Effects of Preoperative Pharmacological Conditioning on the Clinical and Molecular Outcome of Mice after Spinal Cord Ischemia/Reperfusion Sequence

Abstract

Objective: Patients undergoing clamping procedure for thoracoabdominal aortic repair, may suffer postoperatively from the consequences of ischemia/reperfusion (I/R) injury, which can cause paraplegia. Studies show a central role of the endoplasmic reticulum (ER) stress as a result of I/R and the resulting free radicals. Erythropoietin (Epo) and its carbamylated derivate (cEpo-Fc) are able to act as antioxidative and antiapoptotic agents. It was shown that Epo and cEpo-Fc can improve the neurological outcome of animals after spinal cord injury. In this study, we examine the effects of pre-operative use of native erythropoietin (EPO) and its derivative cEPO-Fc on paraplegia and ER stress in vivo. Methods: Ischemia of the spinal cord was induced in male mice (C57BL/6J) by clamping the thoracic aorta and the left subclavian artery. Three study groups (Epo, cEpo-Fc and control) were observed for 96h. The clinical, neurological outcome of the mice was evaluated using the Basso-Mouse-Scale (BMS). The spinal cord was stained in Hematoxylin-Eosin (HE) and Luxol-Fast Blue (LFB). Immunohistochemical staining for ER stress relevant proteins (GRP78 and Caspase 12) was performed as well. Statistical analysis was performed via Kruskal-Wallis-ANOVA and Dunn’s Multiple Comparison Test using SigmaPlot 13. Statistical significance was given at a value of p ≤ 0,05. Results: Animals in the Epo (7,245; +/−3,529) and cEpo-Fc groups (7,171; +/−3,052) show a significantly better clinical, neurological outcome than animals in the control group (1,235; +/−2,457). Hematoxylin-Eosin staining showed less necrotic neurons in the EPO (2,240; +/−0,531) and the cEPO-FC group (2,42 +/−0,870) when compared to control animals (3,790; +/−0,852). LFB-staining showed improved survival of the neurons of the spinal cord as well in the EPO (122,434; +/−82,60) and the cEPO-FC group (143,913; +/−84,225) vs. control group (42,192; +/−40,106). Immunohistochemistry shows a slight increase in protein expression of Caspase 12 in the control group (EPO (20,15; +/−10,451), cEPO-Fc (18,475; +/−9,673), control (26,732; +/−12,914)), while GRP78-Expression was higher in the EPO and cEPO-Fc groups (EPO (16,908; +/−10,761), cEPO-Fc (16,993; +/−9,929), control (13,444; +/−6,900)). Discussion: These results show a significant positive effect of Epo and cEpo-Fc on the clinical neurological and histological outcome of the mice. The increased expression of Caspase12 (control) makes an increased apoptotic rate likely. The increased expression of GRP78 (Epo, cEpo-Fc) might be caused by an increased formation of the UPR. Conclusion: Native Epo and cEpo-Fc can significantly improve the clinical outcome of mice (C57BL/6J). Significant effects are also shown histologically. The molecular mechanisms underlying these effects are being further evaluated.