Pretreatment with Diazoxide Enhances the Neuroprotective Function of Erythropoietin Against Spinal Cord Ischemia and Reperfusion Injury Through Upregulation of Beta Common Receptor
Katsuhiro Yamanaka, Mohamed Eldeiry, Muhammad Aftab, Lisa S. Foley, Joshua Mares, Xianzhong Meng, *Michael J. Weyant, *Joseph C. Cleveland, *David A. Fullerton, *T. Brett Reece
University of Colorado, Aurora, CO
Objective: Paraplegia remains a most terrible complication of thoracoabdominal aortic intervention. While erythropoietin (EPO) has shown the neuroprotective effects in spinal cord ischemia, EPO does not work until the Beta Common Receptor (bcR) subunit of the EPO receptor is induced by ischemia. Diazoxide (DZ) has been shown to mediate the neuroprotective preconditioning effect against ischemic insult. We hypothesized that bcR induced by DZ prior to ischemia amplifies the neuroprotective effects of EPO in mouse with spinal cord ischemia-reperfusion injury.
To determine the optimal time to administer DZ, adult male C57/BL6 received DZ (40 mg/kg) by oral gavage. At time points 0, 12, 24, 36 and 48 hours following the administration of DZ, spinal cords were harvested. The expression of bcR was assesed by western blot analysis (WB). For the optimal dosing, DZ (0, 5, 10, 20, 40 mg/kg) was administered. The expression of bcR was analyzed by WB. To determine the optimal dosage of EPO, one or two daily intraperitoneal injection of EPO (10 or 40 μg/kg) was given 4 hours before surgery. Then spinal cord ischemia was induced by 4-minutes thoracic aortic cross-clamp, and functional scoring (Basso Mouse Score) was assessed after 48 hours reperfusion. Four groups were studied: PBS (pretreatment)+PBS (4 hours before surgery) (n=10), PBS+EPO (n=7), DZ+PBS (n=7), DZ+EPO (n=7), and sham ischemia (operation without crossclamping, n=4). After 4-minutes thoracic aortic cross-clamp, functional scoring was done at 12-hour intervals until 48 hours, and spinal cords were harvested for histological analysis. In these 4 groups, spinal cords were harvested 0, 1, 2, 4 and 6 hours after injection of EPO or PBS, and activation of AKT, STAT3, and CREB were evaluated by WB.
Results:Optimal bcR upregulation occurred 36 hours after administration of DZ. Optimal dosage for bcR induction was 20 mg/kg of DZ. Optimal dosage of EPO was one daily injection of 40 μg/kg. The motor function of DZ+EPO (6.7±1.0) 48 hours after reperfusion was significantly preserved compared to all other groups (Figure), while the DZ+PBS (3.2±0.9) and PBS+EPO (3.4±1.0) groups were significantly better than PBS+PBS (0±0.9) respectively. In DZ+PBS and PBS+EPO group, there were significantly more viable neurons in anterior horn of spinal cord than PBS+PBS. Moreover mice treated with DZ+EPO had significantly more comparing mice treated with DZ+PBS and PBS+EPO. Activation of AKT, STAT3, and CREB in DZ+EPO group peaked 4 hours after injection of EPO. AKT and STAT3 activation in DZ+EPO 4 hours after injection was highest comparing all other groups.
Pharmacological upregulation of bcR with DZ can increase the efficacy of EPO in preventing the spinal cord ischemia and reperfusion injury. Better understanding of this two stage protective mechanism may serve to further prevent ischemic complications for high risk aortic intervention.
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