Knockdown of NADPH oxidase 4 reduces mitochondrial oxidative stress and neuronal pyroptosis following intracerebral hemorrhage

Intracerebral hemorrhage is often accompanied by oxidative stress induced by reactive oxygen species,which causes abnormal mitochondrial function and secondary reactive oxygen species generation.This creates a vicious cycle leading to reactive oxygen species accumulation,resulting in progression of the pathological process.Therefore,breaking the cycle to inhibit reactive oxygen species accumulation is critical for reducing neuronal death after intracerebral hemorrhage.Our previous study found that increased expression of nicotinamide adenine dinucleotide phosphate oxidase 4(NADPH oxidase 4,NOX4)led to neuronal apoptosis and damage to the blood-brain barrier after intracerebral hemorrhage.The purpose of this study was to investigate the role of NOX4 in the circle involving the neuronal tolerance to oxidative stress,mitochondrial reactive oxygen species and modes of neuronal death other than apoptosis after intracerebral hemorrhage.We found that NOX4 knockdown by adeno-associated virus(AAV-NOX4)in rats enhanced neuronal tolerance to oxidative stress,enabling them to better resist the oxidative stress caused by intracerebral hemorrhage.Knockdown of NOX4 also reduced the production of reactive oxygen species in the mitochondria,relieved mitochondrial damage,prevented secondary reactive oxygen species accumulation,reduced neuronal pyroptosis and contributed to relieving secondary brain injury after intracerebral hemorrhage in rats.Finally,we used a mitochondria-targeted superoxide dismutase mimetic to explore the relationship between reactive oxygen species and NOX4.The mitochondria-targeted superoxide dismutase mimetic inhibited the expression of NOX4 and neuronal pyroptosis,which is similar to the effect of AAV-NOX4.This indicates that NOX4 is likely to be an important target for inhibiting mitochondrial reactive oxygen species production,and NOX4 inhibitors can be used to alleviate oxidative stress response induced by intracerebral hemorrhage.

Effects of dexamthasone with different doses on aquaporin-4 in brain of intracerebral hemorrhage rats

Objective：To determine the relationship between the expression of aquaporin-4（AQP4） after intracerebral hemorrhage and dexamethasone treated. Methods：Collagenase Ⅶ was injected in caudate nucleus in a stereotaxis frame to establish the intracerebral hemorrhage（ICH） animal models. The intracerebral hemorrhage（ICH） rats were randomly divided into four groups： the sham group （group A）, the ICH group（group B）, low dose-treated group（group C）, moderate dose group（group D） and high dose group（group E）. The groups were respectively received an intraperitoneal dexamethasone injection with 1 mg/kg, 15 mg/kg, 30 mg/kg, twice a day for three days. The brain water content（BWC）, the permeability of blood-brain barrier（BBB） and the expression of AQP4 were observed. Results：Both the BBB disruption and AQP4 expression decreased in treated groups, and the AQP4 expression had a dose-dependent manner in the dexamethasone treatment. And it seemed that low dose dexamethasone was in favor of brain swelling elimination, but the higher dosage had not similar effect. Conclusion：Dexamethesone may play a critical role on expression of AQP4 in the physiopathology of hemorrhagic edema.

Intranasal insulin ameliorates neurological impairment after intracerebral hemorrhage in mice

In Alzheimer’s disease and ischemic stroke,intranasal insulin can act as a neuroprotective agent.However,whether intranasal insulin has a neuroprotective effect in intracerebral hemorrhage and its potential mechanisms remain poorly understood.In this study,a mouse model of autologous blood-induced intracerebral hemorrhage was treated with 0.5,1,or 2 IU insulin via intranasal delivery,twice per day,until 24 or 72 hours after surgery.Compared with saline treatment,1 IU intranasal insulin treatment significantly reduced hematoma volume and brain edema after cerebral hemorrhage,decreased blood-brain barrier permeability and neuronal degeneration damage,reduced neurobehavioral deficits,and improved the survival rate of mice.Expression levels of p-AKT and p-GSK3βwere significantly increased in the perihematoma tissues after intranasal insulin therapy.Our findings suggest that intranasal insulin therapy can protect the neurological function of mice after intracerebral hemorrhage through the AKT/GSK3βsignaling pathway.The study was approved by the Ethics Committee of the North Sichuan Medical College of China(approval No.NSMC(A)2019(01))on January 7,2019.

Mild hypothermia effects on matrix metalloproteinase-9 expression in the perihematomal region of rats following experimental intracerebral hemorrhage

BACKGROUND： Matrix metalloproteinase-9 （MMP-9） expression increases with intracerebral hemorrhage, and participates in the pathophysiological processes of secondary brain injury after intracerebral hemorrhage. OBJECTIVE： To investigate the effects of mild hypothermia on MMP-9 expression and brain edema in the perihematomal region of experimental intracerebral hemorrhage rats. DESIGN, TIME AND SETTING： The randomized, controlled experiment was performed at the Central Laboratory of Shandong Provincial Hospital between May and September 2007. MATERIALS： Seventy-two, Wistar, male rats, 12-weeks old, were used for this study. Rabbit anti-MMP-9 primary antibody was purchased from Boster, China. METHODS： Wistar rats were equally and randomly divided into normothermia and mild hypothermia groups. The two groups each comprised control, 6-hour intracerebral hemorrhage, 24-hour intracerebral hemorrhage, 48-hour intracerebral hemorrhage, 72-hour intracerebral hemorrhage, and l-week intracerebral hemorrhage subgroups, with six rats in each subgroup. Rat models of intracerebral hemorrhage were established by injecting 100 μL of autologous blood into the rat caudate nucleus. Rats in the mild hypothermia group received four hours of local mild hypothermia immediately following the injection. lntracerebral temperature was maintained at （33 ± 0.5） ℃. Subsequently, intracerebral temperature was spontaneously recovered at 25 ℃. Rats in the control subgroup were not injected with autologous blood and received only with intracerebral hemorrhage. MAIN OUTCOME MEASURES： Brain water content and MMP-9 expression surrounding the hematoma region. RESULTS： MMP-9 expression increased at 6 hours, and brain edema reached a peak at 48 hours after intracerebral hemorrhage. MMP-9 expression was significantly decreased in the mild hypothermia group compared with the normothermia group at each time point （P 〈 0.05）. CONCLUSION： Mild hypothermia can significantly inhibit MMP-9 overexpression and relieve brain edema following intracerebral hemorrhage.

Magnesium:pathophysiological mechanisms and potential therapeutic roles in intracerebral hemorrhage

Intracerebral hemorrhage(ICH) remains the second-most common form of stroke with high morbidity and mortality.ICH can be divided into two pathophysiological stages:an acute primary phase,including hematoma volume expansion,and a subacute secondary phase consisting of blood-brain barrier disruption and perihematomal edema expansion.To date,all major trials for ICH have targeted the primary phase with therapies designed to reduce hematoma expansion through blood pressure control,surgical evacuation,and hemostasis.However,none of these trials has resulted in improved clinical outcomes.Magnesium is a ubiquitous element that also plays roles in vasodilation,hemostasis,and blood-brain barrier preservation.Animal models have highlighted potential therapeutic roles for magnesium in neurological diseases specifically targeting these pathophysiological mechanisms.Retrospective studies have also demonstrated inverse associations between admission magnesium levels and hematoma volume,hematoma expansion,and clinical outcome in patients with ICH.These associations,coupled with the multifactorial role of magnesium that targets both primary and secondary phases of ICH,suggest that magnesium may be a viable target of study in future ICH studies.

Real-time analysis of inflammatory cytokines and regulatory gene expression in tissues surrounding the hematoma after intracerebral hemorrhage

BACKGROUND： Secondary lesions can occur in tissues surrounding the hematoma following intracerebral hemorrhage, with the presence of inflammatory reactions, cytokine expression and apoptosis These have been confirmed in animal studies. Our study sought to determine whether these could be detected in human tissues surrounding the hematoma following intracerebral hemorrhage. OBJECTIVE： To investigate expression of inflammatory cytokines, Bax and Bcl-x, and identify neural cell apoptosis in tissues surrounding the hematoma, and to analyze the correlation between them and pathological damage in intracerebral hemorrhage patients. DESIGN, TIME AND SETTING： This histopathology, controlled study was performed at the Department of Neurosurgery, Sichuan People＇s Hospital, China, from January 2003 to January 2005. PARTICIPANTS： Brain tissues 1 cm from the hematoma in 30 intracerebral hemorrhage patients served as the experimental group. Brain tissues located away from the hematoma in 7 patients served as the control group. METHODS： TUNEL was used to detect neural cell apoptosis. Immunohistochemistry （labeled dextran polymer） and RT-PCR were used to measure tumor necrosis factor- α, interleukin-1 β, interleukin-6, Bax and Bcl-x protein and mRNA expression. Pathological changes in brain tissues surrounding the hematoma were observed following HE staining. MAIN OUTCOME MEASURES： Neural cell apoptosis, inflammatory cytokines, Bax and Bcl-x protein and mRNA expression, pathological changes in brain tissues surrounding the hematoma. RESULTS： Brain tissues surrounding the hematoma were mildly damaged within 6 hours, severely damaged at 24-72 hours, and significantly improved 1 week following intracerebral hemorrhage. Expression of tumor necrosis factor- α protein and mRNA, interleukin-1β and interleukin-6 mRNA was not significant in tissues surrounding the hematoma, which was identical to the control group within 6 hours after intracerebral hemorrhage. This expression was significantly higher compared with the control group from 12-72 hours, and gradually decreased after 72 hours. The number of apoptotic neural cells reached a peak between 12- 72 hours. Tumor necrosis factor-α protein and mRNA, interleukin-1β and interleukin-6 mRNA levels were positively correlated with apoptosis, Bax protein and mRNA levels （P 〈 0.01 ）. CONCLUSION： Tumor necrosis factor-α, interleukin-1β, and interleukin-6 levels are highly correlated with apoptosis. With the decrease in tumor necrosis factor-α, interleukin-1β and interleukin-6 levels, the number of apoptotic cells gradually reduced.

Correlation between aquaporin-4 and brain edema in an animal model of astrocytic oxygen-glucose deprivation and reintroduction

BACKGROUND：Previous studies have demonstrated that aquaporin-4 （AQP4） plays a key role in the formation and resolution of brain edema.However,the molecular mechanisms and role of AQP4 in hypoxia-ischemia-induced brain edema remain poorly understood.OBJECTIVE：To establish a newborn animal model of astrocytic oxygen-glucose deprivation and reintroduction,to observe the correlation between AQP4 and cellular volume,and to investigate the role of AQP4 in the development of brain edema following oxygen deprivation and reintroduction.DESIGN,TIME AND SETTING：A comparative experiment was performed at the Experimental Center of West China Second University Hospital between October 2007 and April 2009.MATERIALS：Astrocytes were derived from the neocortex of Sprague Dawley rats aged 3 days.METHODS：Astrocytes were incubated in glucose/serum-free Dulbecco＇s modified Eagle＇s medium,followed by 1% oxygen for 6 hours.Finally,oxygen-glucose deprivation and reintroduction models were successfully established.MAIN OUTCOME MEASURES：Real-time polymerase chain reaction and Western blot analysis were used to measure expression of AQP4 mRNA and protein in cultured rat astrocytes following oxygen-glucose deprivation and reintroduction.Astrocytic cellular volume,as determined by [3H]-3-O-methyl-D-glucose,was used to represent the extent of astrocytic swelling.RESULTS：During oxygen-glucose deprivation,AQP4 mRNA and protein expression gradually decreased in astrocytes,whereas cellular volume increased in a time-dependent manner （P〈 0.01）.Following oxygen-glucose reintroduction,AQP4 mRNAand protein expression was upregulated,peaked at day 7,and then gradually decreased,but still higher than normal levels （P 〈 0.05）.However,cellular volume gradually decreased （P 〈 0.01）,and then reached normal levels at day 7.CONCLUSION：AQP4 expression highly correlated with cellular volume changes,suggesting that AQP4 played an important role in modulating brain water transport in an astrocytic oxygen-glucose deprivation and reintroduction model.

Aquaporin-4 in the formation of cerebral edema following severe burns What role do arginine vasopressin levels play?

BACKGROUND： Aquaporin-4 （AQP-4）, which is able to rapidly transport water within the brain, is highly expressed in brain tissue. It also plays an important role in the formation of cerebral edema following brain injury. However, the role of AQP-4 in the formation of cerebral edema following severe bums remains unknown. OBJECTIVE： To study changes in AQP-4 protein and mRNA expression during formation of cerebral edema following severe burns, and to explore the correlation between AQP-4 protein and mRNA expression with plasma levels of arginine vasopressin （AVP）. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment was performed at the Research Center of Neuroscience, Chongqing Medical University from 2007 to 2008. MATERIALS： Biotin-labeled goat anti-rabbit antibody was provided by Beijing Zhongshan Biotechnology, China; in situ hybridization kit was provided by Wuhan Boster Biotechnology, China; rabbit anti-AQP-4 polyclonal antibody and horseradish peroxidase-labeled goat anti-rabbit IgG were provided by Chemicon, USA; AVP radioimmunoassay kit was provided by the Research Department of Neurobiology, the Second Military Medical University of Shanghai, China. METHODS： A total of 180 adult, healthy, Wistar rats were randomly assigned to control and burn groups with 30 rats in each group. The burn group was observed at five different time points： 2, 6, 12, 24, and 48 hours after burn. Hair on the mouse back was removed to expose skin on the back. After 1 day, skin with the hair removed was dipped into 100℃ water for 15 seconds to induce grade III bum injury that measures 30% of total bum surface area. MAIN OUTCOME MEASURES： Brain water content was measured using the dry-wet weight method. AQP-4 protein and mRNA expressions were detected using immunohistochemistry, in situ hybridization, Western blot, and reverse transcription-polymerase chain reaction; dynamic changes in plasma AVP were detected using radioimmunoassay. RESULTS： Brain water content gradually increased following severe burn injury. AQP-4 protein and mRNA expressions were upregulated in the supraoptic nucleus, suprachiasmatic nucleus, paraventricular nucleus, hippocampus, choroid plexus, and cerebral cortex. Plasma AVP levels increased following burn injury. AQP-4 protein and mRNA expressions positively correlated with brain water content and AVP levels during formation of cerebral edema （r= 0.870, 0.848, P 〈 0.01）. CONCLUSION： AQP-4 participated in the formation of cerebral edema following burn injury. Plasma AVP upregulated AQP-4 expression in brain tissue, thereby promoting formation of cerebral edema.

Increased expression of aquaporin-4 in brain tissue of amygdala-kindled rats

Recurrent epileptic seizures can lead to brain edema, indicating that water regulation may be perturbed by seizures. We hypothesized that the expression of the brain water channel aquaporin-4 （AQP-4） may be upregulated in the epileptic brain. In the present study, we established the amygdala kindling model of epilepsy, and quantified AQP-4 protein and mRNA levels, using reverse transcription-PCR, immunohistochemistry and western blotting, in epileptic and control rats. We found that AQP-4 was overexpressed in the cerebral cortex of rats with epilepsy compared with controls. These findings show that AQP-4 is highly expressed in the brain of amygdala-kindled rats, suggesting that repeated seizures affect water homeostasis in the brain.

沉默转录激活因子4抑制脑出血后神经元坏死性凋亡的体外实验

背景:脑出血介导的神经元坏死性凋亡是继发性脑损伤的重要原因。转录激活因子4(activating transcription factor 4,ATF4)是转录激活因子家族成员之一,在脑出血后的继发性脑损伤中扮演重要角色。然而,ATF4在脑出血后的神经元坏死性凋亡中的机制有待明确。目的:探索沉默ATF4基因对脑出血后神经元坏死性凋亡的影响。方法:共同培养HT-22小鼠海马神经元细胞系和BV-2小鼠小胶质细胞系,利用氯化血红素刺激神经元构建脑出血体外模型。在0-100μmol/L区间设置氯化血红素干预细胞的浓度梯度,通过MTT实验评估氯化血红素处理24 h后对神经元细胞活力的影响。然后将共培养细胞分为4组:空白对照组不加任何干预,对照组加入50μmol/L氯化血红素处理,其余2组在加入氯化血红素48 h前分别用阴性对照小干扰RNA(NC siRNA)、ATF4小干扰RNA(ATF4 siRNA)干预细胞,再用50μmol/L氯化血红素处理细胞24 h后,利用PI/Hoechst染色检测神经元坏死性凋亡情况,Western blot检测ATF4、RIP3、MLKL的蛋白表达,双重免疫荧光染色定位于神经元,观察神经元坏死性凋亡的发生程度及ATF4的调控作用。结果与结论:①50μmol/L氯化血红素可以较大程度诱导神经元坏死性凋亡;②对照组和NC siRNA组的PI+/Hoechst+细胞数量高于空白对照组(P<0.0001),ATF4 siRNA组PI+/Hoechst+细胞数量低于对照组(P<0.0001);③与对照组相比,ATF4 siRNA组在抑制ATF4蛋白表达(P<0.001)的同时,也抑制了RIP3、MLKL蛋白表达(P<0.001);④通过对神经元荧光染色定位,与对照组相比,ATF4 siRNA组的RIP3、MLKL蛋白表达明显降低(P<0.0001);⑤结果表明,通过沉默ATF4基因可以直接或间接抑制脑出血后神经元坏死性凋亡相关基因的表达,起到缓解继发性脑损伤的作用。

Correlation of aquaporin-4 expression to blood-brain barrier permeability in rats with focal cerebral ischemia

BACKGROUND： Ischemic cerebrovascular disease causes injury to the blood-brain barrier. The occurrence of brain edema is associated with aquaporin expression following cerebral ischemia/reperfusion. OBJECTIVE： To analyze the correlation of aquaporin-4 expression to brain edema and blood-brain barrier permeability in brain tissues of rat models of ischemia/reperfusion. DESIGN, TIME AND SETTING： The randomized control experiment was performed at the Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical College, China from December 2006 to October 2007. MATERIALS： A total of 112 adult, male, Sprague-Dawley rats, weighing 220-250 g, were used to establish rat models of middle cerebral artery occlusion and reperfusion by the suture method. Rabbit anti-aquaporin-4 （Santa Cruz, USA） and Evans blue （Sigma, USA） were used to analyze the tissue. METHODS： The rats were randomized into sham-operated （n = 16） and ischemia/reperfusion （n = 96） groups. There were 6 time points in the ischemia/reperfusion group, comprising 4, 6, 12, 24, 48, and 72 hours after reperfusion, with 16 rats for each time point. Rat models in the sham-operated group at 4 hours after surgery and rat models in the ischemia/reperfusion group at different time points were equally and randomly assigned into 4 different subgroups. MAIN OUTCOME MEASURES： Brain water content on the ischemic side and the control side was measured using the dry-wet weight method. Blood-brain barrier function was determined by Evans Blue. Aquaporin-4 expression surrounding the ischemic focus, as well as the correlation of aquaporin-4 expression with brain water content and Evans blue staining, were measured using immunohistochemistry and Western blot analysis. RESULTS： Brain water content on the ischemic side significantly increased at 12 hours after reperfusion, reached a peak at 48 hours, and was still high at 72 hours. Brain water content was greater on the ischemic hemispheres, compared with the control hemispheres at 6, 12, 24, 48, and 72 hours after reperfusion, as well as both hemispheres in the sham-operated group （P 〈 0.05）. Evans blue content significantly increased on the ischemic side at 4 hours after ischemia/reperfusion, and reached a peak at 48 hours. Evans blue content was greater on the ischemic hemispheres, compared with the control hemispheres at various time points, as well as both hemispheres in the sham-operated group （P 〈 0.05）. Aquaporin-4-positive cells were detected in the cortex and hippocampus, surrounding the ischemic penumbra focus, at 4-6 hours after ischemia/reperfusion. The number of positive cells significantly increased at 12 hours and reached a peak at 48-72 hours. Aquaporin-4 was, however, weakly expressed in the control hemispheres and the sham-operated group. The absorbance ratio of aquaporin-4 to β-actin was greater at 12, 24, 48, and 72 hours following cerebral ischemia/reperfusion, compared with the sham-operated group （P 〈 0.05）. Aquaporin-4 expression positively correlated to brain water content and Evans blue staining following cerebral ischemia/reperfusion （r1 = 0.68, r2 = 0.81, P 〈 0.05）. CONCLUSION： Aquaporin-4 is highly expressed in brain tissues, participates in the occurrence of ischemic brain edema, and is positively correlated to blood-brain barrier permeability following cerebral ischemia/reperfusion.

Effect of Buyanghuanwu Decoction on PI3K/AKT Signaling Pathway and Aquaporin AQP4 in Cerebral Hemorrhage Rats

[Objectives] To explore the effect of Buyanghuanwu decoction on PI3K/AKT signaling pathway and aquaporin AQP4 in cerebral hemorrhage rats and clarify the mechanism to provide clear direction and target for cerebral hemorrhage treatment caused by cerebral edema.[Methods]SD rats were randomly divided into six groups: model group,sham operation group,Buyanghuanwu decoction low,medium and high dose groups,and Ginkgo biloba group. Model group,Buyanghuanwu decoction group,G. biloba group were prepared to be intracerebral hemorrhage rat models by referring to Rosenberg law. While the expression of " polarity" of aquaporin AQP4 was detected by immunofluorescence labeling method,the Evans blue( Evans Blue,EB) content of brain tissue was determined by Spectrophotometry. In addition,the water content of brain tissue was detected by wet and dry weight method. [Results] When compared to the model group,the Buyang Huanwu decoction group,G. biloba group of PI3K and AKT proteins expression increased significantly( P < 0. 05) and AQP4 in Astrocyte end feet membrane concentrated expression significantly increased( P < 0. 05),EB content and water content of brain tissue significantly reduced( P <0. 05).[Conclusions]The protective mechanisms of Buyanghuanwu decoction on cerebral hemorrhage can work might because it can activate PI3K/AKT signaling pathway,regulate AQP4 " polar" expression,and reduce the permeability of the blood brain barrier and cerebral edema.

黄芩素抑制Toll样受体4/核因子-B炎症通路改善小鼠脑出血后脑损伤的作用及机制研究

目的 探讨黄芩素抑制Toll样受体4(TLR4)/核因子-κB(NF-κB)炎症通路改善小鼠脑出血后脑损伤的作用及机制研究。方法 通过胶原酶注射小鼠右侧基底节区诱导小鼠脑出血,模型模型诱导后小鼠腹腔注射不同剂量黄芩素或者同体积溶剂,并通过神经功能学评分及脑水含量分析其保护作用;通过不同时间点给药(模型诱导后6及12h),评估黄芩素治疗时间窗;对TLR4和NF-κB信号通路的变化、小胶质细胞活化及神经细胞退变(FJC)进行检测。结果 黄芩素可减轻脑出血后脑水含量并改善神经功能学评分,并且模型诱导后6 h开始治疗,仍然有效;黄芩素可减少脑出血后神经细胞变性;黄芩素通过抑制小胶质细胞活化及TLR4/NF-κB通路及其下游促炎因子(TNF-α及IL-1β)的产生发挥抗炎作用。结论 黄芩素在脑出血后通过抗细胞死亡和抗炎作用发挥神经保护作用。

The lymphatic drainage systems in the brain:a novel target for ischemic stroke?

Recent studies have proposed three lymphatic drainage systems in the brain,that is,the glymphatic system,the intramural periarterial drainage pathway,and meningeal lymphatic vessels,whose roles in various neurological diseases have been widely explored.The glymphatic system is a fluid drainage and waste clearance pathway that utilizes perivascular space and aquaporin-4 protein located in the astrocyte endfeet to provide a space for exchange of cerebrospinal fluid and interstitial fluid.The intramural periarterial drainage pathway drives the flow of interstitial fluid through the capillary basement membrane and the arterial tunica media.Meningeal lymphatic vessels within the dura mater are involved in the removal of cerebral macromolecules and immune responses.After ischemic stroke,impairment of these systems could lead to cerebral edema,accumulation of toxic factors,and activation of neuroinflammation,while restoration of their normal functions can improve neurological outcomes.In this review,we summarize the basic concepts of these drainage systems,including drainage routes,physiological functions,regulatory mechanisms,and detection technologies.We also focus on the roles of lymphatic drainage systems in brain injury after ischemic stroke,as well as recent advances in therapeutic strategies targeting these drainage systems.These findings provide information for potential novel strategies for treatment of stroke.

高血压脑出血患者血肿周围组织超微结构变化与水通道蛋白-4mRNA表达的关系

目的探讨高血压脑出血(HICH)患者血肿周围组织超微结构变化与水通道蛋白-4 mRNA(AQP-4mRNA)表达的关系。方法选择山东玲珑英诚医院2018年6月至2020年6月行开颅手术治疗的HICH患者68例,术中取血肿周围组织观察超微结构变化、脑水肿病理变化,采用半定量RT-PCR法检测脑水肿组织AQP-4mRNA表达。另取正常脑组织标本30例作为对照组,采用半定量RT-PCR法检测AQP-4mRNA表达。68例HICH患者分别根据出血至手术时间和血肿量分组,比较各组AQP-4mRNA表达。结果脑组织超微结构变化:脑出血至手术时间5 h星形细胞胞体和周边足突肿胀对毛细血管造成压迫,粗面内质网扩张,内有大量水肿液,线粒体融合中的部分嵴和膜局部断裂;脑出血至手术时间36 h神经细胞部分变性,星形细胞明显肿胀,部分细胞变性、坏死;脑出血至手术时间86 h神经细胞变性,轴索内容物溶解,部分双层核膜融合或消失,管腔变窄甚至闭塞。脑组织病理变化:出血至手术时间5 h患者脑组织轻度水肿;出血至手术时间36 h患者脑组织中度水肿,可见部分肿胀细胞;出血至手术时间86 h脑组织重度水肿,细胞核消失,坏死。对照组、<6 h组、6~24 h组、25~72 h组、>72 h组AQP-4mRNA表达差异有统计学意义(F=5.072,P<0.05)。血肿量30~50 mL组、51~70 mL组、71~90 mL组、>90 mL组AQP-4mRNA表达差异无统计学意义(F=0.924,P>0.05)。结论HICH患者出血时间超微结构变化、病理改变、AQP-4mRNA表达有一致性,在72 h后病理显示水肿消退,AQP-4mRNA表达随之降低。

脑部类淋巴系统中水通道蛋白4与脑出血

脑部类淋巴系统(glymphatic system,GS)是脑脊液和间质溶质之间新定义的全脑血管周围转运网络,可促进脑代谢废物的清除。其复杂的网络由动脉周围脑脊液流入通路、星形胶质细胞介导的由水通道蛋白4(aquaporin 4,AQP4)支持的流体和溶质的对流运输以及静脉周流出通路组成。其功能障碍与各种神经系统疾病有关,并在脑出血(intracerebral hemorrhage,ICH)后脑水肿的病理、生理过程中起关键作用。对脑部GS和AQP4在生理和ICH后的相关研究进行分析,可进一步了解其在ICH后的风险评估、诊断、预后和治疗中的潜在用途,以及与中医中风的“玄府”理论的相关性。

三氟拉嗪通过内化水通道蛋白4减轻蛛网膜下腔出血大鼠脑水肿

目的:研究三氟拉嗪(TFP)对蛛网膜下腔出血(SAH)模型大鼠水通道蛋白4(AQP4)细胞定位及脑水肿的影响。方法:利用往小脑延髓池二次注射自体动脉血的方法制备SAH大鼠模型,治疗组通过灌胃给予TFP处理;利用湿干比方法检测大鼠脑含水量;利用伊文思蓝染料渗透实验检测血脑屏障(BBB)的完整性;利用试剂盒分别提取胞膜蛋白和胞浆蛋白,利用Western Blot方法检测AQP4的表达。结果:与SAH组大鼠相比,TFP治疗组大鼠脑含水量减少,脑组织中伊文思蓝染料的含量下降,同时AQP4在细胞膜的分布减少。结论:TFP通过内化AQP4减轻SAH模型大鼠脑水肿。

大黄改善急性脑出血大鼠血脑屏障损伤的水通道蛋白-4机理研究

目的探讨大黄改善急性脑出血血脑屏障损伤的水通道蛋白4(aquaporin4,AQP4)机理。方法采用立体定向注射自体无肝素动脉血制作大鼠脑出血模型,对神经功能缺损、脑含水量进行观察,伊文思蓝染色观察血脑屏障紧密连接(bloodbrainbarriertightjunction,BBBTJ)的损害,电镜观察不同时间点BBB和神经星形胶质细胞足突改变,用RTPCR和Westernblot检测AQP4mRNA和蛋白表达。结果脑出血后,与模型组比较,大黄可明显减轻脑水肿;伊文思蓝染色结果显示血脑屏障从12h开始明显损害;电镜结果提示出血后12h血脑屏障紧密连接被破坏,而且神经星形胶质细胞足突肿胀;大黄可有效使血脑屏障紧密连接的破坏减少、神经星形胶质细胞足突肿胀减轻。脑出血模型组大鼠AQP4表达出现增高(P<0.05);脑出血后1天AQP4mRNA和AQP4蛋白表达增强,第3天达峰值,其后逐渐有所下降。大黄可抑制AQP4基因转录和翻译。结论大黄可通过改善血脑屏障损伤减轻脑水肿;大黄改善血脑屏障破坏的作用可能是通过抑制AQP4基因转录和翻译实现的。

不同介入时间高压氧治疗对实验性脑出血大鼠出血灶周围水肿及水通道蛋白-4表达的影响

目的:观察不同介入时间的高压氧(HBO)治疗对实验性脑出血大鼠出血(ICH)灶周围水肿及水通道蛋白-4(AQP4)表达的影响。方法:应用胶原酶诱导法建立大鼠脑出血模型,将185只雄性Wistar大鼠随机分为正常组(5只)、假手术组(60只)、脑出血对照组(60只)和高压氧治疗组(60只),高压氧治疗组按高压氧介入时间不同分为6h介入组、1d介入组、2d介入组和3d介入组4个亚组,每组15只,再按不同的治疗次数分为高压氧治疗后1d、3d、5d组,每个时间点各5只鼠,高压氧治疗压力2.0ATA,稳压吸氧60min,1次/d,与末次高压氧治疗结束后24h处死。所有大鼠饲养在相同环境中,正常组饲养3d后处死,脑出血对照组与假手术组大鼠不进行任何治疗,参照高压氧治疗组进行分组,并于相应时间点断头取脑,采用干湿比重法测定脑组织的水含量,免疫组化法测定AQP4的表达。结果:高压氧治疗组及脑出血对照组大鼠脑组织的水含量均高于正常组及假手术组(P<0.05);高压氧治疗组较脑出血对照组大鼠脑水含量减少(P<0.05);高压氧治疗组间比较6h介入组较其他治疗组明显减少(P<0.05);高压氧治疗组及脑出血对照组大鼠出血灶周围AQP4的表达均高于正常组及假手术组(P<0.05);高压氧治疗组较脑出血对照组大鼠出血灶周围AQP4表达减少(P<0.05),高压氧治疗组间比较6h介入组较其他组减少明显(P<0.05)。结论:HBO治疗可减少出血灶周围脑组织水含量及AQP4表达,介入治疗时间以6h为最优。

不同压力高压氧对实验性脑出血大鼠出血灶周围水肿及水通道蛋白4表达的影响

目的:观察不同压力高压氧对实验性脑出血大鼠出血灶周围水肿及水通道蛋白-4(AQP4)表达的影响。方法:应用胶原酶诱导法建立大鼠脑出血模型,将90只雄性脑出血SD大鼠随机分为对照组(18只)、常压氧(1.0ATA)治疗组(18只)和高压氧治疗组(54只),高压氧治疗组再按不同的高压氧治疗压力分为1.8ATA组、2.0ATA组、2.2ATA组3个亚组,每组18只,按不同治疗压力1次/d,对照组为脑出血模型组,不进行HBO/常压氧治疗,各组分别于第1、3、5天断头取脑,每个时间点各6只。干湿比重法测定脑组织的水含量,免疫组织化学染色法测定AQP4的表达。结果:脑组织水含量:①常压氧治疗组较脑出血对照组减少,于各时间点比较差异均无显著性意义(P>0.05);②高压氧治疗各组较常压氧组及对照组水肿减轻明显,于第1天时2.0ATA组与常压氧组及对照组比较差异有显著性意义(P<0.05);于第3、5天时高压氧治疗各组与常压氧组及对照组比较均有显著性意义(P<0.05),但2.0ATA组较1.8ATA组及2.2ATA组减轻明显(P<0.05)。AQP4表达:①常压氧治疗组较脑出血对照组表达减少,但于各时间点比较差异均无显著性意义(P>0.05);②高压氧治疗各组较常压氧治疗组及脑出血对照组表达减少,于各时间点比较均有显著性意义(P<0.05),但于3d、5d时2.0ATA组较1.8ATA组及2.2ATA组表达减少更明显(P<0.05)。结论:HBO治疗可减少出血灶周围脑组织水含量及AQP4表达,其中2.0ATA治疗压力优于1.8ATA及2.2ATA。