Ischemic preconditioning protects against spinal cord ischemia-reperfusion injury in rabbits by attenuating blood spinal cord barrier disruption

Abstract

Ischemic preconditioning has been reported to protect against spinal cord ischemia-reperfusion (I-R) injury, but the underlying mechanisms are not fully understood. To investigate this, Japanese white rabbits underwent I-R (30 min aortic occlusion followed by reperfusion), ischemic preconditioning (three cycles of 5 min aortic occlusion plus 5 min reperfusion) followed by I-R, or sham surgery. At 4 and 24 h following reperfusion, neurological function was assessed using Tarlov scores, blood spinal cord barrier permeability was measured by Evan's Blue extravasation, spinal cord edema was evaluated using the wet-dry method, and spinal cord expression of zonula occluden-1 (ZO-1), matrix metalloproteinase-9 (MMP-9), and tumor necrosis factor-α (TNF-α) were measured by Western blot and a real-time polymerase chain reaction. ZO-1 was also assessed using immunofluorescence. Spinal cord I-R injury reduced neurologic scores, and ischemic preconditioning treatment ameliorated this effect. Ischemic preconditioning inhibited I-R-induced increases in blood spinal cord barrier permeability and water content, increased ZO-1 mRNA and protein expression, and reduced MMP-9 and TNF-α mRNA and protein expression. These findings suggest that ischemic preconditioning attenuates the increase in blood spinal cord barrier permeability due to spinal cord I-R injury by preservation of tight junction protein ZO-1 and reducing MMP-9 and TNF-α expression.

Figure 1

IPC improved neurologic function after spinal cord ischemia-reperfusion (I-R) injury. Neurologic function was assessed using Tarlov scores at 4 and 24 h after spinal cord ischemia. Data are presented as individual values for each animal, as well as median values for each group (n = 6/group). **p < 0.01 versus Sham group. ^##p < 0.01 versus I-R group.

Figure 2

IPC inhibited the blood spinal cord barrier breakdown after spinal cord ischemia-reperfusion (I-R) injury. Blood spinal cord barrier permeability was measured at 4 and 24 h after injury using Evan’s Blue dye, and spinal cord edema was measured at 4 and 24 h by tissue water content. (A–F) Representative fluorescence images of Evan’s Blue extravasation in spinal cord parenchyma across groups: (A) Sham 4 h; (B) I-R 4 h; (C) IPC 4 h; (D) Sham 24 h; (E) I-R 24 h; (F) IPC 24 h; (G) Quantification of the content of Evan’s Blue in the injured spinal cord; (H) Quantification of the water content of the spinal cord. All data represent mean ± SEM (n = 6/group). **p < 0.01 versus Sham group. ^#p < 0.05, ^##p < 0.01 versus I-R group.

Figure 3

IPC increases expression of tight junction protein ZO-1 after spinal cord ischemia-reperfusion (I-R) injury. (A) Representative Western blot and quantitative protein analysis of ZO-1 expression in spinal cord at 4 h and 24 h after injury; (B) Real-time PCR analysis of ZO-1 mRNA expression in spinal cord at 4 and 24 h after injury. Levels are expressed as ratios to sham. Data are presented as mean ± SEM (n = 6/group). **p < 0.01 versus Sham group. ^##p < 0.01 versus I-R group; (C) Representative double-labeling immunofluorescence microscopic photographs show that CD31-positive endothelial cells (red) express ZO-1 (green) at 24 h after injury. Bar scale = 20 μm.

Figure 3

IPC increases expression of tight junction protein ZO-1 after spinal cord ischemia-reperfusion (I-R) injury. (A) Representative Western blot and quantitative protein analysis of ZO-1 expression in spinal cord at 4 h and 24 h after injury; (B) Real-time PCR analysis of ZO-1 mRNA expression in spinal cord at 4 and 24 h after injury. Levels are expressed as ratios to sham. Data are presented as mean ± SEM (n = 6/group). **p < 0.01 versus Sham group. ^##p < 0.01 versus I-R group; (C) Representative double-labeling immunofluorescence microscopic photographs show that CD31-positive endothelial cells (red) express ZO-1 (green) at 24 h after injury. Bar scale = 20 μm.

Figure 4

IPC inhibits MMP-9 and TNF-α expression after spinal cord ischemia-reperfusion (I-R) injury. (A) Representative Western blot and quantitative protein analysis of MMP-9 in the spinal cord at 4 and 24 h after injury; (B) Real-time PCR analysis of MMP-9 mRNA expression in the spinal cord at 4 and 24 h after injury; (C) Representative Western blot and quantitative protein analysis of TNF-α in spinal cord at 4 and 24 h after injury. Levels are expressed as ratios to Sham group. Data are presented as mean ± SEM (n = 6/group). **p < 0.01 versus Sham group. ^##p < 0.01 versus I-R group.