Small-molecule protein tyrosine phosphatase inhibition as a neuroprotective treatment after spinal cord injury in adult rats.

Spinal cord injury causes progressive secondary tissue degeneration, leaving many injured people with neurological disabilities. There are no satisfactory neuroprotective treatments. Protein tyrosine phosphatases inactivate neurotrophic factor receptors and downstream intracellular signaling molecules. Thus, we tested whether the peroxovanadium compound potassium bisperoxo(1,10-phenanthroline)oxovanadate (V) [bpV(phen)], a stable, potent and selective protein tyrosine phosphatase inhibitor, would be neuroprotective after a thoracic spinal cord contusion in adult rats. Intrathecal bpV(phen) infusions through a lumbar puncture rescued dorsal column sensory axons innervating the nucleus gracilis and white matter at the injury epicenter. At the most effective dose, essentially all of these axons and most of the white matter at the epicenter were spared (vs approximately 60% with control infusions). bpV(phen) treatments started 4 h after contusion were fully effective. This treatment greatly improved and normalized sensorimotor function in a grid-walking test and provided complete axonal protection over 6 weeks. The treatment rescued sensory-evoked potentials that disappeared after dorsal column transection. bpV(phen) affected early degenerative mechanisms, because the main effects were seen at 7 d and lasted beyond the treatment period. The neuroprotection appeared to be mediated by rescue of blood vessels. bpV(phen) reduced apoptosis of cultured endothelial cells. These results show that a small molecule, used in a clinically relevant manner, reduces loss of long-projecting axons, myelin, blood vessels, and function in a model relevant to the most common type of spinal cord injury in humans. They reveal a novel mechanism of spinal cord degeneration involving protein tyrosine phosphatases that can be targeted with therapeutic drugs.

Dural repair reduces connective tissue scar invasion and cystic cavity formation after acute spinal cord laceration injury in adult rats.

This study examined whether duraplasty after acute cervical laceration spinal cord injury (SCI) in a rat model could (1) improve cerebrospinal fluid (CSF) circulation adjacent to the injury; (2) minimize connective tissue scarring; and (3) reduce post-traumatic inflammation and cystic cavitation. Following a transverse dural/arachnoid incision and C5-6 dorsal spinal hemisection, a 5-mm2 cadaveric dura mater allograft was placed over the lesion and fixed with fibrin glue (n=12). Control animals received an identical dural/arachnoid incision and cervical dorsal hemisection without dural repair (n=12). At 1, 5, and 10 weeks post-injury, plain film myelograms were obtained to characterize CSF circulation, and stereological methods were used to compare the extent of tissue sparing between the two groups. Immunohistochemical studies were performed to assess the degree of inflammation (ED-1), connective tissue scarring (laminin and type IV collagen), and reactive astrogliosis (GFAP). Our results indicate that dural allograft can improve CSF flow adjacent to the site of injury, which may be due to reduced meningeal fibrosis/scarring at the lesion site. Stereological analysis demonstrated that duraplasty resulted in a significant reduction in lesion volume at each time-point (p<0.01) associated with a nearly complete attenuation of post-traumatic cystic cavitation (p<0.001). Immunofluorescence studies demonstrated that duraplasty reduced the infiltration of ED-1-positive macrophages/microglia into and surrounding the lesion site, which may be responsible for the marked reduction in secondary injury following duraplasty. We conclude that duraplasty following acute spinal cord laceration may (1) improve CSF flow by limiting meningeal fibrosis; (2) reduce connective tissue scar formation; and (3) attenuate macrophage accumulation and progressive secondary injury.

Functional redundancy of ventral spinal locomotor pathways.

Identification of long tracts responsible for the initiation of spontaneous locomotion is critical for spinal cord injury (SCI) repair strategies. Pathways derived from the mesencephalic locomotor region and pontomedullary medial reticular formation responsible for fictive locomotion in decerebrate preparations project to the thoracolumbar levels of the spinal cord via reticulospinal axons in the ventrolateral funiculus (VLF). However, white matter regions critical for spontaneous over-ground locomotion remain unclear because cats, monkeys, and humans display varying degrees of locomotor recovery after ventral SCIs. We studied the contributions of myelinated tracts in the VLF and ventral columns (VC) to spontaneous over-ground locomotion in the adult rat using demyelinating lesions. Animals received ethidium bromide plus photon irradiation producing discrete demyelinating lesions sufficient to stop axonal conduction in the VLF, VC, VLF-VC, or complete ventral white matter (CV). Behavior [open-field Basso, Beattie, and Bresnahan (BBB) scores and grid walking] and transcranial magnetic motor-evoked potentials (tcMMEP) were studied at 1, 2, and 4 weeks after lesion. VLF lesions resulted in complete loss or severe attenuation of tcMMEPs, with mean BBB scores of 18.0, and no grid walking deficits. VC lesions produced behavior similar to VLF-lesioned animals but did not significantly affect tcMMEPs. VC-VLF and CV lesions resulted in complete loss of tcMMEP signals with mean BBB scores of 12.7 and 6.5, respectively. Our data support a diffuse arrangement of axons within the ventral white matter that may comprise a system of multiple descending pathways subserving spontaneous over-ground locomotion in the intact animal.

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.

The therapeutic window for spinal cord decompression in a rat spinal cord injury model.

OBJECT: There are no clinically based guidelines to direct the spine surgeon as to the proper timing to undertake decompression after spinal cord injury (SCI) in patients with concomitant stenosis-induced cord compression. The following three factors affect the prognosis: (1) severity of SCI; (2) degree of extrinsic spinal cord compression; and (3) duration of spinal cord compression. METHODS: To elucidate further the relationship between varying degrees of spinal stenosis and a mild contusion-induced SCI (6.25 g-cm), a rat SCI/stenosis model was developed in which 1.13- and 1.24-mm-thick spacers were placed at T-10 to create 38 and 43% spinal stenosis, respectively. Spinal cord damage was observed after the stenosis-SCI that was directly proportional to the duration of spinal cord compression. The therapeutic window prior to decompression was 6 and 12 hours in the 43 and 38% stenosis-SCI lesions, respectively, to maintain locomotor activity. A significant difference in total lesion volume was observed between the 2-hour and the delayed time(s) to decompression (38% stenosis-SCI, 12 and 24 hours, p < 0.05; 43% stenosis-SCI, 24 hours, p < 0.05) indicating a more favorable neurological outcome when earlier decompression is undertaken. This finding was further supported by the animal's ability to support weight when decompression was performed by 6 or 12 hours compared with 24 hours after SCI. CONCLUSIONS: Analysis of the findings in this study suggests that early decompression in the rat improves locomotor function. Prolongation of the time to decompression may result in irreversible damage that prevents locomotor recovery.

A novel vertebral stabilization method for producing contusive spinal cord injury.

Clinically-relevant animal cervical spinal cord injury (SCI) models are essential for developing and testing potential therapies; however, producing reliable cervical SCI is difficult due to lack of satisfactory methods of vertebral stabilization. The conventional method to stabilize the spine is to suspend the rostral and caudal cervical spine via clamps attached to cervical spinous processes.  However, this method of stabilization fails to prevent tissue yielding during the contusion as the cervical spinal processes are too short to be effectively secured by the clamps (Figure 1).  Here we introduce a new method to completely stabilize the cervical vertebra at the same level of the impact injury.  This method effectively minimizes movement of the spinal column at the site of impact, which greatly improves the production of consistent SCIs.  We provide visual description of the equipment (Figure 2-4), methods, and a step-by-step protocol for the stabilization of the cervical 5 vertebra (C5) of adult rats, to perform laminectomy (Figure 5) and produce a contusive SCI thereafter.  Although we only demonstrate a cervical hemi-contusion using the NYU/MASCIS impactor device, this vertebral stabilization technique can be applied to other regions of the spinal cord, or be adapted to other SCI devices.  Improving spinal cord exposure and fixation through vertebral stabilization may be valuable for producing consistent and reliable injuries to the spinal cord.  This vertebral stabilization method can also be used for stereotactic injections of cells and tracers, and for imaging using two-photon microscopy in various neurobiological studies.