tetrodotoxin and Contusions

Focal injection of the sodium channel blocker tetrodotoxin (TTX) into the injury site at either 5 or 15 min after a standardized thoracic contusion spinal cord injury (SCI) reduces white matter pathology and loss of axons in the first 24 hr after injury. Focal injection of TTX at 15 min after SCI also reduces chronic white matter loss and hindlimb functional deficits. We have now tested the hypothesis that the reduction in chronic deficits with TTX treatment is associated with long-term preservation of axons after SCI and compared both acute (24 hr) and chronic (6 weeks) effects of TTX administered at 15 min prior to and 5 min or 4 hr after SCI. Our results indicate a significant reduction of acute white matter pathology in rats treated with TTX at 15 min before and 5 min after injury but no effect when treatment was delayed until 4 hr after contusion. Compared with injury controls, groups treated with TTX at 5 min and 4 hr after injury did not show a significant deficit reduction, nor was there a significant sparing of white matter at 6 weeks compared with injury controls. In contrast, the group treated with TTX at 15 min before SCI demonstrated significantly reduced hindlimb functional deficits beginning at 1 week after injury and throughout the 6 weeks of the study. This was associated with a significantly higher axon density in the ventromedial white matter at 6 weeks. The results demonstrate that blockade of sodium channels preserves axons from loss after SCI and points to the importance of time of administration of such drugs for therapeutic effectiveness.

Topics: Animals; Axons; Cell Count; Contusions; Convalescence; Drug Evaluation, Preclinical; Female; Ion Transport; Myelin Sheath; Neuroprotective Agents; Paraplegia; Rats; Rats, Sprague-Dawley; Sodium; Sodium Channels; Spinal Cord Injuries; Tetrodotoxin; Time Factors; Urinary Bladder, Neurogenic

Focal microinjection of tetrodotoxin (TTX), a potent voltage-gated sodium channel blocker, reduces neurological deficits and tissue loss after spinal cord injury (SCI). Significant sparing of white matter (WM) is seen at 8 weeks after injury and is correlated to a reduction in functional deficits. To determine whether TTX exerts an acute effect on WM pathology, Sprague Dawley rats were subjected to a standardized weight-drop contusion at T8 (10 gm x 2.5 cm). TTX (0. 15 nmol) or vehicle solution was injected into the injury site 5 or 15 min later. At 4 and 24 hr, ventromedial WM from the injury epicenter was compared by light and electron microscopy and immunohistochemistry. By 4 hr after SCI, axonal counts revealed reduced numbers of axons and significant loss of large (>/=5 micrometer)-diameter axons. TTX treatment significantly reduced the loss of large-diameter axons. In addition, TTX significantly attenuated axoplasmic pathology at both 4 and 24 hr after injury. In particular, the development of extensive periaxonal spaces in the large-diameter axons was reduced with TTX treatment. In contrast, there was no significant effect of TTX on the loss of WM glia after SCI. Thus, the long-term effects of TTX in reducing WM loss after spinal cord injury appear to be caused by the reduction of acute axonal pathology. These results support the hypothesis that TTX-sensitive sodium channels at axonal nodes of Ranvier play a significant role in the secondary injury of WM after SCI.

Topics: Animals; Axons; Contusions; Female; Glial Fibrillary Acidic Protein; Injections, Spinal; Microinjections; Myelin Sheath; Necrosis; Neuroglia; Oligodendroglia; Rats; Rats, Sprague-Dawley; Sodium Channel Blockers; Spinal Cord; Spinal Cord Injuries; Tetrodotoxin

Although relatively little is known of the mechanisms involved in secondary axonal loss after spinal cord injury (SCI), recent data from in vitro models of white matter (WM) injury have implicated abnormal sodium influx as a key event. We hypothesized that blockade of sodium channels after SCI would reduce WM loss and long-term functional deficits. To test this hypothesis, a sufficient and safe dose (0.15 nmol) of the potent Na+ channel blocker tetrodotoxin (TTX) was determined through a dose-response study. We microinjected TTX or vehicle (VEH) into the injury site at 15 min after a standardized contusive SCI in the rat. Behavioral tests were performed 1 d after injury and weekly thereafter. Quantitative histopathology at 8 weeks postinjury showed that TTX treatment significantly reduced tissue loss at the injury site, with greater effect on sparing of WM than gray matter. TTX did not change the pattern of chronic histopathology typical of this SCI model, but restricted its extent, tripled the area of residual WM at the epicenter, and reduced the average length of the lesions. Serotonin immunoreactivity caudal to the epicenter, a marker for descending motor control axons, was nearly threefold that of VEH controls. The increase in WM at the epicenter was significantly correlated with the decrease in functional deficits. The TTX group exhibited a significantly enhanced recovery of coordinated hindlimb functions, more normal hindlimb reflexes, and earlier establishment of a reflex bladder. The results demonstrate that Na+ channels play a critical role in WM loss in vivo after SCI.

Topics: Animals; Contusions; Female; Image Processing, Computer-Assisted; Linear Models; Locomotion; Microinjections; Nerve Fibers; Rats; Rats, Sprague-Dawley; Sodium Channel Blockers; Spinal Cord Injuries; Tetrodotoxin; Time Factors