脊髓缺血再灌注损伤动物模型的研究进展

脊髓缺血再灌注损伤(Spinal cord ischemia reperfusion injury,SCIRI)模型对研究临床上SCIRI至关重要。SCIRI动物模型旨在尽可能模拟临床脊髓损伤的病理特点。SCIRI模型因所用动物和方法不同而不同。目前国内外常用的SCIRI模型实验动物包括兔、大鼠和小鼠。大鼠因其脊髓血供和人类相似、相对廉价、繁殖力强且容易获得常常用于制作脊髓再灌注损伤模型。任何模型均有其优缺点。可靠、稳定的动物模型对研究SCIRI的发生机制及评估干预手段的效果和寻求有效的治疗方法具有非常重要的意义。该文就SCIRI动物模型研究进展进行简要综述,为研究者们选择最适合自己研究目标的动物模型提供一定的借鉴。     

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.     

脊髓缺血再灌注损伤的防治概况

脊髓缺血-再灌注损伤(SCII)是一种严重的神经系统损伤,是缺血脊髓组织恢复血液灌注后,脊髓组织的损伤反而加重,表现为其神经损害体征和形态学改变较前更加明显,其发生机制是多因素的综合结果,治疗措施也具有多样性,脊髓缺血后脊髓微血管结构及功能的破坏和脊髓水肿等是脊髓功能损害的主要诱因,至今为止,脊髓缺血再灌注损伤的防治主要有药物及物理治疗等方法,本文作者通过查阅中外文献对脊髓缺血再灌注损伤的特征、发生机制及防治措施作一综述,希望对研究脊髓缺血再灌注损伤防治的学者能有所帮助。     

脊髓分级缺血再灌注损伤模型的建立与病理学评价

目的建立兔脊髓分级缺血再灌注损伤模型和探讨受伤脊髓病理变化可能机制。方法采用肾下腹主动脉阻断法,分别阻断腹主动脉30min、45min和60min后开放,再灌注48h观察神经功能变化以及病理学评价脊髓缺血再灌注损伤程度。结果脊髓缺血时间越长,后肢运动功能损害越明显。伤后2天发现受损脊髓出血、水肿、变性坏死,明显的白细胞浸润以及I-κBα、NF-κBp65、ICAM-l表达增加,脊髓灰质的病理损害严重。再灌注脊髓病理损伤程度依次为缺血60min组>缺血45min组>缺血30min组>假手术组。结论该模型是一种较好的脊髓缺血再灌注损伤模型,阻断肾下腹主动脉血流30min、45min、60min后开放可以较好地反应轻、中、重不同程度缺血再灌注损伤脊髓的病理变化特点。     

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.     

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.     

舒芬太尼预处理对大鼠脊髓缺血再灌注损伤中炎性因子MPO，IL-6，IL-15的影响

目的:探讨舒芬太尼预处理对大鼠脊髓缺血再灌注损伤模型中炎性因子MPO,IL-6,IL-15的影响。方法:健康SD雄性大鼠30只,随机分为假手术组(Sham组,n=10);缺血再灌注组(IR组n=10);舒芬太尼预处理5μg/kg组(Suf5组,n=10)。采用动脉夹夹闭胸主动脉方法制备脊髓缺血再灌注模型。Tarlov法测大鼠运动评分,HE染色观察大鼠脊髓组织细胞形态,Western Blot法测脊髓组织中MPO的表达,ELISA法检测脊髓组织中IL-6,IL-15含量。结果:IR组Tarlov评分高于sham组,Suf5组Tarlov评分低于IR组。HE染色显微镜下见IR组脊髓组织内出现广泛的变性神经元,胞核固缩偏位碎裂,并有有空泡形成;Suf5组脊髓组织神经元损伤坏死数量减少,细胞核形态基本正常。Suf5组中MPO,IL-6,IL-15,含量均低于IR组,IR组中MPO,IL-6,IL-15含量均高于sham组,差异均有统计学意义(P0.05)。结论:舒芬太尼能降低大鼠脊髓缺血再灌注损伤组织中MPO,IL-15,1L-6表达,减轻炎症损害,进而减轻脊髓缺血再灌注损伤。     

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.     

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.     

A Murine Model of Cervical Spinal Cord Injury to Study Post-lesional Respiratory Neuroplasticity

A cervical spinal cord injury induces permanent paralysis, and often leads to respiratory distress. To date, no efficient therapeutics have been developed to improve/ameliorate the respiratory failure following high cervical spinal cord injury (SCI). Here we propose a murine pre-clinical model of high SCI at the cervical 2 (C2) metameric level to study diverse post-lesional respiratory neuroplasticity. The technique consists of a surgical partial injury at the C2 level, which will induce a hemiparalysis of the diaphragm due to a deafferentation of the phrenic motoneurons from the respiratory centers located in the brainstem. The contralateral side of the injury remains intact and allows the animal recovery. Unlike other SCIs which affect the locomotor function (at the thoracic and lumbar level), the respiratory function does not require animal motivation and the quantification of the deficit/recovery can be easily performed (diaphragm and phrenic nerve recordings, whole body ventilation). This pre-clinical C2 SCI model is a powerful, useful, and reliable pre-clinical model to study various respiratory and non-respiratory neuroplasticity events at different levels (molecular to physiology) and to test diverse putative therapeutic strategies which might improve the respiration in SCI patients.     

大鼠脊髓半横断伤联合架桥模型制作及评估

目的:建立大鼠脊髓半横断伤联合架桥模型,为研究脊髓损伤提供动物模型。方法:制作大鼠脊髓半横断伤模型,然后取大鼠前肢正中神经,并于半横断伤两端行正中神经架桥术。术后4周,左心室灌注固定取材,免疫组化染色检测GFAP、RECA、NF-200;另一部分动物行单宁酸-氯化铁灌注;观察移植物内有无血管、血管内有无血流、血管与周边神经纤维的关系。结果:外周神经架桥后4周,移植正中神经贴合于脊髓背侧1/2。移植神经内有RECA阳性的血管存在,而且有血流可以到达移植物内部,且神经纤维(NF-200阳性)与星形胶质细胞(GFAP阳性)关系紧密。结论:大鼠脊髓半横断伤联合正中神经架桥术后,由宿主可以向移植物内长入新生血管,血管有利于神经纤维的存活及生长。本模型为较好的外周神经移植的存活模型,可为进一步的深入研究提供一定的依据。     

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.     

Use of a Hanging Weight System for Coronary Artery Occlusion in Mice

Murine studies of acute injury are an area of intense investigation, as knockout mice for different genes are becoming increasingly available ^1-38. Cardioprotection by ischemic preconditioning (IP) remains an area of intense investigation. To further elucidate its molecular basis, the use of knockout mouse studies is particularly important ^{7, 14, 30, 39}. Despite the fact that previous studies have already successfully performed cardiac ischemia and reperfusion in mice, this model is technically very challenging. Particularly, visual identification of the coronary artery, placement of the suture around the vessel and coronary occlusion by tying off the vessel with a supported knot is technically difficult. In addition, re-opening the knot for intermittent reperfusion of the coronary artery during IP without causing surgical trauma adds additional challenge. Moreover, if the knot is not tied down strong enough, inadvertent reperfusion due to imperfect occlusion of the coronary may affect the results. In fact, this can easily occur due to the movement of the beating heart.Based on potential problems associated with using a knotted coronary occlusion system, we adopted a previously published model of chronic cardiomyopathy based on a hanging weight system for intermittent coronary artery occlusion during IP ³⁹. In fact, coronary artery occlusion can thus be achieved without having to occlude the coronary by a knot. Moreover, reperfusion of the vessel can be easily achieved by supporting the hanging weights which are in a remote localization from cardiac tissues.We tested this system systematically, including variation of ischemia and reperfusion times, preconditioning regiments, body temperature and genetic backgrounds³⁹. In addition to infarct staining, we tested cardiac troponin I (cTnI) as a marker of myocardial infarction in this model. In fact, plasma levels of cTnI correlated with infarct sizes (R2=0.8). Finally, we could show in several studies that this technique yields highly reproducible infarct sizes during murine IP and myocardial infarction^{6, 8, 30, 40, 41}. Therefore, this technique may be helpful for researchers who pursue molecular mechanisms involved in cardioprotection by IP using a genetic approach in mice with targeted gene deletion. Further studies on cardiac IP using transgenic mice may consider this technique.     

miRNAs在脊髓损伤中作用的研究进展

脊髓损伤是一个重要的公共卫生难题,脊髓损伤可划分为三个病理生理阶段:原发性损伤期、继发性损伤期和慢性损伤期。基因表达的改变在脊髓损伤中起到了重要作用,miRNAs可以调控转录后所有基因的表达,所以miRNAs是脊髓损伤中一个很具有研究价值的研究对象。miRNAs是20-25碱基组成的非编码RNA,通过与靶mRNAs 3‘UTR结合下调其表达实现的对mRNA翻译进程的调控。miRNAs与中枢神经系统的发育、功能和疾病有密切关系。脊髓损伤后miRNAs通过调节中性粒细胞和炎性反应通路在炎性应答中起到了重要作用;miRNAs在细胞凋亡中表现出了复杂的功能,其表达的改变可能同时刺激和抑制凋亡;miRNAs可通过增强星形胶质细胞肥大和调节胶质瘢痕的进程;miRNAs的下调可能通过促进轴突靶向作用、神经元存活和轴突生长来促进损伤脊髓部位再生进程。目前脊髓损伤仍是现代医学的难题,对神经系统疾病中miRNAs作用的研究,为脊髓损伤治疗提供了一种新的治疗方案,也是将来研究中的热点。     

Use of a Hanging-weight System for Isolated Renal Artery Occlusion

In hospitalized patients, over 50% of cases of acute kidney injury (AKI) are caused by renal ischemia ^1-3. A recent study of hospitalized patients revealed that only a mild increase in serum creatinine levels (0.3 to 0.4 mg/dl) is associated with a 70% greater risk of death than in persons without any increase ¹. Along these lines, surgical procedures requiring cross-clamping of the aorta and renal vessels are associated with a renal failure rates of up to 30% ⁴. Similarly, AKI after cardiac surgery occurs in over 10% of patients under normal circumstances and is associated with dramatic increases in mortality. AKI are also common complications after liver transplantation. At least 8-17% of patients end up requiring renal replacement therapy ⁵. Moreover, delayed graft function due to tubule cell injury during kidney transplantation is frequently related to ischemia-associated AKI ⁶. Moreover, AKI occurs in approximately 20% of patients suffering from sepsis ⁶.The occurrence of AKI is associated with dramatic increases of morbidity and mortality ¹. Therapeutic approaches are very limited and the majority of interventional trials in AKI have failed in humans. Therefore, additional therapeutic modalities to prevent renal injury from ischemia are urgently needed ^{3, 7-9}. To elucidate mechanisms of renal injury due to ischemia and possible therapeutic strategies murine models are intensively required ^7-13. Mouse models provide the possibility of utilizing different genetic models including gene-targeted mice and tissue specific gene-targeted mice (cre-flox system). However, murine renal ischemia is technically challenging and experimental details significantly influence results. We performed a systematic evaluation of a novel model for isolated renal artery occlusion in mice, which specifically avoids the use of clamping or suturing the renal pedicle ¹⁴. This model requires a nephrectomy of the right kidney since ischemia can be only performed in one kidney due to the experimental setting. In fact, by using a hanging-weight system, the renal artery is only instrumented once throughout the surgical procedure. In addition, no venous or urethral obstruction occurs with this technique. We could demonstrate time-dose-dependent and highly reproducible renal injury with ischemia by measuring serum creatinine. Moreover, when comparing this new model with conventional clamping of the whole pedicle, renal protection by ischemic preconditioning is more profound and more reliable. Therefore his new technique might be useful for other researchers who are working in the field of acute kidney injury.     

Combination of Dexamethasone and Aminoguanidine Reduces Secondary Damage in Compression Spinal Cord Injury

The study was performed to investigate the effect of combination therapy with aminoguanidine (AG) and dexamethasone (DEX) on the compression spinal cord injury (SCI) in rat. Compared to the control group, the combination therapy group with AG (75 mg/kg) and DEX (0.025 mg/kg) significantly reduced the degree of (1) spinal cord edema, (2) the permeability of blood spinal cord barrier (measured by ^99mTc-Albumin), (3) infiltration of neutrophils (MPO evaluation), (4) cytokines expression (tumor necrosis factor-α and interleukin-1β), and (5) apoptosis (measured by Bax and Bcl-2 expression). In addition, we have also clearly demonstrated that the combination therapy significantly ameliorated the recovery of limb function (evaluated by motor recovery score). Taken together, our results clearly indicated for the first time that strategies targeting multiple proinflammatory pathways may be more effective than a single effector molecule for the treatment of SCI.     

Analysis of Rat Hindlimb Muscle Proteins by Two-Dimensional Gels Following Spinal Cord Injury

Proteins from extensor digitorum longus (EDL), plantaris (PLN), and soleus (SOL) muscles of adult, female rats were examined by high resolution two-dimensional gel electrophoresis up to 4 weeks following spinal cord transection. The electrophoretograms were analyzed by computer imaging and densitometry. Reproducible and significant changes in the relative concentrations of several proteins in each muscle type were detected. Whereas changes involving the largest number of proteins were observed in SOL, changes in EDL and PLN were also detected. In SOL, approximately 50% of the altered proteins increased in concentration and the remaining decreased: Actin and myosin light chains LCF-1 and LCF-2 were among those proteins whose concentrations increased, whereas myosin light chains LCS-1 and LCS-2 were among those proteins whose concentrations decreased. The present findings regarding the reversal in myosin light chain composition provide biochemical support for previously published data on changes in contractile properties of muscles following spinalization. In EDL, the relative concentration of only one protein was elevated in a time-dependent manner. The concentrations of two protein species in PLN were increased following cord transection. These findings provide new biochemical markers on the effects of spinal cord on gene expression in specific hindlimb skeletal muscles.     

Experimental Demyelination and Remyelination of Murine Spinal Cord by Focal Injection of Lysolecithin

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system characterized by plaque formation containing lost oligodendrocytes, myelin, axons, and neurons. Remyelination is an endogenous repair mechanism whereby new myelin is produced subsequent to proliferation, recruitment, and differentiation of oligodendrocyte precursor cells into myelin-forming oligodendrocytes, and is necessary to protect axons from further damage. Currently, all therapeutics for the treatment of multiple sclerosis target the aberrant immune component of the disease, which reduce inflammatory relapses but do not prevent progression to irreversible neurological decline. It is therefore imperative that remyelination-promoting strategies be developed which may delay disease progression and perhaps reverse neurological symptoms. Several animal models of demyelination exist, including experimental autoimmune encephalomyelitis and curprizone; however, there are limitations in their use for studying remyelination. A more robust approach is the focal injection of toxins into the central nervous system, including the detergent lysolecithin into the spinal cord white matter of rodents. In this protocol, we demonstrate that the surgical procedure involved in injecting lysolecithin into the ventral white matter of mice is fast, cost-effective, and requires no additional materials than those commercially available. This procedure is important not only for studying the normal events involved in the remyelination process, but also as a pre-clinical tool for screening candidate remyelination-promoting therapeutics.     

富血小板血浆在脊柱脊髓损伤治疗中的应用进展

富血小板血浆是近些年来比较热门的一种血液制品,其来源于自体,且制备方法简单,又富含大量血小板及多种生长因子,能够加速骨愈合,增强骨再生,促进软组织及神经损伤恢复,因此得到了广泛的关注。国内外的研究人员根据富血小板血浆所具有的特点,针对各个方面对其进行了大量的研究实验,并且在临床骨科疾病的治疗中也已经开始了实验性应用,如骨缺损、骨再生,肌腱、韧带及软组织损伤,脊柱脊髓损伤等。尤其是在脊柱脊髓损伤的治疗方面,无论是单独应用富血小板血浆治疗,还是联合应用富血小板血浆与脊髓神经前体细胞、骨髓间充质干细胞等有利于脊髓神经损伤恢复的细胞因子复合物共同治疗,均取得了突破性的进展,为研究脊柱脊髓损伤的治疗提供了新的方向。