Synergistic neuroprotective effects of hyperbaric oxygen and methylprednisolone following contusive spinal cord injury in rat

Abstract

Objective: Recent studies revealed the neuroprotective effects of hyperbaric oxygen (HBO) on spinal cord injury (SCI). Meanwhile, the use of methylprednisolone (MP) is one of the current protocols with limited effects in SCI patients. Accordingly, the aim of the present study was to investigate the effect of combined HBO and MP treatment on SCI.

Design: The present study was conducted on five groups of rats each as follows: Sham group (underwent laminectomy alone at T9 level vertebra); SCI group (underwent moderate contusive SCI); MP group (underwent SCI and received MP); HBO group (underwent SCI and received HBO); HBO + MP group (underwent SCI and simultaneously received MP and HBO). Blood serum and Spinal cord tissue samples were taken 48 h after SCI for analysis of serum ferric reducing antioxidant power (FRAP) and tissue malodialdehyde (MDA) levels as well as immunohistochemistry of caspase-3 and tumor necrosis factor-alpha (TNF-α). Neurological function was evaluated by the Basso-Beattie-Bresnehan (BBB) locomotion scores until the end of experiments. Additionally, histopathology was assessed at the end of the study.

Setting: Mazandaran University of Medical Sciences, Sari, Iran.

Results: Combination therapy with HBO and MP in the HBO + MP group significantly decreased MDA as well as increased FRAP levels compared to other treatment groups. Meanwhile, attenuated TNF-α and Caspase-3 expression could be significantly detected in the HBO + MP group. At the end of treatment, the neurological outcome was significantly improved and the extent of injured spinal tissue was also significantly reduced in the HBO + MP compared to other treatment groups.

Conclusion: The results suggest that combined therapy with MP and HBO has synergistic effects on SCI treatment.

Keywords: Apoptosis; Hyperbaric oxygen; Inflammation; Methylprednisolone; Spinal cord injury.

Figure 1

Effects of HBO and MP on lipid peroxidation. Histogram shows the levels of MDA in the traumatized spinal tissue 48 h after SCI. Values are expressed as nanomole per milligram of protein (nmol/mg-protein). ^aP < 0.001 versus sham group; ^bP < 0.001 versus SCI group; ^cP < 0.01 versus MP group; ^dP < 0.001 versus MP and HBO groups.

Figure 2

Effects of HBO and MP on total antioxidant capacity. Histogram shows the serum levels of FRAP 48 h after SCI. Values are expressed as unit mole per liter Fe²⁺ (umol/LFe²⁺). ^aP < 0.001 versus sham group; ^bP < 0.001 versus SCI group; ^cP < 0.001 versus MP group; ^dP < 0.001 versus MP and HBO groups.

Figure 3

Light Photomicrographs show immunohistochemical expression of TNF-α in the horizontal sections of traumatized spinal tissue. The positive staining of TNF-α is presented by a brown color of cytoplasm (arrows) (Scale bar = 100 µm). Densitometry analysis was performed for immunohistochemical photomicrographs of TNF-α. Data are expressed as a percentage of total tissue area. ^aP < 0.001 versus sham group; ^bP < 0.01 versus sham group; ^cP < 0.05 versus sham group; ^dP < 0.001 versus SCI group; ^eP < 0.05 versus MP and HBO groups.

Figure 4

Light Photomicrographs show immunohistochemical expression of caspase-3 in the horizontal sections of traumatized spinal tissue. The positive staining of caspase-3 is presented by a brown color of cytoplasm (arrows) (Scale bar = 100 µm). Densitometry analysis was performed for immunohistochemical photomicrographs of caspase-3. Data are expressed as a percentage of total tissue area. ^aP < 0.001 versus sham group; ^bP < 0.05 versus sham group; ^cP < 0.01 versus sham group; ^dP < 0.001 versus SCI group; ^eP < 0.01 versus SCI group.

Figure 5

Effects of HBO and MP on neurologic function. Histogram shows the neurologic outcome before SCI and then 6 h, 1, 3 days, 7, and 14 days after SCI in all groups as a BBB score. Values are mean ± SD. ^aP < 0.001 versus sham group; ^bP < 0.05 versus SCI group; ^cP < 0.001 versus SCI group; ^dP < 0.001 versus MP group; ^eP < 0.05 versus HBO group; ^fP < 0.01 versus SCI group; ^gP < 0.05 versus MP group; ^hP < 0.01 versus sham group; ⁱP < 0.05 versus sham group.

Figure 6

Effects of HBO and MP on tissue protection. Photomicrographs taken from the lesion epicenter indicate the destruction of the spinal tissue (arrows) 14 days after SCI in all groups (stained with H&E; magnification, ×40). Histogram shows the mean lesion area as a percentage of the whole spinal cord. Values are mean ± SD. ^aP < 0.001 versus sham group; ^bP < 0.01 versus sham group; ^cP < 0.001 versus SCI group; ^dP < 0.05 versus HBO + MP group; ^eP < 0.01 versus HBO + MP group.