Postischemic hypoxia improves metabolic and functional recovery of the spinal cord

We studied the effect of graded postischemic reoxygenation on the tissue concentrations of adenylates, glucose, and lactate in the rabbit lumbar spinal cord after 10, 20, and 30 minutes of ischemia. In comparison with recirculation without manipulated PaO2, a decrease of PaO2 to 40 to 45 mm Hg upon reestablishment of blood circulation after ischemia led to an amelioration of the energy metabolism in the spinal cord tissue as determined by measuring the ATP concentration and energy charge. The protective effect of postischemic hypoxia was also reflected by the improvement of neurologic functions in animals after 10 and 20 minutes of ischemia.     

不完全性脊髓缺血损伤动物模型的建立及价值

目的 探讨不完全性脊髓缺血损伤动物模型的建立方法,为不完全性脊髓缺血损伤机制研究提供理想的载体. 方法 24只新西兰大白兔按照随机数字表法分为对照组及3根组、4根组,每组8只.对照组用于排除麻醉和手术对运动诱发电位的影响;3根组、4根组分别结扎3根、4根腰动脉.各组麻醉后记录基线诱发电位,手术/结扎后30 min、2d、7d记录诱发电位;麻醉清醒后、手术/结扎后2d、7d进行运动功能评分;手术/结扎后7d后取缺血中心区标本进行HE染色,镜下观察. 结果 3根组动物结扎后30 min诱发电位波幅与对照组比较差异有统计学意义(P＜0.05),结扎后2d、7d与对照组比较差异无统计学意义(P＞0.05);4根组动物结扎后30 min、2d、7d3个时间点诱发电位波幅与对照组比较差异均有统计学意义(P＜0.05).3组动物手术/结扎后30min、2d、7d3个时间点的潜伏期与对照组比较差异均无统计学意义(P＞0.05).各组动物运动功能评分结果与诱发电位波幅变化一致. 结论 结扎3根腰动脉可以造成可逆性不完全性脊髓缺血损伤,结扎4根腰动脉可以造成不可逆性不完全性脊髓缺血损伤.     

Postischemic reperfusion causes a massive calcium overload in the myelinated spinal cord fibers

The visualization of Ca binding in the myelinated axons of lumbosacral segments of rabbit was done at the electron microscopic level using the spinal cord ischemia model. To assess the calcium accumulation, the binding agent pyroantimonate was used. Nonsignificant Ca²⁺ binding was found in the myelinated axons after 40 min of ischemia followed immediately by perfusion fixation. A high concentration of calcium pyroantimonate deposits, seen as electron dense particles, was detected in the myelin interlamellar clefts and axoplasm. The paranodal region was the most affected site.     

Neuroprotective effects of adipose-derived stem cells against ischemic neuronal damage in the rabbit spinal cord

Transplantation of adipose-derived stem cells (ASCs) is one of the possible therapeutic tools for ischemic damage. In this study, we observed the effects of ASCs against ischemic damage in the ventral horn of L(5-6) levels in the rabbit spinal cord. ASCs were isolated from rabbits, and cell type was confirmed by flow cytometry analysis, labeling with CM-DiI dye and differentiation into adipocytes in adipogenesis differentiation medium. ASCs were administered intrathecally into recipient rabbits (2 × 10?) immediately after reperfusion following a 15-min aortic artery occlusion in the subrenal region. Transplantation of ASCs significantly improved functions of the hindlimb and morphology of the ventral horn of spinal cord although CM-DiI-labeled ASCs were not observed in the spinal cord parenchyma. In addition, transplantation of ASCs significantly increased brain-derived neurotrophic factor (BDNF) levels at 72h after ischemia/reperfusion. These results suggest that transplantation of ASCs prevents motor neurons from spinal ischemic damage and reactive gliosis by increasing neurotrophic factors such as BDNF in the spinal cord.     

人参皂甙Rd对兔脊髓缺血性损伤保护作用的量效关系研究

目的探讨人参皂甙Rd对脊髓缺血性损伤保护作用的剂量效应关系。方法 40只雄性新西兰大白兔,随机分为5组（n=8）,采用肾下主动脉阻断法造成脊髓缺血（20min）。对照组：即单纯缺血再灌注组;保护组：即Rd-5组、Rd-10组、Rd-20组和Rd-40组,分别在缺血前1h从耳缘静脉注射人参皂甙Rd5mg/kg、10mg/kg、20mg/kg、40mg/kg;术后观察神经功能变化并记录再灌注4h、8h、12h、24h和48h神经功能学评分,再灌注48h处死动物后取脊髓（L5～7）标本行病理学观察。结果所有动物均存活,再灌注后48h,Rd-5组神经功能学评分和脊髓前角正常运动神经元计数与对照组相比均无显著性差异（P〉0.05）;Rd-10组神经功能学评分与对照组相比无显著性差异（P〉0.05）,但脊髓前角正常运动神经元计数明显高于对照组（P〈0.05）;Rd-20组、Rd-40组神经功能学评分和脊髓前角正常运动神经元计数均明显高于对照组（P〈0.01）,但这二组之间无显著性差异（P〉0.05）。且每组兔神经功能学评分与其对应脊髓前角正常神经元计数之间有显著相关性（r=0.769.P〈0.01）。结论人参皂甙Rd对脊髓缺血性损伤有保护作用,且呈一定的剂量效应关系。     

Differential protein expression in spinal cord tissue of a rabbit model of spinal cord ischemia/reperfusion injury

New Zealand rabbits were randomly divided into an ischemia group(occlusion of the abdominal aorta for 60 minutes),an ischemia-reperfusion group(occlusion of the abdominal aorta for 60 minutes followed by 48 hours of reperfusion) and a sham-surgery group.Two-dimensional gel electrophoresis detected 49 differentially expressed proteins in spinal cord tissue from the ischemia and ischemia/reperfusion groups and 23 of them were identified by mass spectrometry.In the ischemia group,the expression of eight proteins was up regulated,and that of the remaining four proteins was down regulated.In the ischemia/reperfusion group,the expression of four proteins was up regulated,and that of two proteins was down regulated.In the sham-surgery group,only one protein was detected.In the ischemia and ischemia/reperfusion groups,four proteins overlapped between groups with the same differential expression,including three that were up regulated and one down regulated.These proteins were related to energy metabolism,cell defense,inflammatory mechanism and cell signaling.     

Free and peptide-bound amino acids as indicators of ischemic damage of the rabbit spinal cord

The concentrations of free and peptide-bound amino acids aspartate (Asp), glutamate (Glu), glycine (Gly) and gamma-aminobutyric acid (GABA) were studied following ischemia and recirculation in the ventral and dorsal gray matter of the rabbit spinal cord. No changes in the concentration of amino acids following ten minutes (min) of ischemia and four days of recovery were found. The most significant change after 20 min, and especially 40 min, of ischemia was a decrease in free Asp and GABA levels in both parts of the gray matter. The relatively greater decrease in the concentration of free amino acids in the ventral horns corresponds with the greater morphological damage to this part of spinal cord following ischemia. Following 40 min of ischemia and recirculation decrease in peptide-bound Glu in the ventral horns and Asp and Glu in the dorsal horns was found.     

Regional changes of membrane phospholipid concentrations in rabbit spinal cord following brief repeated ischemic insults

Changes in the concentration of membrane-bound phospholipids following single (25-min) spinal cord ischemia and 3 h or reperfusion were determined. These were compared with the changes following brief repeated (8-, 8-, and 9-min) ischemia followed each time by reperfusion for 1h, or the same periods of ischemia followed by 8h, 8h, and 24h of reperfusion, respectively. Phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), and sphingomyelin (SM) were assayed in regions of the spinal cord of the rabbit, including gray matter, white matter, dorsal horns, intermediate zone, and ventral horns. The brief repeated ischemia with 1-h reperfusions produced more extensive degradation of phospholipids in almost all regions compared with the equivalent time of ischemia (25 min) in a single period. After a lengthy reperfusion after repeated ischemia, the phospholipids were resynthesized with the exception of the phosphatidylinositol in the gray matter. The resynthesis was most pronounced in the dorsal horns and in the white matter.     

Relative vulnerability of the brain and spinal cord to ischemia

In this experiment CER and SER were monitored as blood flow was progressively lowered by lowering the systemic arterial pressure below the lower limits of autoregulation (bleeding). Blood flow in the brain and dorsal column of the spinal cord was monitored and recorded with the hydrogen clearance method. Long tract neural conduction in the spinal cord appeared quite refractory to the effects of ischemia and disappeared only after 8--18 min of essentially absolute ischemia. The CER was more sensitive to the effects of ischemia, disappearing first in one animal and returning later in all of the animals. The SER returned in all animals after re-infusion of the blood and re-establishment of the blood flow even after a 13--23 min period of absolute ischemia and a 5 min period of electrical silence.     

择性结扎节段动脉致局灶性脊髓缺血损伤动物模型的建立

背景：以往研究表明采用腹膜后入路结扎兔所有腰动脉的方法能够建立稳定的脊髓缺血损伤动物模型,但动脉的具体结扎部位尚不明确。 目的：采用选择性节段动脉结扎方法建立局灶性脊髓缺血动物模型。 方法：将成年新西兰大白兔16只随机分为对照组和模型组,8只/组。模型组采用腹膜后入路结扎L1~3双侧腰动脉,对照组不结扎血管。建模后第3天,用改良Tarlov评分法评价两组兔神经功能,取结扎血管所对应的脊髓组织并进行苏木精-伊红染色,观察其组织学改变。 结果与结论：模型组后肢出现功能障碍,Tarlov评分低于对照组(P < 0.05)。脊髓组织出现急性炎性改变及神经细胞坏死等脊髓缺血现象。实验结果表明采用腹膜后入路结扎L1~3双侧腰动脉能够建立较稳定的局灶性脊髓缺血损伤模型。     

Localization of NADPHd-exhibiting neurons in the spinal cord of the rabbit

Segmental and laminar distributions of nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd)-exhibiting neurons were examined in the rabbit spinal cord by using horizontal, sagittal, and transverse sections. A large number of NADPHd-positive neurons in the spinal cord of rabbit appeared to fall into six categories (N1-N6), but others could not be classified. Major cell groups of NADPHd-exhibiting neurons were identified in the superficial dorsal horn and around the central canal at all spinal levels and in the intermediolateral cell column at thoracic and upper lumbar levels. NADPHd-exhibiting neurons of the pericentral region were divided into a thin subependymal cell column containing longitudinally arranged, small bipolar neurons with processes penetrating deeply into the intermediolateral cell column and/or running rostrocaudally in the subependymal layer. The second pericentral cell column located more laterally in lamina X contains large, intensely stained NADPHd-exhibiting neurons with long dendrites radiating in the transverse plane. In the pericentral region (lamina X), close association of NADPHd-exhibiting somata and fibers and mostly longitudinally oriented blood vessels were detected. Neurons of the sacral parasympathetic nucleus, seen in segments S1-S3, exhibited prominent NADPHd cellular staining accompanied by heavily stained fibers extending from Lissauer's tract through lamina I along the lateral edge of the dorsal horn to lamina V. A massive dorsal gray commissure, highly positive in NADPHd staining, was found in segments S1-S3. Scattered positive cells were also found in the deeper dorsal horn, ventral horn, and white matter. Fiberlike NADPHd staining was found in the superficial dorsal horn and pericentral region in all the segments studied. Dense, punctate, nonsomatic NADPHd staining was detected in the superficial dorsal horn, in the pericentral region all along the rostrocaudal axis, and in the nucleus phrenicus (segments C4-C5), nucleus dorsalis (segments Th2-L2), Onuf's nucleus (segments S1-S3), and the dorsal part of the dorsal gray commissure (S1-S3).     

Selective vulnerability of spinal cord motor neurons to non-NMDA toxicity

We previously reported that alpha-motor neurons in organotypic cultures of rat spinal cord (OTC-SC) are resistant to excitotoxicity induced through NMDA receptors. Here we describe the effects of non-NMDA glutamate receptor agonists kainic acid (KA) and quisqualic acid (QUIS) on motor neurons in OTC-SC. Large ventral horn acetylcholinesterase-positive neurons (VHANs), most of which are motor neurons, were quite sensitive to QUIS and KA toxicity and displayed losses of 95% and 94%, respectively. Small VHANs were reduced by 41% and 61% only. Identical results were obtained in cultures stained for non-phosphorylated neurofilaments. These observations demonstrate that alpha-motor neurons are considerably more sensitive to KA and QUIS than to NMDA toxicity. The proposed excitotoxic mechanism of ALS, therefore, is most likely mediated through non-NMDA glutamate receptors.     

Inhibition of mononuclear phagocytes reduces ischemic injury in the spinal cord

Transient ischemia to the lumbar and sacral sections of the spinal cord of rabbit leads to a deterioration in neurological function that usually worsens 24 to 48 hours after injury. This decline in movement of the hindlimbs develops simultaneously with the appearance of mononuclear phagocytes in the gray matter of injured neural tissue. Chloroquine and colchicine inhibit phagocytic and secretory functions in mononuclear phagocytes. When given up to 6 hours after an induced ischemic lesion in rabbit, this drug combination decreased the number of mononuclear phagocytes found within the gray matter of damaged spinal cord, improved the recovery of function of the hindlimbs and bladder, preserved spinal somatosensory evoked potentials, and promoted the survival of motor neurons. In contrast, the glucocorticoid dexamethasone, a weak inhibitor of mononuclear phagocytes in vivo, did not reduce the number of inflammatory cells in the spinal cord and did not improve motor and bladder functions. The suppression of mononuclear phagocytes soon after ischemic injury may offer a new approach in the treatment of vascular disease in the central nervous system.     

Immunocomplexes in rat and rabbit spinal cord after injury

The possibility that, following a major lesion of the central nervous system, a humoral immune response could be evoked with formation of immune complexes "in situ" was investigated. For this purpose, an immunohistochemical study on rabbit and rat spinal cord at different times after surgical transection was carried out. The peroxidase-antiperoxidase method showed IgG decoration of the myelin sheaths starting a short time after surgery. The sera of intact and injured animals were then tested both by immunohistochemical methods on intact spinal cord sections and by immunoelectrophoresis on a protein extract of homologous spinal cord. The results showed in the rabbit the absence of antibodies to neural antigens before surgical injury and its appearance within a few days after surgery. On the other hand, in the rat, even before the injury, we found antibodies to neural tissue which decreased in the first few hours after injury, and returned to control values during successive days. The same experiments were conducted after a peripheral nerve lesion (sciatic nerve crush), but no immune response could be detected. The possible role of this immune response in the failure of axonal regeneration in mammalian spinal cord is briefly discussed.     

Spontaneous axonal regeneration in rodent spinal cord after ischemic injury

Here we present evidence for spontaneous and long-lasting regeneration of CNS axons after spinal cord lesions in adult rats. The length of 200 kD neurofilament (NF)-immunolabeled axons was estimated after photochemically induced ischemic spinal cord lesions using a stereological tool. The total length of all NF-immunolabeled axons within the lesion cavities was increased 6- to 10-fold at 5, 10, and 15 wk post-lesion compared with 1 wk post-surgery. In ultrastructural studies we found the putatively regenerating axons within the lesion to be associated either with oligodendrocytes or Schwann cells, while other fibers were unmyelinated. Immunohistochemistry demonstrated that some of the regenerated fibers were tyrosine hydroxylase- or serotonin-immunoreactive, indicating a central origin. These findings suggest that there is a considerable amount of spontaneous regeneration after spinal cord lesions in rodents and that the fibers remain several months after injury. The findings of tyrosine hydroxylase- and serotonin-immunoreactivity in the axons suggest that descending central fibers contribute to this endogenous repair of ischemic spinal cord injury.     

Neuroprotective effects of infliximab in experimental spinal cord ischemic injury

Reactive oxygen species (ROS) have been implicated in the pathogenesis of spinal cord injury after both ischemia–reperfusion (I/R) and trauma. This experimental study was designed to investigate the potential effects of infliximab, an anti-tumor necrosis factor-α agent, on I/R injury of the rabbit spinal cord. Eighteen New Zealand white rabbits were divided into three groups, each consisting of six rabbits: sham (no I/R), I/R, and infliximab (I/R + infliximab). Spinal cord ischemia was induced by applying an infrarenal aortic cross clamp for 30 minutes. At 48 hours after ischemia, animals were functionally evaluated using the Tarlov score. Changes in the spinal cord were observed by measuring tissue levels of malondialdehyde (MDA), glutathione (GSH), advanced oxidation protein products (AOPP), and superoxide dismutase (SOD) and by evaluating hematoxylin–eosin-stained sections. At 48 hours after ischemia, the Tarlov scores in the infliximab group were higher than those of the I/R group, MDA and AOPP levels in the I/R group were significantly higher than those in the sham and infliximab groups (p < 0.05), and SOD levels in the infliximab group were significantly higher than those in the I/R and sham groups (p < 0.05). The sham group had higher GSH levels than the infliximab group; however, the difference was not statistically significant (p > 0.05). Histological examination revealed that the infliximab group had significantly less vascular proliferation, edema, and neuron loss than the I/R group. These results indicate that infliximab may protect the spinal cord against injury in a rabbit I/R model.