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.     

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.     

Poly(ADP-ribose) polymerase during reperfusion after transient forebrain ischemia

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.     

Multiple modes of action of tacrolimus (FK506) for neuroprotective action on ischemic damage after transient focal cerebral ischemia in rats

While the immunosuppressant tacrolimus (FK506) is known to be neuroprotective following cerebral ischemia, the mechanisms underlying its neuroprotective properties are not fully understood. To determine the mode of action by which tacrolimus ameliorates neurodegeneration after transient focal ischemia, we therefore evaluated the effect of tacrolimus on DNA damage, release of cytochrome c, activation of microglia and infiltration of neutrophils following a 60-min occlusion of the middle cerebral artery (MCA) in rats. In this model, cortical brain damage gradually expanded until 24 h after reperfusion, whereas brain damage in the caudate putamen was fully developed within 5 h. Tacrolimus (1 mg/kg) administered immediately after MCA occlusion significantly reduced ischemic damage in the cerebral cortex, but not in the caudate putamen. Tacrolimus decreased both apoptotic and necrotic cell death at 24 h and reduced the number of cytochrome c immunoreactive cells at 8 h after reperfusion in the ischemic penumbra in the cerebral cortex. In contrast, tacrolimus did not show significant neuroprotection for necrotic cell death and reduction of cytochrome c immunoreactive cells in the caudate putamen. Tacrolimus also significantly decreased microglial activation at 8 h and inflammatory markers (cytokine-induced neutrophil chemoattractant and myeloperoxidase [MPO] activity) at 24 h after reperfusion in the ischemic cortex but not in the caudate putamen. These results collectively suggest that tacrolimus ameliorates the gradually expanded brain damage by inhibiting both apoptotic and necrotic cell death, as well as suppressing inflammatory reactions.     

甲烯土霉素在脑缺血动物实验中的治疗作用

目的探讨实验性脑缺血嗜中性粒细胞（ＮＣ）的浸润规律及甲烯土霉素（ＭＣ）对其影响。方法采用线栓法制成大脑中动脉脑缺血模型，测定脑组织中髓过氧化物酶（ＭＰＯ）活性，并观察神经病学评分及脑组织病理学改变。结果脑缺血及再灌组局灶性缺血脑组织有ＭＰＯ活性、ＮＣ浸润。ＭＣ能有效地降低局灶性缺血脑组织ＭＰＯ活性，减少ＮＣ浸润数量，减轻脑组织缺血坏死程度，改善缺血后神经功能损害。结论局灶性缺血脑组织中有ＮＣ浸润，后者参与脑缺血损伤的病理生理过程，ＭＣ可通过减轻ＮＣ介导的脑损伤而发挥重要的脑保护作用。     

牛磺酸降低局灶性脑缺血引起的能量代谢紊乱和氧化损伤

目的观察牛磺酸对大鼠局灶性脑缺血引起的能量代谢紊乱和氧化损伤的影响。方法建立大鼠动脉腔内插线局灶性脑缺血模型,在缺血1h后给予牛磺酸(50mg/kg,iv),测定缺血2h再灌注22h时脑组织内ATP、乳酸和丙二醛(MDA)的含量、髓过氧化物酶(MPO)和超氧化物歧化酶(SOD)的活性及总抗氧化能力。结果牛磺酸明显降低再灌注22h时缺血脑组织内乳酸和MDA的含量及MPO活性,升高ATP的含量、SOD的活性和总抗氧化能力。结论牛磺酸可改善局灶性脑缺血损伤后的能量代谢,降低中性粒细胞的浸润,提高缺血组织的抗氧化能力,减轻局灶性脑缺血引起的过氧化损伤。     

Changes in oxidative stress,iNOS activity and neutrophil infiltration in severe transient focal cerebral ischemia in rats

Oxidative stress, inducible nitric oxide synthase (iNOS) and neutrophils all contribute to post-ischemic brain damage. This study has determined the time courses of these three phenomena after ischemia in parallel with histological and functional outcomes. Ischemia was produced in rats by occluding the left middle cerebral artery and both common carotid arteries for 20 min. Regional cerebral blood flow (rCBF) rapidly decreased to 20% of its preischemic value during occlusion and stabilized at 60% following reperfusion. The striatal infarction was maximal 15 h after reperfusion (50+/-3 mm(3)), whereas the cortical infarction reached its maximum at 48 h (183+/-10 mm(3)). This drastic decrease in rCBF followed by incomplete reperfusion and massive infarction is, thus, extremely severe. The cortical infarction was strongly correlated with the neurologic deficit and loss of body weight. Oxidative stress, evaluated by the decrease in glutathione concentrations, appeared in the striatum at 6 h after reperfusion and in the cortex at 15 h. Calcium-independent NOS activity, considered as inducible NOS activity, was significantly enhanced at 24 h in the striatum and at 48 h in the cortex. Myeloperoxidase activity, a marker of neutrophil infiltration, was significantly increased at 48 h in both the striatum and cortex. These time courses show that the delayed iNOS activity and neutrophil infiltration that occur after the maturation of infarction in severe ischemia may not contribute to ischemic brain damage. By contrast, early oxidative stress may well be implicated in cerebral injury.     

Contribution of poly(ADP-ribose) polymerase to postischemic blood-brain barrier damage in rats.

The nuclear enzyme poly(ADP-ribose) polymerase (PARP) is activated by oxidative stress and plays a significant role in postischemic brain injury. We assessed the contribution of PARP activation to the blood-brain barrier (BBB) disruption and edema formation after ischemia-reperfusion. In male Wistar rats, global cerebral ischemia was achieved by occluding the carotid arteries and lowering arterial blood pressure for 20 mins. The animals were treated with saline or with the PARP inhibitor N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-N, N-dimethylacetamide.HCl (PJ34); (10 mg/kg, i.v.) before ischemia. After 40 mins, 24, and 48 h of reperfusion, the permeability of the cortical BBB was determined after Evans Blue (EB) and Na-fluorescein (NaF) administration. The water content of the brain was also measured. The permeability of the BBB for EB increased after ischemia-reperfusion compared with the nonischemic animals after 24 and 48 h reperfusion but PARP inhibition attenuated this increase at 48 h (nonischemic: 170+/-9, saline: 760+/-95, PJ34: 472+/-61 ng/mg tissue). The extravasation of NaF showed similar changes and PJ34 post-treatment attenuated the permeability increase even at 24 h. PARP inhibition decreased the brain edema seen at 48 h. Because PARP has proinflammatory properties, the neutrophil infiltration of the cortex was determined, which showed lower values after PJ34 treatment. Furthermore, PJ34 treatment decreased the loss of the tight junction protein occludin at 24 and 48 h. The inhibition of PARP activity accompanied by reduced post-ischemic BBB disturbance and decreased edema formation suggests a significant role of this enzyme in the development of cerebral vascular malfunction     

Failure of levemopamil to improve histological outcome following temporary occlusion of the middle cerebral artery in cats

Levemopamil, a novel calcium channel blocker with antagonistic action on serotonin S₂-receptors has been reported to be a promising compound for therapy in cerebral ischemia. This data has been obtained in the rat only, and it is of interest to determine if these beneficial effects are present in other models of ischemia in other species. The present study was therefore designed to examine its effect on histological outcome and changes in EEG after focal cerebral ischemia and reperfusion in the cat. Focal cerebral ischemia was induced by a reversible 1 hour occlusion of the middle cerebral artery followed by reperfusion of the brain. Six hours after the induction of the insult, the brain was perfusion-fixed and evaluated for histological damage by light microscopy. In 8 animals an intravenous infusion of levemopamil was initiated 5 minutes after middle cerebral artery occlusion at a rate of 4 mg/kg/h for 15 min and then at 0.6 mg/kg/h until the end of the study. A control group (n = 7) received a similar infusion of saline. The EEG amplitude did not differ between the two groups at any point of the study. The area of ischemic damage in the sections obtained for histological examination at 1-mm intervals, as well as the total volume of ischemic damage for both groups (treated: 1.33 cm³; untreated: 0.97 cm³) also did not show any significant differences. These results indicate that postischemic treatment with levemopamil at this dose, and in this model of focal cerebral ischemia and reperfusion, does not attenuate the ischemic damage.     

Time-related changes in myeloperoxidase activity and leukotriene B4 receptor binding reflect leukocyte influx in cerebral focal stroke

In previous studies, we have used histological methods to characterize cellular changes, and validated the use of the myeloperoxidase (MPO) activity assay to quantitate increased neutrophil infiltration in ischemic stroke. We also identified increased leukotriene B₄ (LTB₄) binding sites as a potential marker for neutrophil infiltration into, focal ischemic tissue. However, these studies were conducted at only one time-point, 24 h after ischemia. In the present study, we examined the full time-course of MPO activity and LTB₄ receptor binding following middle cerebral artery occlusion (MCAO) made permanently (PMCAO) or transiently (160 min followed by reperfusion; TMCAO) in spontaneously hypertensive rats, and compared the results to previously characterized histologic changes in these models. Ischemic and contralateral (control) cortical tissue samples were assayed for MPO (U/g wet wt) and [³H]LTB₄ receptor binding (fmol/mg protein). Following PMCAO, MPO activity significantly increased as early as 12 h and continued to increase over the next 5 d (p<0.05). Following TMCAO, MPO activity was significantly elevated already after only 6 h of reperfusion and also continued to increase over the next 5 d of reperfusion (p<0.05). LTB₄ receptor binding and MPO activity were highly correlated during periods when both measures increased together (i.e., 0.5–5dp<0.01). However, by 15 d post-MCAO, LTB₄ receptor binding remained elevated after MPO activity levels had returned to normal. This persistent LTB₄ binding was associated with the significant gliosis that was characterized previously to persist in these models. The time-course of increased MPO activity and initially increased LTB₄ binding post-MCAO correspond very well to our previous histological data that characterized the time-course for leukocyte infiltration under these conditions. Therefore, the increased MPO activity over time was associated with initial neutrophil and later mononuclear cell infiltration into ischemic tissue in these models. In addition, the present studies utilized histochemical analysis to demonstrate peroxidase activity in macrophages within the cerebral infarct following MCAO, thus validating that MPO activity originates from the later infiltrating mononuclear cells in addition to the early infiltrating neutrophils that had been previously characterized in the same manner. TMCAO produces a significantly larger and earlier increase in ischemic cortex MPO activity and a similar later increase in MPO activity compared to PMCAO treatment. Clearly, reperfusion of cerebral tissue following ischemia greatly exacerbates the degree of cerebral tissue inflammation. These biochemical assays, especially the MPO activity assay, have now been validated for quantitating the early and late phases of the cerebral inflammatory reaction to tissue injury.     

3-aminobenzamide prevents restraint-evoked immunocompromise

Chronic stressors compromise immune function, which may affect disease state in rats and mice. Although the molecular mechanism(s) underlying the link between psychological stressors and physiological responses remain elusive, one putative mechanism is oxidative stress. DNA damage activates poly(ADP-ribose) polymerase (PARP), a nuclear enzyme that participates in DNA repair; if DNA damage is extensive, however, then PARP becomes cytotoxic. Because PARP-1-/- transgenic mice are resistant to chronic stress-induced immunocompromise, we tested the hypothesis that pre-restraint administration of 3-aminobenzamide (3-AB), a PARP inhibitor, would prevent restraint-evoked suppression of antibody production to the novel protein, keyhole limpet hemocyanin (KLH). Mice were physically restrained for 3 h daily for 14 consecutive days, then immunized with KLH. Daily restraint continued for an additional 21 days and anti-KLH IgG production was assessed. Mice exposed to repeated restraint reduced concentrations of anti-KLH IgG, whereas, mice treated with 3-AB (0.5, 5.0, or 20.0 mg/kg) prior to each bout of restraint displayed anti-KLH IgG concentrations similar to those of unrestrained mice. Treatment with 3-AB (0.5 and 5.0 mg/kg) during the restraint paradigm also facilitated habituation of the corticosterone response to restraint, and 3-AB (0.5 mg/kg) reduced the effect of repeated restraint on body mass. However, the immunoprotective effects of 3-AB and the endocrine and metabolic effects appear to be distinctly regulated because, unlike the endocrine and metabolic effects, the immunoprotective effects of 3-AB were independent of dose. These data suggest that PARP inhibitors may be useful to prevent compromised immune function in response to stressors.     

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.     

Inflammatory cell infiltration after endothelin-1-induced cerebral ischemia: histochemical and myeloperoxidase correlation with temporal changes in brain injury.

Accumulation of neutrophils in brain after transient focal stroke remains controversial with some studies showing neutrophils to be deleterious, whereas others suggest neutrophils do not contribute to ischemic injury. Myeloperoxidase (MPO) has been used extensively as a marker for quantifying neutrophil accumulation, but is an indirect method and does not detect neutrophils alone. To elucidate the interaction of macrophages in the neutrophil inflammatory response, we conducted double-label immunofluorescence in brain sections at 0, 1, 2, 3, 7, and 15 days after ischemia. Each of these results was obtained from the same animal to determine correlations between neutrophil infiltration and ischemic damage. It was found that MPO activity increased up to 3 days after cerebral ischemia. Dual-staining revealed that macrophages engulf neutrophils in the brain and that this engulfment of neutrophils increased with time, with 50% of neutrophils in the brain engulfed at 3 days and approximately 85% at 15 days (N=5, P<0.05). Interestingly, at 7 days the amount of dual-staining was decreased to 20% (N=5, P<0.05). Neutrophil infiltration was positively correlated with ischemic damage in both the cortex and striatum (r(2)=0.86 and 0.80, respectively, P<0.01). The results of this study indicate that the MPO from neutrophils phagocytized by macrophages may continue to contribute to the overall MPO activity, and that previous assessments that have utilized this marker to measure neutrophil accumulation may have mis-calculated the number of neutrophils within the ischemic territory and hence their contribution to the evolution of the infarct at later time points. Thus any biphasic infiltration of neutrophils may have been masked by the accumulation of macrophages.     

大鼠脑缺血再灌注区ICAM-1表达与白细胞浸润的观察及丹参的影响

目的　观察细胞间粘附分子（ Ｉ Ｃ Ａ Ｍ１） 蛋白在大鼠脑缺血再灌注的不同时程脑组织中的表达与中性白细胞浸润程度的关系及丹参对它们的影响。方法　 Ｓ Ｄ大鼠分为３ 组：假手术组、对照组及丹参组。大脑中动脉缺血２ ｈ 再灌注２ ｈ 、１２ ｈ、２４ ｈ 、４８ ｈ 、７２ ｈ 、７ ｄ、１４ ｄ 后,分别进行 Ｉ Ｃ Ａ Ｍ１ 免疫组织化学及组织 Ｈ Ｅ染色。结果　在脑缺血再灌早期,脑微血管内皮细胞 Ｉ Ｃ Ａ Ｍ１ 免疫反应开始逐渐增加,再灌注４８ ｈ 达到高峰,再灌注１４ ｄ 接近正常水平,同时脑缺血区中性白细胞浸润也随之增加,在时程上与 Ｉ Ｃ Ａ Ｍ１ 表达同步。丹参组,再灌注４８ ｈ 后, Ｉ Ｃ Ａ Ｍ１ 免疫阳性血管数及中性白细胞的浸润比同时间对照组明显降低。结论　脑缺血 Ｉ Ｃ Ａ Ｍ１ 的表达与中性白细胞浸润密切相关,丹参能降低 Ｉ Ｃ Ａ Ｍ１ 的表达,抑制中性白细胞的浸润。     

The effect of reperfusion on neuroprotection using an inhibitor of poly(ADP-ribose) polymerase.

The activation of poly(ADP-ribose) polymerase (PARP) by free radical-damaged DNA plays a pivotal role in mediating ischemia-reperfusion injury. The purpose of the present study was to examine the neuroprotective effects of a PARP inhibitor, 3-aminobenzamide (3-ABA), which was administered either prior to or following reperfusion, to determine the importance of PARP inhibition prior to reperfusion. 3-ABA was injected i.p. either 15 min before or 15 min following reperfusion in a transient focal ischemia model in the rat. Treatment prior to the reperfusion led to a significant decrease in the volume of damaged tissue at 24 h (118.7 +/- 18.8 mm3, mean +/- s.d., p < 0.01), compared with the control (176.1 +/- 22.8 mm3). However, treatment after the reperfusion failed to produce a reduction in the damaged volume (171.9 +/- 27.6 mm3). These findings suggest that PARP activation sufficient to produce cellular damage occurs immediately after the reperfusion following cerebral ischemia.     

A hemorrhagic transformation model of mechanical stroke therapy with acute hyperglycemia in mice

Clinical benefit for mechanical thrombectomy (MT) in stroke was recently demonstrated in multiple large prospective studies. Acute hyperglycemia (HG) is an important risk factor of poor outcome in stroke patients, including those that underwent MT. The aim of this therapy is to achieve a complete reperfusion in a short time, given that reperfusion damage is dependent on the duration of ischemia. Here, we investigated the effects of acute HG in a mouse model of ischemic stroke induced by middle cerebral artery occlusion (MCAO). Hyperglycemic (intraperitoneal [ip] injection of glucose) and control (ip saline injection) 10‐week male C57BL6 mice were subjected to MCAO (30, 90, and 180 min) followed by reperfusion obtained by withdrawal of the monofilament. Infarct volume, hemorrhagic transformation (HT), neutrophil infiltration, and neurological scores were assessed at 24 hr by performing vital staining, ELISA immunofluorescence, and behavioral test, respectively. Glucose injection led to transient HG (blood glucose = 250–390 mg/dL) that significantly increased infarct volume, HT, and worsened neurological outcome. In addition, we report that HG promoted blood‐brain barrier disruption as shown by hemoglobin accumulation in the brain parenchyma and tended to increase neutrophil extravasation within the infarcted area. Acute HG increased neurovascular damage for all MCAO durations tested. HTs were observed as early as 90 min after ischemia under hyperglycemic conditions. This model mimics MT ischemia/reperfusion and allows the exploration of brain injury in hyperglycemic conditions.     

Neutrophils do not contribute to infarction,oxidative stress,and NO synthase activity in severe brain ischemia

Polymorphonuclear leukocytes (PMNs) were reported to contribute to ischemia-reperfusion-induced brain damage. The present work examined whether PMN infiltration is deleterious in a severe model of transient focal cerebral ischemia and in which part PMNs contribute to oxidative stress and nitric oxide (NO) production. A 20-min occlusion of the left middle cerebral artery and both common carotid arteries was performed in rats. Infarction was maximal 24 h after reperfusion, while accumulation of PMNs in infarcted tissue was not significant before 48 h. Moreover, neutropenia induced by vinblastine (0.5 mg/kg iv) significantly decreased by 60-80% PMN infiltration 48 h after reperfusion but did not reduce the infarct volume. Thus PMNs do not contribute to cerebral injury in our model. Furthermore, decreased PMN infiltration modified neither oxidative stress evaluated by glutathione concentrations nor NO synthase activities 48 h after reperfusion. In conclusion, our results suggest that PMNs are not involved in severe cerebral ischemia and that anti-PMN strategies may be inefficient in some pathological conditions.     

汉防己碱对缺血—再灌流大鼠脑NOS活性及超微结构的影响

用ＮＡＤＰＨｄ 组织化学和电镜技术研究了汉防己碱（Ｔｅｔ）对大鼠缺血再灌流脑一氧化氮合酶（ＮＯＳ）活性及超微结构变化的作用。结果发现,缺血１５ ｍ ｉｎ,再灌流２ ｈ 后,海马超微结构出现明显的病理学损害,主要包括核周出现髓鞘样变,核周间隙增大,胞核均质化,大量片状软化灶,毛细血管周围水肿。Ｔｅｔ １０ 和２０ｍ ｇ／ｋｇ 缺血前３０ ｍ ｉｎ 腹腔注射可明显改善或避免出现上述缺血组织学改变。缺血１５ ｍ ｉｎ,再灌流２４ ｈ 后,皮层和海马ＮＯＳ阳性细胞数目显著增多,Ｔｅｔ５,１０ 和１５ ｍ ｇ／ｋｇ 缺血前３０ ｍ ｉｎ 腹腔注射,浓度依赖地抑制缺血再灌流所致的ＮＯＳ阳性细胞数目增加。这些结果提示,Ｔｅｔ可抑制缺血再灌流脑ＮＯＳ活性,减少ＮＯ产生,这可能与Ｔｅｔ减轻脑缺血再灌流的损害有关。     

Focal cerebral ischemia in the cat: pretreatment with a competitive NMDA receptor antagonist, D-CPP-ene

The effects of the competitive N-methyl-D-aspartate (NMDA) receptor antagonist D-(E)-4-(3-phosphonoprop-2-enyl)piperazine-2-carboxylic acid (D-CPP-ene; SDZ EAA 494) upon ischemic brain damage have been examined in anesthetized cats. Focal cerebral ischemia was produced by permanent occlusion of the middle cerebral artery (MCA) and the animals were killed 6 h later. The amount of early ischemic brain damage was assessed in coronal sections at 16 predetermined stereotaxic planes. Pretreatment with D-CPP-ene (15 mg/kg i.v. followed by continuous infusion at 0.17 mg/kg/min until death), 15 min prior to MCA occlusion, significantly reduced the volume of ischemic brain damage (from 20.6 +/- 9.9% of the cerebral hemisphere in vehicle-treated cats to 7.2 +/- 4.4% in drug-treated cats; p less than 0.01). The competitive NMDA receptor antagonist D-CPP-ene is as effective as noncompetitive NMDA antagonists in reducing the amount of ischemic brain damage in this model of focal cerebral ischemia in a gyrencephalic species.     

Neuroprotective effect of 4-amino-1,8-napthalimide, a poly(ADP ribose) polymerase inhibitor in middle cerebral artery occlusion-induced focal cerebral ischemia in rat

In the present study, neuroprotective effect of 4-amino-1,8-napthalimide (4-ANI), a poly(ADP-ribose) polymerase (PARP) inhibitor was investigated in middle cerebral artery occlusion (MCAo)-induced focal ischemia. Sprague-Dawley rats were subjected to 2 h of middle cerebral artery occlusion followed by 22 h of reperfusion. After 22 h of reperfusion rats were evaluated for cerebral infarction, neurological deficits, brain NAD levels, and in situ terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL). Focal ischemia produced significant infarct volume (201 +/- 14 mm3), neurological scores (2 +/- 0.5) and 28 +/- 4.5% brain NAD depletion. Ischemia was associated with increased in TUNEL positive cells in brain sections indicating DNA fragmentation. 4-ANI treatment (1 and 3 mg/kg, i.p.) significantly decreased infarct volume to 35 +/- 7% and 70 +/- 6%, respectively. Neurological functions were also significantly improved at these doses. 4-ANI (3 mg/kg) completely reversed brain NAD depletion and significantly reduced the increase in the number of TUNEL positive cells. Nevertheless, 4-ANI treatment did not alter cerebral blood flow and blood pressure. Our study suggests 4-ANI is a potent neuroprotective agent in focal cerebral ischemia and its neuroprotective effects may be attributed to reduction of NAD depletion and DNA fragmentation.