Cytoskeletal disruption following contusion injury to the rat spinal cord

Following experimental spinal cord injury (SCI), there is a delayed loss of neurofilament proteins but relatively little is known regarding the status of other cytoskeletal elements. The purpose of the present study was to compare the extent and time course of the MAP2 loss with that of neurofilament proteins, and to examine tau protein levels and distribution following SCI. Within 1 to 6 hours following SCI, there is rapid loss of MAP2, tau, and nonphosphorylated neurofilament proteins at the injury site. In contrast, the loss of phosphorylated neurofilament proteins was not significant until 1 week postinjury. In addition to the loss of MAP2 protein, there was extensive beading of MAP2-immunoreactive dendrites extending into the white matter. This was most pronounced 1 hour after injury and gradually resolved such that beading was no longer evident 2 weeks after SCI. The time course of beading resolution is similar to that of behavioral recovery following SCI, but the functional significance of the beading remains to be determined. Together, these results demonstrate that there are 2 phases of cytoskeletal disruption following SCI; a rapid loss of MAP2, tau, and nonphosphorylated neurofilament proteins, and a delayed loss of phosphorylated neurofilaments.     

Fetal grafts alter chronic behavioral outcome after contusion damage to the adult rat spinal cord.

In the present experiments, we have examined the capacity of intraspinal transplants to effect alterations in certain locomotor behaviors after spinal contusion injuries. An electromechanical impactor that was sensitive to tissue biomechanical characteristics was used to produce rapid (20 ms) compression injuries to the thoracic spinal cord (T8). Suspensions of fetal spinal tissue (14-day) were placed at 10 days postinjury into the intraspinal cavity created by these reproducible spinal injuries. In the pre- and postinjury period, a number of general and sensitive motor behaviors were used to characterize the immediate and long-term progress of hindlimb behavioral recovery over an extended period of time (73 days). Our data reveal that a lasting alteration in some motor behaviors can be achieved by suspension grafts. While little improvement in some generalized motor tasks (inclined plane analysis, grid walking) takes place, fetal transplants precipitate a rapid and enduring change in certain motivated fine motor behaviors (gait analysis). The base of support and stride length of the hindlimbs were improved by 7 days post-transplantation and the effect was stable over time. The angle of rotation was, however, not altered. The lasting effect in two gait parameters noted was accompanied by the presence of well-developed spinal grafts that often fused with the host spinal parenchyma. These results provide the first documentation of an influence of fetal transplants on motivated locomotor capacity in a well-characterized spinal injury model that mimics lesions seen in the contused adult human spinal cord.     

Comparing deficits following excitotoxic and contusion injuries in the thoracic and lumbar spinal cord of the adult rat

The majority of human spinal cord injuries involve gray matter loss from the cervical or lumbar enlargements. However, the deficits that arise from gray matter damage are largely masked by the severe deficits due to associated white matter damage. We have developed a model to examine gray matter-specific deficits and therapeutic strategies that uses intraspinal injections of the excitotoxin kainic acid into the T9 and L2 regions of the spinal cord. The resulting deficits have been compared to those from standard contusion injuries at the same levels. Injuries were assessed histologically and functional deficits were determined using the Basso, Beattie, and Bresnahan (BBB) 21-point open field locomotor scale and transcranial magnetic motor evoked potentials (tcMMEPs). Kainic acid injections into T9 resulted in substantial gray matter damage; however, BBB scores and tcMMEP response latencies were not different from those of controls. In contrast, kainic acid injections into L2 resulted in paraplegia with BBB scores similar to those following contusion injuries at either T9 or L2, without affecting tcMMEP response latencies. These observations demonstrate that gray matter loss can result in significant functional deficits, including paraplegia, in the absence of a disruption of major descending pathways.     

Chemokine antagonist infusion promotes axonal sparing after spinal cord contusion injury in rat

Spinal cord injury produced by mechanical contusion causes the onset of acute and chronic degradative events. These include blood brain barrier disruption, edema, demyelination, axonal damage and neuronal cell death. Posttraumatic inflammation after spinal cord injury has been implicated in the secondary injury that ultimately leads to neurologic dysfunction. Studies after spinal cord contusion have shown expression of several chemokines early after injury and suggested a role for them in the ordered recruitment of inflammatory cells at the lesion site (McTigue et al. [1998] J. Neurosci. Res. 53:368-376; Lee et al., [2000] Neurochem Int). We have demonstrated previously that infusion of the broad-spectrum chemokine receptor antagonist (vMIPII) in the contused spinal cord initially attenuates leukocyte infiltration, suppresses' gliotic reaction and reduces neuronal damage after injury. These changes are accompanied by increased expression of bcl-2, the endogenous apoptosis inhibitor, and reduced neuronal apoptosis (Ghirnikar et al. [2000] J. Neurosci. Res. 59:63-73). We demonstrate that 2 and 4 weeks of vMIPII infusion in the contusion-injured spinal cord also results in decreased hematogenous infiltration and is accompanied by reduced axonal degeneration in the gray matter. Luxol fast blue and MBP immunoreactivity indicated reduced myelin breakdown in the dorsal and ventral funiculi. Increased neuronal survival in the ventral horns of vMIPII infused cords was seen along with increased bcl-2 staining in them. Immunohistochemical identification of fiber phenotypes showed increased presence of calcitonin gene related peptide, choline acetyl transferase and tyrosine hydroxylase positive fibers as well as increased GAP43 staining in treated cords. These results suggest that sustained reduction in posttraumatic cellular infiltration is beneficial for tissue survival. A preliminary report of this study has been published (Eng et al. [2000] J. Neurochem. 74(Suppl):S67B). In contrast to vMIPII, infusion of MCP-1 (9-76), a N-terminal analog of the MCP-1 chemokine showed only a modest reduction in cellular infiltration at 14 and 21 dpi without significant tissue survival after spinal cord contusion injury. Comparing data on tissue survival obtained with vMIPII and MCP-1 (9-76) further validate the importance of the use of broad-spectrum antagonists in the treatment of spinal cord injury. Controlling the inflammatory reaction and providing a growth permissive environment would enhance regeneration and ultimately lead to neurological recovery after spinal cord injury. J. Neurosci. Res. 64:582-589, 2001. Published 2001 Wiley-Liss, Inc.     

Functional gait analysis in a spinal contusion rat model

Evaluating functional performance of spinal cord injury (SCI) rat models is essential for the development of novel treatments and breakthroughs. However, due to the variety of functional analysis methods available – each with its own strengths and weaknesses – it can be challenging to choose the most appropriate functional analysis test for the animal model. Therefore, we analyzed the strengths and weaknesses of five methods in order to determine which test is not only accurate and easily reproducible, but also relatively inexpensive so that it can be adopted universally. When comparing the Basso, Beattie, and Bresnahan (BBB) test, Ladder walking test, CatWalk test, Rotating Rod test, Microsoft Kinect system and VICON, we used the criteria of sensitivity, quality of data generated, statistical analysis of data, and rate of human error. These specific tests were chosen in order to compare the advantages and disadvantages of simple yet effective methods (BBB, Ladder test, and Rotating Rod test) to more complex and computerized methods (Catwalk, Microsoft Kinect and VICON).     

Senegenin inhibits neuronal apoptosis after spinal cord contusion injury

Senegenin has been shown to inhibit neuronal apoptosis, thereby exerting a neuroprotective effect. In the present study, we established a rat model of spinal cord contusion injury using the modiifed Allen’s method. Three hours after injury, senegenin (30 mg/g) was injected into the tail vein for 3 consecutive days. Senegenin reduced the size of syringomyelic cavities, and it substantially reduced the number of apop-totic cells in the spinal cord. At the site of injury, Bax and Caspase-3 mRNA and protein levels were decreased by senegenin, while Bcl-2 mRNA and protein levels were increased. Nerve ifber density was increased in the spinal cord proximal to the brain, and hindlimb motor function and electrophysiological properties of rat hindlimb were improved. Taken together, our results suggest that senegenin exerts a neuroprotective effect by suppressing neuronal apoptosis at the site of spinal cord injury.     

Serotonergic fiber sprouting to external anal sphincter motoneurons after spinal cord contusion

The present study analyzed the anatomical plasticity of serotonergic immunoreactive projections to external anal sphincter (EAS) motoneurons, and the behavioral plasticity of EAS reflexes, penile erection, and locomotion in rats with spinal contusion injury (SCI) or complete spinal cord transection (TX). Electromyographic activity of the EAS, penile erection latency, and BBB locomotor score exhibited parallel recovery over the 6-week recovery period after contusion SCI. This pattern of recovery was not observed in TX animals. While locomotor scores demonstrated a small increase after TX, erectile and anorectal function remained at abnormal levels established immediately after injury. Serotonergic immunofluorescent (5-HT-IF) staining at the lesion site identified a small number of fibers spared after SCI that may provide a substrate for functional recovery. Pixel density measurements of 5-HT-IF in the vicinity of retrogradely labeled EAS and unlabeled pudendal motoneurons necessary for penile erection provide indirect evidence of serotonergic sprouting that parallels the observed functional recovery in animals with SCI. No 5-HT-IF was detected caudal to the injury site in TX animals. These studies indicate: (1) lumbosacral eliminative and reproductive reflexes provide a valid means of studying the mechanisms of post-SCI plasticity; (2) the similar recovery curves suggest similar return of descending control, perhaps through sprouting of descending serotonergic fibers; (3) the observed deficits after TX likely represent the permanent removal of descending inhibition and reflect reorganization of segmental circuitry.     

Central necrosis following contusion to the sheep's spinal cord

This paper presents the results of a study on the pathological changes associated with post traumatic central spinal cord necrosis.     

Spinal cord contusion in the rat: morphometric analyses of alterations in the spinal cord

Morphometric analyses were carried out on rat spinal cords which were injured by a weight drop technique. A 10-g weight was dropped 0.0, 2.5, 5.0, 7.5, 10.0, or 17.5 cm onto the dura which was exposed at the T8 vertebral level. Four weeks after injury, lesion volume, lesion length, and the dimensions of the tissue at the epicenter (lesion area, area of gray matter, and area of white matter) were measured and correlated with the height from which the weight was dropped and the results from tests of motor and sensory functional deficit. The results, based on linear regression analyses, indicated significant correlations between certain morphologic parameters (lesion volume, lesion length, and the area of gray and white matter at the epicenter) and both the height from which the weight was dropped and behavioral scores. Because the area of white matter at the epicenter is a very simple measurement which correlates well (r = 0.91) with behavioral outcome, this morphologic feature is a useful quantitative measure of the histopathologic consequences of spinal cord injury.     

Elevation and clearance of extracellular K following contusion of the rat spinal cord

The elevation and clearance of extracellular potassium following a standard contusion injury was studied in the thoracic spinal cord of rats. Animals were anesthetized, paralyzed, laminectomized at T9-T11, then artificially ventilated. A 10-g rod was released 5.0 cm above the cord with the dura intact. After impact, the dura-arachnoid and pial membranes were incised to allow penetration of K(+)-selective microelectrodes. Electrodes utilized a valinomycin ionophore and were double-barreled, with tip diameters of 3-5 microns. Extracellular potassium activity ([K+]o) increased with the depth of penetration. The maximum values of [K+]o occurred at depths greater than 500 microns, and remained so with time after injury. These data indicate that a dorsal-ventral gradient of [K+]o develops in spinal cords contused from the dorsal surface, with the greatest elevation of [K+]o in the gray matter. In 8 preparations, the maximum [K+]o was 65 +/- 8 mM (mean +/- S.E.M.) at 5 +/- 1 min after injury. The [K+]o peak values decayed with a half-time of 11.0 +/- 3.4 min. Compared with data available for the injured cat spinal cord, the peak [K+]o recovered relatively rapidly. Although a simple diffusion model could account for the rapid clearance of [K+]o, the persistence of dorsal-ventral [K+]o gradients could not be explained by such a model. It is postulated that secondary injury processes contributed to the persistent [K+]o gradients.     

Effect of tractotomy on the reflex activity of the lumbar segment of the spinal cord severed after sectioning of the sciatic nerve in the rat

Electrical responses of the spinal cord roots of rats evoked by the preliminary cutting of nerve were investigated in 1, 3, 7 and 14 days after tractotomy. Mean amplitude of the monosynaptic reflex discharges in ventral roots on the side of nerve section is substantially higher 7 and 14 days after the tractotomy, than that of these responses 1 and 3 days after it.     

Forelimb motor performance following cervical spinal cord contusion injury in the rat.

The purpose of this study was to examine the degree, persistence, and nature of forelimb behavioral deficits following cervical spinal cord contusion injury in the rat. Forelimb reaching and pellet retrieval, forehead adhesive sticker removal, and vibrissae-induced forelimb placing were examined for 16 weeks following a weight-drop injury (10.0 g-2.5 cm) at the C4-C5 spinal level. Nine of 13 rats studied were unable to perform the pellet retrieval task due to pronounced forelimb extension hypometria. However, these animals did carry out the forehead sticker removal and vibrissae-induced placing tasks. Therefore, the loss of reaching ability related to pellet retrieval was not due to generalized paralysis. This interpretation was further supported by evaluation of the rostrocaudal extent of relative motoneuron loss from 1-mm divisions through the lesion zone. The extent of motoneuron pathology ranged from 2 to 6 mm but was largely confined to the C4-C5 spinal segments. Morphometric assessments of axonal sparing revealed that pellet retrieval performance during the last month of observation was significantly correlated with fiber sparing in the dorsal columns and ventral white matter, whereas no significant correlation could be demonstrated with regard to dorsolateral white matter. While there were no conspicuous differences in qualitative assessments of damage to interneuron pools (i.e., laminae V to VII) between the nonreaching and retrieval-recovered rats, the possibility of combined white and gray matter pathology contributing to this deficit still exists. These initial findings thus demonstrate that the weight-drop contusion injury model can be adopted to studies of cervical spinal cord trauma in the rat. Such lesions yield permanent deficits in forelimb function lending to future studies of possible therapeutic interventions. Furthermore, performance deficits observed at 1 week postinjury in the placing and forehead sticker removal tasks can be predictive of any potential for long-range spontaneous recovery in pellet retrieval ability.     

Angiotensin II receptors in the lumbar spinal cord of the rat

Locations of cells responsive to microiontophoretically applied angiotensin II (AII) were compared to distributions of AII receptor binding sites identified by autoradiography in the lumbar enlargement region of the rat spinal cord. Angiotensin II receptor binding sites were densely concentrated in the superficial layers of the dorsal horn. Considerably lower densities of binding sites were present in the remaining gray matter. Effects of microiontophoretically applied AII on lumbar spinal cord cells did not vary with location within the gray matter. AII facilitated firing of most cells in the lumbar cord whether the cells were in superficial or deeper laminae of the dorsal horn or in the ventral horn. The distribution of AII binding sites and the distribution of cells that were responsive to AII suggest that AII may play a role in modulating both sensory and motor functions of the spinal cord.     

Temporal-spatial pattern of acute neuronal and glial loss after spinal cord contusion

The secondary loss of neurons and glia over the first 24 h after spinal cord injury (SCI) contributes to the permanent functional deficits that are the unfortunate consequence of SCI. The progression of this acute secondary cell death in specific neuronal and glial populations has not previously been investigated in a quantitative manner. We used a well-characterized model of SCI to analyze the loss of ventral motoneurons (VMN) and ventral funicular astrocytes and oligodendrocytes at 15 min and 4, 8, and 24 h after an incomplete midthoracic contusion injury in the rat. We found that both the length of lesion and the length of spinal cord devoid of VMN increased in a time-dependent manner. The extent of VMN loss at specified distances rostral and caudal to the injury epicenter progressed symmetrically with time. Neuronal loss was accompanied by a loss of glial cells in ventral white matter that was significant at the epicenter by 4 h after injury. Oligodendrocyte loss followed the same temporal pattern as that of VMN while astrocyte loss was delayed. This information on the temporal-spatial pattern of cell loss can be used to investigate mechanisms involved in secondary injury of neurons and glia after SCI.     

Co-induction of HSP70 and heme oxygenase-1 in macrophages and glia after spinal cord contusion in the rat

HSP70 and heme oxygenase-1 (HO-1) are thought to be markers of cell injury and oxidative stress, respectively. We have immunolocalized these proteins in the spinal cord at 1-14 days after contusion. HSP70 and HO-1 were co-induced in glia and macrophages within the injured segment at all time points. This co-induction may reflect complementary functions that serve to protect these cells as they respond to the postcontusional environment.     

Functional and electrophysiological changes after graded traumatic spinal cord injury in adult rat

A graded contusion spinal cord injury (SCI) was created in the adult rat spinal cord using the Infinite Horizons (IH) impactor to study the correlation between injury severity and anatomical, behavioral, and electrophysiological outcomes. Adult Fisher rats were equally divided into five groups and received contusion injuries at the ninth thoracic level (T9) with 100, 125, 150, 175, or 200 kdyn impact forces, respectively. Transcranial magnetic motor-evoked potentials (tcMMEPs) and BBB open-field locomotor analyses were performed weekly for 4 weeks postinjury. Our results demonstrated that hindlimb locomotor function decreased in accordance with an increase in injury severity. The locomotor deficits were proportional to the amount of damage to the ventral and lateral white matter (WM). Locomotor function was strongly correlated to the amount of spared WM, which contains the reticulospinal and propriospinal tracts. Normal tcMMEP latencies were recorded in control, all of 100-kdyn-injured and half of 125-kdyn-injured animals. Delayed latency responses were recorded in some of 125-kdyn-injured and all of 150-kdyn-injured animals. No tcMMEP responses were recorded in 175- and 200-kdyn-injured animals. Comparison of tcMMEP responses with areas of WM loss or demyelination identified the medial ventrolateral funiculus (VLF) as the location of the tcMMEP pathway. Immunohistochemical and electromicroscopic (EM) analyses showed the presence of demyelinated axons in WM tracts surrounding the lesion cavities at 28 days postinjury. These data support the notion that widespread WM damage in the ventral and lateral funiculi may be a major cause for locomotor deficits and lack of tcMMEP responses after SCI.