NMDA受体NR2A亚单位在凝血酶诱导的大鼠脑出血后脑损伤作用机制的研究

目的探讨N-甲基-D天冬氨酸受体(NMDA)受体NR2A亚单位在凝血酶诱导的大鼠脑出血后脑损伤中的作用机制。方法将健康雄性SD大鼠随机分为生理盐水对照组、凝血酶组、凝血酶+阿加曲班组,建立大鼠脑出血模型。造模后48h,采用伊文思蓝法检测血脑屏障(BBB)的通透性,干-湿重法测脑组织的含水量。采用Western-blot方法观察出血后各时间点(0h、0.5h、6h、24h、72h、120h)血肿周围脑组织NR2A分布及动态变化规律。结果 (1)阿加曲班可以明显改善大鼠脑出血BBB通透性(P<0.05),血肿周围脑水肿明显减轻(P<0.05)。(2)Western-blot结果显示,NR2A在6h时开始增加,72h达到高峰,在120h时基本恢复。阿加曲班可以明显减少NR2A在72h时的表达(P<0.05)。结论 (1)NR2A参与了凝血酶诱导的脑出血后脑损伤的病理生理过程。(2)凝血酶可能通过上调NR2A表达引起脑出血后脑组织的损伤。     

大鼠大脑皮层损伤后N-甲基D-天门冬氨酸受体亚单位NR1、NR2A和NR2B的表达研究

目的 分析研究急性损伤后大鼠大脑皮层N-甲基D-天门冬氨酸受体(NMDA)亚单位NR1，NR2A和NR2B的表达。方法 建立开放式挫伤型机械脑损伤大鼠模型，采用NMDA受体的NR1，NR2A和NR2B3种亚单位的特异性抗体，对不同时间点的皮层组织3种亚单位蛋白作定量免疫印迹分析。结果 以健侧大脑皮层作为对照组，NR1亚单位在各时间点的含量保持基本稳定。NR2A的含量在3h内升高明显，至3h达到高峰，随后降至正常；NR2B在1h时略有上升后随即下降，至3h时最低，尔后又明显升高，于6h时达高峰，随后又恢复正常。结论 大鼠脑皮层损伤NMDA受体NR2A和NR2B亚单位的表达发生了改变。     

阿加曲班抑制脑出血大鼠血肿周围组织蛋白酶激活受体-1的表达

目的：探讨凝血酶与脑出血后蛋白酶激活受体-1（PAR-1）表达的关系,以及观察阿加曲班对脑出血后PAR-1表达的影响.方法：60只大鼠随机分为5组：假手术对照组,脑出血组（ICH组）,脑出血＋阿加曲班干预组（ICH＋Arg 组）,凝血酶注入组（TH组）和凝血酶＋阿加曲班干预组（TH＋Arg组）,每组6只.建立大鼠自体血脑出血模型以及凝血酶注入模型,术后3 和12 h经腹腔分别注入阿加曲班0.9 mg·kg-1体重.术后24 h处死大鼠取脑,分别用免疫组化、West ern blot和RT-PCR检测PAR-1的表达.结果： ICH组血肿周围和TH组尾状核区PAR-1阳性细胞数明显高于假手术对照组,ICH＋Arg和TH＋Arg组PAR-1阳性细胞数明显低于ICH和TH组.Western blot和RT-PCR法检测显示ICH＋Arg和TH＋Arg组的PAR-1蛋白和PAR-1 mRNA表达水平明显低于ICH和TH组（P〈0.01或P 〈0.05）.结论：脑出血后PAR-1的表达水平与凝血酶有关,阿加曲班可抑制脑出血后的PAR-1表达.     

经腹腔应用阿加曲班对脑出血后凝血酶神经毒性的抑制作用

目的:探讨经腹腔应用阿加曲班治疗脑出血（ICH）后凝血酶神经毒性损伤的可能性。方法:①研究阿加曲班对ICH及凝血酶注入后脑水肿、细胞损伤的影响。Wistar大鼠60只,随机分为假手术对照组（只进针不注血）;ICH组（50μL自体尾血注入右尾状核）;ICH+阿加曲班干预组(50μL自体尾血注入大鼠右侧尾状核,分别于术后3和12h经腹腔给予阿加曲班0.9mg·kg^-1,总量0.6mL);凝血酶组(10U·2μL^-1凝血酶注入大鼠右侧尾状核);凝血酶+阿加曲班干预组(10U·2μL^-1凝血酶注入大鼠右侧尾状核,分别于术后3和12h经腹腔给予阿加曲班0.9mg·kg^-1,总量为0.6mL)。各组均n=12,术后24h处死各组大鼠,每组中6只用于检测脑组织水含量,6只检测caspase-3免疫反应细胞及TUNEL阳性细胞数。②经腹腔注射阿加曲班对血肿容积的影响:建立胶原酶ICH大鼠模型组:注入Ⅰ型胶原酶+肝素;阿加曲班组:胶原酶ICH模型成功后3及12h分别经腹腔注入阿加曲班溶液0.9mg·kg^-1,每次注入液体量为0.6mL,测定两组大鼠脑组织血肿血红蛋白的含量(测定血红蛋白A₅₅₀值)以评价阿加曲班对血肿容量的影响。结果:自体血ICH及凝血酶模型大鼠在阿加曲班干预后,ICH组及凝血酶组的TUNEL阳性细胞数、caspase-3阳性细胞数明显下降（P<0.01或P<0.05）,脑组织水肿含量百分比明显降低（P<0.05）。胶原酶ICH血红蛋白A值为（0.45±0.12）,阿加曲班干预组为（0.46±0.09）,差异无统计学意义（P>0.05）。结论:ICH后3～12h经腹腔注入阿加曲班可减轻ICH后的脑水肿及细胞凋亡性损伤,且没有血肿增大的不良反应。     

神经干细胞分化的神经元离子型谷氨酸受体NMDA亚单位的mRNA和蛋白表达

目的在体外研究由大鼠神经干细胞(NSCs)分化而来神经元细胞中离子型谷氨酸NMDA受体表达。方法分离培养孕14～16d胎鼠皮质和海马神经干细胞，对NSCs进行nestin和分化鉴定。通过RT—PCR、Western blot免疫印迹和免疫组化检测NSCs分化的神经元细胞中离子型谷氨酸NMDA受体亚单位NR1、NR2A和NR2B的mRNA和蛋白表达。结果从孕14～16d胎鼠大脑中分离培养出NSCs，NSCs分化后的神经元可以表达离子型谷氨酸NMDA受体亚单位NR1、NR2A和NR2B。结论由NSCs分化而来的神经元能表达离子型谷氨酸NMDA受体。     

单耳气传导阻滞模型大鼠下丘脑N-甲基-D-门冬氨酸受体各亚基mRNA的表达

实验利用单耳外耳道皮下缝合的方法建立单侧气传导持续阻滞大鼠模型，观察环境变化对生后9，23，37 d的SD大鼠听觉中枢神经系统下丘脑NMDA受体NR1，NR2A，NR2B和NR2C mRNA基因表达的影响。PT-PCR结果显示，单耳缝合后缝耳对侧下丘脑NR1，NR2A，NR2B亚单位与缝耳同侧下丘脑NR1，NR2B依赖的听觉神经元发育临界期均在23 d附近，但同侧下丘脑NR2A亚单位依赖的听觉神经元发育的临界期的结束可能接近出生后37 d。结果证实了下丘脑NMDA受体亚基可受听觉环境调控的假设。     

阿加曲班对实验性大脑出血后脑组织N-κB表达变化的研究

目的 研究实验性大鼠大脑出血(ICH)后脑组织核转录因子kB(NF-kB)的表达在阿加曲班(argatroban)注入前后的变化及意义.方法 采用立体定向技术将自体不凝血注入大鼠尾状核区制备不同时间段的大脑出血模型,另设加药组,即术后30 min及术后每天给予同等量的阿加曲班(3.0 mg/kg,腹腔注射给药),用免疫组化染色法检测加药组及未加药组脑组织NF-kB表达的变化.结果 未加药组在出血第2 d表达的阳性细胞数较高,而加药组在出血后12 h较高;出血后12 h NF-kB的表达较加药前显著增加(P<0.05).加药组及未加药组NF-kB均在神经元、神经胶质细胞及星形胶质细胞足突广泛表达;在脉络丛及室管膜细胞亦见阳性表达.结论 进行阿加曲班治疗后NF-kB在出血后12 h表达显著增加,NF-kB可能在脑水肿形成过程中起到双重作用.     

局部亚低温对脑出血后水肿影响的实验研究

目的探讨局部亚低温对大鼠脑出血后水肿形成的影响及其可能机制。方法雄性Wistar大鼠230只随机分为:对照组;脑出血组;脑出血加局部亚低温组;凝血酶加局部亚低温组。应用Evans-Blue测定血脑屏障(BBB)通透性,应用干湿重法测定脑水含量。结果与对照组相比,大鼠注血后6h开始出现脑组织水含量和BBB通透性的增加,在72h达到高峰,然后逐渐消退。不同时程局部亚低温均可以显著降低脑出血后72h时脑组织水含量及BBB通透性(P<0.01),其中给以4h局部亚低温时,降低最明显。注射凝血酶6h后,脑组织水含量及BBB通透性显著增高(P<0.01),于24~48h达高峰,然后逐渐下降。凝血酶 局部亚低温组在各个时间点与凝血酶组相比,脑组织水含量及BBB通透性明显降低(P<0.01)。结论局部亚低温可能是通过抑制凝血酶的毒性作用来减轻脑出血后水肿的形成及血脑屏障的破坏。     

脑缺血再灌注大鼠皮质NMDA受体亚单位NR2A/B蛋白的表达变化

目的 采用免疫细胞化学技术，探讨急性脑缺血再灌注不同时间段大鼠脑顶皮质NMDA受体亚单位NR2A及NR2B蛋白的表达变化。方法 雄性Wistar大鼠70只,随机分成正常对照组、假手术对照组、脑缺血再灌注对照组；以颈动脉引流法行全脑缺血7min再灌注造模，术后分6h、24h、72h3个时间段取脑．脑组织恒冷箱连续冠状切片,免疫细胞化学ABC反应，图像分析系统行顶皮质Ⅰ区V层免疫阳性面积检测。结果 （1）麻醉及假手术可导致顶皮质NR2A、NR2B蛋白表达短暂增多，24h内恢复正常；（2）缺血再灌注后6h前后形成表达高峰,24h恢复正常，随后表达急剧减少，持续至72h以后。结论（1）脑缺血再灌注可导致顶皮质神经元NR2A、NR2B蛋白表达变化，且表达存在明显的时间依赖性；（2）缺血再灌注早期皮质NR2A、NR2B蛋白高度表达可能是导致迟发性神经元丢失的重要原因。     

阿加曲班对大鼠脑出血后脑水肿作用的研究

目的探讨阿加曲班对大鼠脑出血模型脑水肿的作用。方法取雄性SD大鼠32只,体重300~350g,随机分为A、B、C、D,4组,每组8只。大鼠经立体定向分别向右侧尾状核区注入生理盐水60ul、无肝素自体血50ul+生理盐水10ul、生理盐水50ul+阿加曲班生理盐水溶液10ul、无肝素自体血50ul+阿加曲班生理盐水溶液10ul。实验大鼠于术后48小时断头处死,测定脑含水量,HE染色,观察脑组织学变化。结果D组脑组织含水量降低,与B组比较有明显差别(P<0.05);C组脑组织含水量与A组比较无明显差别(P>0.05);HE染色观察:B组可见血肿周围脑组织水肿,炎症细胞渗出,脑组织疏松;D组上述改变减轻;A、C两组未见明显病理学改变。结论阿加曲班可减轻大鼠脑出血后血肿周围脑组织脑水肿。     

Brain Storming in Brain Trauma

Neurocritical Care -     

Brain Tissue Oxygenation in Children Diagnosed With Brain Death

Background  

Diagnosing brain death in children is challenging. Guidelines recommend using confirmatory testing to provide ancillary information to support the diagnosis. Brain tissue oxygenation (PbtO₂) is being increasingly used in the adult neurocritical care for continuous monitoring of the adequacy of brain oxygenation; however, data in pediatrics is limited. Evidence from adult studies suggests that persistent PbtO₂ of 0 mmHg is associated with brain death, but this relationship has not yet been demonstrated in children; therefore, we examined our experience with PbtO₂ monitoring and brain death in children with acute neurological pathology.     

Noninvasive Brain Stimulation to Modulate Neuroplasticity in Traumatic Brain Injury

Objective: To review the use of noninvasive brain stimulation (NBS) as a therapeutic tool to enhance neuroplasticity following traumatic brain injury (TBI). Materials and Methods: Based on a literature search, we describe the pathophysiological events following TBI and the rationale for the use of transcranial magnetic stimulation (TMS) and transcranial direct current stimulation (tDCS) in this setting. Results: The pathophysiological mechanisms occurring after TBI vary across time and therefore require differential interventions. Theoretically, given the neurophysiological effects of both TMS and tDCS, these tools may: 1) decrease cortical hyperexcitability acutely after TBI; 2) modulate long‐term synaptic plasticity as to avoid maladaptive consequences; and 3) combined with physical and behavioral therapy, facilitate cortical reorganization and consolidation of learning in specific neural networks. All of these interventions may help decrease the burden of disabling sequelae after brain injury. Conclusions: Evidence from animal and human studies reveals the potential benefit of NBS in decreasing the extent of injury and enhancing plastic changes to facilitate learning and recovery of function in lesioned neural tissue. However, this evidence is mainly theoretical at this point. Given safety constraints, studies in TBI patients are necessary to address the role of NBS in this condition as well as to further elucidate its therapeutic effects and define optimal stimulation parameters.     

Brain abscess in children

During that last 6 years, 100 children have been treated for brain abscess at the National Institute of Mental Health and Neurosciences (NIMHANS), Bangalore. Of these, 69 abscesses were otogenic. The treatment included aspiration of the abscess (84% of cases) and antibiotic therapy. CT scan as a diagnostic tool was available only for a few cases in the last year. In 16% of the cases the abscess was excised. The overall mortality was 21%. Of the surviving children, 76% recovered totally. Epilepsy as a sequel occurred in 21% of the cases. The fact that mortality continues to be high is attributed to the degree of impairment of consciousness of the children at the time of admission. Early detection and successful treatment of brain abscess in children still remain a clinical challenge.