Syed Faisal Hashmi, The University of Manchester MD Thesis 2016Effects of CO2 insufflation during endoscopic vein harvesting on the structural and functional viability of saphenous vein endothelium: The role of Calcium mobilisation and nitric oxide production.Endoscopic vein harvesting (EVH) has been reported to decrease saphenectomy related wound complications, improved patient satisfaction, shorter hospital stay and reduced postoperative pain compared with open conventional vein harvesting (OCVH) technique. Criticism of this technique centres on the risk of injury at the time of vein harvest with its potential detrimental effect on the structural viability and long term graft patency. Our hypothesis was to investigate the effect of surgical manipulation and carbon dioxide (CO2) insufflation used as an aid in visualisation, on endothelial preservation and functional viability (calcium mobilisation and eNOS related nitric oxide generation) of human saphenous vein graft (HSV).In our histological analysis by haematoxylin and eosin staining, we demonstrated superiority of closed tunnel EVH (CT-EVH) technique over OCVH method. The expression of CD31, eNOS, HSP90 and CaM in immunohistochemical analysis, showed a similar trend among the 3 groups, with preserved expression of all endothelial markers in CT-EVH group. Both, CD31 and CaM were significantly preserved in CT-EVH group, making it the most favourable technique. We have performed an ex vivo experiment by exposing HSV segments harvested by "No touch" technique to 100% CO2 for 40 minutes to investigate the level of cellular damage by Live/Dead assay. CO2 did not cause any immediate structural endothelial damage and there was no statistical difference between control and CO2 groups. We have used Ca2+ (FLUO3 AM) and NO (DAF-FM) specific indicators to investigate calcium mobilisation and eNOS related nitric oxide (NO) production. After CO2 exposure and bradykinin stimulation, Ca2+ mobilisation was significantly attenuated and NO generation was also markedly reduced in endothelial cells. We believe, these results are purely due to CO2 insufflation, which can compromise the entire length of vein graft. However, more research is required to establish, whether these findings are transient in nature or they have long term implications on graft survival and clinical outcome.