Comparative Efficacy of GABAA and GABAB Receptor Agonists in Pain Alleviation in a Spinal Cord Injury Model of Neuropathic Pain

Neurophysiology - We investigated the effectiveness of intrathecal introduction of GABAA and GABAB receptor agonists in reversing pain modalities in a central model of neuropathic pain. In adult...     

Rapamycin Plays a Neuroprotective Effect after Spinal Cord Injury via Anti‐Inflammatory Effects

Whether rapamycin has neuroprotective effects in spinal cord injury remains controversial. The present study shows that rapamycin protects neurons from death after spinal cord injury by inhibiting the secondary inflammatory response. The effects of rapamycin were tested using a myeloperoxidase assay, Western blotting, immunohistochemistry, and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The experimental results showed that after spinal cord injury, rapamycin reduced the numbers of activated microglia and neutrophils in the damage zone, lowered the expression levels of TNF‐α and IL‐1β, reduced the apoptotic cells, and increased the survival of neurons. The above data proved that rapamycin diminishes inflammatory cell activation and proliferation, downregulates the expression of inflammatory factors, reduces the microenvironmental damage effects on neurons in the acute injury phase, and thus promotes the survival of neurons. Therefore, we believe that rapamycin has neuroprotective effects in spinal cord injury.     

Upregulation of G-protein Coupled Receptor 120 in Rats Following Spinal Cord Injury

Neurochemical Research - G protein-coupled receptors (GPCRs) are fundamental mediators of a wide array of processes including proliferation, immune cell function and neural signaling. GPR120 is a...     

Calibrated Forceps Model of Spinal Cord Compression Injury

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The calibrated forceps model of compression injury is a convenient, low cost, and very reproducible animal model for SCI. We used a pair of modified forceps in accordance with the method published by Plemel et al. (2008) to laterally compress the spinal cord to a distance of 0.35 mm. In this video, we will demonstrate a dorsal laminectomy to expose the spinal cord, followed by compression of the spinal cord with the modified forceps. In the video, we will also address issues related to the care of paraplegic laboratory animals. This injury model produces mice that exhibit impairment in sensation, as well as impaired hindlimb locomotor function. Furthermore, this method of injury produces consistent aberrations in the pathology of the SCI, as determined by immunohistochemical methods. After watching this video, viewers should be able to determine the necessary supplies and methods for producing SCI of various severities in the mouse for studies on SCI and/or treatments designed to mitigate impairment after injury.     

糖尿病介导的周细胞损伤对脊髓损伤后血脊屏障破坏的作用

糖尿病是一种常见的慢性代谢异常性疾病,可通过血糖异常诱导体内内环境紊乱,引起一系列急性或慢性并发症。慢性高血糖可引起大血管和微血管病变,该过程由错综复杂的分子机制协同调控,例如炎症反应、细胞内应激作用、细胞焦亡和细胞铁死亡等。糖尿病可抑制脊髓损伤后血脊屏障修复,加重神经功能损伤,从而不利于运动功能恢复。周细胞是神经血管单元的重要组成部分,参与调控血管再生、毛细血管血流量以及血脊屏障渗透性。脊髓损伤后,血脊屏障遭到破坏,周细胞覆盖率显著降低,血管正常功能受到巨大影响。糖尿病不仅参与调控周细胞的收缩表型和信号传导,而且改变周细胞分泌基因组谱,影响周细胞正常功能。此外,有研究证实,糖尿病促进脊髓损伤后周细胞丢失。本综述系统阐述了糖尿病对血管系统中周细胞的调控作用,及其介导的周细胞损伤对脊髓损伤后血脊屏障修复影响的研究进展。     

Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) below the site of injury. The secondary ischemia that develops following the initial mechanical insult is a serious complication of the SCI and severely impairs the function and viability of surviving neuronal and non-neuronal cells in the SC. In addition, ischemia is also responsible for the growth of lesion during chronic phase of injury and interferes with the cellular repair and healing processes. Thus there is a need to develop a spinal cord ischemia model for studying the mechanisms of ischemia-induced pathology. Focal ischemia induced by photothrombosis (PT) is a minimally invasive and very well established procedure used to investigate the pathology of ischemia-induced cell death in the brain. Here, we describe the use of PT to induce an ischemic lesion in the spinal cord of mice. Following retro-orbital sinus injection of Rose Bengal, the posterior spinal vein and other capillaries on the dorsal surface of SC were irradiated with a green light resulting in the formation of a thrombus and thus ischemia in the affected region. Results from histology and immunochemistry studies show that PT-induced ischemia caused spinal cord infarction, loss of neurons and reactive gliosis. Using this technique a highly reproducible and relatively easy model of SCI in mice can be achieved that would serve the purpose of scientific investigations into the mechanisms of ischemia induced cell death as well as the efficacy of neuroprotective drugs. This model will also allow exploration of the pathological changes that occur following SCI in live mice like axonal degeneration and regeneration, neuronal and astrocytic Ca²⁺ signaling using two-photon microscopy.     

右旋氯胺酮对大鼠脊髓损伤后BDNF和炎症反应的影响

摘要 目的：探讨脊髓损伤后,使用右旋氯胺酮对BDNF和炎症因子TNF-α、IL-1β、IL-10、IL-13表达的影响。方法：将60只成年雄性大鼠随机分入5组,损伤组（A组）5 mg/kg右旋氯胺酮组（B组）、10 mg/kg右旋氯胺酮组（C组）和20 mg/kg右旋氯胺酮组（D组）,假手术组（S组）,每组12只。除S组外,其余4组使用脊髓打击法制备脊髓损伤模型,于脊髓损伤后4 h按照相应的给药剂量以5 mL/h的速度泵注右旋氯胺酮,S组仅进行手术操作,不损伤脊髓,手术后4h以相同的方法泵注等量的0.9 %氯化钠溶液。脊髓损伤后7、14、21和28天使用BBB法进行神经功能缺陷评分。采用HE染色法观察脊髓损伤后存活的神经元数量,ELISA法测定BDNF、Trk B、TNF-α、IL-1β、IL-10、IL-13、的表达水平。结果：与S组比较,其余四组BBB评分升高,存活神经元数量减少,BDNF、TrkB表达显著增加,促炎因子TNF-α、IL-1β表达上调,抑炎因子IL-10、IL-13表达均显著下调（P<0.05）;与A组比较,B组大鼠BBB评分、神经元数量、BDNF、TrkB、炎症因子的表达无明显差异（P>0.05）,C组、D组大鼠BBB评分升高,存活神经元数量减少,BDNF、TrkB表达增多,促炎因子TNF-α、IL-1β上调,抑炎因子IL-10、IL-13表达下调,且差异有统计学意义(P<0.05),C组与D组以上指标差异均无统计学意义（P>0.05）。结论：脊髓损伤后4 h给予10或20 mg/kg右旋氯胺酮可以减轻SCI后神经元损伤,其机制与上调原肌球蛋白受体激酶B（TrkB）的表达,增加BDNF含量,从而下调促炎因子IL-1β,TNF-α,上调抑炎因子 IL-10、IL-13有关,在本研究中右旋氯胺酮最佳作用剂量为10 mg/kg。     

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.     

An Upregulation of SENP3 After Spinal Cord Injury: Implications for Neuronal Apoptosis

SENP3 (SUMO-specific proteases 3), a member of the small ubiquitin-like modifier specific protease family, was identified as a molecule that deconjugates SUMOylation of modified protein substrates and functions as an isopeptidase by disrupting SUMO homeostasis to facilitate cancer development and progression. However, its expression and function in nervous system injury and repair are still unclear. In this study, we employed an acute spinal cord injury (SCI) model in adult rats and investigated the dynamic changes of SENP3 expression in the spinal cord. Western blot analysis indicated a gradual increase in SENP3 expression, which peaked 3?days after SCI, and then declined over the following days. Immunohistochemistry results further confirmed that SENP3 was expressed at low levels in the gray and white matter in the non-injured condition and increased after SCI. Moreover, immunofluorescence double-labeling showed that SENP3 was co-expressed with the neuronal marker, NeuN. Furthermore, the SENP3-positive cells that were co-expressed with NeuN had also expressed active caspase-3 after injury. To investigate whether SENP3 plays a role in neuronal apoptosis, we applied H₂O₂ to induce neuronal apoptosis in vitro. Western blot analysis showed a significant upregulation of SENP3 and active caspase-3 following H₂O₂ stimulation. Taken together, these results suggest that SENP3 may play important roles in the pathophysiology of SCI.     

促凋亡基因Bax在大鼠脊髓损伤后的表达及意义

目的:研究促凋亡基因Bax表达与脊髓损伤(spinal cord injury,SCI)程度的关系.方法:Wistar大鼠36只,随机分成3组,为正常对照组、轻(中)度损伤组和重度损伤组.大鼠在脊髓损伤后14天处死,HE和Nissel染色观察脊髓组织形态结构和病理学变化,免疫组织化学S-P法检测脊髓中Bax表达情况.结果:Bax蛋白在大鼠脊髓损伤前后表达阳性率分别为5.6%和58.3%,有显著性差异(P＜0.05);轻(中)度脊髓损伤和重度脊髓损伤中的Bax的阳性率分别为18.5%和59.3%,有显著性差异(P＜0.05).结论:Bax基因表达与大鼠脊髓损伤有密切关系,且随着损伤程度加重Bax表达也增强.     

Effect of Microcirculation Reserves on Spinal Cord Recovery after Injury

Local capillary blood flow was studied in and around the spinal cord compression focus in humans with spinal injuries in the acute and early periods of the trauma. The effect of the capillary blood flow in the perimedullary network in the region of spinal cord compression on the degree of motor and sensory disturbances was analyzed. The relationship of the increase in capillary blood flow after spinal cord decompression with increases in leg muscle strength and pain threshold was determined.     

Relationship between Spinal Cord Volume and Spinal Cord Injury due to Spinal Shortening

Vertebral column resection is associated with a risk of spinal cord injury. In the present study, using a goat model, we aimed to investigate the relationship between changes in spinal cord volume and spinal cord injury due to spinal shortening, and to quantify the spinal cord volume per 1-mm height in order to clarify a safe limit for shortening. Vertebral column resection was performed at T10 in 10 goats. The spinal cord was shortened until the somatosensory-evoked potential was decreased by 50% from the baseline amplitude or delayed by 10% relative to the baseline peak latency. A wake-up test was performed, and the goats were observed for two days postoperatively. Magnetic resonance imaging was used to measure the spinal cord volume, T10 height, disc height, osteotomy segment height, and spinal segment height pre- and postoperatively. Two of the 10 goats were excluded, and hence, only data from eight goats were analyzed. The somatosensory-evoked potential of these eight goats demonstrated meaningful changes. With regard to neurologic function, five and three goats were classified as Tarlov grades 5 and 4 at two days postoperatively. The mean shortening distance was 23.6 ± 1.51 mm, which correlated with the d-value (post-pre) of the spinal cord volume per 1-mm height of the osteotomy segment (r = 0.95, p < 0.001) and with the height of the T10 body (r = 0.79, p = 0.02). The mean d-value (post-pre) of the spinal cord volume per 1-mm height of the osteotomy segment was 142.87 ± 0.59 mm³ (range, 142.19–143.67 mm³). The limit for shortening was approximately 106% of the vertebral height. The mean volumes of the osteotomy and spinal segments did not significantly change after surgery (t = 0.310, p = 0.765 and t = 1.241, p = 0.255, respectively). Thus, our results indicate that the safe limit for shortening can be calculated using the change in spinal cord volume per 1-mm height.     

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.     

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.     

Arylsulfatase B Improves Locomotor Function after Mouse Spinal Cord Injury

Bacterial chondroitinase ABC (ChaseABC) has been used to remove the inhibitory chondroitin sulfate chains from chondroitin sulfate proteoglycans to improve regeneration after rodent spinal cord injury. We hypothesized that the mammalian enzyme arylsulfatase B (ARSB) would also enhance recovery after mouse spinal cord injury. Application of the mammalian enzyme would be an attractive alternative to ChaseABC because of its more robust chemical stability and reduced immunogenicity. A one-time injection of human ARSB into injured mouse spinal cord eliminated immunoreactivity for chondroitin sulfates within five days, and up to 9 weeks after injury. After a moderate spinal cord injury, we observed improvements of locomotor recovery assessed by the Basso Mouse Scale (BMS) in ARSB treated mice, compared to the buffer-treated control group, at 6 weeks after injection. After a severe spinal cord injury, mice injected with equivalent units of ARSB or ChaseABC improved similarly and both groups achieved significantly more locomotor recovery than the buffer-treated control mice. Serotonin and tyrosine hydroxylase immunoreactive axons were more extensively present in mouse spinal cords treated with ARSB and ChaseABC, and the immunoreactive axons penetrated further beyond the injury site in ARSB or ChaseABC treated mice than in control mice. These results indicate that mammalian ARSB improves functional recovery after CNS injury. The structural/molecular mechanisms underlying the observed functional improvement remain to be elucidated.     

脊髓损伤的基因治疗

基因治疗脊髓损伤（SCI）既不存在胎儿神经组织移植的组织来源问题,且比外周神经组织移植引起的排异性低,是目前脊髓损伤治疗中最有前途的方法.基因治疗的转基因方式有两种：一是将目的基因直接导入体内靶细胞令其表达;二是将基因在体外导入适当的细胞内,并筛选出高效表达的移植细胞作为转基因中介移植到体内靶组织.不论采用何种方式,将基因导入细胞又可用多种手段实现：如微注射、脂质体等物理或化学手段;利用缺陷病毒作为载体感染细胞的生物学手段.因为用生物学手段转基因的细胞移植方法空间定位明确,所以目前最常采用它作为基因治疗效果的研究.虽然SCI基因治疗目前仍停留在实验探索阶段,一些问题尚待解决,但随着基因治疗技术方法的不断提高,它的临床应用前景可以预见.     

Spinal Autofluorescent Flavoprotein Imaging in a Rat Model of Nerve Injury-Induced Pain and the Effect of Spinal Cord Stimulation

Nerve injury may cause neuropathic pain, which involves hyperexcitability of spinal dorsal horn neurons. The mechanisms of action of spinal cord stimulation (SCS), an established treatment for intractable neuropathic pain, are only partially understood. We used Autofluorescent Flavoprotein Imaging (AFI) to study changes in spinal dorsal horn metabolic activity. In the Seltzer model of nerve-injury induced pain, hypersensitivity was confirmed using the von Frey and hotplate test. 14 Days after nerve-injury, rats were anesthetized, a bipolar electrode was placed around the affected sciatic nerve and the spinal cord was exposed by a laminectomy at T13. AFI recordings were obtained in neuropathic rats and a control group of naïve rats following 10 seconds of electrical stimulation of the sciatic nerve at C-fiber strength, or following non-noxious palpation. Neuropathic rats were then treated with 30 minutes of SCS or sham stimulation and AFI recordings were obtained for up to 60 minutes after cessation of SCS/sham. Although AFI responses to noxious electrical stimulation were similar in neuropathic and naïve rats, only neuropathic rats demonstrated an AFI-response to palpation. Secondly, an immediate, short-lasting, but strong reduction in AFI intensity and area of excitation occurred following SCS, but not following sham stimulation. Our data confirm that AFI can be used to directly visualize changes in spinal metabolic activity following nerve injury and they imply that SCS acts through rapid modulation of nociceptive processing at the spinal level.