Stretch in Brain Microvascular Endothelial Cells (cEND) as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying mechanisms involved in it are useful for treatment. There are both in vivo and in vitro methods available for this purpose. In vivo models can mimic actual head injury as it occurs during TBI. However, in vivo techniques may not be exploited for studies at the cell physiology level. Hence, in vitro methods are more advantageous for this purpose since they provide easier access to the cells and the extracellular environment for manipulation.Our protocol presents an in vitro model of TBI using stretch injury in brain microvascular endothelial cells. It utilizes pressure applied to the cells cultured in flexible-bottomed wells. The pressure applied may easily be controlled and can produce injury that ranges from low to severe. The murine brain microvascular endothelial cells (cEND) generated in our laboratory is a well-suited model for the blood brain barrier (BBB) thus providing an advantage to other systems that employ a similar technique. In addition, due to the simplicity of the method, experimental set-ups are easily duplicated. Thus, this model can be used in studying the cellular and molecular mechanisms involved in TBI at the BBB.     

Traumatic Brain Injury-Induced Excitotoxicity Assessed in a Controlled Cortical Impact Model

Using a controlled cortical impact model of traumatic brain injury (TBI) coupled with tissue microdialysis, interstitial concentrations of aspartate and glutamate (together with serine and glutamine) were assessed in rat frontal cortex. Histological analysis indicated that the severity of injury following severe TBI (depth of deformation = 3.5 mm) was approximately twice that occurring following moderate TBI (depth of deformation = 1.5 mm). Both groups demonstrated significant postinjury maximal increases in excitatory amino acid (EAA) concentration, which were proportional to the severity of injury. The mean ± SEM fold increase in dialysate concentrations of aspartate was 38 ± 13 (n = 5) for moderate TBI and 74 ± 12 (n = 5) for severe TBI. Fold increases in glutamate concentrations were 81 ± 26 and 144 ± 23 for moderate and severe TBI, respectively. Although these increases normalized within 20–30 min following moderate TBI, concentrations of aspartate and glutamate took >60 min to normalize after severe TBI. Changes in levels of nontransmitter amino acids were much smaller. Fold increases for serine concentrations were 4.6 ± 0.6 and 7.6 ± 1.7 in moderate and severe TBI, respectively; glutamine concentrations had similar small fold increases (2.6 ± 0.2 and 4.1 ± 0.6, respectively). Calculation of interstitial concentrations following severe TBI indicated that aspartate and glutamate maximally increased to 123 ± 20 and 414 ± 66 μM, respectively. To determine the extent to which such tissue concentrations of EAAs could contribute to the injury seen in TBI, the EAA receptor agonists N-methyl-d - aspartate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid were slowly injected into rat cortex. Remarkably similar histological injuries were produced by this procedure, supporting the notion that TBI is an excitotoxic injury.     

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and mortality. Based on the nature of primary injury following TBI, complex and heterogeneous secondary consequences result, which are followed by regenerative processes ^1,2. Primary injury can be induced by a direct contusion to the brain from skull fracture or from shearing and stretching of tissue causing displacement of brain due to movement ^3,4. The resulting hematomas and lacerations cause a vascular response ^3,5, and the morphological and functional damage of the white matter leads to diffuse axonal injury ^6-8. Additional secondary changes commonly seen in the brain are edema and increased intracranial pressure ⁹. Following TBI there are microscopic alterations in biochemical and physiological pathways involving the release of excitotoxic neurotransmitters, immune mediators and oxygen radicals ^10-12, which ultimately result in long-term neurological disabilities ^13,14. Thus choosing appropriate animal models of TBI that present similar cellular and molecular events in human and rodent TBI is critical for studying the mechanisms underlying injury and repair.Various experimental models of TBI have been developed to reproduce aspects of TBI observed in humans, among them three specific models are widely adapted for rodents: fluid percussion, cortical impact and weight drop/impact acceleration ¹. The fluid percussion device produces an injury through a craniectomy by applying a brief fluid pressure pulse on to the intact dura. The pulse is created by a pendulum striking the piston of a reservoir of fluid. The percussion produces brief displacement and deformation of neural tissue ^1,15. Conversely, cortical impact injury delivers mechanical energy to the intact dura via a rigid impactor under pneumatic pressure ^16,17. The weight drop/impact model is characterized by the fall of a rod with a specific mass on the closed skull ¹⁸. Among the TBI models, LFP is the most established and commonly used model to evaluate mixed focal and diffuse brain injury ¹⁹. It is reproducible and is standardized to allow for the manipulation of injury parameters. LFP recapitulates injuries observed in humans, thus rendering it clinically relevant, and allows for exploration of novel therapeutics for clinical translation ²⁰.We describe the detailed protocol to perform LFP procedure in mice. The injury inflicted is mild to moderate, with brain regions such as cortex, hippocampus and corpus callosum being most vulnerable. Hippocampal and motor learning tasks are explored following LFP.     

创伤性脑损伤中神经炎症相关细胞的研究进展

创伤性脑损伤(traumatic brain injury, TBI), 亦称颅脑损伤或头部外伤, 专指由外伤引起的脑组织损害。然而,从轻度到重度的TBI,改善TBI患者预后的治疗方法都十分匮乏。神经炎症可引起脑外伤后急性继发性损伤,并与慢性神经退行性疾病有关,因此,系统了解参与TBI后神经炎性反应的细胞显得尤为重要。主要对TBI中参与炎症反应的细胞（如小胶质细胞、星形胶质细胞、少突细胞、中性粒细胞和淋巴细胞）的启动以及相互作用的最新研究进展进行了综述,以期为临床研究提供新的策略。     

Cerebrospinal Fluid Taurine after Traumatic Brain Injury

In the experimental setting, taurine is known to be released from swollen cells to reestablish their normal volume. However, its clinical relevance has not been fully understood. This study was undertaken to reveal changes in cerebrospinal fluid (CSF) amino acids concentration in patients with severe traumatic brain injury (TBI). The study included eight patients, in whom a ventricular catheter was inserted to measure intracranial pressure and obtain CSF samples for 5 days. CSF obtained from patients with normal pressure hydrocephalus served as a control. CSF taurine concentration increased 1.8 times control (P < 0.05) after TBI and returned to control value approximately 67 h after injury. Taurine decreased further and remained lower than control thereafter. Phosphoethanolamine showed similar increase, whereas glutamine decreased transiently and arginine remained close to control value. The present data support the period of astrocytic swelling observed after TBI in other morphological studies. The mechanism and consequences of CSF taurine decrease in the subacute stage of TBI need to be elucidated.     

创伤性脑损伤与肠道菌群关系研究进展

创伤性脑损伤是一种高致死率的疾病，严重危害人类生命健康。肠脑轴是大脑和胃肠道系统之间主要的双向通讯途径。近年来，创伤性脑损伤与肠道菌群的相互作用关系逐渐被揭示。肠道菌群通过肠脑轴参与了创伤性脑损伤后急性病理损伤的调节过程并发挥重要作用。本文综述了创伤性脑损伤的发生、对人类健康的巨大影响，肠脑轴的含义及其在颅脑损伤中的病理调节机制，并在此基础上提出可能的治疗手段，包括粪便微生物菌群移植、使用益生菌、刺激迷走神经、摄入多酚类物质以及靶向免疫调节策略，以期为临床治疗创伤性脑损伤提供新的思路。     

高压氧治疗重型颅脑损伤的临床疗效观察

目的:观察高压氧辅助治疗重型颅脑损伤的临床疗效。方法:68例重型颅脑损伤患者随机分为观察组和对照组各34例,所有患者均根据病情选择手术或保守治疗,观察组在患者病情稳定后加用高压氧辅助治疗,治疗结束后比较两组患者的临床疗效。结果:观察组治疗有效率为91.2%,显著高于对照组的76.5%(P<0.05);治疗后1周和2周观察组的GCS评分均显著高于对照组(P<0.05),治疗后6个月观察组的GOS评分显著高于对照组(P<0.05);治疗后两组患者脑动脉血流速度均较治疗前有明显的改善(P<0.05),且观察的的改善情况优于对照组。结论:选择高压氧辅助治疗重型颅脑损伤的临床疗效好,可改善患者的临床情况和预后,值得临床应用。     

血必净注射液对重型颅脑损伤患者的神经保护及其机制研究

目的:探讨血必净注射液对重型颅脑损伤患者的神经保护作用及其机制。方法:将2012年1月至2014年12月我院收治的200例重型颅脑损伤患者分为研究组(100例)和对照组(100例),另选取100例同期在我院体检的健康者为正常组。对照组给予常规治疗,研究组在对照组基础上静脉滴注血必净注射液,治疗后第1天、第3天、第5天、第7天时观察各组TNF-α及IL-6水平变化。结果:对照组、研究组各时间点TNF-α及IL-6水平均高于正常组(均P0.05),且研究组的TNF-α及IL-6水平均低于对照组(均P0.05)。结论:血必净注射液对重型颅脑损伤患者的神经具有保护作用,其作用机制可能跟降低炎症反应有关。     

Astrocytic Ephrin-B1 Regulates Synapse Remodeling Following Traumatic Brain Injury

Traumatic brain injury (TBI) can result in tissue alterations distant from the site of the initial injury, which can trigger pathological changes within hippocampal circuits and are thought to contribute to long-term cognitive and neuropsychological impairments. However, our understanding of secondary injury mechanisms is limited. Astrocytes play an important role in brain repair after injury and astrocyte-mediated mechanisms that are implicated in synapse development are likely important in injury-induced synapse remodeling. Our studies suggest a new role of ephrin-B1, which is known to regulate synapse development in neurons, in astrocyte-mediated synapse remodeling following TBI. Indeed, we observed a transient upregulation of ephrin-B1 immunoreactivity in hippocampal astrocytes following moderate controlled cortical impact model of TBI. The upregulation of ephrin-B1 levels in hippocampal astrocytes coincided with a decline in the number of vGlut1-positive glutamatergic input to CA1 neurons at 3 days post injury even in the absence of hippocampal neuron loss. In contrast, tamoxifen-induced ablation of ephrin-B1 from adult astrocytes in ephrin-B1^loxP/yERT2-Cre^GFAP mice accelerated the recovery of vGlut1-positive glutamatergic input to CA1 neurons after TBI. Finally, our studies suggest that astrocytic ephrin-B1 may play an active role in injury-induced synapse remodeling through the activation of STAT3-mediated signaling in astrocytes. TBI-induced upregulation of STAT3 phosphorylation within the hippocampus was suppressed by astrocyte-specific ablation of ephrin-B1 in vivo, whereas the activation of ephrin-B1 in astrocytes triggered an increase in STAT3 phosphorylation in vitro. Thus, regulation of ephrin-B1 signaling in astrocytes may provide new therapeutic opportunities to aid functional recovery after TBI.     

丙泊酚镇静对颅脑损伤患者脑氧供需平衡的影响

目的:探讨丙泊酚实施不同程度镇静对颅脑损伤患者脑氧供需平衡的影响。方法:选择急性闭合性颅脑损伤需行机械通气患者46例,随机分为轻度镇静组(A组),设定目标脑电双频谱指数(BIS)值75%;中度镇静组(B组),设定目标BIS值65%。主要观察达设定目标BIS值时丙泊酚靶控输注(TCI)浓度、Ramsay镇静评分、脑氧供需平衡指标颈内静脉血氧饱和度(SjvO_2)和脑氧摄取率(CERO_2)以及心率(HR)、平均动脉压(MAP)。结果:两组设定镇静目标需丙泊酚TCI浓度有明显差异(P0.05),但Ramsay评分比较差异无统计学意义;中度镇静组SjvO_2较基础值增加约12%(P0.05),CERO_2较基础值下降约15%(P0.05);而轻度镇静组对SjvO_2和CERO_2基础值没有影响。两组HR均较基础值减慢(P0.05),但对MAP均没有影响。结论:颅脑损伤患者维持目标镇静BIS值65%,调控丙泊酚靶浓度1.5-1.6μg/mL,更有利于改善脑氧供需平衡。     

A Study on the Finite Element Model for Head Injury in Facial Collision Accident

In order to predict and evaluate injury mechanism and biomechanical response of the facial impact on head injury in a crash accident. With the combined modern medical imaging technologies, namely computed tomography (CT) and magnetic resonance imaging (MRI), both geometric and finite element (FE) models for human head-neck with detailed cranio-facial structure were developed. The cadaveric head impact tests were conducted to validate the headneck finite element model. The intracranial pressure, skull dynamic response and skull-brain relative displacement of the whole head-neck model were compared with experimental data. Nine typical cases of facial traffic accidents were simulated, with the individual stress wave propagation paths to the intracranial contents through the facial and cranial skeleton being discussed thoroughly. Intracranial pressure, von Mises stress and shear stress distribution were achieved. It is proved that facial structure dissipates a large amount of impact energy to protect the brain in its most natural way. The propagation path and distribution of stress wave in the skull and brain determine the mechanism of brain impact injury, which provides a theoretic basis for the diagnosis, treatment and protection of craniocerebral injury caused by facial impact.     

吸氢对大鼠颅脑损伤引起的急性炎症反应的抑制作用

为了探讨吸氢对大鼠创伤性颅脑损伤（traumatic brain injury,TBI）急性期炎症反应的影响,将6周龄雄性SD大鼠随机分为假手术组、TBI组和吸氢治疗组。采用悬浮芯片技术检测TBI后2、6和24 h的血清细胞因子水平;TBI后24 h采用改良的神经功能缺失评分法（modified neurological severity score,mNss）评估吸氢的神经保护作用,同时取脑组织进行尼氏染色分析并对血清生化指标进行检测。神经功能评分表明,TBI大鼠吸氢后24 h内神经功能就有显著改善,尼氏染色进一步验证了吸氢对神经元的保护作用;血清细胞因子的检测表明,吸氢对TBI引起的急性炎症反应具有很好的抑制作用,表现为7种促炎因子的血清水平在TBI后2 h明显降低。此外,吸氢还可明显降低血清中心脏和肝脏标志物水平,提示吸氢对TBI急性期心脏和肝脏功能损伤具有保护作用。研究提示吸氢可能通过抑制TBI急性期的炎症反应发挥其神经保护作用。     

亚低温治疗对重型颅脑损伤患者颅内压、脑血流及氧代谢的影响

目的:探讨亚低温治疗对重症颅脑损伤(sTBI)患者颅内压(ICP)、脑血流及氧代谢的影响。方法:收集50例sTBI患者随机分为实验组和对照组,每组25例,均给予常规治疗,观察组在常规治疗基础上给予亚低温辅助治疗,检测患者治疗前、治疗第3、5、7天ICP动态变化以及治疗前和治疗7天后脑血流和氧代谢等指标变化。结果:治疗第3、5、7天ICP组间差异均具有统计学意义(P0.05),随着治疗时间增加两组ICP均逐渐降低,差异具有统计学意义(P0.05);治疗前Qmean、Vmean、Wv、DR等组间差异无统计学意义(P0.05),治疗7天后Qmean、Vmean均升高,Wv、DR均降低,差异具有统计学意义(P0.05);治疗前SjvO_2、CjvO_2、CaO_2、CERO_2组间差异无统计学意义(P0.05),治疗7天后SjvO_2、CjvO_2、CERO_2均升高,CaO_2降低,差异具有统计学意义(P0.05)。结论:亚低温治疗可以显著降低患者颅内压,改善脑血流和氧代谢水平。     

Administration of DHA Reduces Endoplasmic Reticulum Stress-Associated Inflammation and Alters Microglial or Macrophage Activation in Traumatic Brain Injury

We investigated the effects of the administration of docosahexaenoic acid (DHA) post-traumatic brain injury (TBI) on reducing neuroinflammation. TBI was induced by cortical contusion injury in Sprague Dawley rats. Either DHA (16 mg/kg in dimethyl sulfoxide) or vehicle dimethyl sulfoxide (1 ml/kg) was administered intraperitonially at 5 min after TBI, followed by a daily dose for 3 to 21 days. TBI triggered activation of microglia or macrophages, detected by an increase of Iba1 positively stained microglia or macrophages in peri-lesion cortical tissues at 3, 7, and 21 days post-TBI. The inflammatory response was further characterized by expression of the proinflammatory marker CD16/32 and the anti-inflammatory marker CD206 in Iba1⁺ microglia or macrophages. DHA-treated brains showed significantly fewer CD16/32⁺ microglia or macrophages, but an increased CD206⁺ phagocytic microglial or macrophage population. Additionally, DHA treatment revealed a shift in microglial or macrophage morphology from the activated, amoeboid-like state into the more permissive, surveillant state. Furthermore, activated Iba1⁺ microglial or macrophages were associated with neurons expressing the endoplasmic reticulum (ER) stress marker CHOP at 3 days post-TBI, and the administration of DHA post-TBI concurrently reduced ER stress and the associated activation of Iba1⁺ microglial or macrophages. There was a decrease in nuclear translocation of activated nuclear factor kappa-light-chain-enhancer of activated B cells protein at 3 days in DHA-treated tissue and reduced neuronal degeneration in DHA-treated brains at 3, 7, and 21 days after TBI. In summary, our study demonstrated that TBI mediated inflammatory responses are associated with increased neuronal ER stress and subsequent activation of microglia or macrophages. DHA administration reduced neuronal ER stress and subsequent association with microglial or macrophage polarization after TBI, demonstrating its therapeutic potential to ameliorate TBI-induced cellular pathology.     

重型颅脑损伤后颅内高压的治疗研究进展

重型颅脑损伤后颅内压增高预示着不良的神经功能预后和极高的死亡率,一直是临床治疗中的研究热点,可采取高渗性脱水,亚低温疗法,巴比妥昏迷治疗及外科手术干预等治疗措施控制颅内压。由于亚低温治疗会增加患者发生肺炎的风险,巴比妥类药物副作用较大,现均已少用。近来研究发现,监测颅内压、脑灌注压、脑组织氧分压并指导临床治疗,可降低死亡率与改善预后。也有研究发现去骨瓣减压术治疗顽固性颅内高压与神经功能预后较差有关。目前关于颅内高压治疗的最佳方案仍存在争议,未来还需根据患者病情,为其制定规范化与个体化的治疗方案,预防继发性颅脑损伤,降低颅内压。本文就近年来重型颅脑损伤后颅内高压的治疗进展进行阐述。     

3D ASL在急性轻度创伤性脑损伤中的应用

为了探讨3D动脉自旋标记(arterial spin labeling,ASL)技术对急性创伤性脑损伤(traumatic brain inju-ry,TBI)病人的诊断价值,将43例急性轻度TBI患者和20例健康志愿者进行了常规MRI(magnetic resonanceimaging)和3D ASL扫描。结果表明,3D ASL能显示常规MRI所不能显示的脑内血流灌注情况。3D ASL结果发现,健康志愿者组双侧前、中、后动脉供血区的脑血流量(cerebral brain flow,CBF)值比较均无差异(P值均>0.05);TBI患者未出现明显低灌注区的脑实质CBF较志愿者脑实质CBF值明显降低(P值<0.01);TBI患者脑内局部低灌注区较对侧镜面区的CBF值明显减低(P值<0.01)。3D ASL技术能检测出急性轻度TBI患者脑实质灌注减低情况,对于临床诊治有重要意义,值得在临床推崇。     

重型颅脑损伤患者血清Arc水平及其与程序性坏死关系的研究

目的:探讨重型颅脑损伤(s TBI)患者血清Arc蛋白表达及其与神经元程序性坏死的关系。方法:采用回顾性研究的方法,选取中国人民解放军第123医院神经外科自2013年6月至2017年4月收治的s TBI患者55例(s TBI组)和同期该院体检中心健康体检者55例(Control组),将s TBI组患者分为Arc蛋白低表达组(小于150.37 pg/m L,s TBI-L)和Arc蛋白高表达组(大于150.37pg/m L,s TBI-H)。采用酶联免疫吸附法(ELISA)检测受试者外周血Arc蛋白含量,采用蛋白质印迹法(Western blot)检测程序性坏死相关蛋白受体相互作用蛋白激酶1(RIPK1)、受体相互作用蛋白激酶3(RIPK3)和混合系激酶区域样蛋白(MLKL)的表达,采用ELISA法检测炎症因子白介素1β(IL-1β)、肿瘤坏死因子α(TNF-α)和白介素10(IL-10)的表达。结果:s TBI组血清Arc蛋白含量明显高于Control组(150.37±21.08 pg/m L vs.87.65±13.43 pg/m L),差异有统计学意义(P0.001)。与Control组比较,s TBI-L组、s TBI-H组RIPK1、RIPK3和MLKL表达均明显增高(P0.001);与s TBI-H组比较,s TBI-L组RIPK3和MLKL表达明显增高(P0.001),而RIPK1表达无差异(P=0.181)。与Control组比较,s TBI-L组、s TBI-H组血清IL-1β、TNF-α和IL-10表达均明显增高(P0.001);与s TBI-H组比较,s TBI-L组血清IL-1β和TNF-α表达明显增高(P=0.027,0.008),而IL-10表达明显降低(P=0.015)。结论:重型颅脑损伤患者血清Arc蛋白表达增加,可能介导了神经元程序性坏死和炎症反应。     

Foxj1 在脑外伤后的表达变化

目的:探讨脑外伤后Foxj1在脑组织中的表达变化及其意义。方法:建立大鼠脑外伤模型,利用Western blot和免疫组织化学方法检测脑外伤后Foxj1在脑组织中表达的变化。结果:Western blot显示大鼠脑外伤后,Foxj1的表达逐步增高,伤后3 d升至最高点,之后逐渐降低;免疫组织化学的结果与Western blot一致。结论:脑外伤后Foxj1在脑组织中的表达增高,这种增高的表达参与了脑外伤后脑组织的病理生理和生化变化。     

A Novel Model of Mild Traumatic Brain Injury for Juvenile Rats

Despite growing evidence that childhood represents a major risk period for mild traumatic brain injury (mTBI) from sports-related concussions, motor vehicle accidents, and falls, a reliable animal model of mTBI had previously not been developed for this important aspect of development. The modified weight-drop technique employs a glancing impact to the head of a freely moving rodent transmitting acceleration, deceleration, and rotational forces upon the brain. When applied to juvenile rats, this modified weight-drop technique induced clinically relevant behavioural outcomes that were representative of post-concussion symptomology. The technique is a rapidly applied procedure with an extremely low mortality rate, rendering it ideal for high-throughput studies of therapeutics. In addition, because the procedure involves a mild injury to a closed head, it can easily be used for studies of repetitive brain injury. Owing to the simplistic nature of this technique, and the clinically relevant biomechanics of the injury pathophysiology, the modified weight-drop technique provides researchers with a reliable model of mTBI that can be used in a wide variety of behavioural, molecular, and genetic studies.     

Detection and Structural Characterization of Ether Glycerophosphoethanolamine from Cortical Lysosomes Following Traumatic Brain Injury Using UPLC‐HDMSE

The use of ultra performance liquid chromatography coupled to data independent tandem mass spectrometry with traveling wave ion mobility for detection and structural identification of ether‐linked glycerophosphoethanolamine is described. The experimental design generates 4D data (chromatographic retention time, precursor accurate mass, drift time with associated calculated collisional cross‐section, and time‐aligned accurate mass diagnostic product ions) for each ionization mode. Confident structure identification depends on satisfying 4D data confirmation in both positive and negative ion mode. Using this methodology, a number of ether‐linked glycerophosphoethanolamine lipids are structurally elucidated from mouse brain lysosomes. It is further determined that several ether‐linked glycerophosphoethanolamine structures are differentially abundant between lysosomes isolated from mouse cortex following traumatic brain injury as compared to that of sham animals. The combined effort of aligning multi‐dimensional mass spectrometry data with a well‐defined traumatic brain injury model lays the foundation for gaining mechanistic insight in the role lysosomal membrane damage plays in neuronal cell death following brain injury.