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.     

Role of Na(+)-Ca(2+) exchanger after traumatic or hypoxic/ischemic injury to spinal cord white matter.

BACKGROUND CONTEXT: Spinal cord injury is a devastating condition in which clinical disability results from demyelination of white matter tracts. Changes in glial-axonal signaling, and enhanced Ca(2+) channel activity with excessive accumulation of intracellular Ca(2+), is a common phenomenon after hypoxia/ischemia or mechanical trauma to spinal cord dorsal column white matter tracts leading to irreversible injury. PURPOSE: In the present study we examined the role of Na(+)-Ca(2+) exchanger (NCX) at physiological temperatures after hypoxia/ischemia and compressive injury to spinal cord dorsal column white matter in vitro. STUDY DESIGN: A 30-mm length of dorsal column was isolated from the spinal cord of adult rats, pinned in an in vitro recording chamber (maintained at 37 degrees C) and injured by exposure to a hypoxic atmosphere for 60 minutes or compressed with a modified aneurysm clip (2-gm closing force) for 15 seconds. The functional integrity of the dorsal column was monitored electrophysiologically by quantitatively measuring the compound action potential (CAP) with glass microelectrodes. RESULTS: The mean CAP decreased to 49.5 +/- 5.7% and 49.4 +/- 2.6% of control (p<.05) after hypoxia/ischemia and compressive injury, respectively. KB-R7943, a potent, selective NCX reverse mode inhibitor, significantly promoted greater recovery of CAP amplitude to 82.0 +/- 10.0% and 70.8 +/- 10.7% of control (p<.05) after hypoxic/ischemic or compressive injury to dorsal column white matter, respectively, when applied at 10 microM concentration. Bepridil (Research Biochemical Inc., Natick, MA, USA) (a less selective NCX inhibitor), when applied at 10 microM and 50 microM concentration promoted CAP amplitude recovery only to 46.8 +/- 7.8% and 29.9 +/- 3.3% of control, respectively, after hypoxic/ischemic injury to dorsal column white matter. Western blot analysis identified NCX presence with positive immunolabeling of 160 kD and 120 kD NCX proteins in the spinal cord white matter. CONCLUSION: In conclusion, at physiological temperature NCX activation plays an important role in intracellular calcium overload after hypoxic/ischemic and compressive injury to spinal cord dorsal column white matter in vitro.     

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.     

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。     

An evaluation of white matter injury after spinal cord ischemia in rats: a comparison with gray matter injury

We quantitatively assessed both gray and white matter injury after spinal cord ischemia in rats, and the relationship between the magnitude of gray and white matter injury was determined. Twenty-five male rats were anesthetized with isoflurane, and spinal cord ischemia (SCI) was induced by balloon intraaortic occlusion combined with hypotension. The animals were randomly allocated to one of the following three groups: animals with SCI for 12 min (SCI-12; n = 8), 15 min (SCI-15; n = 9), or those with sham operation (n = 8). Twenty-four hours after reperfusion, hindlimb motor function was assessed using the Basso-Beattie-Bresnahan scale scoring. Gray matter damage was assessed on the basis of the number of normal neurons in the ventral horn. White matter damage was assessed on the basis of the extent of vacuolation and amyloid precursor protein immunoreactivity in the ventral and ventrolateral white matter. There were significantly less normal neurons in the SCI-15 group compared with those in the SCI-12 and sham groups (P < 0.05). There was a significant positive correlation between the Basso-Beattie-Bresnahan scores and the number of normal neurons. The percentages of vacuolation areas in the SCI-15 group were significantly larger compared with those in the SCI-12 and sham groups (30% +/- 10% versus 9% +/- 7%, 0% +/- 0%, P < 0.05). Immunohistochemical analysis revealed increased amyloid precursor protein immunoreactivity in the swollen axons, especially in the SCI-15 group. There was a significant negative correlation between the number of normal neurons and percentages of vacuolation areas. These results indicate that both gray and white matter were injured after SCI in rats and the degree of white mater injury was correlated with the severity of gray matter injury after a relatively short recovery period.     

Effects of glycine administration on canine experimental spinal spasticity and the levels of glycine, glutamate, and aspartate in the lumbar spinal cord.

Twelve female mongrel dogs were made paraplegic by midthoracic spinal cord transection. Beginning at 9 weeks posttransection, either glycine (50 mg/kg) or saline was injected intramuscularly each day and the signs of spinal spasticity were assessed clinically. After treating the dogs for 3 weeks, we removed the lumbar enlargement of each dog and microdissected it into gray and white areas which we assayed for glycine, glutamate, and aspartate content. Some of the clinical signs of spasticity improved in the animals injected with glycine compared to the saline-injected controls. The content of glycine was significantly elevated in the central gray matter and ventral medial white matter of the glycine-treated dogs. The levels of glutamate were also significantly elevated in the central, lateral ventral, and medial ventral gray matter and in the dorsal lateral and ventral medial white matter of the glycine-treated dogs. The possible role of these segmental putative neurotransmitters in spinal spasticity is discussed.     

Effects of xenon on ischemic spinal cord injury in rabbits: a comparison with propofol

Background: Xenon has been shown to reduce cellular injury after cerebral ischemia. However, the neuroprotective effects of xenon on ischemic spinal cord are unknown. The authors compared the effects of xenon and propofol on spinal cord injury following spinal cord ischemia in rabbits. Methods: Thirty‐two male New Zealand white rabbits were randomly assigned to one of three groups. In the xenon and propofol group, 70% of xenon and 0.8 mg/kg/min of propofol were administered 30 min before an aortic occlusion and maintained until the end of the procedure. The aortic occlusion was performed for 15 min. In the sham group, the aorta was not occluded. After an assessment of the hind limb motor function using the Tarlov score (0=paraplegia, 4=normal) at 48 h after reperfusion, gray and white matter injuries were evaluated based on the number of normal neurons in the anterior spinal cord and the percentage areas of vacuolation in the white matter, respectively. Results: In the xenon and propofol groups, the Tarlov score and the number of normal neurons were significantly lower than those in the sham group, whereas the percentage areas of vacuolation were similar among the three groups. There were no significant differences in Tarlov scores and the number of normal neurons between the xenon and the propofol groups. Conclusion: The results indicated that 70% of xenon has no additional neuroprotective effects on ischemic spinal cord injury in rabbits compared with propofol.     

The microvasculature in transitory traumatic paraplegia. An electron microscopic study in the monkey

Fine structural alterations in the microvasculature, primarily of the gray matter, occur as one aspect of experimental spinal cord contusion. A force of 300 gm-cm, shown by the authors to produce a transitory paraplegia, was applied to the T-10 level of exposed primate spinal cord. At 5 min post-contusion, the muscular venules of the central gray matter were distended with erythrocytes. Erythrocytes were seen within the perivascular spaces of the post-capillary venules and muscular venules at 15 and 30 min post-contusion, and there was hemorrhage into the gray matter at 1 hour post-contusion. The appearance of erythrocytes within the perivenular spaces was apparently due to small ruptures in the walls of the muscular venules, which were first demonstrated by electron microscopy 15 min after contusion. Alterations in capillary and post-capillary venule endothelium of both gray and white matter were present at 4 hours post-contusion and consisted of vacuolation and endothelial swelling. In conclusion, following experimental contusion of the spinal cord sufficient to cause a transitory paraplegia, the principal changes were early perivascular and parenchymal hemorrhages followed by later evidence of ischemic endothelial injury in the microvasculature.     

Diffusion tensor imaging predicts hyperacute spinal cord injury severity

Experimental strategies that focus on ventral white matter (VWM) preservation during the hyperacute phase hold great potential for our improved understanding of functional recovery following traumatic spinal cord injury (SCI). Critical comparisons of human SCI to rapidly accumulating data derived from rodent models are limited by a basic lack of in vivo measures of subclinical pathophysiologic changes and white matter damage in the spinal cord. Spinal cord edema and intraparenchymal hemorrhage demonstrated with routine MR sequences have limited value for predicting functional outcomes in SCI animal models and in human patients. We recently demonstrated that in vivo derived diffusion tensor imaging (DTI) parameters are sensitive and specific biomarkers for spinal cord white matter damage. In this study, non-invasive in vivo DTI was utilized to evaluate the white matter of C57BL/6 mice 3 h after mild (0.3 mm), moderate (0.6 mm), or severe (0.9 mm) contusive SCI. In the hyperacute phase, relative anisotropy maps provided excellent gray-white matter contrast in all degrees of injury. In vivo DTI-derived measurements of axial diffusion differentiated between mild, moderate, and severe contusive SCI with good histological correlation. Cross-sectional regional measurements of white matter injury severity between dorsal columns and VWM varied with increasing cord displacement in a pattern consistent with spinal cord viscoelastic properties.     

Experimental spinal epidural abscess: a pathophysiological model in the rabbit

To define the pathophysiology of spinal cord dysfunction associated with spinal epidural abscess formation, we developed an experimental model. Spinal epidural abscesses were produced in rabbits by injecting Staphylococcus aureus into the posterior thoracolumbar epidural space under direct vision. Progressive neurological deficits were detected in 18 of 20 animals; severe paraparesis or paraplegia occurred in 75%, and sphincter dysfunction occurred in 55%. Clinical data, including the results of plain spine roentgenography, myelography, and biochemical and bacteriological examination of the cerebrospinal fluid, were recorded. Epidural abscesses with varying degrees of spinal cord compression were confirmed pathologically in 95% of the experimental group. Spinal cord white matter changes included vacuolization, loss of myelin, and axonal swelling. The gray matter of the spinal cords was relatively preserved. There was no microscopic evidence of thrombosis or vasculitis in the major blood vessels supplying the spinal cords. Histopathological changes detected in the spinal cords were more consistent with direct compression of neural tissue than with infarction. The progressive clinical course and the histopathological changes in the spinal cord after compression by abscess closely resembled those of experimental compression of the spinal cord by epidural neoplasm.     

Synaptic blockade plays a major role in the neural disturbance of experimental spinal cord compression

We analyzed dynamic processes of neural excitation propagation in the experimentally compressed spinal cord using a high-speed optical recording system. Transverse slices of the juvenile rat cervical spinal cord were stained with a voltage-sensitive dye (di-4-ANEPPS). Two components were identified in the depolarizing optical responses to dorsal root electrical stimulation: a fast component of short duration corresponding to pre-synaptic excitation and a slow component of long duration corresponding to post-synaptic excitation. In the directly compressed dorsal horn, the slow component was attenuated more (attenuated to 37.4 +/- 9.1% of the control) than the fast component (to 70.5 +/- 14.9%) (p < 0.01) at 400 msec after stimulation. Depolarizing optical responses to compression and to chemical synaptic blockade were similar. There was a regional difference between white matter (attenuated to 86.2 +/- 10.5%) and gray matter (to 72.6 +/- 10.4%) (p < 0.03) in compression-induced changes of the fast components; neural activity in the white matter was resistant to compression, especially in the dorsal root entry zone. Depolarizing optical signals in the region adjacent to the directly compressed site were also attenuated; the fast component was attenuated to 77.6 +/- 10.4% and the slow component to 31.8 +/- 11.3% of the control signals (p < 0.01). Spinal cord dysfunction induced by purely mechanical compression without tissue destruction was virtually restored with early decompression. We suggest that a disturbance of synaptic transmission plays an important role in the pathophysiological mechanisms of spinal cord compression, at least under in vitro experimental conditions of juvenile rats.     

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.     

大鼠脊髓三种不同损伤后神经细胞凋亡的实验比较

目的了解三种不同脊髓机械性损伤对继发性神经细胞凋亡的影响。方法采用脊髓挫伤、持续性占位、脊髓横断三种大鼠损伤模型,在伤后1、4、7d观察损伤断面神经细胞凋亡现象。结果脊髓挫伤组:灰质三个时间组的凋亡指数波动明显,灰质中凋亡指数4d时最高;白质中凋亡细胞除背侧束外腹侧束也存在;另外细胞凋亡以少突胶质细胞凋亡为主。脊髓持续性占位损伤组:灰质、白质细胞凋亡指数在4d和7d组中差异无显著性意义(P>0.05),白质凋亡细胞在受压的背侧束、外侧束为主。脊髓横断损伤组:灰质、白质在三个时间组中均有大量凋亡细胞。结论大鼠脊髓损伤后神经细胞虽然存在主动死亡(细胞凋亡)方式,但凋亡在时间和部位上与损伤类型、损伤部位、损伤程度有关。     

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.     

Microsurgical anterior cervical myelotomy.

Cervical myelotomy utilizing the anterior approach and the operating microscope in a patient with an acute spinal cord injury is described. The procedure overcomes the technical difficulties previously encountered. The ease with which the hemorrhagic gray matter may be separated from the normal gray and surrounding white matter is emphasized.     

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.     

Spinal cord diffusion tensor imaging and fiber tracking can identify white matter tract disruption and glial scar orientation following lateral funiculotomy

Diffusion tensor magnetic resonance imaging (DTI) provides data concerning water diffusion in the spinal cord, from which white matter tracts may be inferred, and connectivity between spinal cord segments may be determined. We evaluated this potential application by imaging spinal cords from normal adult rats and rats that received cervical lateral funiculotomies, disrupting the rubrospinal tract (RST). Vitrogen and fibroblasts were transplanted into the surgical lesion at time of injury in order to fill the cavity. At 10 weeks, animals were sacrificed; the spinal cords were dissected out and then imaged in a 9.4-Tesla magnet. DTI tractography demonstrated the disruption of the rubrospinal tract axons while indicating which axon tracts were preserved. Additionally, DTI imaging could identify the orientation of glial processes in the gray matter adjacent to the site of injury. In the injured animals, reactive astrocytes in adjacent gray matter appeared to orient themselves perpendicular to white matter tracts. In summary, DTI identified not only white matter disruption following injury, but could distinguish the orientation of the accompanying glial scar.     

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.     

The effect of thalidomide on spinal cord ischemia/reperfusion injury in a rabbit model

STUDY DESIGN: Randomized study. OBJECTIVES: To evaluate the effects of thalidomide on spinal cord ischemia/reperfusion injury via reduced TNF-alpha production. SETTING: Animal experimental laboratory, Clinical Research Institute of Seoul National University Hospital, Seoul, Korea. METHODS: Spinal cord ischemia was induced in rabbits by occluding the infrarenal aorta. Rabbits in group N did not undergo ischemic insult, but rabbits in groups C (the untreated group), THA, and THB underwent ischemic insult for 15 min. The THA and THB groups received thalidomide (20 mg/kg) intraperitoneally (i.p.) before ischemia, but only the THB group received thalidomide (i.p., 20 mg/kg) after 24 and 48 h of reperfusion. After evaluating neurologic functions at 1.5 h, 3, and 5 days of reperfusion, rabbits were killed for histopathologic examination and Western blot analysis of TNF-alpha. RESULTS: The THA and THB groups showed significantly less neurologic dysfunction than the C group at 1.5 h, 3, and 5 days of reperfusion. The number of normal spinal motor neurons in ventral gray matter was higher in THA and THB than in C, but no difference was observed between THA and THB. Western blot analysis showed a significantly higher level of TNF-alpha in C than in THA and THB at 1.5 h of reperfusion, but no difference was observed between C, THA, or THB at 3 or 5 days of reperfusion. CONCLUSION: Thalidomide treatment before ischemic insult reduces early phase ischemia/reperfusion injury of the spinal cord in rabbits.     

Control of blood flow in the cat spinal cord

Spinal cord blood flow (SCBF) and the effect of end-tidal CO2 concentration (ETCO2) on SCBF (CO2 reactivity) were studied in the lumbar spinal cord of cats by means of the hydrogen-clearance technique Hydrogen gas was administered by inhalation, and its level in spinal cord tissue was estimated amperometrically with small (75 micrometers) platinum electrodes. The average SCBF's at normocapnia (ETCO2 = 4%) of the ventral horn gray matter and of the white matter at several locations were 43.2 and 16.2 ml . 100 gm-1 . min-1, respectively. For gray and white matter, the values of CO2 reactivity, estimated by the coefficient of the regression of SCBF (ml . 100 gm-1 . min-1) on ETCO2 (ml . 100 ml-1) were 11.6 and 2.1, respectively. No differences in SCBF or CO2 reactivity were observed between intact animals kept under N2O-O2 ventilation and decerebrated animals with no anesthesia. After an acute spinal section, ventral horn SCBF and CO2 reactivity (measured eight segments below the cordotomy) were not altered, in spite of the profound neural depression present (that is, spinal shock). Orthodromic (dorsal root) stimulation of the ventral horn neurons induced an average increase in blood flow of 128% above control values. Antidromic (ventral root) motoneuron activation failed to produce any significant changes in ventral horn blood flow.