The effect of blockade of the vasa vasorum upon cholesterol-induced atherosclerosis in rabbits

의과대학/박사[한글] The Effect of blockade of the Vasa Vasorum upon Cholesterol-induced Atherosclerosis in Rabbits by Chung Ho Huh, M.D, Department of Pathology Yonsei University College of Medicine, Seoul, Korea Directed by Professors: Dong Sik Kim, M.D., Kwang sik Min, M.D. Introduction since the vasa vasorum furnish most of the oxygen and nutrients for the arterial wall, many investigators consider them to have an important role in the genesis of degenerative disease of the arteries (Winternitz et al. 1938, Wartman 1938, 1955). Among the degenerative disease of the arteries, the most serious and important one is atherosclerosis. Atherosclerosis has been produced experimentally by the excessive feeding of cholesterol (Duff 1935, Jobling and Meeker 1936, Duff and McMillan 1951). Hypercholesterolemia is regarded as a prime factor in the development of atherosclerosis. However, previous investigations by the authors (Huh and Kim 1965, and chang 1965), as well as other reports in the literature, strongly indicate that many factors other than increased lipids in the blood also play an important role in atherogenesis (Wartman 1955). These factors include local vascular wall changes and such as degeneration of the media, intimal hyperplasia, local anoxia, and local alteration of hemodynamics, etc. Local injuries of the vascular wall have been induced by various methods; injection of bacterial toxins or chemical agents, irradiation, crushing injuries, freezing and by electric cauterization. Any type of local alteration of the vascular wall enhances the development of atheroma formation. However, the mechanism by which local alterations accelerate atheroma formation has not been elaborated. The present investigation was undertaken to investigate the normal distribution of the vasa vasorum in the aorta of rabbit, effect of the blockade of the vasa vasorum on the normal aortic wall, and finally the influence of the blockade of the vasa vasorum upon cholesterol-induced atherosclerosis in rabbits. Materials and Methods Albino rabbits, around 1.8kg of body weight, were divided into four groups and treated as follows. Group Ⅰ consisted of 5 normal rabbits which were subjected to the demonstration of the aortic vasa vasorum using the lead acetate and potassium dichromate impregnation method of williams (1948) to study the normal distribution of the vasa vasorum. Group Ⅱ of 14 rabbits were subjected to blockade of the aortic vasa vasorum to investigate the effect of the blockade of vasa vasorum on the normal aorta. Group Ⅲ of 5 rabbits were fed excessive amounts of cholesterol to induce atherosclerosis. Group Ⅳ of 20 rabbits were fed an excessive amount of cholesterol after blockade of the vasa vasorum to study the effect of the blockade of vasa vasorum upon cholesterol-induced atherosclerosis. The blockade of vasa vasrum was achieved by stripping off the adventitia of the abdominal aorta between the renal arteries and the iliac bifurcation. Then the stripped segment of the aorta was wrapped with polyethylene tubing to prevent the formation of new vasa vasorum from the surrounding tissue. The operation was performed under ether anesthesia with aseptic precautions, and 0.25Gm. of penstreptomycin was given for 3 days to prevent postoperative infection. All animals were fed with a basic diet of a bean-curd residue, 200 Gms. per day per animal. Cholesterol, mixed with a small amount of been-curd residue, was given in a fasting state (1.5 Gm. per animal per day). Serum total cholesterol determination was made once a month. Animals in group Ⅱ and Ⅳ were killed at set intervals to make serial studies on the effect of blockade of the vasa vasorum after the operation. The longest observation period lasted 80 days. At the necropsy, a section from the heart, kidneys, adrenals, liver, and thyroid was taken, and sections were taken from the ascending, arch, thoracic, abdominal and operative site from the aorta. All sections were embedded in paraffin after fixation with 4% neutral formalin. Microsections were cut at 5-6 μ. thickness. Hematoxylin-esoin, Verhoeff Van Gieson, and colloidal-iron stains were applied to all aortic sections and hematoxylin-eosin stain alone to the sections from other organs. Results and Summary All animals fed with cholesterol (group Ⅲ and Ⅳ) showed marked and rapid elevation of serum cholesterol concentration during the first month after the beginning of the experiment. However, thereafter, continuous feeding of cholesterol raised the serum cholesterol level more slowly and to a lesser degree than during the first month. There was no statistical difference between the serum cholesterol levels in group Ⅲ and Ⅳ. The elevation of serum cholesterol in both groups was mainly due to the increase in the ester fraction. The distribution of the vasa vasorum in the aorta of nomal rabbits was confined to the outer adventitia with short ramifications into the inner adventitia. No case showed penetration of the vasa vasorum into the media. No direct opening from the intima was noted. Blockade of the vasa vasorum caused a complete ischemic necrosis of the entire thickness of the aortic segment and an acute inflammatory reaction followed by calcification. Finally the aortic wall was reestablisbed by a newly formed hyperplastic fibromuscular intima which later resulted in the narrowing of the lumen. After 80 days rabbits fed cholesterol alone showed a relatively mild degree of atheroma formation at the ascending and arch portions of the aorta. The thoracic and abdominal portions of the aorta showed a minimal amount of atheroma formation around the ostia of the arterial branchings from the aorta. However, at all parts of the aorta as well as in the coronary arteries, atheroma formation in the animals fed with cholesterol after the blockade of the vasa vasorum was greater than that of cholesterol fed animals which did not have blockade of the vasa vasorum. Accentuated atheroma formation was noted in the segment of the aorta where the vasa vasorum were blocked. Atheroma at the site of the vasa vasorum blockade showed of proliferative intimal thickening, medial necrosis, medial calcification, and lipid deposition in the thickened intima and degenerated media, simulating the atheroma seen in the human aorta. The enhancement of atheroma formation at the site of vasa vasroum blockade was considered due to the accelerating effect of lipid deposition followed by a fibromuscular proliferation in the intima. Also the blockade of venous vasa vasorum impaired lipid clearance from the aortic wall. The increase of atheroma formation in the remaining areas of the aorta was probably due to increased blood pressure secondary to localized stricture of the aorta at the site at which the vasa vasorum was blocked. [영문] Introduction since the vasa vasorum furnish most of the oxygen and nutrients for the arterial wall, many investigators consider them to have an important role in the genesis of degenerative disease of the arteries (Winternitz et al. 1938, Wartman 1938, 1955). Among the degenerative disease of the arteries, the most serious and important one is atherosclerosis. Atherosclerosis has been produced experimentally by the excessive feeding of cholesterol (Duff 1935, Jobling and Meeker 1936, Duff and McMillan 1951). Hypercholesterolemia is regarded as a prime factor in the development of atherosclerosis. However, previous investigations by the authors (Huh and Kim 1965, and chang 1965), as well as other reports in the literature, strongly indicate that many factors other than increased lipids in the blood also play an important role in atherogenesis (Wartman 1955). These factors include local vascular wall changes and such as degeneration of the media, intimal hyperplasia, local anoxia, and local alteration of hemodynamics, etc. Local injuries of the vascular wall have been induced by various methods; injection of bacterial toxins or chemical agents, irradiation, crushing injuries, freezing and by electric cauterization. Any type of local alteration of the vascular wall enhances the development of atheroma formation. However, the mechanism by which local alterations accelerate atheroma formation has not been elaborated. The present investigation was undertaken to investigate the normal distribution of the vasa vasorum in the aorta of rabbit, effect of the blockade of the vasa vasorum on the normal aortic wall, and finally the influence of the blockade of the vasa vasorum upon cholesterol-induced atherosclerosis in rabbits. Materials and Methods Albino rabbits, around 1.8kg of body weight, were divided into four groups and treated as follows. Group Ⅰ consisted of 5 normal rabbits which were subjected to the demonstration of the aortic vasa vasorum using the lead acetate and potassium dichromate impregnation method of williams (1948) to study the normal distribution of the vasa vasorum. Group Ⅱ of 14 rabbits were subjected to blockade of the aortic vasa vasorum to investigate the effect of the blockade of vasa vasorum on the normal aorta. Group Ⅲ of 5 rabbits were fed excessive amounts of cholesterol to induce atherosclerosis. Group Ⅳ of 20 rabbits were fed an excessive amount of cholesterol after blockade of the vasa vasorum to study the effect of the blockade of vasa vasorum upon cholesterol-induced atherosclerosis. The blockade of vasa vasrum was achieved by stripping off the adventitia of the abdominal aorta between the renal arteries and the iliac bifurcation. Then the stripped segment of the aorta was wrapped with polyethylene tubing to prevent the formation of new vasa vasorum from the surrounding tissue. The operation was performed under ether anesthesia with aseptic precautions, and 0.25Gm. of penstreptomycin was given for 3 days to prevent postoperative infection. All animals were fed with a basic diet of a bean-curd residue, 200 Gms. per day per animal. Cholesterol, mixed with a small amount of been-curd residue, was given in a fasting state (1.5 Gm. per animal per day). Serum total cholesterol determination was made once a month. Animals in group Ⅱ and Ⅳ were killed at set intervals to make serial studies on the effect of blockade of the vasa vasorum after the operation. The longest observation period lasted 80 days. At the necropsy, a section from the heart, kidneys, adrenals, liver, and thyroid was taken, and sections were taken from the ascending, arch, thoracic, abdominal and operative site from the aorta. All sections were embedded in paraffin after fixation with 4% neutral formalin. Microsections were cut at 5-6 μ. thickness. Hematoxylin-esoin, Verhoeff Van Gieson, and colloidal-iron stains were applied to all aortic sections and hematoxylin-eosin stain alone to the sections from other organs. Results and Summary All animals fed with cholesterol (group Ⅲ and Ⅳ) showed marked and rapid elevation of serum cholesterol concentration during the first month after the beginning of the experiment. However, thereafter, continuous feeding of cholesterol raised the serum cholesterol level more slowly and to a lesser degree than during the first month. There was no statistical difference between the serum cholesterol levels in group Ⅲ and Ⅳ. The elevation of serum cholesterol in both groups was mainly due to the increase in the ester fraction. The distribution of the vasa vasorum in the aorta of nomal rabbits was confined to the outer adventitia with short ramifications into the inner adventitia. No case showed penetration of the vasa vasorum into the media. No direct opening from the intima was noted. Blockade of the vasa vasorum caused a complete ischemic necrosis of the entire thickness of the aortic segment and an acute inflammatory reaction followed by calcification. Finally the aortic wall was reestablisbed by a newly formed hyperplastic fibromuscular intima which later resulted in the narrowing of the lumen. After 80 days rabbits fed cholesterol alone showed a relatively mild degree of atheroma formation at the ascending and arch portions of the aorta. The thoracic and abdominal portions of the aorta showed a minimal amount of atheroma formation around the ostia of the arterial branchings from the aorta. However, at all parts of the aorta as well as in the coronary arteries, atheroma formation in the animals fed with cholesterol after the blockade of the vasa vasorum was greater than that of cholesterol fed animals which did not have blockade of the vasa vasorum. Accentuated atheroma formation was noted in the segment of the aorta where the vasa vasorum were blocked. Atheroma at the site of the vasa vasorum blockade showed of proliferative intimal thickening, medial necrosis, medial calcification, and lipid deposition in the thickened intima and degenerated media, simulating the atheroma seen in the human aorta. The enhancement of atheroma formation at the site of vasa vasroum blockade was considered due to the accelerating effect of lipid deposition followed by a fibromuscular proliferation in the intima. Also the blockade of venous vasa vasorum impaired lipid clearance from the aortic wall. The increase of atheroma formation in the remaining areas of the aorta was probably due to increased blood pressure secondary to localized stricture of the aorta at the site at which the vasa vasorum was blocked.restrictio