The Effects Of Denervation On The Collateral Vessel Growth And The Possible Mechanisms

Objective:To make investigations into the possible mechanism and the functions of denervation on collateral vessel growth induced by ligature of femoral artery in rat/rabbit hind limb, enriching and perfecting the theories of collateral vessel growth mechanism, and searching for a novel way of promoting the collateral vessel growth for the therapy of ischemia disease.Methods:Animal Models:Fifty-two adult Sprague-Dawley (SD) rats and eight adult New Zealand white rabbits. The right legs of the rats/rabbits were first denervated by sectioning the sciatic nerve high in the thigh, after a7-day survival, the animals were re-anesthetized, acute bilateral femoral artery ligature was performed with two knots. For the right leg, the femoral nerve and obturator nerve were sectioned. In addition, the branches from the proximal femoral artery were carefully exposed and topically painted with10%phenol solution using a cotton bud. The simply ligated contralateral side was used as control, building up the model of collateral vessel growth induced by femoral artery ligature plus sciatic and femoral nerve incise. The simply denervation group is the same with the above except ligature;Experimental methods and Testing Index:Part Ⅰ:To study the effect of denervation on the collateral vessel growth induced by ligature of femoral artery in rat hind limb. With thirty-two animals' survival for1and8weeks respectively (The stages for active growth and maturation respectively) and by post-mortem angiography, histology and confocal immunofluorescence, the effect of denervation on collateral vessel growth was observed.Part Ⅱ:To study the effect of denervation on the activation of collateral vessel growth induced by ligature of femoral artery in rat/rabbit hind limb. On the basis of successfully building up the model of collateral vessel growth induced by femoral artery ligature plus sciatic and femoral nerve incise, The expression of the following molecules which are important for collateral vessel growth were detected in collateral vessels by confocal immunofluorescence, intercellular adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM), monocyte chemoattractant protrin-1(MCP-1), the marker of macrophages(CD11b), the marker of cell proliferation (Ki67), the marker of fibroblasts in the adventitia and an indicator of dedifferentiated phenotype in SMC (Vimentin), matrix metalloproteinase-2(MMP-2) and endothelial nitric oxide synthase (eNOS)Part III:To investigate the possible mechanism for innervation on the collateral vessel growth, the involvement of secretoneurin (SN) in collateral vessel growth was studied in both vitro and vivo:①Animal Models:To observe the expression of SN during the collateral vessel growth; To investigate the role of SN in promoting collateral vessel growth, A dose of10μg/d SN local injection in rat hindlimb gracilis of femoral artery ligated rats was carried out, After a7-day's survival, cell proliferation and infiltration of inflammatory cells wre detected by confocal immunofluorescence.②Cell culture:since VSMC is the main component of collateral vessel, and the fact that VSMC in the collateral vessel close to adventitia express VCAM and MCP-1, the cultures of rat aortic smooth muscle cells were employed, cells from passage five or six were used in the experiments. After identification with specific α-SM actin antibodies, the cells were divided into following groups:normal control,1ng/ml SN, lOng/ml SN and100ng/ml SN group. The effect of SN on VSMC proliferation and expression of MCP-1and VCAM-1was assessed with MTT, immunoblotting and immunocytochemistry respectively.Results:Part I:In only femoral artery ligature side, intensive arterial collaterals were noticed in angiograph, histology and confocal immunofluorescence demonstrated remodeling process occurred in all three layers of the vascular wall. The collateral vessel growth was characterized by outward growth showing a larger lumen and a relatively thin wall. Furthermore, the sleletal muscles were well-preserved. In the ligated with denervation side, the number of collateral vessels decreased compared to ligature side. The collateral vessels growth was characterized by inward growth showing a narrowing lumen and a much thicker wall. Moreover, degeneration and loss of sleletal muscles were apparent.Part II:After dennervation, there was no significant difference in expression of ICAM and VCAM in endothelial cells, but epressions of VCAM and MCP-1was obviously decreased in the adventitia and tunica media close to adventitia. The number of CD11b decreased in the vascular wall. The expression of vimentin and MMP-2in the adventitia was lower as compared to only femoral artery occluded side, but the expression of vimentin in the tunica media and eNOS in the endothelium had no significant change.Part Ⅲ:①SN-immunoreactive nerve fibers were mainly distributed in the adventitia of the collateral vessel in only ligated side, SN positive staining was decreased and disappeared after denervation. The number of Ki67and CDllb positive cells was significantlly increased after SN injection.②MTT assay showed that SN augmented VSMC proliferation obviously.Immunoblotting and immuocytochemistry showed the SN stimulated VSMC to express VCAM and MCP-1Conclusion:①Collateral vessel growth induced by femoral artery ligation in rat/rabbit hind limb is impaired by denervation is characterized by showing a thicker wall and narrowing lumen.②Denervation resulted in loss of SN in the adventitia and insufficient activation of the adventitia including lower level of VCAM, MCP-1and MMP-2, and less number of macrophages and fibroblasts.③The neural regulation is an important factor that contributes to collateral vessels growth by releasing neuropeptide. SN, on one hand, could act as an inflammation mediator to regulate collateral vessel growth, on the other, it could directly stimulate cell proliferation of the vascular wall to promote the collateral vessel growth.