Effects of dantrolene on apoptosis and immunohistochemical expression of NeuN in the spinal cord after traumatic injury in rats

Abstract

Dantrolene has been shown to be neuroprotective by reducing neuronal apoptosis after brain injury in several animal models of neurological disorders. In this study, we investigated the effects of dantrolene on experimental spinal cord injury (SCI). Forty-six male Wistar rats were laminectomized at T13 and divided in six groups: GI (n = 7) underwent SCI with placebo and was euthanized after 32 h; GII (n = 7) underwent laminectomy alone with placebo and was euthanized after 32 h; GIII (n = 8) underwent SCI with dantrolene and was euthanized after 32 h; GIV (n = 8) underwent SCI with placebo and was euthanized after 8 days; GV (n = 8) underwent laminectomy alone with placebo and was euthanized after 8 days; and GVI (n = 8) underwent SCI with dantrolene and was euthanized after 8 days. A compressive trauma was performed to induce SCI. After euthanasia, the spinal cord was evaluated using light microscopy, TUNEL staining and immunochemistry with anti-Caspase-3 and anti-NeuN. Animals treated with dantrolene showed a smaller number of TUNEL-positive and caspase-3-positive cells and a larger number of NeuN-positive neurons, both at 32 h and 8 days (P ≤ 0.05). These results showed that dantrolene protects spinal cord tissue after traumatic SCI by decreasing apoptotic cell death.

Figure 1

Means ± SD of the average number per field (100×) of TUNEL-positive cells in the spinal cord of Wistar rats. GI [spinal cord injury (SCI) with placebo - 32 h]; GII (laminectomy only with placebo - 32 h); GIII (SCI with dantrolene - 32 h); GIV (SCI with placebo - 8 days); GV (laminectomy only with placebo – 8 days) and GVI (SCI with dantrolene - 8 days). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

Means ± SD of the average number per field (100×) of caspase-3-positive cells in the spinal cord of Wistar rats. GI (SCI with placebo - 32 h); GII (laminectomy only with placebo - 32 h); GIII [spinal cord injury (SCI) with dantrolene - 32 h]; GIV (SCI with placebo - 8 days); GV (laminectomy only with placebo – 8 days) and GVI (SCI with dantrolene - 8 days). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

Means ± SD of the average number per field (100×) of NeuN-positive neurons in the spinal cord of Wistar rats. GI (SCI with placebo - 32 h); GII (laminectomy only with placebo - 32 h); GIII [spinal cord injury (SCI)with dantrolene - 32 h]; GIV (SCI with placebo - 8 days); GV (laminectomy only with placebo – 8 days) and GVI (SCI with dantrolene - 8 days). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

Photomicroscopy of spinal cord cross-sections adjacent to the epicentre of injury in Wistar rats. Immunohistochemistry with anti-NeuN. (a) Nuclei staining of intact neuronal cell bodies (NeuN-positive) in the grey matter in animal from GII (laminectomy only with placebo - 32 h) - 23.5×; (b) NeuN-positive neurons in the animals from GV (laminectomy only with placebo - 8 days) - 26×; (c) Mild staining of NeuN-positive neurons in animals from GI [spinal cord injury (SCI) with placebo - 32 h] - 24×; (d) Moderate staining of NeuN-positive neurons in animal from GIII (SCI with dantrolene - 32 h) – 21.8×; (e) Mild staining of NeuN-positive neurons in animal from GIV (SCI with placebo - 8 days) - 23.9×; (f) NeuN-positive neurons similar to the groups without injury. Animal VI (SCI with dantrolene - 8 days) - 23.3×; (g) Detail of NeuN-positive neurons (arrows) with nucleus staining by monoclonal anti-NeuN - 374.6×.