) in the hippocampal levels of vitamin E. No loss of vitamin E was observed in the cortex at the same time. It is hypothesized that the selective ischemic vulnerability of the hippocampus is mechanistically related to a selective post-ischemic burst in ·OH in that region. Moreover, this may be based upon an intrinsically higher level of oxidative stress in that region as a by-product of greater arachidonic acid turnover. © 1993 Wiley-Liss, Inc.     

脑缺血再灌注时鼠脑纹状体区细胞外液半胱氨酸含量变化及丹参的影响

目的动态观察脑缺血再灌注时鼠脑纹状体区细胞外液半胱氨酸（Ｃｙｓ）含量变化及丹参的影响，探讨Ｃｙｓ对缺血性神经元的损害作用。方法应用脑内微透析技术，采用高灵敏度的高压液相色谱－电化学检测手段，动态观察沙土鼠前脑缺血３０分钟再灌注１２０分钟时，纹状体区细胞外液Ｃｙｓ的变化。结果前脑缺血３０分钟时，细胞外液Ｃｙｓ浓度迅速升高，达缺血前的１８倍。再灌注３０、６０分钟时，分别为缺血前的５倍和２倍，９０分钟时基本恢复到缺血前的水平。丹参治疗组脑缺血时细胞外液Ｃｙｓ的含量较对照组低。结论Ｃｙｓ参与了脑缺血再灌注引起的损伤，而丹参可降低Ｃｙｓ水平，从而对缺血脑组织起到保护作用。     

The highly selective cyclooxygenase-2 inhibitor DFU is neuroprotective when given several hours after transient cerebral ischemia in gerbils

Several studies suggest that cyclooxygenase-2 contributes to the delayed progression of ischemic brain damage. In this study we examined whether the highly selective cyclooxygenase-2 inhibitor DFU reduces neuronal damage when administered several hours after 5 min of transient forebrain ischemia in gerbils. The extent of ischemic injury was assessed behaviorally by measuring the increases in locomotor activity and by histopathological evaluation of the extent of CA1 hippocampal pyramidal cell injury 7 days after ischemia. DFU treatment (10 mg/kg, p.o.) significantly reduced hippocampal neuronal damage even if the treatment is delayed until 12 h after ischemia. These results suggest that selective cyclooxygenase-2 inhibitors may be a valuable therapeutic strategy for ischemic brain injury.