The functional and neuroprotective actions of Neu2000, a dual-acting pharmacological agent, in the treatment of acute spinal cord injury

Abstract

The goal of the present study was to examine the neuroprotective and functional significance of targeting both N-methyl-D-aspartate (NMDA) receptor-mediated excitotoxicity and oxidative stress using a dual-acting compound, Neu2000, in rat model of moderate spinal cord injury (SCI). An initial set of experiments was conducted in uninjured rats to study the pharmacokinetic profile of Neu2000 following intraperitoneal and intravenous administration. A second experiment measured free radical production in mitochondria isolated from sham or injured spinal cords of animals receiving vehicle or Neu2000 treatment. A third set of animals was divided into three treatment groups consisting of vehicle treatment, a single dose of Neu2000 (50 mg/kg) administered at 10 min following injury, or a repeated treatment paradigm consisting of a single bolus of Neu2000 at 10 min following injury (50 mg/kg) plus a maintenance dose (25 mg/kg) administered every 24 h for an additional 6 days. Animals were tested once a week for a period of 6 weeks for evidence of locomotor recovery in an open field and kinematic analysis of fine motor control using the DigiGait Image Analysis System. At the end of the testing period, spinal cord reconstruction was performed to obtain nonbiased stereological measures of tissue sparing. The results of this study demonstrate that Neu2000 treatment significantly reduced the production of mitochondrial free radicals and improved locomotor outcomes that were associated with a significant increase in the volume of spared spinal cord tissue.

FIG. 1.

Basal and oligomycin-induced reactive oxygen species (ROS) production in mitochondria isolated from sham, spinal cord injury (SCI) + vehicle, and SCI + Neu2000 animals. Animals received 50 mg/kg Neu2000 at 10 min after SCI, and spinal cord mitochondria were obtained 24 h later to examine ROS production. The relative levels of ROS produced under basal conditions were unaffected by Neu2000, while ROS production induced by oligomycin was significantly reduced compared to vehicle. *p < 0.01 compared to sham, **p < 0.05 compared to vehicle and sham. DCF, dichlorodihydrofluorescein.

FIG. 2.

Group mean (±SEM) Basso, Beattie, and Bresnahan (BBB) scores in sham and spinal cord–injured animals treated with vehicle or Neu2000. All injured animals showed an initial deficit in locomotor scores that improved over time in a pattern consistent with the level of injury. However, there was a significant improvement (*p < 0.05) in the group mean BBB scores in animals receiving repeated (but not single) treatment with Neu2000 compared to vehicle treatment.

FIG. 3.

(A) Representative DigiGait traces of sham and injured rats treated with vehicle. Effects of Neu2000 treatment was observed on several indices of locomotor function at 42 days following injury including (B) percentage of stride in swing, (C) swing to stance ratio, (D) degree of hindpaw external rotation, and (E) hindpaw area. *p < 0.05 compared to sham and Neu2000 treatment, **p < 0.01 compared to sham and Neu2000 treatment.

FIG. 4.

Representative photomicrographs of spinal cord sections demonstrating the effects of vehicle or repeated or single Neu2000 treatment on tissue sparing (upper set of panels). Note the pronounced effect of repeated Neu2000 treatment (Group B) on tissue sparing compared to vehicle (Group A) and single Neu2000 treatment (Group C). The images across the treatment groups correspond to spinal cord sections equidistant rostral and caudal to the injury epicenter. Panels A–D demonstrate the results of the stereological analysis of total lesion volume and spared white matter. A 20 mm segment from each animal was used for the stereological studies. The X axis refers to the spinal cord section being analyzed relatively to the injury epicenter, with 1 referring to the most rostral section and 20 the most caudal section. (A) The lesion area and (C) spared white matter area at each millimeter along the 20 mm segment demonstrate the pattern of tissue sparing due to vehicle (Group A), repeated (Group B), or single (Group C) Neu2000 treatment. Calculation of (B) total lesion volume and (D) spared white matter volume demonstrate a significant decrease (*p < 0.05) in lesion volume in both Neu2000 treatment groups, while only the repeated Neu2000 treatment showed a significant increase (*p < 0.05) in white matter sparing.