Hyperlipidemia affects neuronal nitric oxide synthase expression in brains of focal cerebral ischemia rat model★

BACKGROUND: Hyperlipidemia, a risk factor for ischemic cerebrovascular disease, may mediate production of neuronal nitric oxide synthase (nNOS) to induce increased nitric oxide levels, resulting in brain neuronal injury. OBJECTIVE: To investigate effects of hyperlipidemia on brain nNOS expression, and to verify changes in infarct volume and pathology during reperfusion, as well as neuronal injury following ischemia/reperfusion in a rat model of focal cerebral ischemia. DESIGN, TIME AND SETTING: Complete, randomized grouping experiment was performed at the Laboratory of Physiology, Shanxi Medical University from March 2005 to March 2006. MATERIALS: A total of 144 eight-week-old, male, Wistar rats, weighing 160-180 g, were selected. A rat model of middle cerebral artery occlusion was established by suture method after 4 weeks of formulated diet. Nitric oxide kit and rabbit anti-rat nNOS kit were respectively purchased from Nanjing Jiancheng Bioengineering Institute, China and Wuhan Boster Biological Technology, Ltd., China. METHODS: The rats were equally and randomly divided into high-fat diet and a normal diet groups. Rats in the high-fat diet group were fed a high-fat diet, consisting of 10% egg yolk powder, 5% pork fat, and 0.5% pig bile salt combined with standard chow to create hyperlipidemia. Rats in the normal diet group were fed a standard rat chow. A total of 72 rats in both groups were randomly divided into 6 subgroups: sham-operated, 4-hour ischemia, 4-hour ischemia/2-hour reperfusion, 4-hour ischemia/4-hour reperfusion, 4-hour ischemia/6-hour reperfusion, and 4-hour ischemia/12-hour reperfusion, with 12 rats in each subgroup. MAIN OUTCOME MEASURES: nNOS expression was measured by immunohistochemistry, and pathomorphology changes were detected by hematoxylin-eosin staining. Infarct volume and nitric oxide levels were respectively measured using 2, 3, 5-triphenyltetrazolium chloride (TTC) and immunohistochemistry. RESULTS: In the ischemic region, pathology changes were significant in the 4-hour ischemia/4-hour, 4-hour ischemia/6-hour reperfusion, and 4-hour ischemia/12-hour reperfusion subgroups fed on a high-fat diet compared to the same groups fed on a normal diet. In each ischemia subgroup, nNOS expression in brain tissues was higher than in the sham-operated subgroups fed on either the high-fat diet or normal diet (P< 0.01). At each ischemia/reperfusion time point, rats fed on a high-fat diet expressed higher levels of nNOS compared to rats fed on the normal diet (P<0.05). When tissue was stained with TTC, a white infarction area was detected in the ischemic hemisphere, demonstrating that the infarct volume gradually increased with prolonged reperfusion time in each ischemia subgroup. At each ischemia/reperfusion time point, the infarct volume was larger in rats fed on a high-fat diet compared to those fed on a normal diet. CONCLUSION: nNOS expression was greater in hyperlipidemia rats following ischemia/reperfusion. Cerebral ischemia/reperfusion injury is aggravated with prolonged reperfusion time.     

Functional deterioration of endothelial nitric oxide synthase after focal cerebral ischemia

Endothelial nitric oxide synthase (eNOS) dysfunction is related to secondary injury and lesion expansion after cerebral ischemia. To date, there are few reports about postischemic alterations in the eNOS regulatory system. The purpose of the present study was to clarify eNOS expression, Ser1177 phosphorylation, and monomer formation after cerebral ischemia. Male Wistar rats were subjected to transient focal cerebral ischemia. Endothelial nitric oxide synthase messenger RNA (mRNA) and protein expression increased ∼8-fold in the ischemic lesion. In the middle cerebral artery core, eNOS-Ser1177 phosphorylation increased 6 hours after ischemia; however, there was an approximately 90% decrease in eNOS-Ser1177 phosphorylation observed 24 hours after ischemia that continued until at least 7 days after ischemia. Endothelial nitric oxide synthase monomer formation also increased 24 and 48 hours after ischemia (P<0.05), and protein nitration progressed in parallel with monomerization. To assess the effect of a neuroprotective agent on eNOS dysfunction, we evaluated the effect of fasudil, a Rho-kinase inhibitor, on eNOS phosphorylation and dimerization. Postischemic treatment with fasudil suppressed lesion expansion and dephosphorylation and monomer formation of eNOS. In conclusion, functional deterioration of eNOS progressed after cerebral ischemia. Rho-kinase inhibitors can reduce ischemic lesion expansion as well as eNOS dysfunction in the ischemic brain.     

Delayed treatment with sildenafil enhances neurogenesis and improves functional recovery in aged rats after focal cerebral ischemia

Increasing age decreases the number of new neurons in the dentate gyrus and the subventricular zone (SVZ). Sildenafil, a phosphodiesterase type 5 (PDE5) inhibitor, enhances neurogenesis in young rats. The present study tested the hypothesis that sildenafil augments neurogenesis in aged rats after focal cerebral ischemia. Nonischemic aged (18 months, n = 6) Wistar rats exhibited a significant reduction of actively proliferating and relatively quiescent cells in the SVZ measured by the number of minichromosome maintenance protein-2-positive (MCM-2+) cells, a marker of the proliferating cells, compared with nonischemic young (3-4 months, n = 8) rats. Occlusion of the middle cerebral artery did not increase the number of MCM-2+ cells in the SVZ of aged rats at 3 months after focal ischemia. However, treatment with sildenafil at a dose of 3 mg/kg (n = 8) daily for 7 consecutive days starting 7 days after focal ischemia significantly increased the number of MCM-2+ cells in the SVZ of aged rats compared with aged rats treated with saline (n = 8). Double immunostaining revealed that substantially more Ki67+ cells (a marker of proliferating cells) were doublecortin+ (a marker of migrating neuroblasts) in sildenafil-treated than in saline-treated aged animals. In addition, treatment with sildenafil significantly improved functional recovery compared with saline-treated rats. These data suggest that inhibition of PDE5 activity by sildenafil augments neurogenesis in the SVZ of aged ischemic rats, although these rats have reduced numbers of neural progenitor and stem cells in the SVZ.     

Nitric oxide synthase in cerebral ischemia

The results of our continuing studies on the role of nitric oxide (NO) in cellular mechanisms of ischemic brain damage as well as related reports from other laboratories are summarized in this paper. Repetitive ip administration ofN ^G-nitro-L-arginine (L-NNA), a NO synthase (NOS) inhibitor, protected against neuronal necrosis in the gerbil hippocampal CA1 field after transient forebrain ischemia with a bell-shaped response curve, the optimal dose being 3 mg/kg. Repeated ip administration of L-NNA also mitigated rat brain edema or infarction following permanent and transient middle cerebral artery (MCA) occlusion with a U-shaped response. The significantly ameliorative dose-range and optimal dose were 0.01–1 mg/kg and 0.03 mg/kg, respectively. Studies using a NO-sensitive microelectrode revealed that NO concentration in the affected hemisphere was remarkably increased by 15–45 min and subsequently by 1.5–4 h after MCA occlusion. Restoration of blood flow after 2 h-MCA occlusion resulted in enhanced NO production by 1–2 h after reperfusion. Administration of L-NNA (1 mg/kg, ip) diminished the increments in NO production during ischemia and reperfusion, leading to a remarkable reduction in infarct volume. In brain microvessels obtained from the affected hemisphere, Ca²⁺-dependent constitutive NOS (cNOS) was activated significantly at 15 min, and Ca²⁺-independent inducible NOS (iNOS) was activated invariably at 4 h and 24 h after MCA occlusion. Two hour reperfusion following 2 h-MCA occlusion caused more than fivefold increases in cNOS activity with no apparent alterations in iNOS activity. Thus, we report here based on available evidence that there is good reason to think that NOS activation in brain microvessels may play a role in the cellular mechanisms underlying ischemic brain injury.     

一氧化氮及一氧化氮合酶抑制剂在大鼠局灶性脑缺血时作用研究

目的：观察一氧化氮及一氧化氮合酶抑制剂在大鼠局灶性脑缺血时的作用方法;民凝大鼠大脑中动脉制成脑缺血模型,选择脑缺血３０ｍｉｎ,６０ｍｉｎ,１２０ｍｉｎ,１８０ｍｉｎ为研究时点,观察各时点用选择性,非选择性一氧化氮合酶抑制剂亚硝酸盐含量测定,缺血脑组织坏死体积测定及损伤海马ＣＡ１电镜观察。     

L-NAME对大鼠局灶性脑缺血灌注损伤Fos蛋白表达的影响

目的观察一氧化氮含量的变化对缺血再灌注损伤后Fos蛋白表达的影响。方法采用线拴法制作大鼠局灶性脑缺血再灌注损伤模型,利用NADPH组化和Fos蛋白免疫组化双标技术研究NOS抑制剂L－NAME对大鼠局灶性脑缺血再灌注损伤脑皮层Fos蛋白表达的影响。结果缺血60min再灌注3h后损伤侧脑组织皮质一氧化氮合酶阳性神经元较正常增多并深染,Fos蛋白表达增加,L－NAME（3mg／kg）治疗组脑皮质神经元Fos蛋白的表达量较对照组减少,L－NAME（10mg/kg）治疗组脑皮质神经元Fos蛋白的表达量较对照组明显减少,同时也可见给予L－NAME后脑组织皮质内NOS阳性神经元无论在数量上还是在细胞着色、胞体突起均明显减少。结论c－fos基因表达也可能部分参与了NO的致神经细胞损伤过程。     

依达拉奉预处理对小鼠脑缺血再灌注损伤后皮质一氧化氮合酶表达的影响

目的探讨依达拉奉预处理对小鼠脑缺血再灌注(IR)损伤后皮质一氧化氮合酶(NOS)表达的影响。方法 48只健康ICR小鼠被分为假手术组、对照组和依达拉奉组。依达拉奉组和对照组分别给予依达拉奉3 mg/(kg.d)和同等体积的生理盐水腹腔注射共7 d,然后建立小鼠IR模型;缺血1 h、再灌注24 h时应用2,3,5-氯化三苯基四氮唑(TTC)染色法测量各组脑梗死体积,应用免疫组化法检测各组小鼠皮质神经元型、、诱导型和内皮型NOS(nNOS、iNOS、eNOS)阳性细胞数。结果与假手术组比较,对照组小鼠皮质nNOS、iNOS和eNOS阳性细胞数明显增多(均P<0.05);与对照组比较,依达拉奉组脑梗死体积明显缩小,皮质nNOS和iNOS阳性细胞数明显减少,eNOS阳性细胞数明显增多(均P<0.05)。结论依达拉奉预处理可以影响IR小鼠皮质nNOS、iNOS和eNOS的表达,发挥神经保护作用。     

一氧化氮在脑缺血再灌流神经损伤中作用的实验研究

采用改良的Ｇｒｉｅｓｓ法，测定了脑缺血再灌流大鼠血清和脑组织中一氧化氮（ＮＯ）代谢产物ＮＯｘ（ＮＯ２＋ＮＯ３）的含量。结果表明，在脑缺血再灌流过程中实验大鼠血清和脑组织中ＮＯｘ含量的变化表现出独特的双峰现象，其第二高峰的出现时间与迟发性神经元损伤的发生相吻合。这提示，ＮＯ在脑缺血再灌流神经损伤中可能具有重要作用。     

Time dependent changes of striatal interneurons after focal cerebral ischemia in rats

Summary. The cellular damage over time and the alterations of neuronal subtypes was characterized in the striatum after 90-min middle cerebral artery occlusion and reperfusion in rats. We investigated the immunohistochemical alterations of choline acetyltransferase (ChAT)-positive (cholinergic-positive), γ-aminobutyric acid (GABA)ergic parvalbumin (PV)-positive, GABAergic nNOS (neuronal nitric oxide synthase)-positive interneurons, neuronal nuclei (NeuN)-positive spiny projection neurons, glial fibrillary acidic protein (GFAP)-positive strocytes and microglial response factor-1 (MRF-1)-positive microglia in the striatum after focal cerebral ischemia in rats. In the present study, transient focal cerebral ischemia in rats caused severe damage against interneurons as well as spiny projection neurons in the striatum. In contrast, a significant increase in the number of GFAP-immunopositive astrocytes was observed in the ipsilateral striatum 15 days after focal cerebral ischemia. Furthermore, a significant increase of MRF-1 immunoreactivity was observed in microglia of the ipsilateral striatum 7 days and 15 days after focal cerebral ischemia. Among three types of cholinergic interneurons, GABAergic PV-positive interneurons and GABAergic nNOS-positive interneurons, the severe damage of cholinergic and GABAergic PV-positive interneurons was more pronounced than that of GABAergic nNOS-positive interneurons after transient focal cerebral ischemia in rats. Furthermore, the present results suggest that GABAergic nNOS-positive interneurons in the striatum after focal cerebral ischemia undergo cellular death in a delayed manner. Correspondence: Tsutomu Araki, Department of Neurobiology and Therapeutics, Graduate School and Faculty of Pharmaceutical Sciences, The University of Tokushima, 1-78 Sho-machi, Tokushima 770-8505, Japan     

Cervical sympathetic trunk transection affects inducible nitric oxide synthase expression in rat hippocampus following focal cerebral ischemia/reperfusion injury

BACKGROUND： The stellate ganglion block （SGB） plays a protective role in focal cerebral ischemia/reperfusion injury. The human SGB can be simulated by transection of the cervical sympathetic trunk （TCST） in rats. OBJECTIVE： To observe the effects of TCST on inducible nitric oxide synthase （iNOS） levels and cerebral infarct volume in the hippocampus of rats with cerebral ischemia/reperfusion injury, and to analyze the mechanism of action. DESIGN, TIME AND SETTING： A completely randomized, controlled, neuropathological experiment was performed at the Institute of Neurological Disease, Taihe Hospital, Yunyang Medical College between March and September 2006. MATERIALS： A total of 93 Wistar rats, aged 1718 weeks, of either gender, were used for this study. 2, 3, 5-triphenyl tetrazolium chloride was purchased from Changsha Hongyuan Biological Reagent Company China. Rabbit iNOS antibody and goat anti-rabbit IgG antibody were the products of Wuhan Boster Biological Reagent Co., Ltd., China. METHODS： Ten rats were randomly selected for the sham-operated group. Cerebral ischemia/reperfusion injury was induced by middle cerebral artery occlusion （MCAO） using the suture method in the remaining rats. Forty successful rat models were randomly and equally divided into the following two groups： （1） TCST group： subsequent to TCST, MCAO was performed for 2 hours, followed by 24 hours reperfusion; （2） model group： rats underwent experimental procedures similar to the TCST group, with the exception of TCST. Rats in the sham-operated group were subjected to experimental procedures similar to the model group; however, the thread was only introduced to a depth of 10 mm. MAIN OUTCOME MEASURES： Following 24 hours of reperfusion, functional neurological deficits were scored. Brain tissue sections from ten rats of each group were used to measure cerebral infarct volume by TTC staining. Hippocampal tissue sections of an additional ten rats from each group were used to detect iNOS levels using the s     

神经元型一氧化氮合酶的神经生物学研究进展(英文)

神经元型一氧化氮合酶(neuronal nitric oxide synthase,nNOS)主要表达于神经元,在星形胶质细胞和神经干细胞中也有一定水平的表达。不同的mRNA拼接形式产生了nNOS蛋白的5种亚型,包括nNOS- 、nNOS- 、nNOS- 、nNOS- 和nNOS-2。nNOS单体不具催化活性,二聚体是其活性形式。nNOS单体发生二聚化需要四氢生物蝶呤、血红素以及L-精氨酸的结合。nNOS的表达在很大程度上依赖于cAMP反应元件结合蛋白的活化,其催化活性的调节与热休克蛋白90/ 热休克蛋白70、钙调节蛋白、PIN 蛋白,以及自身Ser847和Ser1412位点的磷酸化和脱磷酸化相关。能与nNOS相互作用的蛋白主要有9种,包括突触后密度蛋白95(post-synaptic density protein 95, PSD95)、CALM、CAMKIIA、DLG4、DLG2、PFK-M、CAPON、syntrophin和dynein轻链。其中 PSD95、CAPON和PFK-M是神经元中最重要的 nNOS 调节蛋白。PSD95与nNOS 的相互作用能介导突触形成,并参与N-甲基-D-天冬氨酸诱导的神...     

神经节苷脂对不完全性脑缺血后海马CA1区NOS阳性神经元表达的影响

目的 :探讨神经节苷脂 (GM1)对不完全性脑缺血及再灌注不同时间后海马CA1区一氧化氮合酶 (NOS)的影响及对神经元的保护作用。方法 :用双侧颈总动脉夹闭加放血的方法制成大鼠不完性脑缺血及再灌注模型 ,以还原尼克酰胺腺嘌呤二核苷酸脱氢酶 (NADPH d)组织化学方法观察缺血及再灌注后海马CA1区NOS阳性神经细胞变化及GM1对其影响。结果 :海马CA1区神经细胞受损 ,在缺血 30min时NOS阳性细胞数最高 (44 .5±7.4 ) ,为对照组的 2倍 ,再灌注 2h ,12h ,2 4h ,3d后逐渐下降 ,5d时恢复正常水平。而GM1能防止脑缺血及再灌注后神经细胞受损和NOS阳性神经细胞变化。 结论 :GM1对大鼠不完全性脑缺血及再灌注不同时间后海马CA1区NOS的表达有抑制作用 ,并对神经元具有保护作用。     

NOS抑制剂对局灶性缺血再灌流大鼠脑神经细胞凋亡的影响研究

目的 研究一氧化氮合酶（ＮＯＳ）抑制剂在局灶性脑缺血再灌流过程中对神经细胞的作用机理,特别是ＮＯＳ抑制剂（ＮＮＬＡ）与细胞凋亡的关系。方法 利用末端标记法,测定ＮＮＬＡ干预后局灶性脑缺血鼠脑组织中神经细胞的凋亡情况。结果 大、小剂量的ＮＯＳ抑制剂分别具有增加或减少阳性凋亡细胞出现率的作用。结论 给予不同剂量的ＮＯＳ抑制剂与单纯缺血再灌流相比,对脑神经细胞的凋亡出现率有不同的影响。     

Effects of tetrahydrobiopterin on cerebral infarction after transient focal ischemia in rats

Abstract

Objectives: The present study investigated the effects of tetrahydrobiopterin (BH4) on cerebral infarction after transient focal ischemia in rats.

Methods: Focal ischemia (1·5 hours) was created in male Sprague-Dawley rats (250-280 g) by middle cerebral artery occlusion. Some rats were treated with 20 mg/kg tetrahydrobiopterin by intraperitoneal injection 30 minutes before reperfusion. At 2, 6, and 12 hours of reperfusion, the brains were harvested for the nitric oxide synthase (NOS) activity and nitric oxide (NO) level assays. At 12 hours of reperfusion, the brains were harvested for infarct size measurement.

Results: NOS activity and NO level were all augmented after reperfusion. BH4 treatment significantly further increased NOS activity and NO level. Cerebral infarct size was significantly bigger in BH4 treatment group compared to that in no treatment group.

Conclusions: The data indicate that BH4 enhances cerebral infarction after transient focal ischemia in rats, through NOS and NO pathway.     

Upregulation of neuronal nitric oxide synthase and mRNA, and selective sparing of nitric oxide synthase-containing neurons after local cerebral ischemia in rat

Nitric oxide synthase-containing neurons are presumed to be resistant to neurodegeneration and neurotoxicity, however this resistance has not been demonstrated after focal cerebral ischemia. We therefore measured the temporal profile of neuronal nitric oxide synthase (NOS-I) mRNA and immunoreactivity and NADPH-diaphorase reactivity over a one week period after permanent middle cerebral artery (MCA) occlusion in 48 male Wistar rats and compared these data to ischemic cell damage as evaluated on hematoxylin and eosin (H & E) stained sections by light microscopy. NOS-I mRNA increased as early as 15 min after MCA occlusion in the ipsilateral striatum and maximal expression of NOS-I was found in the ipsilateral cortex and striatum 1 h after MCA occlusion. The numbers of NOS-I-containing neurons in the ipsilateral cortex and striatum were significantly greater (P < 0.05) than NOS-I-containing neurons in the contralateral hemisphere at 2–48 h after the onset of ischemia. The number of NOS-I-containing neurons peaked at 4 h after MCA occlusion. Neurons exhibited shrinkage or were swollen at 1 to 4 h after MCA occlusion. At 24–48 h after ischemia, neurons in the ischemia lesion appeared to be eosinophilic or ghost like on H & E stained sections. However, some of these neurons retained morphological integrity on the NOS-I immunohistochemical sections. At 168 h after ischemia, all neurons within the lesion appeared necrotic on H & E stained sections; however, scatterred neurons expressed NOS-I and NADPH-diaphorase. The rapid upregulation of NOS-I and mRNA in the ischemic lesion suggests that NOS-I is involved in focal cerebral ischemic injury; the expression of NOS-I by neurons that retain their morphological structure in the area of the infarct suggests that NOS-I-containing neurons are more resistant to the ischemic insult. Our data also indicate a close association of NOS-I immunoreactivity and NADPH-diaphorase reactivity in ischemic brain.     

局灶性鼠脑缺血时一氧化氮及一氧化氮合酶抑制剂的作用机制

目的研究一氧化氮在鼠脑局灶性脑缺血再灌注损伤中的作用。方法用线栓法建立大鼠大脑中动脉区缺血再灌注模型，分别用选择性和非选择性诱导型一氧化氮合酶抑制剂对鼠脑局灶性缺血再灌注过程中脑组织一氧化氮的变化规律及可能作用进行探讨。结果非选择性一氧化氮合酶抑制剂（Ｌ－ＮＡＭＥ）可加重局灶性脑缺血性损害，而选择性诱导型一氧化氮合酶抑制剂（ａｍｉｎｏｇｕａｎｉｄｉｎｅ，ＡＧ）具有明确的脑保护作用。结论不同类型的一氧化氮合酶所产生的一氧化氮在脑局灶性缺血性损害中具有不同的作用。     

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;     

大鼠脑缺血再灌流后海马区一氧化氮合酶活性的变化

目的 :观察鼠全脑缺血再灌流后海马区NOS活性的变化。方法 :采用大鼠 4血管关闭方法制作全脑缺血再灌流模型。实验动物分为假手术组、缺血 10min组、再灌注 1、2、3d组 ,测定脑缺血再灌流后海马区NOS活性的变化。结果 :全脑缺血再灌注后海马组织NOS活性被激活上调。结论 :NO可能参与了海马CA1区迟发性神经元死亡 (DND)的发生。