Use of ebselen as a neuroprotective agent in rat spinal cord subjected to traumatic injury

Abstract

Spinal cord injury (SCI) causes disturbances of motor skills. Free radicals have been shown to be essential for the development of spinal cord trauma. Despite some progress, until now no effective pharmacological therapies against SCI have been verified. The purpose of our experiment was to investigate the neuroprotective effects of ebselen on experimental SCI. Twenty-two rats subjected to SCI were randomly subjected to SCI with no further treatment (n = 10) or intragastric administration of ebselen (10 mg/kg) immediately and 24 hours after SCI. Behavioral changes were assessed using the Basso, Beattie, and Bresnahan locomotor scale and footprint test during 12 weeks after SCI. Histopathological and immunohistochemical analyses of spinal cords and brains were performed at 12 weeks after SCI. Magnetic resonance imaging analysis of spinal cords was also performed at 12 weeks after SCI. Rats treated with ebselen presented only limited neurobehavioral progress as well as reduced spinal cord injuries compared with the control group, namely length of lesions (cysts/scars) visualized histopathologically in the spinal cord sections was less but cavity area was very similar. The same pattern was found in T2-weighted magnetic resonance images (cavities) and diffusion-weighted images (scars). The number of FluoroGold retrogradely labeled neurons in brain stem and motor cortex was several-fold higher in ebselen-treated rats than in the control group. The findings suggest that ebselen has only limited neuroprotective effects on injured spinal cord. All exprimental procedures were approved by the Local Animal Ethics Committee for Experiments on Animals in Katowice (Katowice, Poland) (approval No. 19/2009).

Keywords: antioxidant; ebselen; functional recovery; neuroprotection; neuroregeneration; spinal cord injury; spinal cord repair.

Figure 1

Angle of rotation of the hind paw in spinal cord injury rats treated (n = 12; at 12 weeks n = 8) or not (n = 10; at 12 weeks n = 6) with ebselen. All data are presented as the mean ± SD.

Figure 2

Inter-limb coordination (cm) in spinal cord injury rats treated or not with ebselen. All data are presented as the mean ± SD. *P < 0.05 (Student’s t-test). n = 12 and 10 in the ebselen and control groups, respectively, at 1–7 weeks, and n = 8 and 6 in both groups at 12 weeks.

Figure 3

Basso, Beattie, and Bresnahan (BBB) locomotor scale scores after spinal cord injury in rats treated or not with ebselen. All data are presented as the mean ± SD. n = 12 and 10 in the ebselen and control groups, respectively, at 1–7 weeks, and n = 8 and 6 in both groups at 12 weeks.

Figure 4

Magnetic resonance images from the representative injured spinal cord after ebselen treatment at 12 weeks. (A) T2-weighted image. Diffusion-weighted image with diffusion gradient applied along phase direction (B) and (C) slice direction. Scar/hemorrhage pathological change present in the white matter (indicted by arrow) and at the border between the white and gray matter of lateral funiculus of spinal cord (marked with the circle).

Figure 5

Magnetic resonance imaging – average value of the longitudinal diffusion coefficient in spinal cord white matter, respectively, in healthy, ebselen, and control groups. All data are presented as the mean ± SD from n = 3 per group. *P < 0.05 (one-way analysis of variance followed by Tukey’s test).

Figure 6

Microphotographs obtained 12 weeks after spinal cord injury in rats treated with ebselen. (A) Spinal cord, toluidine blue staining; numerous cystic cavitations (vacuolar structures). Lined area of anterior horn of spinal cord; ASA: anterior spinal artery; arrows: axon spheroids (swollen axons). (B) Formation of glial fibrillary acidic portein primary-labeled cavity in the injured spinal cord ventral white column. Asterisk: microcyst with astrogliosis. (C) FluoroGold retrograde labeling of regenerating neurons. Neuronal cytoplasm is labeled by fluorogold in transverse sections of the raphe nuclei in the brain stem.

Figure 7

Area of lesion cavity 12 weeks after spinal cord injury in rats treated or not with ebselen. All data are presented as the mean ± SD. n = 8 and 6 in the ebselen and control groups, respectively.

Figure 8

Measurement of the length of the spinal cord lesion in rats treated or not with ebselen. All data are presented as the mean ± SD. *P < 0.05 (Student’s t-test). n = 8 and 6 in the ebselen and control groups, respectively.

Figure 9

Number of FluoroGold (FG)-positive brain stem and motor cortex neurons 12 weeks after spinal cord injury in rats treated or not with ebselen. All data are presented as the mean ± SD from 4 rats per group. *P < 0.05 (Student’s t-test).