大鼠放射性脊髓损伤脊髓血流量变化规律

目的:放射性脊髓损伤(Radiation spinal cord injury,RSCI)是头颈部、胸部及上腹部肿瘤放射治疗和射线意外照射时的常见并发症,一般认为,白质坏死、脱髓鞘为其主要的病理学变化.然而,越来越多的证据表明血-脊髓屏障破裂和血管通透性增加等血管损伤远早于白质坏死和脱髓鞘改变.所以本文阐明大鼠放射性脊髓损伤病理生理过程中脊髓血流量变化规律.方法:将60只Sprague-Dawley (SD)大鼠随机分为12组,1组为对照,其余11组采用60Co放射治疗机行30 Gy大鼠颈髓C2-T2单次照射,剂量率为153 cGy/min,源皮距为80 cm,照射时长为1153 s,照射范围为2.0× 1.0 cm,对照组大鼠于麻醉后置于60Co放射治疗机下,佯照,照射前及照射后分别采用激光多普勒法测量脊髓血流量,11组大鼠于照射前以及照射后1、3、7、14、21、30、60、90、120、150、180天进行测量,以照射前测量值为基数,各时间点以基数的百分比表示该时间点脊髓血流量.结果:大鼠放射性脊髓损伤后,脊髓血流量在照射早期即有降低,照射后90天达到最低,随后脊髓血流量进入平台期.结论:阐明了大鼠放射性脊髓损伤后脊髓血流量的变化规律.大鼠放射性脊髓损伤可影响脊髓血流量,导致脊髓长期处于持续低灌流、缺血缺氧状态,最终导致脊髓不可逆性损伤.临床上放射性脊髓损伤的病人感到疲乏无力,出现神经系统的症状体征,通常死于脑疝.本文为临床上疲乏无力,出现神经系统的症状体征,死于脑疝放射性脊髓损伤的病人的早期防治提供病理生理基础.     

A Contusion Model of Severe Spinal Cord Injury in Rats

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is described to obtain a consistent model of severe SCI. Use of a stereotactic frame and computer controlled impactor allows for creation of reproducible injury. Hypothermia and urinary tract infection pose significant challenges in the post-operative period. Careful monitoring of animals with daily weight recording and bladder expression allows for early detection of post-operative complications. The functional results of this contusion model are equivalent to transection models. The contusion model can be utilized to evaluate the efficacy of both neuroprotective and neuroregenerative approaches.     

炎性小体在诱导脊髓损伤神经炎症中的作用

脊髓损伤的治疗与康复一直是医学领域的重大难题,尤其是在改善损伤的神经功能方面进展甚微。继发性损伤是造成脊髓损伤后神经功能障碍的主要原因,炎症反应是继发性损伤阶段最重要的病理过程。急性期通过抑制神经炎症来减轻继发性损伤被认为可减轻神经功能损害而达到神经保护作用。炎性小体是一类蛋白质复合体,由模式识别受体中的NLRs家族和PHYIN家族的受体蛋白质作为主要框架组装并命名,常见的炎性小体包括NLRP1、NLRP3、NLRC4(IPAF)、AIM2等。在感染或受到损伤刺激时,炎性小体在细胞质内组装,并激活促炎症蛋白酶胱天蛋白酶1(caspase-1),活化的胱天蛋白酶1一方面促进促炎症细胞因子IL-1β和IL-18的前体成熟和分泌,另一方面介导细胞焦亡。细胞焦亡以细胞肿胀破裂并释放细胞内容物为特征,是在炎症和应激的病理条件下诱导的程序性细胞死亡方式。促炎症细胞因子和焦亡释放的胞内物质都可作为促炎信号引发炎症反应。近期发现,炎性小体通过诱导促炎因子释放以及介导细胞焦亡等途径, 参与激活脊髓损伤后的炎症级联反应,加重继发性神经炎症。靶向抑制炎性小体的激活可减轻炎症反应,促进神经细胞存活,达到神经保护作用。因此,炎性小体有望成为脊髓损伤治疗的新靶点。本文拟从炎性小体的结构及其在脊髓损伤中的作用、激活机制和治疗前景进行综述,以期为后续研究提供思路。     

Cyclosporin-A Inhibits Constitutive Nitric Oxide Synthase Activity and Neuronal and Endothelial Nitric Oxide Synthase Expressions after Spinal Cord Injury in Rats

Nitric oxide (NO) plays a role in the pathophysiology of spinal cord injury (SCI). NO is produced by three types of nitric oxide synthase (NOS) enzymes: The constitutive Ca²⁺/calmodulin-dependent neuronal NOS (nNOS) and endothelial NOS (eNOS) isoforms, and the inducible calcium-independent isoform (iNOS). During the early stages of SCI, nNOS and eNOS produce significant amounts of NO, therefore, the regulation of their activity and expression may participate in the damage after SCI. In the present study, we used Cyclosporin-A (CsA) to further substantiate the role of Ca-dependent NOS in neural responses associated to SCI. Female Wistar rats were subjected to SCI by contusion, and killed 4 h after lesion. Results showed an increase in the activity of constitutive NOS (cNOS) after lesion, inhibited by CsA (2.5 mg/kg i.p.). Western blot assays showed an increased expression of both nNOS and eNOS after trauma, also antagonized by CsA administration.     

Neuroprotective Effects of Ebselen on Experimental Spinal Cord Injury in Rats

Spinal cord injury (SCI) results in rapid and significant oxidative stress. This study was aimed to investigate the possible beneficial effects of Ebselen in comparison with Methylprednisolone in experimental SCI. Thirty six Wistar albino rats (200–250 g) were divided in to six groups; A (control), B (only laminectomy), C (Trauma; laminectomy + spinal trauma), D (Placebo group; laminectomy + spinal trauma + serum physiologic), E (Methylprednisolone group; laminectomy + spinal trauma + Methylprednisolone treated), F (Ebselen group; laminectomy + spinal trauma + Ebselen treated), containing 6 rats each. Spinal cord injury (SCI) was performed by placement of an aneurysm clip, extradurally at the level of T11–12. After this application, group A, B and C were not treated with any drug. Group D received 1 ml serum physiologic. Group E received 30 mg/kg Methylprednisolone and, Group F received 10 mg/kg Ebselen intraperitoneally (i.p.). Rats were neurologically examined 24 h after trauma and spinal cord tissue samples had been harvested for both biochemical and histopathological evaluation. All rats were paraplegic after SCI except the ones in group A and B. Neurological scores were not different in traumatized rats than that of non-traumatized ones. SCI significantly increased spinal cord tissue malondialdehyde (MDA) and protein carbonyl (PC) levels and also decreased superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) enzyme activities compared to control. Methylprednisolone and Ebselen treatment decreased tissue MDA and PC levels and prevented inhibition of the enzymes SOD, GSH-Px and CAT in the tissues. However, the best results were obtained with Ebselen. In groups C and D, the neurons of the spinal cord tissue became extensively dark and degenerated with picnotic nuclei. The morphology of neurons in groups E and F were very well protected, but not as good as the control group. The number of neurons in the spinal cord tissues of the groups C and D were significantly less than the groups A, B, E and F. We concluded that the use of Ebselen treatment might have potential benefits in spinal cord tissue damage on clinical grounds.     

Glucocorticoid Receptors and Enzyme Induction in the Spinal Cord of Rats: Effects of Acute Transection

The spinal cord is a glucocorticoid-responsive tissue, as demonstrated by hormonal effects on enzyme induction and by the presence of type II and type I glucocorticoid receptors in cytoplasmic extracts of this CNS region. Using microdissection techniques, we have found in the present investigation that glucocorticoid type II receptors are the most abundant class detected in gray (ventral and dorsal horns) and white (lateral funiculus) matter and that the distribution of type II sites among these regions was quantitatively similar. Type I sites were also quantified, with a slight prevalence in gray matter as opposed to white matter. Furthermore, stimulation of an inducible enzyme, ornithine decarboxylase (ODC), was found in ventral horn and lateral funiculus but not in dorsal horn after administration of dexamethasone (DEX), a type II receptor ligand. We also found that surgical transection of the spinal cord, while markedly increasing ODC activity per se, did not prevent the stimulatory effect of DEX administration on ODC activity measured in the lumbar enlargement of the spinal cord located below the surgical lesion. Taken together, the results suggest a direct effect of glucocorticoids on ODC activity in the spinal cord of rats, probably mediated by glucocorticoid receptors (type II) found in target cells of the ventral horn and lateral funiculus. The results also indicate that glucocorticoid receptors of the dorsal horn were not involved in ODC induction, and a function for these receptors awaits the results of further experimentation.     

Long-Term Changes in Spinal Cord Evoked Potentials After Compression Spinal Cord Injury in the Rat

SUMMARY 1. After traumatic spinal cord injury (SCI), histological and neurological consequences are developing for several days and even weeks. However, little is known about the dynamics of changes in spinal axonal conductivity. The aim of this study was to record and compare repeated spinal cord evoked potentials (SCEP) after SCI in the rat during a 4 weeks’ interval. These recordings were used: (i) for studying the dynamics of functional changes in spinal axons after SCI, and (ii) to define the value of SCEP as an independent outcome parameter in SCI studies.2. We have used two pairs of chronically implanted epidural electrodes for stimulation/recording. The electrodes were placed below and above the site of injury, respectively. Animals with implanted electrodes underwent spinal cord compression injury induced by epidural balloon inflation at Th8–Th9 level. There were five experimental groups of animals, including one control group (sham-operated, no injury), and four injury groups (different degrees of SCI).3. After SCI, SCEP waveform was either significantly reduced or completely lost. Partial recovery of SCEPs was observed in all groups. The onset and extent of recovery clearly correlated with the severity of injury.There was good correlation between quantitated SCEP variables and the volumes of the compressing balloon. However, sensitivity of electropohysiological parameters was inferior compared to neurological and morphometric outcomes.4. Our study shows for the first time, that the dynamics of axonal recovery depends on the degree of injury. After mild injury, recovery of signal is rapid. However, after severe injury, axonal conductivity can re-appear after as long as 2 weeks postinjury.In conclusion, SCEPs can be used as an independent parameter of outcome after SCI, but in general, the sensitivity of electrophysiological data were worse than standard morphological and neurological evaluations.     

器官型培养的脊髓与体内脊髓的对比

旨在观察体外器官型培养的脊髓薄片是否与同龄大鼠体内生长的脊髓具有相似的形态和恒定的前角a运动神经元数目,建立能模拟体内生长环境的稳定的脊髓器官培养模型。利用出生后8天乳鼠的腰段脊髓组织切片建立脊髓器官型培养模型,用神经元的特异性免疫组化染色SMI-32对脊髓前角a运动神经元加以鉴定并与同龄大鼠体内生长的脊髓做比较。结果发现脊髓体外生长良好,形态完整,a运动神经元数目恒定,与同龄大鼠比较无显著差异,并可长期存活达2个月。脊髓的器官培养技术为研究脊髓生理、病理改变及神经保护提供了有效的方法。     

Spatiotemporal Expression of Dexras1 After Spinal Cord Transection in Rats

Dexras1, a brain-enriched member of the Ras subfamily of GTPases, as a novel physiologic nitric oxide (NO) effector, anchor neuronal nitric oxide synthase (nNOS) that increased after spinal cord injury (SCI), to specific targets to enhance NO signaling, and is strongly and rapidly induced during treatment with dexamethasone. It is unknown how the central nervous system (CNS) trauma affects the expression of Dexras1. Here we used spinal cord transection (SCT) model to detect expression of Dexras1 at mRNA and protein level in spinal cord homogenates by real-time PCR and Western blot analysis. The results showed that Dexras1 mRNA upregulated at 3 day, 5 day, and 7 day significantly (P < 0.05) that was consistent with the protein level except at 7 day. Immunofluorescence revealed that both neurons and glial cells showed Dexras1 immunoreactivivty (IR) around SCT site, but the proportion is different. Importantly, injury-induced expression of Dexras1 was co-labeled by caspase-3 (apoptotic marker) and Tau-1 (marker for pathological oligodendrocyte). Furthermore, colocalization of Dexras1, carboxy-terminal PSD95/DLG/ZO-1 (PDZ) ligand of nNOS (CAPON) and nNOS was observed in neurons and glial cells, supporting the existence of ternary complexes in this model. Thus, the results that the transient high expression of Dexras1 which localized in apoptotic neurons and pathological oligodendrocytes might provide new insight into the secondary response after SCT. Xin Li, Chun Cheng, and Min Fei contributed equally to this work.     

MiR-144通过靶向抑制GRK5表达促进脊髓星形胶质细胞活化

已知miR-144与细胞活化和增殖有关,然而其具体分子机制尚不明确。本研究发现miR-144通过靶向GRK5促进脊髓星形胶质细胞的活化。运用real-time PCR检测脊髓损伤和正常大鼠的脊髓组织及其脊髓星形胶质细胞中miR-144的表达,发现与正常的组织和细胞相比,miR-144在脊髓损伤组织和星形胶质细胞中的表达水平显著降低;Western印迹检测到脊髓损伤大鼠的星形胶质细胞中GFAP蛋白的表达显著低于正常大鼠,而GRK5蛋白的表达高于正常大鼠;MTT分析结果显示转染miR-144可显著提高脊髓损伤大鼠的星形胶质细胞活性,但对细胞增殖无明显作用;酶活性试剂盒分析发现miR-144显著提高了SOD和GSH活性;生物学信息分析和萤光素酶报告基因检测结果显示miR-144能靶向结合GRK5,并下调GRK5的表达;MiR-144 mimic转染或miR-144 mimic与pcDNA-GRK5共转染脊髓损伤的星形胶质细胞,发现miR-144转染能通过激活NF-κB通路消除pcDNA-GRK5引起的细胞活化抑制。综上所述,miR-144通过靶定结合癌基因GRK5来促进脊髓星形胶质细胞细胞的活化。     

超早期高压氧对脊髓完全横断损伤大鼠血液生化及后肢运动功能的影响

目的探讨超早期高压氧（HBO）治疗对脊髓完全横断损伤模型血液生化及后肢运动功能的影响。方法 55只SD大鼠随机分为A组（假手术组,15只）、B组（模型组,20只）及C组（高压氧组,20只）,A组仅行椎板切除术,其余2组均行T10椎板水平脊髓完全横向切断术。B、C组均予常规护理,C组于术后3 h置于动物舱内开始高压氧治疗,10 d一疗程,共3疗程。分别于建立模型后第1～6周末,用BBB运动功能评分法评价并比较两组大鼠后肢运动功能恢复程度,术后第6周过量麻醉处死大鼠,以40 g/L多聚甲醛行心室-主动脉灌注,取脊髓损伤区标本,光镜观察损伤脊髓的组织病理学改变。检测血钙（Ca）、血磷（P）、血清碱性磷酸酶（ALP）改变情况。结果 B、C两组大鼠术后第1～6周BBB运动功能评分逐渐增高,C组在3～6周末的BBB运动功能评分均明显高于B组。B组、C组血钙、血磷在术后1、3周高于A组,血清碱性磷酸酶（ALP）术后1、3周低于A组;C组血钙、血磷在术后5、6周低于B组。病理组织切片观察C组较B组组织水肿减轻,炎性细胞浸润减轻。结论超早期高压氧治疗能促进脊髓完全横断损伤大鼠后肢运动功能的部分恢复,降低血钙、血磷含量,对脊髓完全横断损伤大鼠具有保护和治疗作用。     

铁死亡参与脊髓损伤调控的研究进展

铁死亡是一种铁依赖性的,以细胞内脂质活性氧堆积为特征的细胞程序性死亡方式。广泛存在于肿瘤、癌症、急性肾损伤等多种疾病当中。脊髓损伤(spinal cord injury, SCI) 是一种严重的创伤性神经系统疾病,具有高发病率、高死亡率、高致残率的特点。目前,脊髓损伤的具体发生机制及高效治疗方法仍在探索当中,这也是亟待解决的世界性难题。研究表明,脊髓损伤后调控神经细胞的程序性死亡是治疗SCI的重点。然而,对于铁死亡参与脊髓损伤的分子生物学机制尚缺乏系统和深入的认识。收集和整理了近几年国内外有关脊髓损伤后铁死亡方面的相关文献,针对铁死亡参与脊髓损伤的调控机制和研究进展进行了综述,以期为治疗脊髓损伤带来新的思路。     

Contrast Enhanced Ultrasound Imaging for Assessment of Spinal Cord Blood Flow in Experimental Spinal Cord Injury

Reduced spinal cord blood flow (SCBF) (i.e., ischemia) plays a key role in traumatic spinal cord injury (SCI) pathophysiology and is accordingly an important target for neuroprotective therapies. Although several techniques have been described to assess SCBF, they all have significant limitations. To overcome the latter, we propose the use of real-time contrast enhanced ultrasound imaging (CEU). Here we describe the application of this technique in a rat contusion model of SCI. A jugular catheter is first implanted for the repeated injection of contrast agent, a sodium chloride solution of sulphur hexafluoride encapsulated microbubbles. The spine is then stabilized with a custom-made 3D-frame and the spinal cord dura mater is exposed by a laminectomy at ThIX-ThXII. The ultrasound probe is then positioned at the posterior aspect of the dura mater (coated with ultrasound gel). To assess baseline SCBF, a single intravenous injection (400 µl) of contrast agent is applied to record its passage through the intact spinal cord microvasculature. A weight-drop device is subsequently used to generate a reproducible experimental contusion model of SCI. Contrast agent is re-injected 15 min following the injury to assess post-SCI SCBF changes. CEU allows for real time and in-vivo assessment of SCBF changes following SCI. In the uninjured animal, ultrasound imaging showed uneven blood flow along the intact spinal cord. Furthermore, 15 min post-SCI, there was critical ischemia at the level of the epicenter while SCBF remained preserved in the more remote intact areas. In the regions adjacent to the epicenter (both rostral and caudal), SCBF was significantly reduced. This corresponds to the previously described “ischemic penumbra zone”. This tool is of major interest for assessing the effects of therapies aimed at limiting ischemia and the resulting tissue necrosis subsequent to SCI.     

Novel Therapeutic Effects in Rat Spinal Cord Injuries: Recovery of the Definitive and Early Spinal Cord Injury by the Administration of Pentadecapeptide BPC 157 Therapy

Recently, marked therapeutic effects pertaining to the recovery of injured rat spinal cords (1 min compression injury of the sacrocaudal spinal cord (S2-Co1) resulting in tail paralysis) appeared after a single intraperitoneal administration of the stable gastric pentadecapeptide BPC 157 at 10 min post-injury. Besides the demonstrated rapid and sustained recovery (1 year), we showed the particular points of the immediate effect of the BPC 157 therapy that began rapidly after its administration, (i) soon after injury (10 min), or (ii) later (4 days), in the rats with a definitive spinal cord injury. Specifically, in counteracting spinal cord hematoma and swelling, (i) in rats that had undergone acute spinal cord injury, followed by intraperitoneal BPC 157 application at 10 min, we focused on the first 10–30 min post-injury period (assessment of gross, microscopic, and gene expression changes). Taking day 4 post-injury as the definitive injury, (ii) we focused on the immediate effects after the BPC 157 intragastric application over 20 min of the post-therapy period. Comparable long-time recovery was noted in treated rats which had definitive tail paralysis: (iii) the therapy was continuously given per orally in drinking water, beginning at day 4 after injury and lasting one month after injury. BPC 157 rats presented only discrete edema and minimal hemorrhage and increased Nos1, Nos2, and Nos3 values (30 min post-injury, (i)) or only mild hemorrhage, and only discrete vacuolation of tissue (day 4, (ii)). In the day 4–30 post-injury study (iii), BPC 157 rats rapidly presented tail function recovery, and no demyelination process (Luxol fast blue staining).     

VEGF165 Therapy Exacerbates Secondary Damage Following Spinal Cord Injury

Vascular endothelial growth factor (VEGF) demonstrates potent and well-characterized effects on endothelial cytoprotection and angiogenesis. In an attempt to preserve spinal microvasculature and prolong the endogenous neovascular response observed transiently following experimental spinal cord injury (SCI), exogenous recombinant human VEGF (rhVEGF₁₆₅) was injected into the injured rat spinal cord. Adult female Fischer 344 rats were subjected to moderate SCI (12.5 g-cm) using the NYU impactor. At 72 h after injury, animals were randomly assigned to three experimental groups receiving no microinjection or injection of saline or saline containing 2 g of rhVEGF₁₆₅. Acutely, VEGF injection resulted in significant microvascular permeability and infiltration of leukocytes into spinal cord parenchyma. 6 weeks postinjection, no significant differences were observed in most measures of microvascular architecture following VEGF treatment, but analysis of histopathology in spinal cord tissue revealed profound exacerbation of lesion volume. These results support the idea that intraparenchymal application of the proangiogenic factor VEGF may exacerbate SCI, likely through its effect on vessel permeability.     

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6 weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2 days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.     

Effect of Propionyl-L-Carnitine On Rat Spinal Cord Ischaemia and Post-Ischaemic Reperfusion Injury

In this study we have examined the effect of propionyl-L-carnitine (PC) on rat spinal cord ischaemia and post-ischaemic reperfusion injury by evaluating two lipid peroxidation indices, thiobarbituric acid reactive substances (TBARS) and diene conjugation, before and after the addition of an ADP-Fe⁺² complex to spinal cord homogenates. Aerobic, ischaemic, and post ischaemic reperfusion rat spinal cord homogenates from PC treated and untreated animals did not show any statistically significant difference in their TBARS and conjugated diene content. The addition of the ADP-Fe⁺² complex to these homogenates resulted in an increased production of both the lipid peroxidation indices, though the magnitude of such formation was related to the type of experimental intervention. The post-ischaemic reperfusion samples of untreated rats showed the highest TBARS and conjugated diene content, while ischaemic samples in either treated and untreated rats did not show any statistically significant difference with respect to the aerobic samples. The post-ischaemic reperfusion samples of treated rats showed a statistically significant decrease of TBARS and conjugated diene production in comparison to the untreated samples. In addition, PC was also able to partially inhibit TBARS and conjugated diene formation in linoleic acid micelles exposed to hemoglobin, though it did not protect albumin fragmentation from the irradiation of water with an X-ray source.     

Effect of Propionyl-L-Carnitine On Rat Spinal Cord Ischaemia and Post-Ischaemic Reperfusion Injury

In this study we have examined the effect of propionyl-L-carnitine (PC) on rat spinal cord ischaemia and post-ischaemic reperfusion injury by evaluating two lipid peroxidation indices, thiobarbituric acid reactive substances (TBARS) and diene conjugation, before and after the addition of an ADP-Fe⁺² complex to spinal cord homogenates. Aerobic, ischaemic, and post ischaemic reperfusion rat spinal cord homogenates from PC treated and untreated animals did not show any statistically significant difference in their TBARS and conjugated diene content. The addition of the ADP-Fe⁺² complex to these homogenates resulted in an increased production of both the lipid peroxidation indices, though the magnitude of such formation was related to the type of experimental intervention. The post-ischaemic reperfusion samples of untreated rats showed the highest TBARS and conjugated diene content, while ischaemic samples in either treated and untreated rats did not show any statistically significant difference with respect to the aerobic samples. The post-ischaemic reperfusion samples of treated rats showed a statistically significant decrease of TBARS and conjugated diene production in comparison to the untreated samples. In addition, PC was also able to partially inhibit TBARS and conjugated diene formation in linoleic acid micelles exposed to hemoglobin, though it did not protect albumin fragmentation from the irradiation of water with an X-ray source.