Microvascular perfusion experimental spinal cord injury

Microperfusion of the spinal cords in cats was studied using a colloidal carbon perfusion technique following compression injury at 1/2 hour, 2 hours, 4 hours, 8 hours, 24 hours, and 51 days. Quantitative estimates of vascular filling were determined at these post-compression intervals. Microperfusion diminished in both the gray and white matter at 1/2 hour following injury and severe lack of perfusion was evident at 8 and 24 hours. Diminished filling of the vessels of gray and white matter seemed to parallel the degree of hemorrhagic necrosis of the gray matter. An increased number of vessels were evident in the spinal cords of long term survivals. The observation that microperfusion in the white matter of the spinal cord was diminished at 24 hours is at variance with some previous investigations. The hypoperfusion of the white matter found in this study suggests that ischemia plays a role in paraplegia resulting from experimental compression injury of the spinal cord.     

Gray matter of the bovine cervical spinal cord is mechanically more rigid and fragile than the white matter

The gray matter of the cervical spinal cord has been thought to be equally or less rigid than the white matter. Based on this assumption, various studies have been conducted on the changes of stress distributions within the spinal cord under mechanical compression, although the mechanical properties of the white and gray matters had not been fully elucidated. The present study measured the mechanical properties of the white and gray matter of bovine spinal cords. For both the white and gray matter, the stress-strain curves had a nonlinear region, followed by a linear region, and then a region where the stresses plateaued before failure. In the nonlinear region, stress was not significantly different between the gray and white matter samples (strain approximately 0-10%), while stress and Young's modulus in the gray matter was significantly higher than the white matter in the linear part of the curve. The gray matter ruptured at lower strains than the white matter. These findings demonstrated the gray matter is more rigid and fragile than the white matter, and the conventional assumption (i.e., the white matter is more rigid than the gray matter) is not correct. We then applied our data to computer simulations using the finite element method, and confirmed that simulations agreed with actual magnetic resonance imaging findings of the spinal cord under compression. In future computer simulations, including finite element method using our data, changes in stress and strain within the cervical spinal cord under compression would be clarified in more detail, and our findings would also help to elucidate the area which can easily receive histologic damage or which could have hemodynamic disorders under mechanical compression, as well as severity and location of biochemical and molecular biological changes.     

Relationship between posttraumatic ischemia and hemorrhage in the injured rat spinal cord as shown by colloidal carbon angiography

Adult female Wistar rats underwent extradural clip compression injury of the spinal cord at T-1. A force of 40 g was applied for 1 minute; this moderately severe injury renders animals paraparetic. We performed colloidal carbon angiography with Aquablak on four animals at 15 minutes, 2 hours, and 24 hours after injury. The entire spinal cord was then removed, frozen, and sectioned serially at 250 micron. The sections were examined microscopically for patterns of ischemia and hemorrhage at the site of compression injury and at adjacent and remote sites in the spinal cord. There was a marked lack of filling of the arterioles, capillaries, and venules at the injury site. In addition, there was a specific anatomical distribution of the ischemic areas in that ischemia of the white matter occurred in areas supplied by arteries that traversed adjacent hemorrhagic gray matter. For example, ischemia of the ventral funiculus was consistently seen adjacent to hemorrhage in the ventral gray matter. Similarly, ischemia of the dorsal white columns was related to hemorrhagic lesions surrounding the feeding vessels in the dorsal gray matter. This study also demonstrates the usefulness of a new colloidal carbon suspension (Aquablak) for documenting the pathophysiology of posttraumatic ischemia of the spinal cord. The results suggest that ischemic lesions in the white matter are anatomically related to hemorrhagic lesions in the gray matter. The pathophysiology of this relationship is unknown, but may include thrombosis, vasospasm, or direct injury of the feeding vessels.     

Comparison of inflammation in the brain and spinal cord following mechanical injury

Inflammation in the CNS predominantly involves microglia and macrophages, and is believed to be a significant cause of secondary injury following trauma. This study compares the microglial and macrophage response in the rat brain and spinal cord following discrete mechanical injury to better appreciate the degree to which these cells could contribute to secondary damage in these areas. We find that, 1 week after injury, the microglial and macrophage response is significantly greater in the spinal cord compared to the brain. This is the case for injuries to both gray and white matter. In addition, we observed a greater inflammatory response in white matter compared to gray matter within both the brain and spinal cord. Because activated microglia and macrophages appear to be effectors of secondary damage, a greater degree of inflammation in the spinal cord is likely to result in more extensive secondary damage. Tissue saving strategies utilizing anti-inflammatory treatments may therefore be more useful in traumatic spinal cord than brain injury.     

Oxygenated fluorocarbon nutrient solution in the treatment of experimental spinal cord injury

We employed an extravascular perfusion system through the subarachnoid space of the traumatized spinal cord of the cat for the delivery of oxygen utilizing a fluorocarbon emulsion containing essential nutrients, termed the oxygenated fluorocarbon nutrient solution (OFNS). Animals perfused for 2 hours with saline after impact injury of the spinal cord had significantly less edema at 1 cm below this site of injury than injured, untreated animals. However, in injured animals perfused with OFNS there was significant protection from spinal cord edema at both 1 and 2 cm below the site of injury. OFNS perfusion reduced the magnitude of hemorrhagic necrosis in both the gray and the white matter and protected the anterior horn cells against lysis at the site of injury. Adenosine triphosphate (ATP) is decreased within 1 minute and remains suppressed for 1 hour in gray and white matter of unperfused, injured animals. The level of ATP in both gray and white matter was significantly higher in injured OFNS-perfused animals than in saline-treated animals at the site below the spinal cord injury. Our data show that OFNS perfusion of the injured spinal cord reduced necrosis and edema and tended to normalize the levels of high energy ATP and intact anterior horn cells. These results demonstrate the feasibility of treating ischemic hypoxia of the spinal cord after trauma through an extravascular perfusion route that utilizes a fluorocarbon emulsion as a vehicle for the delivery of oxygen and other cellular nutrients.     

脊髓急性损伤后神经细胞凋亡的时相和空间分布特点

目的 研究脊髓急性损伤后神经细胞的凋亡及其时相和空间特点。方法 大鼠脊髓（T8,9）经中度压迫损伤后,分别在30min、2h、4h、8h、24h、48h、72h、7d、14d、和21d处死取材（n=4)。应用HE、Nissl染色及凋亡细胞原位末端标记法对脊髓组织进行标记。结果 损伤4h后,在损伤段及邻近段可见末端标记阳性神经细胞,损伤段灰质中阳性细胞数8h达高峰,24h白质中阳性胶质细胞数量达高峰。相邻节段阳性细胞数量在72h达高峰。阳性细胞以白质中胶质细胞为主,主要分布于相邻节段。结论 脊髓损伤后神经细胞凋亡是继发损伤期的重要病理变化,并有其时相和空间分布特点。     

Reevaluation of gray and white matter injury after spinal cord ischemia in rabbits

BACKGROUND: Although gray matter injury has been well characterized, the available data on white matter injury after spinal cord ischemia (SCI) in rabbits are limited. The current study was conducted to investigate the evolution of ischemia induced injury to gray and white matter and to correlate this damage to hind-limb motor function in rabbits subjected to SCI. METHODS: Thirty-eight rabbits were randomly assigned to 24-h, 4-day, or 14-day reperfusion groups or a sham group (n = 9 or 10 per group). SCI was induced by occlusion of the infrarenal aorta for 16 min. Hind-limb motor function was assessed using the Tarlov scale (0 = paraplegia, 4 = normal). The gray matter damage was assessed on the basis of the number of normal neurons in the anterior spinal cord. White matter damage was assessed on the basis of the extent of vacuolation and accumulation of amyloid precursor protein immunoreactivity. RESULTS: Tarlov scores gradually decreased and reached a nadir 14 days after reperfusion. There were no significant differences in the number of normal neurons among the 24-h, 4-day, and 14-day groups. The extent of vacuolation, expressed as a percent of total white matter area, was significantly greater in the 4-day and 14-day groups in comparison with the sham group. By contrast, there was no difference in vacuolation between the sham and 24-h groups. Amyloid precursor protein immunoreactivity was greater in the 4-day and 14-day groups. CONCLUSION: The results in the current study show that SCI induced white matter injury as well as gray matter injury in a rabbit model of SCI. The time course for 14 days after reperfusion may differ among the gray and white matter damages and hind-limb motor function in rabbits subjected to SCI.     

A vertebral dislocation model of spinal cord injury in rats

A new model of spinal cord injury (SCI) has been developed in the rat, which produces axonal and vascular injury within the spinal cord through lateral displacement of the vertebral column. An electromechanical feedback-controlled device produces the injury by displacing the vertebral column to the left hand side. The speed and lateral displacement is controllable by the user, and the resulting injury ranges from no histologically evident injury, to total disruption of the vertebral column with associated widespread axonal and vascular damage. Histological and immunohistological techniques were employed to correlate mechanical parameters with the extent of pathological injury of spinal cord. Axonal injury was most severe in the left lateral white matter, and vascular injury was concentrated in the gray matter.     

Experimental spinal cord injury: qualitative and quantitative histopathologic evaluation

This study involved a morphometric analysis of an experimental model of spinal cord injury. The spinal cords of rats were injured by a weight drop at T8 level. Animals were sacrificed 4 weeks after injury, and histopathologic examination of the spinal cords was carried out qualitatively and also quantitatively with the aid of computer-assisted morphometry. Total cross-sectional areas of residual gray and white matter were determined at five regularly spaced intervals through the injured cord segment. The histologic findings were correlated with height of weight-drop and motor recovery in the hind limbs at 4 weeks postinjury. The weight-drop injury was found to produce a longitudinally asymmetrical cavitary defect, which was better assessed by a series of cross-sectional profiles than by a single histologic cross-section through the epicenter (site of maximal impact) of the cord injury. There was a strong correlation between height of weight-drop and amount of residual tissue (gray and white matter) at the epicenter. A correlation was also found between height of weight-drop and a composite of residual tissue evaluated at multiple levels through the injury site. By comparison with cross-sectional morphometry at the epicenter, multiple cross-sections, reflecting volume of residual tissue in the longitudinal extent of injury, showed greater statistical correlation with functional (behavioral) outcome. This "volumetric" assessment of the total region of injury is therefore recommended as preferable to a histopathologic evaluation limited to the epicenter.     

Chronic spinal compression model in minipigs: a systematic behavioral, qualitative, and quantitative neuropathological study

The goal of the present study was to develop a porcine spinal cord injury (SCI) model, and to describe the neurological outcome and characterize the corresponding quantitative and qualitative histological changes at 4-9 months after injury. Adult Gottingen-Minnesota minipigs were anesthetized and placed in a spine immobilization frame. The exposed T12 spinal segment was compressed in a dorso-ventral direction using a 5-mm-diameter circular bar with a progressively increasing peak force (1.5, 2.0, or 2.5?kg) at a velocity of 3?cm/sec. During recovery, motor and sensory function were periodically monitored. After survival, the animals were perfusion fixed and the extent of local SCI was analyzed by (1) post-mortem MRI analysis of dissected spinal cords, (2) qualitative and quantitative analysis of axonal survival at the epicenter of injury, and (3) defining the presence of local inflammatory changes, astrocytosis, and schwannosis. Following 2.5-kg spinal cord compression the animals demonstrated a near complete loss of motor and sensory function with no recovery over the next 4-9 months. Those that underwent spinal cord compression with 2 kg force developed an incomplete injury with progressive partial neurological recovery characterized by a restricted ability to stand and walk. Animals injured with a spinal compression force of 1.5?kg showed near normal ambulation 10 days after injury. In fully paralyzed animals (2.5?kg), MRI analysis demonstrated a loss of spinal white matter integrity and extensive septal cavitations. A significant correlation between the magnitude of loss of small and medium-sized myelinated axons in the ventral funiculus and neurological deficits was identified. These data, demonstrating stable neurological deficits in severely injured animals, similarities of spinal pathology to humans, and relatively good post-injury tolerance of this strain of minipigs to spinal trauma, suggest that this model can successfully be used to study therapeutic interventions targeting both acute and chronic stages of SCI.     

Biomechanics of spinal cord injury: a multimodal investigation using ex vivo guinea pig spinal cord white matter

The primary injury phase of traumatic spinal cord injury (SCI) was investigated using a novel compression injury model. Ventral white matter from adult guinea pigs was crushed to 25%, 50%, 70%, and 90% ex vivo. During compression, the physical deformation, applied force and the compound action potentials (CAP) were simultaneously recorded. In addition, axonal membrane continuity was analyzed with a horseradish peroxidase (HRP) exclusion assay. Experimental results showed both a CAP decrease and increased HRP uptake as a function of increased compression. The percent CAP reduction was also consistent to the percent HRP uptake, which implies that either metric could be used to assess acute axon damage. Analysis of the HRP stained axon distribution demonstrated a gradient of damage, with the highest levels of staining near the gray matter. The patterns of axon damage revealed by histology also coincided with higher levels of von Mises stress, which were predicted with a recently developed finite element model of ventral white matter. Numerical values obtained from the finite element model suggest stress magnitudes near 2 kPa are required to initiate anatomical tissue injury. We believe that data from this study could further elucidate the deformation-function relationship in acute spinal cord injury.     

大鼠脊髓急性损伤后神经细胞凋亡及相关基因表达△

目的研究脊髓急性损伤后神经细胞的凋亡及相关基因的表达.方法大鼠脊髓(T8、T9)经中度压迫损伤后,分别在30min、2h、4h、8h、24h、48h、72h、7d、14d和21d处死取材(各时间组n=4).应用HE染色、免疫组化及凋亡细胞原位末端标记法对脊髓组织进行标记.结果损伤4h后,在损伤段及邻近段可见末端标记阳性神经元,损伤段灰质中阳性细胞数8h达高峰,24h白质中阳性胶质细胞数量达高峰.相邻节段阳性细胞数72h达高峰.损伤后P53及Bax大量表达,而Bcl-2仅少量表达.结论脊髓损伤后神经细胞的凋亡是继发损伤期的重要病理变化.     

椎板外冲击对脊髓血流影响的实验研究

目的应用一种闭合性脊髓损伤模型,通过不同强度冲击力作用于椎板,研究这种间接性冲击伤对脊髓的影响,并探讨血管机制在脊髓损伤中的作用。方法选用雄性SD大鼠120只,随机分为四组,每组30只,A组为假手术对照组,B、C、D、三组分别给予10、15、20N的椎板外冲击负荷,在相应的时间点进行心率、血压及脊髓血流测量观察。结果各组在各个时段的血压和心率无明显变化。C、D组血流表现为规律性的变化。结论椎板外冲击负荷能改变脊髓微循环血流,从而对脊髓产生影响;其对脊髓灰质微循环血流的改变更明显。     

Morphologic change and astrocyte response to unilateral spinal cord compression in rabbits

In myelopathy, unilateral compression of the spinal cord in cases of disc herniation would be expected to produce Brown-Séquard syndrome. However, a transverse lesion syndrome occurs in most clinical cases. In order to reveal the mechanism by which unilateral compression induces transverse damage to the spinal cord, damage of the gray and white matter in each half of the spinal cord were evaluated quantitatively to determine the density of GFAP-positive astrocytes. The cervical spinal cord in rabbits was unilaterally compressed with a small screw. The area of each half of the damaged cord and the density of GFAP-positive astrocytes of the compressed and contralateral halves were investigated one week after the surgery. No apparent paralysis was observed during the period of observation. As the compression increased, the area of the compressed half of the spinal cord decreased significantly compared to the contralateral half. The densities of GFAP-positive astrocytes in the gray matter and the anterior funiculus increased significantly in the compressed half. There were no significant differences in the densities at the lateral and dorsal funiculi between the compressed and contralateral halves. The tissue damage in the gray matter of the compressed half was markedly higher. No significant difference between the two halves in damage was seen in the lateral funiculus, where in the lateral pyramidal and the dorsal spinocerebellar tracts are found. These findings provide evidence of the mechanistic basis for the spinal cord damage that leads to transverse lesion syndrome in unilateral compression myelopathy.     

Effects of a contusion injury on spinal cord blood flow in the sheep

The effect of experimental trauma on the blood flow in the central (essentially gray matter) and peripheral (essentially white matter) regions of the sheep's spinal cord was studied using a radioactive microsphere technique. In seven out of eight animals, a progressive fall in blood flow occurred in both the peripheral and central regions of the cord within 2 hours following injury and remained reduced over the period of recording (up to 12 hours). Changes in local vascular resistance indicated that in approximately 60% of our animals, changes in arterial pressure alone contributed highly significantly to the decreased spinal blood flow. There remains the possibility that early therapeutic intervention could sustain neuronal function where local blood flow would otherwise be inadequate in the damaged spinal cord.     

Toxic substances in spinal cord injury.

Several investigators have implicated norepinephrine and other toxic substances released in the region of a spinal cord injury in the genesis of the progressive pathological and clinical changes that follow spinal trauma. To test this hypothesis, we subjected cats to T-10 to T-12 laminectomy and monitored epidural spinal evoked potentials from sciatic nerve stimulation. The spinal subarachnoid space was perfused with normal saline, with norepinephrine solution, or with heparinized autologous blood or the pial surface of the spinal cord was exposed to macerated gray matter taken from the upper cervical cord. During 1- to 2-hour exposure periods, we noted no significant changes in the base line spinal evoked potentials. In another series of cats, we have shown that norepinephrine perfused over the spinal cord in this manner diffuses rapidly into the subpial white matter. Therefore, its failure to affect spinal evoked potentials does not represent a failure to penetrate the spinal cord. Putative toxins must originate either in extravasated blood or damaged neural tissue in the region of the spinal cord injury. The failure of ascending spinal tracts to react to blood or cord tissue in our experiment suggests that toxins are not involved in the spinal cord dysfunction that occurs soon after injury.     

In vitro investigation of heat transfer in calf spinal cord during polymethylmethacrylate application for vertebral body reconstruction

The objective of this experimental study was to investigate the temperature variations within the spinal cord of calf cadavers during polymethlymethacrylate (PMMA) application for vertebral body reconstruction. Cervical spines including the cervical spinal cord of ten fresh cadavers were used. Corpectomy and laminectomy were performed and dura was exposed at the same level for proper placement of thermal sensors. Sensors were placed in multiple holes in the spinal cord at depths of 3, 6, 9 and 12 mm, respectively. Whether the thermal sensors were placed in the gray or white matter was determined by computerized tomography. The white and gray matters of the spinal cord exhibited different thermal properties. The white matter was more conductive and absorbed less heat than the gray matter. The heat sensor nearest to PMMA exhibited temperatures of 42–44°C. The second heat sensor placed at 9 mm depth within the gray matter showed 44°C. The third sensor, which was placed at 6 mm depth within the spinal cord recorded the same temperature as the first, i.e., nearest to PMMA sensor. The fourth heat sensor, which was at the farthest location from PMMA demonstrated 37–39°C. The temperature distribution within the gray matter was inversely proportional to the distance from the heat source. The temperature at the dorsal white matter, which was distant from the heating source, remained nearly constant and was not elevated. Our data suggest that thermal injury to the spinal cord during PMMA application may be expected to be more significant in the gray matter when compared with other neural tissues.     

Functional consequences of lumbar spinal cord contusion injuries in the adult rat

Our understanding of the substrates of locomotion, and hence our understanding of the causes of deficits following spinal cord injury, is still incomplete. While severe locomotor deficits can be induced by either contusion or laceration injuries or demyelination of thoracic spinal cord ventral and ventrolateral white matter, loss of mid-thoracic gray matter (intraspinal kainic acid injection) has no impact on locomotion. In contrast, loss of gray matter from the rostral lumbar segments induces severe locomotor deficits. This study examines the histological and locomotor outcomes following contusion injuries involving the rostral segments of the lumbar enlargement in the adult rat. Adult Sprague-Dawley rats received contusion injuries centered on the T13/L1, L2, or L3/4 spinal cord segments. Moderately severe injuries centered on the T13/L1 and L2 spinal cord segments induced more severe locomotor deficits than those centered on the L3/4 segments, despite a significantly smaller total gray matter volume loss (1.7 vs. 2.7 mm3). Moderately-severe injuries at T13/L1, L2, and L3/4 showed 21%, 31%, and 39% white matter sparing, respectively, with 6-week BBB scores of 10, 10, and 15.7, respectively. These data suggest that moderately-severe contusion injuries centered on the rostral segments of the lumbar enlargement induce more severe locomotor deficits than would be predicted by the histological outcome (spared white matter), suggesting that gray matter loss may play a role in functional deficits following some lumbar contusion injuries.     

A new rabbit model for the study on cervical compressive myelopathy.

Development process and pathology of myelopathy due to chronic spinal cord compression have not been fully elucidated. This study was conducted in order to establish an experimental model which can efficiently produce myelopathy and be useful in the studies on myelopathy due to chronic spinal cord compression. Under electrophysiological monitoring of the spinal cord, anterior compression was produced on C5 using a plastic screw. Two weeks later, a plastic plate was inserted under the C5 arch. For the subsequent 10 months on average, walking pattern and MR images were periodically monitored. Before the sacrifice, electrophysiological test was performed and then histopathological examination was done. Palsy appeared at 5 months on average after the addition of posterior compression. Mean compression ratio of the spinal cord calculated on MR images was 34%. All animals with compression showed a high intramedullary signal intensity, and the mean contrast-to-noise ratio (CNR) in the compressed area was 49%. Electrophysiological test showed a significant decrease in the amplitude of spinal cord evoked potentials (SCEPs) at the given compression level. Histology showed flattening of the anterior horn, disappearance and necrosis of anterior horn cells in the gray matter; and demyelination and axonal degeneration in the white matter. The antero-posterior compression produces the condition of spinal canal stenosis. Repeated antero-posterior compression to the spinal cord is important in establishing myelopathy. The present animal model was evaluated to be useful in the studies on myelopathy.     

Reevaluation of Gray and White Matter Injury after Spinal Cord Ischemia in Rabbits

Background: Although gray matter injury has been well characterized, the available data on white matter injury after spinal cord ischemia (SCI) in rabbits are limited. The current study was conducted to investigate the evolution of ischemia induced injury to gray and white matter and to correlate this damage to hind-limb motor function in rabbits subjected to SCI.

Methods: Thirty-eight rabbits were randomly assigned to 24-h, 4-day, or 14-day reperfusion groups or a sham group (n = 9 or 10 per group). SCI was induced by occlusion of the infrarenal aorta for 16 min. Hind-limb motor function was assessed using the Tarlov scale (0 = paraplegia, 4 = normal). The gray matter damage was assessed on the basis of the number of normal neurons in the anterior spinal cord. White matter damage was assessed on the basis of the extent of vacuolation and accumulation of amyloid precursor protein immunoreactivity.

Results: Tarlov scores gradually decreased and reached a nadir 14 days after reperfusion. There were no significant differences in the number of normal neurons among the 24-h, 4-day, and 14-day groups. The extent of vacuolation, expressed as a percent of total white matter area, was significantly greater in the 4-day and 14-day groups in comparison with the sham group. By contrast, there was no difference in vacuolation between the sham and 24-h groups. Amyloid precursor protein immunoreactivity was greater in the 4-day and 14-day groups.