大鼠海马胶质细胞培养牵张损伤模型的建立

目的 建立大鼠海马胶质细胞培养牵张损伤模型.方法 提取出生时间24h到48h的SD大鼠海马组织行星形胶质细胞培养,采用CIC Ⅱ型细胞损伤装置、根据Ellis方法建立改良的大鼠海马胶质细胞体外牵张损伤模型,损伤程度分轻、中、重三级.对照组不予损伤.分别在2h和24h检测细胞乳酸脱氧酶释放量,并通过碘化丙啶荧光染色观察细胞损伤情况.结果 细胞培养液中乳酸脱氧酶释放量随着损伤程度加重而增高.PrI红染细胞数随着牵张损伤的程度增高而增加.结论 本实验建立的牵张损伤模型使用方便,可重复性好,适于进行神经细胞机械件体外损伤的研究.     

术前高分辨率磁共振血管壁成像评估对症状性大脑中动脉狭窄支架成形术的作用

目的探讨术前应用高分辨率磁共振血管壁成像(HRMR-VWI)评估对症状性大脑中动脉M1段狭窄支架成形术的应用价值。方法回顾性分析2011年8月至2019年5月天津市环湖医院神经外科收治的35例症状性大脑中动脉M1段动脉粥样硬化性狭窄患者的临床资料。所有患者均行支架成形术治疗。术前均采用HRMR-VWI评估动脉粥样斑块的位置和稳定性。通过临床随访明确患者术后30 d的病死率、致残率以及术后12个月的缺血性卒中发生情况,采用影像学随访评估再狭窄率。结果35例患者的术前HRMR-VWI评估结果提示,15例斑块位于大脑中动脉M1段腹侧壁,12例位于下壁,8例位于上壁;其中7例大脑中动脉M1段存在不稳定斑块。35例患者均成功置入支架,支架均贴壁良好,血流通畅。2例(5.7%)患者术中出现技术性并发症。术后30 d 1例(2.8%)患者因脑疝死亡,1例(2.8%)患者遗留神经功能障碍。术后12个月,34例患者无一例发生缺血性卒中,获影像学随访的5例患者未见再狭窄发生。结论HRMR-VWI对于评估大脑中动脉狭窄处斑块的位置、稳定性等具有较高的诊断价值,有利于提高支架成形术治疗症状性大脑中动脉M1段狭窄的安全性和有效性。     

体外培养大鼠脑皮质神经元离心损伤模型的建立

目的建立体外培养大鼠脑皮质神经元离心损伤模型，观察离心损伤后神经元的形态学变化和培养液中乳酸脱氢酶（LDH）的含量变化。方法把培养神经元的六孔培养板的孔内，置入与孔大小相当的200目钢网，并在钢刚上放置6g重物，通过变速离心培养板，造成神经元损伤的作用。使用倒置相差显微镜，电镜观察损伤后神经元的形态改变，并测量培养液中乳酸脱氢酶（LDH）的含量变化。结果光镜可见神经元破碎、轴索肿胀、轴索球、轴索部分崩解。电镜可见细胞骨架发生变化。从神经元损伤15min后，培养上清液中LDH活性增高。损伤组LDH升高有两个时相，第一峰在30min出现，第二峰在72h出现，损伤后30min，24h，48h，72h，损伤组与对照组同时间相比，LDH含量均有显著统计学意义（P〈0．01），第一峰值可能是原发性损伤造成的细胞膜破损，LDH释放量升高引起的，第二峰值可能是继发性损伤导致细胞膜破损，LDH漏出所引起的。结论实验建立的神经元损伤模型操作简便，能较好地模拟脑外伤。     

壁面切应力与颅内动脉瘤的研究进展

壁面切应力(WSS)是血流对血管壁的切向摩擦力,平行作用于血管壁.WSS被认为是与颅内动脉瘤发生、生长和破裂关系最密切的血流动力学参数.由于缺乏统一标准,WSS这一参数如何影响动脉瘤仍存在争议.本文结合文献,对WSS在颅内动脉瘤中的作用研究进展作一综述.     

Dynamics of early locomotor network dysfunction following a focal lesion in an in vitro model of spinal injury

It is unclear how a localized spinal cord injury may acutely affect locomotor networks of segments initially spared by the lesion. To investigate the process of secondary damage following spinal injury, we used the in vitro model of the neonatal rat isolated spinal cord with transverse barriers at the low thoracic–upper lumbar region to allow focal application of kainate in hypoxic and aglycemic solution (with reactive oxygen species). The time‐course and nature of changes in spinal locomotor networks downstream of the lesion site were investigated over the first 24 h, with electrophysiological recordings monitoring fictive locomotion (alternating oscillations between flexor and extensor motor pools on either side) and correlating any deficit with histological alterations. The toxic solution irreversibly suppressed synaptic transmission within barriers without blocking spinal reflexes outside. This effect was focally associated with extensive white matter damage and ventral gray neuronal loss. Although cell losses were < 10% outside barriers, microglial activation with neuronal phagocytosis was detected. Downstream motor networks still generated locomotor activity 24 h later when stimulated with N‐methyl‐d ‐aspartate (NMDA) and serotonin, but not with repeated dorsal root stimuli. In the latter case, cumulative depolarization was recorded from ventral roots at a slower rate of rise, suggesting failure to recruit network premotoneurons. Our data indicate that, within the first 24 h of injury, locomotor networks below the lesion remained morphologically intact and functional when stimulated by NMDA and serotonin. Nevertheless, microglial activation and inability to produce locomotor patterns by dorsal afferent stimuli suggest important challenges to long‐term network operation.     

脊髓损伤大鼠损伤早期电位变化与损伤程度的关系

To investigate characteristics of injury potentials after different degrees of spinal cord injury in rats,the present study established models of spinal cord contusion with severe,moderate,and mild degrees of injury.Injury potential was measured in vivo using a direct current voltage amplification system.Results revealed that in the first 4 hours after acute spinal cord injury,initial amplitude of injury potential was greatest after severe injury,followed by moderate and mild injuries.Amplitude of injury potential decreased gradually with injury time,and the recession curve was logarithmic.Under the same degree of injuries,amplitude of rostral injury potential was generally less than caudal injury potential.Results suggested that injury potential reflected injury severity,because large initial amplitude of injury potential during the early injury stage implied severe injury.     

Assessment of axonal dysfunction in an in vitro model of acute compressive injury to adult rat spinal cord axons

An in vitro model of spinal cord injury was developed to study the pathophysiology of posttraumatic axonal dysfunction. A 25 mm length of thoracic spinal cord was removed from the adult male rat (n = 27). A dorsal column segment was isolated and pinned in a recording chamber and superfused with oxygenated (95%O₂/5% CO₂) Ringer. The cord was stimulated with a bipolar electrode, while two point responses were recorded extracellularly. Injury was accomplished by compression with a modified aneurysm clip which applied a 2 g force for 15 s. With injury the compound action potential (CAP) amplitude decreased to 53.7 ± 5.4% (P < 0.001), while the latency increased to 115.6 ± 3.1% (P < 0.0025) of control values. The absolute refractory period increased with injury from 1.7 ± 0.1 ms to 2.1 ± 0.1 ms (P < 0.001). With train stimulation (200 and 400 Hz), injured axons showed evidence of high frequency conduction failure (P < 0.05). The infusion of 5 mM 4-aminopyridine (4-AP), a blocker of voltage-sensitive ‘fast’ K channels confined to internodal regions, resulted in broadening of the CAP of injured axons to 114.9 ± 3.1% of control (P < 0.05). Ultrastructural analysis of the injured dorsal column segments revealed marked axonal and myelin pathology, including considerable myelin disruption.In conclusion, we have developed and characterized an in vitro model of mammalian spinal cord injury which simulates many of the features of in vivo trauma. Injured axons display characteristic changes in physiological function including a shift in refractory period and high frequency conduction failure. The ultrastructural data and response of injured axons to 4-AP suggest that myelin disruption with exposure of ‘fast’ K⁺ channels contributes to posttraumatic axonal dysfunction.     

Aquaporin-4 expression after experimental contusional injury in an ovine impact-acceleration head injury model

Cerebral contusions are one of the principal lesions of traumatic brain injury and the attendant oedema formation contributes substantially to the clinicopathological manifestations. While it is now recognised that the membrane channel protein aquaporin-4 (AQP-4) plays a major role in the development and resolution of cerebral oedema, assessments of its expression in and around contusions have produced conflicting results. We used an ovine impact-acceleration model of closed head injury to examine contusion-related AQP-4 expression and found that there was a heterogeneous AQP-4 response within contusions, with some astrocytes being nonviable and immunonegative, while others showed increased AQP-4 expression. Pericontusional astrocytes in the penumbra region generally showed more robust AQP-4 immunopositivity than intracontusional glia. Thus, manipulation of AQP-4 expression could have therapeutic applications in controlling cerebral oedema associated with contusions.     

Spontaneous extrusion of peritoneal catheter through intact abdominal wall

A rare occurrence of spontaneous extrusion of a Denver peritoneal catheter through the right flank in a 14-year-old boy is reported. The firm texture and short length of the catheter coupled with its movements with respiration produced a hammer effect and eroded the abdominal wall. Disconnection of the peritoneal catheter from the chamber and pulling out the extruded catheter is suggested as a simple and effective method of removal of the shunt.     

Immediate short-duration hypothermia provides long-term protection in an in vivo model of traumatic axonal injury

A prospective, multicenter, randomized trial did not demonstrate improved outcomes in severe traumatic brain injured patients treated with mild hypothermia [Clifton, G.L., Miller, E.R., Choi, S.C., Levin, H.S., McCauley, S., Smith, K.R., Jr., Muizelaar, J.P., Wagner, F.C., Jr., Marion, D.W., Luerssen, T.G., Chesnut, R.M., Schwartz, M., 2001. Lack of effect of induction of hypothermia after acute brain injury. N. Engl. J. Med. 344, 556-563.]. However, the mean time to target temperature was over 8 h and patient inclusion was based on Glasgow Coma Scale score so brain pathology was likely diverse. There remains significant interest in the benefits of hypothermia after traumatic brain injury (TBI) and, in particular, traumatic axonal injury (TAI), which is believed to significantly contribute to morbidity and mortality of TBI patients. The long-term beneficial effect of mild hypothermia on TAI has not been established. To address this issue, we developed an in vivo rat optic nerve stretch model of TAI. Adult male Sprague-Dawley rats underwent unilateral optic nerve stretch at 6, 7 or 8 mm piston displacement. The increased number of axonal swellings and bulbs immunopositive for non-phosphorylated neurofilament (SMI-32) seen four days after injury was statistically significant after 8 mm displacement. Ultrastructural analysis 2 weeks after 8 mm displacement revealed a 45.0% decrease (p < 0.0001) in myelinated axonal density in the optic nerve core. There was loss of axons regardless of axon size. Immediate post-injury hypothermia (32 °C) for 3 h reduced axonal degeneration in the core (p = 0.027). There was no differential protection based on axon size. These results support further clinical investigation of temporally optimized therapeutic hypothermia after traumatic brain injury.     

Pharmacology of N-methyl-D-aspartate-induced brain injury in an in vivo perinatal rat model

Intrastriatal injection of the glutamate analogue N-methyl-D-aspartate (NMDA, 25 nmol) in postnatal day (PND) 7 rats provides a rapid, sensitive, and reproducible assay in which potential neuroprotective strategies against excitotoxic neuronal injury can be examined in vivo. Brain injury is quantified 5 days postinjection by comparison of the weights of the injected and contralateral cerebral hemispheres. Intraperitoneal injections (15 minutes post-NMDA) of competitive and noncompetitive NMDA receptor antagonists attenuated the severity of NMDA-induced brain injury. The rank order of neuroprotective potency of these antagonists was CGS-19755 greater than DOIPG greater than dextromethorphan greater than HA-966. Of these compounds only the competitive antagonist CGS-19755 provided complete neuroprotection. NMDA-mediated brain injury was also reduced by the specific sigma receptor ligands +PPP and haloperidol (35% reduction). In contrast, drugs that reduce presynaptic neurotransmitter release (adenosine) or enhance neuronal inhibition (baclofen) were not effective against NMDA toxicity. Although all five of the anticonvulsants tested limited NMDA-induced seizure activity, only carbamazepine reduced NMDA-mediated brain injury (36% reduction). These findings extend earlier observations that NMDA receptor antagonists can limit NMDA-induced toxicity in vivo and suggest that sigma receptors contribute to the pathophysiology of NMDA-mediated brain injury in vivo. Furthermore, NMDA-induced seizures and brain injury appear dissociable in this in vivo model. The results illustrate important practical limitations of neuroprotection in vivo vs. in vitro.     

Intimatan (dermatan 4,6-O-disulfate) prevents rethrombosis after successful thrombolysis in the canine model of deep vessel wall injury

INTRODUCTION: Intimatan (dermatan 4,6-O-disulfate), a heparin cofactor II (HCII) agonist, inhibits both the fluid phase and thrombus bound thrombin. The efficacy of Intimatan as an adjunctive anticoagulant during thrombolysis was evaluated in the canine model of arterial injury. MATERIALS AND METHODS: After forming an occlusive thrombus in the right carotid artery (RCA), twenty-one dogs were administered recombinant tissue plasminogen activator (rt-PA) intra-arterially to achieve thrombolysis in the presence of either 0.9% NaCl or Intimatan (9 mg/kg bolus+300 mug/kg/min i.v. infusion). Next, the left carotid arteries (LCA) of the same animals were injured in the presence of either Intimatan or 0.9% NaCl. RESULTS: The incidence of RCA rethrombosis between the Intimatan and control groups was 2/9 and 8/12, respectively. The quality of RCA blood flow, i.e., patency score (Scale of 0-3, i.e., no flow to high flow, respectively), was 2.3+/-0.4 (Intimatan) versus 0.9+/-0.4 (0.9% NaCl). The incidence of primary thrombosis was determined among the groups as 0/9 (Intimatan) versus 7/12 (0.9% NaCl); the patency score was 2.8+/-0.1 (Intimatan) versus 0.9+/-0.4 (0.9% NaCl). Intimatan resulted in a >90% ex vivo inhibition of gamma-thrombin-induced platelet aggregation whereas 0.9% NaCl had no inhibitory effect. Clot-bound thrombin activity was reduced significantly by Intimatan. Intimatan induced <2-fold change in aPTT and bleeding time (BT) when corrected for the 0.9% NaCl group. CONCLUSIONS: Intimatan significantly reduces the incidence of both primary and secondary arterial thrombosis while maintaining a high-grade vessel patency score with only moderate increases in BT and aPTT.     

Pattern of cerebrospinal immediate early gene c-fos expression in an ovine model of non-accidental head injury

Expression of the immediate early gene, c-fos, was examined in a large animal model of non-accidental head injury (“shaken baby syndrome”). Lambs were used because they have a relatively large gyrencephalic brain and weak neck muscles resembling a human infant. Neonatal lambs were manually shaken in a manner similar to that believed to occur with most abused human infants, but there was no head impact. The most striking c-fos expression was in meningothelial cells of the cranial cervical spinal cord and, to a lesser degree, in hemispheric, cerebellar, and brainstem meninges. Vascular endothelial cells also frequently showed c-fos immunopositivity in the meninges and hemispheric white matter. It was hypothesised that this c-fos immunoreactivity was due to mechanical stress induced by shaking, with differential movement of different craniospinal components.     

An in vitro model of traumatic brain injury utilising two-dimensional stretch of organotypic hippocampal slice cultures

Traumatic brain injury (TBI) is caused by rapid deformation of the brain, resulting in a cascade of pathological events and ultimately neurodegeneration. Understanding how the biomechanics of brain deformation leads to tissue damage remains a considerable challenge. We have developed an in vitro model of TBI utilising organotypic hippocampal slice cultures on deformable silicone membranes, and an injury device, which generates tissue deformation through stretching the silicone substrate. Our injury device controls the biomechanical parameters of the stretch via feedback control, resulting in a reproducible and equi-biaxial deformation stimulus. Organotypic cultures remain well adhered to the membrane during deformation, so that tissue strain is 93 and 86% of the membrane strain in the x- and y-axis, respectively. Cell damage following injury is positively correlated with strain. In conclusion, we have developed a unique in vitro model to study the effects of mechanical stimuli within a complex cellular environment that mimics the in vivo environment. We believe this model could be a powerful tool to study the acute phases of TBI and the induced cell degeneration could provide a good platform for the development of potential therapeutic approaches and may be a useful in vitro alternative to animal models of TBI.     

Myeloid cell tissue factor does not contribute to venous thrombogenesis in an electrolytic injury model

Introduction

Tissue factor (TF) is a potent initiator of the extrinsic coagulation cascade. The role and source of TF in venous thrombotic disease is not clearly defined. Our study objective was to identify the contribution of myeloid cell TF to venous thrombogenesis in mice.

Materials and methods

The mouse electrolytic inferior vena cava model was used to induce thrombosis. The following groups of mice were used (1) TF^flox/floxLysMCre⁺ mice that have reduced TF expression in myeloid cells, (2) TF^flox/floxLysMCre^- littermate controls, (3) Wild type mice given a monoclonal anti-mouse TF antibody (1H1) to inhibit TF activity, and (4) Wild type mice given rat IgG. Evaluations at baseline, day 2, and day 6 post thrombosis included thrombus weight, vein wall inflammatory cell migration, vein wall TF mRNA, and plasma D-dimer levels.

Results

Inhibition of TF significantly decreased thrombus weight 2 days post venous thrombosis. In contrast, TF^flox/floxLysMCre⁺ had no change in thrombus weight when compared to littermate controls. The absence of myeloid cell TF did not affect infiltration of neutrophils or monocytes into the vein wall. TF mRNA expression in the vein wall decreased at 2 days but then returned to baseline levels by 6 days post thrombosis. D-dimer levels peaked at 2 days post thrombosis in mice with or without myeloid cell TF.

Conclusions

TF is important in the formation of venous thrombi in the macrovasculature. However, TF expression by myeloid cells does not significantly contribute to venous thrombogenesis in this model.     

大鼠颅脑爆震伤模型的建立

目的建立稳定、可靠的大鼠颅脑爆震伤模型。方法 SD大鼠96只随机分为8、10、12cm组和对照组(n=24),将大鼠麻醉后分别放置距电雷管(600mgTNT当量)垂直距离8 cm、10 cm、12 cm处进行爆炸,于伤后24h行头颅MRI检查,6h、24h、3d、7d行检测损伤区光、电镜下病理变化。结果 8 cm组大鼠几乎在当天全部死亡,10 cm组大鼠存活时间也较短,少有存活7天,12 cm组大鼠存活时间在7天以上,且伤后24小时MRI提示挫伤区存在血肿及挫裂伤,病理学提示皮质、脑膜、脑室周围、神经元胞体、线粒体等均出现不同程度水肿。结论 600mgTNT电雷管在距离大鼠头部垂直12 cm处可制作稳定的大鼠颅脑爆震伤模型。     

不同剂量尼莫地平对大鼠多发性硬化轴索损伤的保护作用

目的探讨尼莫地平对大鼠多发性硬化轴索损伤保护作用的最佳剂量。方法 70只Wistar大鼠随机分为正常组、MS模型对照组和MS模型治疗组,治疗组按给药剂量又分为0.25mg/kg、0.5mg/kg、1mg/kg、2mg/kg、4mg/kg组,分别在治疗前及治疗后14d、28d进行神经丝蛋白(NF)免疫组化分析及髓鞘组化染色分析;每天进行神经功能评分。结果尼莫地平1mgl/kg、2mg/kg、4mg/kg组的轴索损伤明显减轻,与模型对照组及0.25mg/kg、0.5mg/kg组比较差异有显著性(均P<0.01);1mgl/kg、2mg/kg、4mg/kg组之间差异无显著性(均P>0.05)。结论尼莫地平对多发性硬化轴索损伤有保护作用,尼莫地平1mgl/kg是对大鼠多发性硬化轴索损伤保护作用的最佳剂量。     

Gliovascular disruption and cognitive deficits in a mouse model with features of small vessel disease

Cerebral small vessel disease (SVD) is a major cause of age-related cognitive impairment and dementia. The pathophysiology of SVD is not well understood and is hampered by a limited range of relevant animal models. Here, we describe gliovascular alterations and cognitive deficits in a mouse model of sustained cerebral hypoperfusion with features of SVD (microinfarcts, hemorrhage, white matter disruption) induced by bilateral common carotid stenosis. Multiple features of SVD were determined on T2-weighted and diffusion-tensor magnetic resonance imaging scans and confirmed by pathologic assessment. These features, which were absent in sham controls, included multiple T2-hyperintense infarcts and T2-hypointense hemosiderin-like regions in subcortical nuclei plus increased cerebral atrophy compared with controls. Fractional anisotropy was also significantly reduced in several white matter structures including the corpus callosum. Investigation of gliovascular changes revealed a marked increase in microvessel diameter, vascular wall disruption, fibrinoid necrosis, hemorrhage, and blood–brain barrier alterations. Widespread reactive gliosis, including displacement of the astrocytic water channel, aquaporin 4, was observed. Hypoperfused mice also demonstrated deficits in spatial working and reference memory tasks. Overall, gliovascular disruption is a prominent feature of this mouse, which could provide a useful model for early-phase testing of potential SVD treatment strategies.     

星形胶质细胞体外缺血缺氧损伤后炎症模型的建立及应用

目的 建立SD大鼠星形胶质细胞缺血缺氧损伤后炎症模型,探讨亚低温对星形胶质细胞缺血缺氧及损伤后炎症反应的影响.方法 体外原代培养新生SD大鼠星形胶质细胞,免疫荧光染色检测胶质纤维酸性蛋白(GFAP)进行鉴定.实验分正常对照组(C组)、单纯缺氧组(H组)和缺氧+白细胞组(H+W组),以无糖DMEM培养基、5%CO2+95%N2混合气体培养4 h诱导细胞缺氧模型.H+W组加入1 mLSD大鼠外周血白细胞(1×106/mL),C组加入等量培养液.将3组细胞分别置入37℃、34℃、32℃、30 ℃ CO2孵箱中作用24 h,应用速率法和LIVE/DEAD染色分别检测3组细胞在不同温度下乳酸脱氢酶(LDH)释放率的变化和形态学变化.结果 缺氧4 h即可造成星形胶质细胞的损伤.37℃时C组、H组、H+W组细胞LDH释放率依次升高,差异有统计学意义(P<0.05);与37℃相比,亚低温状态(34℃、32℃)下H组、H+W组细胞LDH释放率均降低;但在30℃时,则有明显升高,与32℃相比差异具统计学意义(P<0.05).结论 亚低温状态可明显降低星形胶质细胞的缺血缺氧性损伤及炎症性损伤,其机制可能并非通过单纯的抑制代谢来实现的.