巴曲酶对局灶性脑缺血再灌流大鼠成纤维细胞生长因子样免疫…

以往实验研究曾发现巴曲酶对脑血管病具有良好的神经保护作用，本文研究的目的为：巴曲酶是否还通过影响成纤维细胞生长因子起修复作用，用大鼠中大脑动脉（ＭＣＡ）线栓法脑缺血再灌注模型及免疫组化方法发现在缺血９０ｍｉｎ再灌流４８ｈ后，缺血侧皮层，尾壳核及海马区ｂＦＧＦ样免疫反应细胞增加及神经细胞变性，在缺血后给予巴曲酶（８ＢＵ／ｋｇ），ｂＦＧＦ样免疫反应加强，并且缺血对侧相应ＭＣＡ供血脑区也可见轻度的ｂＦＧ     

丹参在缺血再灌注时的神经保护作用──成纤维细胞生长因子上调

丹参作为传统中药治疗缺血性脑血管病具有良好的疗效，本实验研究丹参是否通过影响成纤维细胞生长因子（ｂＦＧＦ）起修复作用。用大鼠线栓法缺血再灌注模型及免疫组化方法，发现大鼠缺血９０ｍｉｎ、再灌流４８ｈ后，缺血侧皮层、尾壳核及海马区ｂＦＧＦ样免疫反应增加及神经细胞变性；缺血后给予丹参（１５ｇ／　ｋｇ）大鼠，ｂＦＧＦ样免疫反应加强，并且缺血对应侧脑区也可见轻度的ｂＦＧＦ样免疫反应改变，且神经细胞变性程度较轻。提示丹参对脑缺血再灌流的保护作用可能与它加强ｂＦＧＦ的修复作用有关。     

丹参在缺血再灌注时的神经保护作用——成纤维细…

丹参作为传统中药治疗缺血性脑血管病具有良好的疗效，本实验研究丹参是否通过影响成纤维细胞生长因子起修复作用，用大鼠线栓法缺血再灌注模型及免疫组化方法，发现大鼠缺血９０ｍｉｎ，再灌流４８ｈ后，缺血侧皮层，尾壳核及海马区ｂＦＧＦ样免疫反应增加及神经细胞变性；缺血后给予丹参大鼠，ｂＦＧＦ样免疫反应加强，并且缺血对应侧脑区也可见轻度的ｂＦＧＦ样免疫反应改变，且神经细胞变性程度较轻，提示丹参对脑缺血再灌流的保     

巴曲酶对脑缺血再灌注细胞外液单胺类及其代谢产物的影响——微透析研究

目的巴曲酶对脑缺血再灌流损伤的保护机理。方法采用脑内微透析技术结合高灵敏度的高压液相色谱－电化学检测手段（ＨＰＬＣ－ＥＤ），测定前脑缺血３０ｍｉｎ再灌注１２０ｍｉｎ时的纹状体细胞外液（ＥＣＦ）的ＤＡ、５－ＨＴ和ＮＥ及其代谢产物（５－ＨＩＡＡ）和ＨＶＡ的变化和巴曲酶的影响。结果显示脑缺血时，ＥＣＦＤＡ、ＮＥ及５－ＨＴ明显升高，巴曲酶能显著地降低脑缺血时ＥＣＦＤＡ及再灌注时ＥＣＦＨＶＡ和５－ＨＩＡＡ的水平。结论巴曲酶影响单胺神经递质是对脑缺血再灌注损伤起保护作用的机理之一     

巴曲酶对大鼠大脑缺血及缺血再灌注ET1基因表达的影响

本实验采用大鼠急性脑缺血及缺血再灌注模型，研究巴曲酶对脑缺血及脑缺血再灌注时内皮素（ET1）基因表达的影响。用中大脑动脉（MCA）线检法大鼠模型，共12只，分为缺血组及缺血再灌注组（每组各6只），每组又分为巴曲酶组及盐水组（对照组）。缺血组在缺血后24h，再灌注组则在缺血1．5h及再灌注24h后用原位杂交，并采用IBHS图像分析系统研究ET1基因表达。发现巴曲酶组或对照组手术侧大脑皮质及尾壳核ET1mRNA表达均显著高于对侧（非手术侧）。但是巴曲酶组手术侧的ET1mRNA表达显著低于对照组。结果提示，巴曲酸可使缺血及缺血再灌注ET1基因表达下调。这可能是巴曲酶对缺血再灌注的脑保护作用机理之一。     

巴曲酶对大鼠脑缺血再灌注损伤保护作用机理的研究

用Ｓｍｉｔｈ等的方法制备大鼠前脑短暂缺血再灌注模型，在缺血１０分钟后，再灌注６小时缺血前脑ＯＨ、ＧＳＳＧ及ＧＳＳＧ／总ＧＳＨ比值明显增高；海马区组织病理学检查显示神经元有不同程度的变性及轻度坏死；海马ＣＡ１区细胞计数显示存活细胞明显减少。巴曲酶（１．６ＢＵ／ｋｇ）可明显地逆转上述氧应激状态，海马ＣＡ１区存活细胞比缺血组明显增多。巴曲酶对缺血神经元的保护作用可能是缓解氧自由基损伤的结果，似乎不是诱导热休克蛋白７０基因表达的结果。     

抗TNF—αmAb治疗局灶性脑缺血／再灌注动物实验

脑缺血过程有大量ＴＮＦ－α的表达，ＴＮＦ－α参与和促进了脑损害的过程。本研究的目的为用抗ＴＮＦ－αｍＡｂ抑制ＴＮＦ－α的表达及其生物活性，从而达到保护脑组织的作用，采用大白鼠ＭＣＡ堵塞模型。将缺血６小时大白鼠１６只随机地分为两组，一组给予抗ＴＮＦ－αｍＡｂ，另一组给予等量生理盐水作对照；观察梗塞体积大小，白细胞附壁、聚集及组织浸润情况。结果发现抗ＴＮＦ－αｍＡｂ显著减少短暂性脑缺血的梗塞体积，显微镜观察发现能显著减少再灌注时白细胞聚集和粘附，从而保护脑组织。结论：抗ＴＮＦ－αｍＡｂ能减轻短暂性脑缺血损害     

巴曲酶对脑缺血再灌注细胞外液单胺类及其代谢产物的影响 …

目的 巴曲酶对脑缺血再灌流损伤的保护机理。方法 采用脑内微透析技术结合高灵敏度的高压液相色谱－电化学检测手段（ＨＰＬＣ－ＥＤ），测定前脑缺血３０ｍｉｎ再灌注１２０ｍｉｎ时的纹状体细胞外液（ＥＣＦ）的ＤＡ、５－ＨＴ和ＮＥ及其代谢产物（５－ＨＩＡＡ）和ＨＶＡ的变化和巴曲酶的影响。结果 显示脑缺血时，ＥＣＦ ＤＡ、ＮＥ及５－ＨＴ明显升高，巴曲酶能显著地降低脑缺血时ＥＣＦ ＤＡ及再灌注时ＥＣＦ ＨＶＡ和５     

大鼠脑缺血再灌注损伤后脑组织STAT3变化的免疫组织化学研究

目的 探讨信号转导和转录激活子（ＳＴＡＴ）３在大鼠局灶性脑缺血再灌注损伤中的表达及其与缺血性神经细胞损伤的关系方法 用ＡＢＣ免疫组化方法观察大鼠局灶性脑缺血再灌注损伤后脑组织中的ＳＴＡＴ３蛋白免疫反应阳性细胞分布。结果 正常和假手术大鼠脑内以及脑缺血后的非缺血半球脑组织中未发现有ＳＴＡＴ３免疫反应阳性细胞,脑缺血再灌注损伤后１２小时在栓塞侧梗死区可见少量ＳＴＡＴ３免疫阳性细胞,２４小时后阳性细胞显著增多达高峰,在缺血侧纹状体和缺血皮质周边区表达最明显,１周后梗死周边区少数神经细胞仍有阳性表达。差异有显著意义（Ｐ〈０．０１）。结论 ＳＴＡＴ３活化及超量表达可能介导了缺血神经细胞信号转导过程,并参与了脑缺血神经细胞损伤与修复的病理生理过程。     

实验性脑缺血及再灌注大鼠脑组织bFGF表达的研究

目的　探讨大鼠脑缺血２ ｈ 后不同时间再灌注脑组织碱性成纤维细胞生长因子（ｂＦＧＦ）的表达程度。方法　采用线栓法栓堵一侧大鼠大脑中动脉,建立脑缺血及再灌注模型。用免疫组化的方法,对缺血脑组织ｂＦＧＦ的表达进行观察。用统计学分析ｂＦＧＦ阳性细胞的数据。结果　正常脑组织中只有微量ｂＦＧＦ的表达;脑缺血２ ｈ 后再灌注０．５ ｈ 起,ｂＦＧＦ少量表达,为神经元表达;脑缺血２ ｈ 后再灌注２２ ｈ,ｂＦＧＦ表达达到高峰,神经元及神经胶质细胞均有表达。脑缺血２ ｈ 后再灌注１６６ ｈ,ｂＦＧＦ仍有持续表达。结论　ｂＦＧＦ参与缺血脑组织的修复过程     

Increase in basic fibroblast growth factor-like immunoreactivity in rat brain after forebrain ischemia

Using immunohistochemical techniques, a study was conducted to determine whether basic fibroblast growth factor (bFGF) is generated as one of the 'self-repair' responses in rat brain following transient forebrain ischemia. In normal brain, slight bFGF-like immunoreactivity was observed. However, in rats exposed to 20 min of forebrain ischemia, intense bFGF-like immunoreactivity was observed in the CA1 subfield of the hippocampus and the caudate putamen, and marked activity was evident in the temporal cortex, corpus callosum and the CA4 subfield of the hippocampus. Marked neuronal degeneration was also observed in these brain regions following forebrain ischemia. These results suggest that induction of bFGF-like immunoreactivity may be related to the healing which follows brain ischemia.     

Induction of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression in rat brain after focal ischemia/reperfusion.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme, has been recently identified to be involved in the initiation of neuronal apoptosis. To investigate the serial changes and cellular localization of GAPDH expression, and its role in ischemia/reperfusion-induced neuronal apoptosis, the authors analyzed immunohistochemically brain areas of rats subjected to middle cerebral artery occlusion (MCAO) and reperfusion. Nuclear overexpression of GAPDH was noted in the ischemic core area after 2 hours of MCAO without reperfusion. During the subsequent reperfusion, nuclear accumulation of GAPDH in this area decreased in a time-dependent manner. However, cytoplasmic and nuclear GAPDH immunoreactivity was detected in neurons of the penumbra area of the parietal cortex, in rats subjected to 2-hour MCAO followed by 3-hour reperfusion. The increase of nuclear GAPDH immunoreactivity was persistently noted up to 48 hours of reperfusion, whereas cytoplasmic immunoreactivity correlated inversely with the duration of reperfusion. Moreover, double staining revealed colocalization of nuclear GAPDH and TUNEL in the penumbra area. The authors' study demonstrated that overexpression of GAPDH and nuclear translocation occurred in both the ischemic core and penumbra area soon after focal ischemia. These processes could be viewed as an early marker of ischemia/reperfusion-induced apoptotic neuronal death. The results suggest that GAPDH may play a critical role in the progression and spread of ischemic neuronal damage.     

Changes in immunoreactivity of HSP60 and its neuroprotective effects in the gerbil hippocampal CA1 region induced by transient ischemia

Heat shock proteins act as molecular chaperones and are involved in protein folding, refolding, transport, and translocation. In the present study, we observed changes in heat shock protein 60 (HSP60) immunoreactivity and protein level in the gerbil hippocampal CA1 region after 5 min of transient forebrain ischemia and its neuroprotective effect against ischemic damage. HSP60 immunoreactivity in the CA1 region began to increase in the stratum pyramidale at 30 min after ischemia/reperfusion, and peaked 24 h after ischemia/reperfusion. Thereafter, HSP60 immunoreactivity was decreased in the CA1 region with time. Seven days after ischemia/reperfusion, HSP60 immunoreactivity was increased again in the CA1 region: at this time point after ischemia/reperfusion, HSP60 immunoreactivity was expressed in glial cells in the ischemic CA1 region. HSP60 immunoreactive glial cells were astrocytes containing glial fibrillar acidic protein. In contrast, change in HSP60 immunoreactivity in the ischemic CA2/3 region was not significant compared with that in the ischemic CA1 region. In Western blot study, HSP60 protein level in the CA1 region was increased after ischemia/reperfusion and highest 24 h after ischemia/reperfusion. Animals treated with recombinant adenoviruses expressing Hsp60 (Ad-Hsp60) showed the neuroprotection of CA1 pyramidal neurons from ischemic damage. These results suggest that HSP60 may be associated with delayed neuronal death of CA1 pyramidal neurons after transient ischemia, and the induction of HSP60 protects the neurons from ischemic damage.     

Betaxolol attenuates retinal ischemia/reperfusion damage in the rat

This study was performed to elucidate the protection afforded by post-treatment with Betoptic (0.25% betaxolol) against neuronal cell damage after ischemia/reperfusion insult in rats. Betaxolol was applied topically after the start of reperfusion and its effect was evaluated by morphometry and choline acetyltransferase immunoreactivity of retinas at 7 days after reperfusion. In non-treated eyes, the thickness of the inner plexiform layer decreased markedly after a reperfusion period of both 3 and 7 days. However, when eyes were treated with betaxolol after ischemia/reperfusion injury, both the reduction of the inner plexiform layer thickness and the retinal choline acetyltransferase immunoreactivity were significantly attenuated. These findings suggest that betaxolol is an efficient neuroprotective agent and prevents the retinal cell damage induced by ischemic injury in rats.     

Protection of rat hippocampus against ischemic neuronal damage by pretreatment with sublethal ischemia

We examined whether preconditioning with sublethal ischemia protects against neuronal damage following subsequent lethal ischemic insults. Forebrain ischemia for 3 min in Wistar rats increased heat shock protein-70 immunoreactivity in the hippocampal CA1 subfield but produced no neuronal damage. Preconditioning with 3 min of ischemia followed by 3 days of reperfusion protected against hippocampal CA1 neuronal damage following 6 and 8 min of ischemia but not damage after 10 min of ischemia. The result strongly suggests that stress response induced by sublethal ischemia protects against ischemic brain damage.     

巴曲酶、尿激酶联合应用对大鼠局灶性脑缺血再灌注损伤保护作用的研究

目的　探讨降纤、溶栓疗法联合应用对局灶性脑缺血再灌注损伤的保护作用。方法　采用线栓法制备大鼠局灶性脑缺血模型 (MCAO) ,分为尿激酶 (U K)、巴曲酶单药治疗组及巴曲酶与 UK合并用药治疗组和空白对照组 ,观察缺血 2 h再灌注后神经功能缺损评分、死亡率、梗死体积、组织病理学改变。结果　神经损伤及出血改变以 U K组为最重 (其中死亡 6只 ,出血 5只 ) ,空白组其次 (其中死亡 5只 ,出血 5只 ) ,巴曲酶单药治疗组及合并用药组均较前两组减轻 (其中巴曲酶单药治疗组死亡 4只 ,出血 2只 ,合并用药组的死亡及出血的例数均相同 ,各为死亡 3只 ,出血 5只 )。结论　巴曲酶与 U K联合应用对局灶性脑缺血再灌注损伤有保护作用 ;联合用药后对出血的影响较单一 U K治疗未见明显加重。巴曲酶可能具有减轻脑缺血 /再灌注损伤的神经保护作用 ,且使用安全。     

Distribution of Ca(2+)/calmodulin-dependent protein kinase I beta 2 in the central nervous system of the rat

We have examined the distribution of transforming growth factor-beta1 (TGF-beta1) and bone morphogenetic protein-6 (BMP-6) in the brain of rats subjected to a mild and reversible ischemic damage produced by a 20-min occlusion of both carotid arteries without occlusion of the vertebral arteries. We have selected this model to study how the expression of trophic factor of the TGF-beta superfamily changes in neurons that recover from a transient insult. Immunocytochemical analysis showed a loss of TGF-beta1 in neurons of all hippocampal subfields immediately after the ischemic period, followed by a recovery of immunoreactivity in CA1 and CA3 neurons after reperfusion. BMP-6 immunoreactivity was also lost in most hippocampal neurons, but immunostaining became particularly intense in the interstitial space after both ischemia and reperfusion. An interstitial localization of BMP-6 was also observed in the cerebral cortex, particularly after reperfusion. Mild ischemia also induced substantial changes in the expression of TGF-beta1 and BMP-6 within the cerebellar cortex. In control animals, these factors appeared to be localized in granule cells (TGF-beta1) and Purkinje cells (both), whereas the molecular layer was not immunopositive. Both TGF-beta1 and BMP-6 were highly expressed in the interstitial spaces of the cerebellar cortex either 20 min after ischemia or 20 min after reperfusion. Taken collectively, these results suggest that a mild and reversible ischemia stimulates the release of BMP-6 from neurons into the interstitial space. We speculate that BMP-6, besides functioning during brain development, may also regulate neuronal resistance to insults of the adult brain.     

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.     

Change in endothelial nitric oxide synthase in the rat retina following transient ischemia

Patterns of endothelial nitric oxide synthase (eNOS) expression in retinal ischemia were studied utilizing a transient high intraocular pressure (HIOP) model. We investigated neuronal cell damage and changes in eNOS immunoreactive expression in the ischemic retina, and its relationship to the neuroprotection of betaxolol treatment after ischemic injury. Immunohistochemical staining for eNOS was performed at 3, 7, 14 and 28 days after ischemia/reperfusion. In controls, eNOS immunoreactivity was detected in retinal vessels, but was not detected in neurons. After ischemia/reperfusion, the intensity of eNOS immunoreactivity increased in both retinal vessels and the ganglion cell layer (GCL) compared with controls. eNOS-positive neurons were induced first in the inner nuclear layer (INL) 7 days after reperfusion. However, when experiments were carried out on animals that had been treated with betaxolol after ischemia/reperfusion, the intensity of eNOS immunoreactivity decreased compared to the untreated ischemic retinas. These results suggest that an increase in eNOS expression could be associated with the degenerative changes in the ischemic retina, and that betaxolol treatment appears to protect retinal tissue from ischemic damage.