Experimental Study Of Preventing Intimal Hyperplasia And Stenosis Of Autogenous Vein Graft In Rabbits

Autogenous vein graft has been more and more common in clinical practice since autogenous vein was first used for artery reconstruction by Gluck in 1898. Heretofore autogenous vein graft continues to be the primary method for the reconstruction of the artery defect and coronary artery bypass graft. However, the long-term therapeutic efficacy is always limited by the intimal hyperplasia and stenosis of vein graft. Raja reported that patency rate of vein graft was no more than 50% 10 years after surgery. So it is always a focus of study for the researcher of orthopaedics, vascular surgery and cardiac surgery to prevent intimal hyperplasia and stenosis of vein graft. Intimal hyperplasia is the result of remodeling for vein graft to adapt itself to a charged environment, the pathologic process comprised thrombogenesis, activating of endothelial cell, leukocyte adhesion, Leukocytic infiltrate,smooth muscle cells migration, proliferation and apoptosis, extracellular matrix formation and migration, activating cytokine and signal transducting, it is a complex and continuously changing pathogenesis influenced by multiple factor and interfered by multiple cytokine and gene. Accordingly, it has been being a pressing difficult problem to prevent the occurrence of stenosis of vein graft.It is the critical way of preventing stenosis to inhibit the intimal hyperplasia of vein graft because intimal hyperplasia is the primary and leading pathological change of stenosis of vein graft. For this reason, we designed a project to answer 3 questions: First, to assess the effect of 5-Fluorouracil(5-FU) applied topically on intimal hyperplasia of the vein grafts; Second, to assess the effect of the autologous single-layer and double-layer venous external stents on intimal hyperplasia; Third, to assess the effect of preventing peripheral adhesion of vein graft on intimal hyperplasia. Part 1 Inhibitory effects of topical application of 5-fluorouracil on intimal hyperplasia of the vein grafts in rabbitsThe excessive proliferation and migration of vascular smooth muscle cell are the critical pathogenesis of intimal hyperplasia, so it is a immediate way of preventing intimal hyperplasia and stenosis to inhibit excessive proliferation and migration of vascular smooth muscle cell and to induce apoptosis and to regulate the remodeling of vein graft. 5-Fluorouracil(5-FU) is a antimetabolite belonging to miazines, it can inhibit cell proliferation and induce cell apoptosis, and it is very safe applied locally. Cragg reported that 5-Fluorouracil can effectively inhibit proliferation of vascular smooth muscle cell cultured in vitro. So, external jugular vein grafts was interposed into the carotid artery of rabbits and applied 5-Fluorouracil to test the effect of 5-Fluorouracil in inhibiting proliferation of vascular smooth muscle cell and inducing apoptosis of vascular smooth muscle cell in vivo and regulating the remodeling and preventing stenosis of vein graft.Objective To assess the effect of 5-Fluorouracil(5-FU) applied topically on intimal hyperplasia of the vein grafts in rabbits.Methods Sixty-four male New Zealand white rabbits, aged 5 months and weighing 2.8 to 3.0kg, were randomly divided into 4 groups: Group A, B, C and D (n=16 rabbits per group). Artery defect model was established by cutting about 1 cm artery from the middle part of the dissociated left common carotid artery. A section about 3 cm was cut from the right external jugular vein, and the harvested vein was inverted and end-to-end anastomosed to the artery defect with 9-0 non-traumatic suture. After anastomosis, the extima of the grafted veins in group A, B, and C was completely wrapped with cotton sheet (12mm×30mm×1mm in size) immersed by 5-FU at a concentration of 50.0, 25.0, and 12.5 mg/mL, respectively, and each vein was treated 5 times (1 minute at a time). In group D, the extima of the graft veins was treated with normal sal ine instead of 5-FU. The grafted veins were obtained 1, 2, 4, and 6 weeks after operation, HE staining and Masson staining were preformed for histological changes of grafted vein wall, proliferating cell nuclear antigen (PCNA) immunohistochemistry staining and terminal deoxymudeotityl transferase mediated dUTP nick end labeling (TUNEL) staining were conducted for proliferation and apoptosis of smooth muscle cell of the grafted vein, and transmission electron microscope observation was performed for cellular ultrastructure.Result The HE staining, Masson staining showed that the thickness of intima in group A and B was obviously less than that in group C and D at 1, 2, 4, and 6 weeks after operation. PCNA immunohistochemistry staining and TUNEL labeling staining showed that the proliferation cells in group A and B were less than that in group C and D at 1, 2, and 4 weeks after operation and the apoptotic cells in group A and B were more than that in group C and D at 1 and 2 weeks after operation. The thickness of the intima, the degree of intima hyperplasia, the degree of vessel lumen stenosis of four groups at different time points were as follows: at 1 week after operation, group A [(12.69±1.68)μm, 0.73±0.05, 0.025±0.003], group B [(17.52±2.01)μm, 0.86±0.06, 0.027±0.004], group C [(21.92±1.85)μm, 1.06±0.09, 0.036±0.006] and group D [(26.45±3.86)μm, 1.18±0.08, 0.041±0.005]; at 2 weeks after operation, group A [(24.61±2.91)μm, 0.86±0.06, 0.047±0.003], group B [(37.28±2.78)μm, 1.17±0.09, 0.060±0.004], group C [(46.52±2.25)μm, 1.44±0.08, 0.073±0.003], and group D [(52.07±3.29)μm, 1.45±0.05, 0.081±0.006]; at 4 weeks after operation, group A [(61.09±6.84)μm, 1.38±0.08, 0.106±0.007], group B [(63.61±8.25)μm, 1.40±0.07, 0.107±0.010], group C [(80.04±7.65)μm, 1.64±0.07, 0.129±0.011], and group D [(84.45±9.39)μm, 1.68±0.10, 0.138±0.014]; at 6 weeks after operation, group A [(65.27±5.25)μm, 1.46±0.07, 0.113±0.005], group B [(65.82±7.12)μm, 1.45±0.05, 0.112±0.011], group C [(84.45±6.39)μm, 1.69±0.09, 0.135±0.007], and group D [(87.27±8.96)μm, 1.76±0.05, 0.140±0.012]. Group A and B were inferior to group C and D in terms of the above three parameters 1, 2, 4 and 6weeks after operation (P<0.05). Cell proliferation index and cell apoptosis index of intima and that of media were as follows: at 1 week after operation, group A (4.83±1.96, 30.51±4.01, 14.61±2.17, 9.23±1.40), group B (8.01±2.18, 29.27±5.18, 20.25±3.18, 6.93±1.05), group C (9.09±1.27, 19.21±3.16, 26.80±3.13, 3.89±1.11) and group D (12.25±2.13, 28.71±4.17, 19.01±4.12, 3.83±1.34); at 2 weeks after operation, group A (18.92±3.73, 36.16±2.27, 22.31±3.94, 10.72±2.32), group B (19.16±3.26, 32.41±3.74, 30.51±4.01, 5.13±2.22), group C (25.97±2.17, 29.24±2.11, 37.81±4.50, 5.94±1.79), and group D (27.06±2.91, 27.15±2.51, 42.71±3.60, 5.21±1.91); at 4 weeks after operation, group A (10.17±1.21, 11.73±3.21, 20.00±3.16, 5.19±1.83), group B (11.18±2.01, 11.23±2.15, 21.13±2.62, 4.99±1.41), group C (15.01±2.03, 11.04±2.84, 24.64±2.98, 4.15±2.31), and group D (15.96±2.44, 12.21±3.68, 28.70±2.08, 4.86±1.69); at 6 weeks after operation, group A (6.67±2.10, 5.69±2.31, 2.51±1.53, 3.84±1.41), group B (6.67±1.81, 5.04±1.71, 2.47±1.51, 3.53±1.34), group C (6.88±2.01, 5.99±2.13, 3.40±0.98, 3.74±1.03), and group D (8.21±2.27, 6.01±2.03, 3.28±1.09, 3.64±1.08). Group A and B were inferior to group C and D in terms of cell proliferation index of intima and media 1, 2 and 4 weeks after operation (P<0.05). Group A and B were superior to group C and D in terms of cell apoptosis index of intima and media 1 and 2 weeks after operation (P<0.05). Transmission electron microscope observation showed that the synthetic cell organelles such as rough endoplasmic reticulum, golgi apparatus, and ribosome in group A and B were obviously less than those in group C and D.Conclusion Topical application of 5-FU can inhibits proliferation of vascular smooth muscle cell and induces cell apoptosis of vascular smooth muscle cell, so that it can effectively inhibit intima hyperplasia and stenosis of the vein grafts.Part 2 The effects of the autologous venous external stents on intimal hyperplasia of the vein grafts in rabbitsIn 1963, Parsonne first reported that external stent can inhibit intimal hyperplasia and stenosis of vein graft. After that, many researcher proved that the application of external stent was a promising and effective means of preventing stenosis of vein graft. External stent can counteract the early distension of vein grafted, and it made hemodynamic index of vein graft approaching that of the host artery by the means of prevoking neoadventitia forming and its counteraction, so that the pathologic process induced by the change of hemodynamic index was inhibited. At present the material of most external stent is artificial synthetics, which was likely to evoke inflammation, local infection and adventitial fibrosis of vein graft if implanted. In addition, the mechanical properties of artificial synthetics were far from artery, so this kind of external stents cannot enough inhibit the change of hemodynamic index of vein grafted. Accordingly, the autologous venous external stents, whose mechanical properties were approaching the artery and unlikely to trigger reject reaction, were selected and tested. Single-layer and double-layer venous external stents were applied to vein graft in rabbits to test the effect of autologous venous external stents in counteracting the early distension of veingrafted and preventing intimal hyperplasia and stenosis of vein graft. Objective To assess the effect of the single-layer and double-layer autologous venous external stents on intimal hyperplasia of the vein grafts in rabbits.Methods Thirty-six male New Zealand white rabbits, aged 5 months and weighing 2.8 to 3.0kg, were randomly divided into 3 groups: Group A, B and C, with 12 rabbits in each group. First, a section about 6cm long of vein was cut from the right external jugular vein of each rabbit and severed to have 3 equal-length segments. Next, each distal segment prepared for anastomosis. The proximal segment invaginating middle segment in group A and only middle segment in group B were used for the external stent. Later, the left common carotid artery was separated from surrounding tissue, from it a section about 0.5 cm long was cut away. Finally, the vein graft was inverted and end-to-end anastomosed to the two ends of the artery with a 9-0 suture. After bloodstream re-established, the diameter of each vein graft was measured. At 2, 4 weeks postoperative,the graft veins were cut off and histologically examined by the means of HE staining and Masson staining. The smooth muscle cells(SMC) proliferation was studied by the immunohistochemical detection of proliferating cell nuclear antigen(PCNA). Result After bloodstream re-established, the diameters of vein graft of group A, group B and group C were 1.62±0.31mm, 2.21±0.40mm and 2.65±0.58mm respectively. The diameter of vein graft of group A was smaller than of group B and group C, and that of group B was smaller than group C. (P<0.05). At 2 and 4 weeks after operation, HE staining, Masson staining and PCNA immunohistochemistry staining showed that the thickness of intima in group A and group B was obviously less than that in group C and the proliferation cells of intimal and media in group A and group B were less than that in group C. The thickness of the intima, the degree of intima hyperplasia, the degree of vessel lumen stenosis of 3 groups at different time points were as follows: at 2 week after operation, group A [(35.13±3.24)μm, 0.88±0.03, 0.046±0.004], group B [(44.53±3.55)μm, 1.08±0.02, 0.058±0.005] and group C [(51.62±4.72)μm, 1.21±0.04, 0.068±0.007]; at 4 weeks after operation, group A [(51.73±6.08)μm, 1.01±0.07, 0.076±0.008], group B [(69.29±6.33μm), 1.32±0.08, 0.101±0.009] and group C [(85.00±7.36)μm, 1.55±0.03, 0.134±0.003]. Group A was inferior to group B and C and Group B was inferior to group C in terms of the above three parameters 2 and 4weeks after operation (P<0.05). Cell proliferation index of intima and that of media were as follows: at 2 weeks after operation, group A (16.98±3.96, 28.39±6.02), group B (24.07±4.13, 36.41±6.93) and group C (29.90±4.68, 48.45±5.60); at 4 weeks after operation, group A (6.84±1.98, 15.65±5.12), group B (11.01±2.61, 23.31±4.30) and group C (14.96±4.14, 29.64±4.15). Group A and B were inferior to group C and Group B was inferior to group C in terms of cell proliferation index of intima and media 2 and 4 weeks after operation (P<0.05).Conclusion The autologous venous two-layer external stents inhibit intimal hyperplasia and relieve the stenosis of the vein gratfs effectively. Part 3 The effects of inhibit perivascular adhesion on intimal hyperplasia of the vein grafts in rabbitsThere was apparent adhesion between vein grafted and surrounding tissue. Perivascular adhesion was the way which vasa vasorum enter through to and hyperplasia if to prevent perivascular adhesion and vasa vasorum going in. However, Fogelstrand and Zhang et al reported that vascular smooth muscle cell derived from surrounding tissue played a very important role in intimal hyperplasia, which was proved by animal experiment. Vascular smooth muscle cell derived from surrounding tissue migrated to vein grafted through perivascular adhesion, so it can inhibit intimal hyperplasia to prevent perivascular adhesion. Accordingly, opposite actions on intimal hyperplasia were caused by preventing adhesion. It should be studied which action was dominant and what the effect of preventing adhesion on intimal hyperplasia was. Local application of sodium hyaluronate can prevent adhesion, but also it didn't inflenced intimal hyperplasia of vein graft by biomechanical mechanism because it was liquid. For these reasons, sodium hyaluronate was applied locally to adventitia of vein graft in rabbits to test the effect of sodium hyaluronate on cell proliferation, expression and distribution of PDGF and intimal hyperplasia of vein graft.Objective To assess the effect of sodium hyaluronate applied topically on cell proliferation, expression and distribution of PDGF and intimal hyperplasia of vein graft in rabbits.Methods twenty-four male New Zealand white rabbits, aged 5 months and weighing 2.8 to 3.0kg, were randomly divided into 3 groups: Group A and B (n=12 rabbits per group). Artery defect model was established by cutting about 1 cm artery from the middle part of the dissociated left common carotid artery. A section about 3 cm was cut from the right external jugular vein, and the harvested vein was inverted and end-to-end anastomosed to the artery defect with 9-0 non-traumatic suture. After anastomosis, the adventitia and two anastomosis of the grafted veins in group A was applied 0.2ml sodium hyaluronate locally to. The grafted veins were obtained 1, 2 and 4 weeks after operation, HE staining and Masson staining were preformed for histological changes of grafted vein wall, proliferating cell nuclear antigen (PCNA) and platelet-derived growth factor (PDGF) immunohistochemistry staining were conducted for proliferation and expression and distribution of PDGF of the grafted vein.Result The macroscopic observation showed that there was not apparent adhesion in group B at 1 and 2 weeks and was slight adhesion at 4 weeks after operation in group A and there was apparent adhesion in group B. HE staining, Masson staining showed that the thickness of intima in group A was obviously less than that in group B at 2 and 4 weeks after operation. PCNA and PDGF immunohistochemistry staining showed that the proliferation cells in group A was less than that in group B at 1, 2, and 4 weeks after operation and the positive cells of PDGF immunohistochemistry staining of intimal, media and adventitia in group A was less than that in group B at 1, 2 and 4 weeks after operation. The thickness of the intima, the degree of intima hyperplasia, the degree of vessel lumen stenosis of 2 groups at different time points were as follows: at 1 week after operation, group A [(25.51±3.93)μm, 1.18±0.07, 0.041±0.005] and group B [(26.18±4.16)μm, 1.19±0.09, 0.042±0.006]; at 2 weeks after operation, group A [(44.27±2.53)μm, 1.22±0.06, 0.062±0.003] and group B [(50.99±3.76)μm, 1.40±0.03, 0.078±0.004]; at 4 weeks after operation, group A [(69.85±6.76)μm, 1.49±0.07, 0.114±0.009] and group B [(84.43±6.41)μm, 1.69±0.09, 0.137±0.007]. Group A was inferior to group B in terms of the above three parameters 2 and 4weeks after operation (P<0.05). Cell proliferation index and cell apoptosis index of intima and that of media were as follows: at 1 week after operation, group A (7.43±2.21, 21.53±3.24) and group B (11.41±2.01, 28.63±4.48); at 2 weeks after operation, group A (20.01±3.21, 35.81±3.41) and group B (26.78±4.14, 42.63±4.15); at 4 weeks after operation, group A (11.41±2.01, 22.09±2.65) and group B (15.52±2.37, 28.64±3.90). Group A was inferior to group B in terms of cell proliferation index of intima and media 2 and 4 weeks after operation (P<0.05). The percentage of PDGF-positive cells of intima, media and adventitia was as follows: at 1 week after operation, group A (7.67±1.61, 19.59±3.66, 2.46±1.53) and group B (7.60±2.42, 20.58±4.39, 10.25±2.31); at 2 weeks after operation, group A (11.37±2.55, 19.81±3.09, 12.90±3.26) and group B (19.45±3.48, 30.63±5.16, 30.47±5.84); at 4 weeks after operation, group A (6.15±1.94, 11.09±2.83, 10.19±2.44) and group B (10.51±2.03, 18.64±3.21, 26.51±3.84). Group A was inferior to group B in terms of the percentage of PDGF-positive cells of intima, media and adventitia 2 and 4 weeks after operation (P<0.05) and Group A was inferior to group B that of adventitia 1 week after operation (P<0.05).Conclusion Topical application of sodium hyaluronate prevents perivascular adhesion and inhibits intima hyperplasia. It doesn't promote intima hyperplasia and stenosis of the vein graft to prevent perivascular adhesion.