一氧化氮／一氧化氮合酶与神经创伤

一氧化氮是一种简单的气体分子,可在哺乳类神经细胞内经一氧化氮合酶作用产生。ＮＯ在神经创伤修复中的多重作用近年来已受到越来越多的重视。本文对ＮＯ／ＮＯＳ与神经创伤和再生之间的关系作一综述。     

Expression of nitric oxide synthase in the cerebral microvasculature after traumatic brain injury in the rat

Reduced nicotinamide adenine dinucleotide phosphate-diaphorase (NADPHd) histochemistry and nitric oxide synthase (NOS) immunocytochemistry were performed on sections of brain after moderate traumatic brain injury. There was a pronounced increase in NADPHd reactivity and an induction of the endothelial NOS (eNOS) isoform in microvessels surrounding the cortical contusion by 24 h post-injury. This altered microvascular state may contribute to barrier breakdown and hyperemia which characterize traumatic brain injury.     

L-NIL在大鼠创伤性脑损伤中的应用研究

目的探讨选择性诱导型一氧化氮合酶(iNOS)抑制剂L-N6-亚氨乙基-赖氨酸(L-NIL)在大鼠创伤性脑损伤(TBI)模型中对大鼠伤后学习记忆功能、海马神经元的影响.方法 24只SD大鼠随机分为假创伤性脑损伤组、创伤性脑损伤组、非选择性一氧化氮合酶(NOS)抑制剂N-硝基-L-精氨酸(L-NNA)组、选择性诱导型一氧化氮合酶抑制剂L-NIL组.其中L-NNA组、L-NIL组分别于伤后即刻、24 h、48 h经腹腔注射L-NNA或L-NIL.进行避暗回避试验以评价伤后各组大鼠的学习记忆功能恢复情况.伤后第7 d处死大鼠取脑,采用Nissl染色及神经元抗核抗体(NeuN)免疫组化染色观察测量伤侧海马CA2区锥体细胞层面积.结果①L-NIL组避暗回避试验结果显著好于创伤性脑损伤组(P<0.05).②L-NIL组海马CA2区锥体细胞层面积与创伤性脑损伤组比较显著增加(P<0.05).③L-NIL组避暗回避试验结果及海马CA2区锥体细胞层面积值均优于L-NNA组.结论适当地应用选择性iNOS抑制剂L-NIL可促进大鼠脑损伤后学习记忆功能的恢复,保护海马神经元,其效果优于非选择性NOS抑制剂.     

Inhaled nitric oxide reduces secondary brain damage after traumatic brain injury in mice

Ischemia, especially pericontusional ischemia, is one of the leading causes of secondary brain damage after traumatic brain injury (TBI). So far efforts to improve cerebral blood flow (CBF) after TBI were not successful because of various reasons. We previously showed that nitric oxide (NO) applied by inhalation after experimental ischemic stroke is transported to the brain and induces vasodilatation in hypoxic brain regions, thus improving regional ischemia, thereby improving brain damage and neurological outcome. As regional ischemia in the traumatic penumbra is a key mechanism determining secondary posttraumatic brain damage, the aim of the current study was to evaluate the effect of NO inhalation after experimental TBI. NO inhalation significantly improved CBF and reduced intracranial pressure after TBI in male C57 Bl/6 mice. Long-term application (24 hours NO inhalation) resulted in reduced lesion volume, reduced brain edema formation and less blood–brain barrier disruption, as well as improved neurological function. No adverse effects, e.g., on cerebral auto-regulation, systemic blood pressure, or oxidative damage were observed. NO inhalation might therefore be a safe and effective treatment option for TBI patients.     

大鼠实验性脑损伤血浆中NO及NOS的动态变化研究

目的 测定实验性脑损伤后血浆中一氧化氮含量、一氧化氮合酶活性,研究其与脑水肿之间的关系.方法 大鼠随机分组,用硝酸还原酶法测定血清中NO含量、NOS活性,及测定脑组织含水量.并行还原型辅酶Ⅱ依赖性黄递酶(NADPⅡ-d)组化染色检测皮层及脑底NOS阳性细胞.结果 (1)TBI后血浆内NO含量、NOS活力即有升高,与对照组比较有明显差异(P＜0.05).(2)脑组织含水量在外伤后升高,与对照组比较有明显差异(P＜0.05).与血浆中NO含量、NOS活力变化趋势一致.(3) NADPⅡ-d组化染色显示TBI皮层NOS阳性细胞明显多于正常对照组,伤灶脑底也出现了染色块及浓染的细胞群与阳性纤维束.结论 大鼠TBI后NO含量、NOS活性的升高,与脑水肿的发生有关.     

糖皮质激素干预肺源性脑损伤大鼠血清一氧化氮合酶活性和一氧化氮含量的变化

实验观察地塞米松及Gi蛋白抑制剂百日咳毒素,转录抑制剂放线杆菌素干扰后肺源性脑损伤大鼠模型大鼠血清一氧化氮合酶活性,一氧化氮含量变化。 发现高剂量地塞米松(13 mg/kg)和地塞米松联合放线菌干预肺源性脑损伤模型大鼠肺含水量降低,血清一氧化氮合酶活性和一氧化氮含量明显增高,肺含水量降低,肺泡间质内炎细胞浸润减少,脑膜血管充血明显减轻,浸润的胶质细胞较少。结果说明,高剂量糖皮质激素可通过提高血清一氧化氮合酶活性,增加一氧化氮含量在肺源性脑损伤中发挥保护作用。     

大鼠重型颅脑损伤急性期水通道蛋白4的表达

目的探讨水通道蛋白（AQP4）在大鼠重型脑外伤急性期的表达变化及其与脑水肿间的关系。方法49只成年雄性SD大鼠,随机分为对照组及实验组（伤后4h、8h、12h、24h、5d共5组）。制作重度冲击加速性损伤模型,分别于伤后4h、8h、12h、24h、72h、5d采用干湿比重法测脑组织含水量,原子吸收分光光度法测定Na^＋、K^＋含量,Evans Blue（EB）测定法观察大鼠血-脑屏障（BBB）通透性变化,半定量逆转录聚合酶链反应（RT-PCR）检测脑组织AQP4 mRNA表达及其变化。结果脑组织AQP4 mRNA在伤后4h开始表达上调,8h、12h依次增高,24h达到峰值（P〈0.05）,3d时仍维持较高水平,伤后5d有所降低。脑含水量、Na^＋含量的变化与AQP4 mRNA表达变化一致。经相关性分析,AQP4 mRNA的表达与脑含水量及脑EB含量均呈正相关（P〈0.05）。结论重型脑损伤急性期,AQP4 mRNA表达的变化与颅脑损伤后BBB的破坏及脑水肿的形成和发展密切相关。AQP4可能参与重型脑损伤后脑水肿的形成并起重要作用。     

青白散对慢性软组织损伤模型大鼠骨骼肌一氧化氮合酶系统和一氧化氮的影响*☆

背景：对慢性软组织损伤后一氧化氮合酶系统和一氧化氮的研究目前较少。 目的：观察青白散对大鼠慢性软组织损伤模型骨骼肌中一氧化氮合酶系统和一氧化氮的影响。 方法：雄性 SD 大鼠随机分为对照组、模型组、氨基胍组、青白散组。后3组采用机械损伤法制备慢性骨骼肌损伤动物模型,分别予以生理盐水 10 mL/kg,0.10 g/kg氨基胍,0.54 g/kg青白散,1次/d,连续 14 d。于给药后1,2,3周,分别检测大鼠肌组织一氧化氮含量、总一氧化氮合酶和诱导型一氧化氮合酶的活性。 结果与结论：骨骼肌损伤修复过程中,模型组大鼠骨骼肌中一氧化氮含量、总一氧化氮合酶和诱导型一氧化氮合酶的活性较对照组显著增高;而青白散组和氨基胍组大鼠骨骼肌中一氧化氮含量、总一氧化氮合酶和诱导型一氧化氮合酶的活性均较模型组显著降低。说明青白散可能通过阻抑诱导型一氧化氮合酶诱导过量一氧化氮的产生,为慢性软组织损伤的修复创造了有利条件。     

小剂量rhIL—2对实验性颅脑伤中神经功能的保护作用

目的：为探讨白细胞介素－２（ＩＬ－２）对脑损伤后神经功能恢复的作用效应及机理。方法：采用侧方液压大鼠脑损伤模型，观察侧脑室注射（ｉ．ｃ．ｖ．）小剂量ｒｈＩＬ－２对大鼠神经功能和组织病理变化的影响。实验选用雄性ＳＤ大鼠，伤后１～３天连续ｉ．ｃ．ｖ．ｒｈＩＬ－２，分３组（３０，６０，３００Ｕ），等量生理盐水治疗为对照组。体重测定，平衡试验，行走试验评价神经功能。结果：发现ｒｈＩＬ－２能明显改善大鼠脑伤后神经功能障碍，减缓体重丢失，促进运动功能和平衡功能恢复（Ｐ＜０．０１），尤以６０Ｕ治疗效果最显著；并且能缩小皮层空洞。６０Ｕ治疗伤后２周时的空洞为０．０７ｍｍ×０．１０ｍｍ，对照组为０．５１ｍｍ×１．００ｍｍ，相差显著（Ｐ＜０．０１），ｒｈＩＬ－２治疗组海马ＣＡ２，ＣＡ３区神经元较对照组死亡少。结论：小剂量ｒｈＩＬ－２具有显著的神经保护作用，参与脑创伤后的修复，具有潜在的临床应用价值。     

r—HuEPO对外伤性脑损伤大鼠凋亡基因表达的影响

目的探讨重组人促红细胞生成素（r—HuEPO）对外伤性脑损伤大鼠凋亡基因表达的影响。方法将48只健康成年雄性SD大鼠随机分成4组：r-HuEPO1000U／kg、3000U／kg、5000U／kg治疗组和生理盐水对照组。采用改良的Feeney氏法制作大鼠自由落体脑创伤模型,r—HuEPO干预。一周后麻醉取脑冻存：免疫组织化学法测定脑组织中NF—κB、C-myc和Fas／Fasl的阳性细胞及凋亡细胞数。结果与对照组相比,各组中NF—KB、Fas和Fasl阳性细胞及凋亡细胞数均有显著减少（P〈0．05）,尤其是5000U／kgr—HuEPO组减少最显著（P〈0．01）。结论r—HuEPO可抑制创伤性脑损伤大鼠NF—KB、Fas／Fasl的促凋亡作用,减轻迟发性神经元损伤,从而起到神经保护的作用。     

黄体酮对创伤性脑损伤大鼠神经细胞凋亡的影响

目的 观察黄体酮对脑外伤后神经细胞凋亡的影响,探讨其对脑外伤(TBI)继发性脑损伤是否存在保护作用.方法 雄性Wistar 大鼠随机分为无脑损伤的假手术(sham)组、脑损伤(TBI)组、脑损伤后注射黄体酮治疗(P-TBI)组及注射二甲基亚砜(DMSO)溶剂(D-TBI)组,每大组24 只,再分1 d、3 d、5 d 和7 d 四个小组,每小组6 只.采用Freeney 法造成鼠脑挫裂伤模型,在伤后四个不同时相点用TUNEL 染色法分别检测四组大鼠脑组织中神经细胞凋亡情况.结果 创伤性脑损伤后凋亡细胞数量在TBI 第1 d 明显增加,TBI后第7 d 神经细胞凋亡达到高峰.伤后注射黄体酮治疗组神经细胞凋亡指数明显下降(P ＜0.05).结论 大鼠TBI 后周围脑组织神经细胞凋亡在伤后持续增加,注射黄体酮能抑制细胞凋亡,对脑组织有一定保护作用.     

Role of nitric oxide in the brain during lipopolysaccharide-evoked systemic inflammation

Although the inducible isoform of nitric oxide synthase (iNOS) is a well-established source of nitric oxide (NO*) during inflammation of the central nervous system (CNS), little is known about the involvement of constitutive isoforms of NOS (cNOS) in the inflammatory process. The aim of this study was to compare the responses of the expression and activity of iNOS and the two cNOS isoforms, neuronal and endothelial (nNOS and eNOS, respectively), in the brain to systemic inflammation and their roles in the cascade of events leading to degeneration and apoptosis. A systemic inflammatory response in C57BL/6 mice was induced by intraperitoneal injection of lipopolysaccharide [LPS; 1 mg/kg body weight (b.w.)]. The relative roles of the NOS isoforms were evaluated after injection of NG-nitro-L-arginine (NNLA; 30 mg/kg b.w.), which preferentially inhibits cNOS, or 1400W (5 mg/kg b.w.), an inhibitor of iNOS. Biochemical and morphological alterations were analyzed up to 48 hr after administration of LPS. Systemic LPS administration evoked significant ultrastructural alterations in brain capillary vessels, neuropils, and intracellular organelles of neurons, astrocytes, and microglia. Apoptotic/autophagic processes occurred in many neurons of the substantia nigra (SN), which coincided with exclusive enhancement of iNOS expression and activity in this brain region. Moreover, inhibitors of both iNOS and cNOS prevented LPS-evoked release of apoptosis-inducing factor (AIF) from SN mitochondria. Collectively, the results indicate that synthesis of NO* by both the inducible and constitutive NOS isoforms contribute to the activation of apoptotic pathways in the brain during systemic inflammation.     

癫痫患儿血清一氧化氮和一氧化氮合酶含量的变化及意义

目的　探讨癫疒间 患儿血清一氧化氮 (NO)、一氧化氮合酶 (NOS)的变化及意义。方法　利用ELISA方法 ,测定 5 8例癫疒间 患儿 (癫疒间 组 )和 2 3名健康儿童 (对照组 )血清中NO、NOS的含量 ,并分组比较不同条件下其含量的变化。结果　癫疒间 组血清NO、NOS的含量分别为 (5 .86± 1.2 1) μmol/ml和 (2 8.2 6± 8.4 9)U/ml,较对照组的 (3.78± 0 .74 ) μmol/ml及 (17.86± 4 .5 8)U/ml明显升高 (P <0 0 1) ;发作近期为 (7.31± 1.2 7)μmol/ml和 (31.2 5± 11.35 )U/ml,明显高于发作间期 (4 .2 7± 0 .6 6 ) μmol/ml和 (2 4 .15± 7.85 )U/ml(P <0 0 1) ;癫疒间 组EEG异常者为 (7.18± 1.35 ) μmol/ml和 (34.4 8± 8.5 6 )U/ml,明显高于EEG正常者 (4 .0 4± 0 .75 ) μmol/ml和 (2 2 .85± 7.4 5 )U/ml(P <0 0 1) ;但与发作类型、病程及是否接受治疗无关 (P >0 0 5 )。结论　癫疒间 发作近期血中NO、NOS生成增加 ,NO作为内源性调质参与癫疒间 发作病理生理过程     

Role of endothelial nitric oxide synthase for early brain injury after subarachnoid hemorrhage in mice

The first few hours and days after subarachnoid hemorrhage (SAH) are characterized by cerebral ischemia, spasms of pial arterioles, and a significant reduction of cerebral microperfusion, however, the mechanisms of this early microcirculatory dysfunction are still unknown. Endothelial nitric oxide production is reduced after SAH and exogenous application of NO reduces post-hemorrhagic microvasospasm. Therefore, we hypothesize that the endothelial NO-synthase (eNOS) may be involved in the formation of microvasospasms, microcirculatory dysfunction, and unfavorable outcome after SAH. SAH was induced in male eNOS deficient (eNOS^–/–) mice by endovascular MCA perforation. Three hours later, the cerebral microcirculation was visualized using in vivo 2-photon-microscopy. eNOS^–/– mice had more severe SAHs, more severe ischemia, three time more rebleedings, and a massively increased mortality (50 vs. 0%) as compared to wild type (WT) littermate controls. Three hours after SAH eNOS^–/– mice had fewer perfused microvessels and 40% more microvasospasms than WT mice. The current study indicates that a proper function of eNOS plays a key role for a favorable outcome after SAH and helps to explain why patients suffering from hypertension or other conditions associated with impaired eNOS function, have a higher risk of unfavorable outcome after SAH.     

Inhibitors of advanced glycation end-products prevent loss of enteric neuronal nitric oxide synthase in diabetic rats.

Gastrointestinal dysfunction is common in diabetes, and several studies indicate that loss of neuronal nitrergic inhibition may play an important role in its pathogenesis. However, the mechanisms responsible for this effect remain largely unknown. We have previously shown that advanced glycation end-products (AGEs) formed by non-enzymatic glycation dependent processes, can inhibit the expression of intestinal neuronal nitric oxide synthase (nNOS) in vitro acting via their receptor, receptor for AGEs. We now hypothesized that this effect may also be important in experimental diabetes in vivo. We aimed to evaluate the role of AGEs on duodenal nNOS expression and the effects of aminoguanidine (a drug that prevents AGE formation) and ALT-711 (AGE cross-link breaker) in experimental diabetes. Streptozotocin induced diabetic rats were randomized to no treatment, treatment with aminoguanidine (1 g L(-1) daily through drinking water) at the induction of diabetes, or treatment with ALT-711 (3 mg kg(-1) intraperitoneally), beginning at week 6. A fourth group was used as healthy controls. We performed real time polymerase chain reaction, Western blotting and immunohistochemistry to detect nNOS expression. AGE levels were analysed using sandwich ELISA. Diabetes enhanced accumulation of AGEs in serum, an effect that was prevented by treatment with aminoguanidine and ALT-711. Further, diabetic rats showed a significant reduction in duodenal nNOS expression by mRNA, protein and immunocytochemistry, an effect that was prevented by aminoguanidine. ALT-711 had similar effects on nNOS protein and immunohistochemistry (but not on mRNA levels). The generation of AGEs in diabetes results in loss of intestinal nNOS expression and may be responsible for enteric dysfunction in this condition. This study suggests that treatment directed against AGEs may be useful for the treatment of gastrointestinal complications of diabetes.     

Delayed increase in neuronal nitric oxide synthase immunoreactivity in thalamus and other brain regions after hypoxic-ischemic injury in neonatal rats

We examined the response of neuronal nitric oxide synthase (nNOS)-containing CNS neurons in rats exposed to a unilateral hypoxic-ischemic insult at 7 days of age. Animals were sacrificed at several time points after the injury, up to and including 7 days (Postnatal Day 14). Brain regions ipsilateral to the injury (including cerebral cortex, caudate-putamen, and thalamus) exhibited delayed, focal increases in nNOS immunoreactivity. The increase in nNOS immunoreactive fiber staining was prominent in areas adjacent to severe neuronal damage, especially in the cortex and the thalamus, regions that are also heavily and focally injured in term human neonates with hypoxic-ischemic encephalopathy. In cerebral cortex, these increases occurred despite modest declines in nNOS catalytic activity and protein levels. Proliferation of surviving nNOS immunoreactive fibers highlights regions of selective vulnerability to hypoxic-ischemic insult in the neonatal brain and may also contribute to plasticity of neuronal circuitry during recovery.     

大剂量伽玛刀照射后脑组织一氧化氮合酶亚型表达改变及其与急性脑水肿的关系

目的探讨大剂量伽玛刀(γ刀)照射后脑组织一氧化氮合酶(NOS)亚型表达改变及其与急性脑水肿的关系。方法200Gy量γ刀照射正常大鼠脑,采用光镜、电镜、免疫组化及原位杂交技术研究照射后急性脑水肿的发生发展及脑组织NOS亚型表达变化。结果①照射后30min出现急性脑水肿病理改变,照射后2h出现明显血管源性脑水肿,照射后6h出现明显细胞性脑水肿,照射后3d急性脑水肿达高峰。②脑组织NOS亚型表达在照射后30min开始增高,内皮型一氧化氮合酶(eNOS)及诱导型一氧化氮合酶(iNOS)表达分别于照射后2h及6h显著增高,神经元型一氧化氮合酶(nNOS)表达亦增高,但nNOS阳性表达细胞数量较少。3种NOS亚型表达增高持续时间长,伴行于脑水肿的急性发展阶段。结论大剂量γ刀照射后脑组织NOS亚型表达增高与急性脑水肿的发生发展有关。     

能量限制对大鼠创伤性脑损伤的保护作用

目的能量限制（CR）可产生脑缺血耐受作用,但详细机制尚不清楚。本研究主要探讨CR是否对脑外伤引起的脑损伤起到脑保护作用,及其作用是否与一氧化氮（NO）产生有关。方法SD大鼠分为两组：CR和自由饮食（AL）组,喂养4W后,行大鼠脑损伤模型,进行神经功能评分,并监测造模前后大鼠脑组织及血清中的内皮型一氧化氮合酶（eNOS）和NO的含量。结果①CR组脑组织和血清内eNOS表达和NO含量明显高于AL组（P〈0．05）;②神经功能评分显示,CR组大鼠神经功能评分明显好于AL组（P〈0．05）。结论CR可通过上调eNOS表达和促进NO合成对脑外伤引起的脑损伤起到脑保护作用。     

Hyaluronidase reduced edema after experimental traumatic brain injury

Cerebral edema and the subsequent increased intracranial pressure are associated with mortality and poor outcome following traumatic brain injury. Previous in vitro studies have shown that the Gibbs-Donnan effect, which describes the tendency of a porous, negatively charged matrix to attract positive ions and water, applies to brain tissue and that enzymatic reduction of the fixed charge density can prevent tissue swelling. We tested whether hyaluronidase, an enzyme that degrades the large, negatively charged glycosaminoglycan hyaluronan, could reduce brain edema after traumatic brain injury. In vivo, intracerebroventricular injection of hyaluronidase after controlled cortical impact in mice reduced edema in the ipsilateral hippocampus at 24 h by both the wet-weight/dry-weight method (78.15 ± 0.65% vs. 80.4 ± 0.46%; p < 0.01) and T₂-weighted magnetic resonance imaging (13.88 ± 3.09% vs. 29.23 ± 6.14%; p < 0.01). Hyaluronidase did not adversely affect blood–brain-barrier-integrity measured by dynamic contrast-enhanced magnetic resonance imaging, nor did hyaluronidase negatively affect functional recovery after controlled cortical impact measured with the rotarod or Morris water maze tasks. Reduction of fixed charge density by hyaluronidase was confirmed in cortical explants in vitro (5.46 ± 1.15 µg/mg vs. 7.76 ± 1.87 µg/mg; p < 0.05). These data demonstrate that targeting the fixed charge density with hyaluronidase reduced edema in an in vivo mouse model of traumatic brain injury.     

Neuroprotective effects of bloodletting atJing points combined with mild induced hypothermia in acute severe traumatic brain injury

Bloodletting at Jing points has been used to treat coma in traditional Chinese medicine. Mild induced hypothermia has also been shown to have neuroprotective effects. However, the therapeutic effects of bloodletting at Jing points and mild induced hypothermia alone are limited. Therefore, we investigated whether combined treatment might have clinical effectiveness for the treatment of acute severe traumatic brain injury. Using a rat model of traumatic brain injury, combined treatment substantially alleviated cerebral edema and bloodbrain barrier dysfunction. Furthermore, neurological function was ameliorated, and cellular necrosis and the inflammatory response were lessened. These findings suggest that the combined effects of bloodletting at Jing points(20 μL, twice a day, for 2 days) and mild induced hypothermia(6 hours) are better than their individual effects alone. Their combined application may have marked neuroprotective effects in the clinical treatment of acute severe traumatic brain injury.