Pregabalin as a neuroprotector after spinal cord injury in rats: biochemical analysis and effect on glial cells

Abstract

As one of trials on neuroprotection after spinal cord injury, we used pregabalin. After spinal cord injury (SCI) in rats using contusion model, we observed the effect of pregabalin compared to that of the control and the methylprednisolone treated rats. We observed locomotor improvement of paralyzed hindlimb and body weight changes for clinical evaluation and caspase-3, bcl-2, and p38 MAPK expressions using western blotting. On histopathological analysis, we also evaluated reactive proliferation of glial cells. We were able to observe pregabalin's effectiveness as a neuroprotector after SCI in terms of the clinical indicators and the laboratory findings. The caspase-3 and phosphorylated p38 MAPK expressions of the pregabalin group were lower than those of the control group (statistically significant with caspase-3). Bcl-2 showed no significant difference between the control group and the treated groups. On the histopathological analysis, pregabalin treatment demonstrated less proliferation of the microglia and astrocytes. With this animal study, we were able to demonstrate reproducible results of pregabalin's neuroprotection effect. Diminished production of caspase-3 and phosphorylated p38 MAPK and as well as decreased proliferation of astrocytes were seen with the administration of pregabalin. This influence on spinal cord injury might be a possible approach for achieving neuroprotection following central nervous system trauma including spinal cord injury.

Keywords: Apoptosis; Astrocytes; Microglia; Pregabalin; Spinal Cord Injuries.

Fig. 1

Motor scores for the each group. The rats in all the groups show a sequential recovery of hindlimb function. At the postoperative 7th day, the pregabalin treated group show a more significant recovery compared to that of the other groups. Horizontal line at motor score means the score in the sham-operated rats.

Fig. 2

(A) The effect of intraperitoneal pregabalin (30 mg/kg) on the expression of caspase-3 in contusive spinal cord injury. One-way ANOVA reveals significant differences between the groups for the expression of caspase-3 expression in the spinal cord tissue and the post hoc comparison (Bonferroni) test revealed that pregabalin treatment induced the least expression of caspase-3.This difference showed a statistical significance compared to the control group and the methylprednisolone treated group (P < 0.05, n = 10 for the experimental group, n = 3 for sham group). (B) The effect of intraperitoneal pregabalin (30 mg/kg) on the expression of bcl-2 in contusive spinal cord injury. One-way ANOVA test and post hoc analysis reveal no significant difference between the groups for the expression of bcl-2 expression in the spinal cord tissue (f = 0.52, P = 0.67, n = 10 for the experimental group, n = 3 for the sham group).

Fig. 3

The effect of intraperitoneal injection of pregabalin (30 mg/kg) on the expression of phosphorylated p38 MAPK in contusive spinal cord injury. The values were examined on post-injury day 7. The methylprednisolone and pregabalin treatment groups demonstrate a reduced expression of phosphorylated p38 MAPK as compared to that of the control group. However, ANOVA indicated no statistically significant difference (f = 0.65, P = 0.58, n = 10 for the experimental group, n = 3 for the sham group). The following Bonferroni post hoc analysis also indicated no statistical difference between the groups.

Fig. 4

Immunofluorescence detection of the activated astrocyte labeled with antibody GFAP. Abundant activation of astrocytes is observed in the most of the specimen of the injured groups. The control group demonstrate a higher number of activated astrocytes than that of the MP and the GP groups. The astrocytes are mostly located around the periphery of the injured site, and the astrocytes also show a tendency of recruitment from the surrounding gray and white matter (magnification × 200, scale bar 20 µm).

Fig. 5

Immunofluorescence detection of the activated microglia labeled with antibody OX-42. The injured group demonstrates a significantly elevated number of activated microglia. However, there was no statistical difference among the injured groups (magnification × 200, scale bar 20 µm).