Activation of SK/KCa Channel Attenuates Spinal Cord Ischemia-Reperfusion Injury via Anti-oxidative Activity and Inhibition of Mitochondrial Dysfunction in Rabbits

Abstract

Spinal cord ischemia-reperfusion injury (SCI/R) is a rare but devastating disorder with a poor prognosis. Small conductance calcium-activated K⁺ (SK/K_Ca) channels are a family of voltage-independent potassium channels that are shown to participate in the pathological process of several neurological disorders. The aim of this study was to investigate the role of SK/K_Ca channels in experimental SCI/R in rabbits. The expression of SK/K_Ca1 protein significantly decreased in both cytoplasm and mitochondria in spinal cord tissues after SCI/R. Treatment with 2 mg/kg NS309, a pharmacological activator for SK/K_Ca channel, attenuated SCI/R-induced neuronal loss, spinal cord edema and neurological dysfunction. These effects were still observed when the administration was delayed by 6 h after SCI/R initiation. NS309 decreased the levels of oxidative products and promoted activities of antioxidant enzymes in both serum and spinal cord tissues. The results of ELISA assay showed that NS309 markedly decreased levels of pro-inflammatory cytokines while increased anti-inflammatory cytokines levels after SCI/R. In addition, treatment with NS309 was shown to preserve mitochondrial respiratory complexes activities and enhance mitochondrial biogenesis. The results of western blot analysis showed that NS309 differentially regulated the expression of mitochondrial dynamic proteins. In summary, our results demonstrated that the SK/K_Ca channel activator NS309 protects against SCI/R via anti-oxidative activity and inhibition of mitochondrial dysfunction, indicating a therapeutic potential of NS309 for SCI/R.

Keywords: NS309; SK/KCa channels; mitochondrial dynamic; mitochondrial dysfunction; spinal cord ischemia and reperfusion.

FIGURE 1

SCI/R decreases the expression of SK/K_Ca1 subtypes. The animals were exposed to spinal I/R, and the expression of SK/K_Ca1 protein in spinal cord tissues was detected by western blot analysis at 3, 6, 12, or 24 h after reperfusion (A). Immunoblot analysis of whole-cell extract (depicted as “Cell”), cytosol supernatant (depicted as “Cyto”) or crude mitochondrial pellets (depicted as “Mito”) were also performed (B), and calculated (C) to determine subcellular expression of SK/K_Ca1 protein. Data are shown as mean ± SEM (n = 8). ^∗p < 0.05 vs. Sham.

FIGURE 2

The SK/K_Ca channel activator NS309 protects against SCI/R. The animals were treated with 2 mg/kg NS309 or vehicle at the beginning of SCI/R operation. Hematoxylin and eosin staining was performed in the ventral horn of L4 spinal cord segment at 72 h after reperfusion (A), and the number of normal motor neurons was countered (B). The spinal cord edema was assayed by measuring spinal cord water content at 72 h (C). Neurological function scores were assessed at 72 h after reperfusion by an independent observer (D). Arrowheads indicate normal neurons. The data were represented as means ± SEM (n = 8). ^∗p < 0.05 vs. Vehicle.

FIGURE 3

Time window of NS309-induced neuroprotection. The animals were exposed to SCI/R and treated with 2 mg/kg NS309 at different time points (3, 6, or 9 h after SCI/R beginning). The spinal cord water content (A), number of normal motor neurons (B) and neurological function scores (C) were measured 72 h later. The data were represented as means ± SEM (n = 8). ^∗p < 0.05 vs. Vehicle.

FIGURE 4

NS309 reduces the expression of oxidative products after SCI/R. The animals were treated with 2 mg/kg NS309 or vehicle at the beginning of SCI/R. The expression levels of MDA (A) and 8-iso-PGF2α (B) in serum were measured at different time points (0, 3, 6, 12, 24, and 48 h). The expression levels of MDA (C) and 8-iso-PGF2α (D) in spinal cord tissues were detected at 72 h after reperfusion. The data were represented as means ± SEM (n = 8). ^∗p < 0.05 vs. Sham. ^#p < 0.05 vs. Vehicle.

FIGURE 5

NS309 preserves the activity of antioxidant enzymes after SCI/R. The animals were treated with 2 mg/kg NS309 or vehicle at the beginning of SCI/R. The enzymatic activities of SOD (A) and CAT (B) in serum were measured at different time points (0, 3, 6, 12, 24, and 48 h). The enzymatic activities of SOD (C) and CAT (D) in spinal cord tissues were detected at 72 h after reperfusion. The data were represented as means ± SEM (n = 8). ^∗p < 0.05 vs. Sham. ^#p < 0.05 vs. Vehicle.

FIGURE 6

NS309 regulates inflammatory cytokines after SCI/R. The animals were treated with 2 mg/kg NS309 or vehicle at the beginning of SCI/R. The levels of TNF-α (A), IL-1β (B), IL-10 (C), and TGF-β1 (D) in spinal cord tissues were determined at different time points (0, 12, 24, and 48 h). The data were represented as means ± SEM (n = 8). ^∗p < 0.05 vs. Sham. ^#p < 0.05 vs. Vehicle.

FIGURE 7

NS309 preserves mitochondrial complex activity and mitochondrial biogenesis after SCI/R. The animals were treated with 2 mg/kg NS309 or vehicle at the beginning of SCI/R. The activities of mitochondrial complex I (A), II (B), III (C), and IV (D) were observed in spinal cord, respectively. Real-time RT-PCR was used to detect mtNDA ratio (E) and the expression of mitochondrial biogenesis factors (F). The data were represented as means ± SEM (n = 8). ^∗p < 0.05 vs. Sham. ^#p < 0.05 vs. Vehicle.

FIGURE 8

NS309 differently regulates the expression of mitochondrial dynamic proteins. The animals were treated with 2 mg/kg NS309, and the expression of Opa-1, Mfn-1, Drp-1, and Fis-1 at different time points (0, 3, 6, 12, and 24 h) was detected by western blot analysis (A,B). The animals were treated with 2 mg/kg NS309 or vehicle at the beginning of SCI/R. The expression of Opa-1, Mfn-1, Drp-1, and Fis-1 was detected by western blot analysis (C) and calculated (D). The data were represented as means ± SEM (n = 8). ^∗p < 0.05 vs. Sham. ^#p < 0.05 vs. Vehicle.