Morphologic change in rabbit femoral arteries induced by storage at four degrees Celsius and by subsequent reperfusion Daniel Crowe, BSc(Hons), Kevin.

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Morphologic change in rabbit femoral arteries induced by storage at four degrees Celsius and by subsequent reperfusion Daniel Crowe, BSc(Hons), Kevin O'Loughlin, MD, Laura Knox, MD, Geraldine Mitchell, PhD, John Hurley, MD, PhD, Roslind Romeo, Dip.Appl.Sci., Wayne Morrison, MD, BS Journal of Vascular Surgery Volume 22, Issue 6, Pages 769-779 (December 1995) DOI: 10.1016/S0741-5214(95)70068-4 Copyright © 1995 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 1 Summary of morphologic changes to rabbit femoral arteries induced by storage at 4° C and by subsequent 24 hours reperfusion. Longitudinal view of rabbit femoral arteries. A, Normal artery, single layer of EC rest on IEL. Typically 15 to 20 layers of SMC occupy rabbit femoral artery media, although only six layers of SMC are drawn in this diagram. B, Short-term cold storage, In arteries cold-stored for short term (24 hours to 1 week), there is patchy cell necrosis and an increase in media thickness. EC are contracted or lost, and subendothelium is exposed. Pycnotic nuclei and mildly damaged cells (as depicted by fibroblast) are visible in media and adventitia. C, Long-term cold storage, After long period of cold storage (4 weeks to 6 months), all cells are necrotic, but extracellular framework remains intact. Media returns to normal thickness. Myelin figures (as seen replacing fibroblast) are occasionally present. D, Normal artery reperfused for 24 hours, Normal artery grafts are infiltrated with WBC, of which neutrophils and macrophages are mostly present. WBC are visible beneath endothelium, in media and in adventitia. E, Short-term cold-stored artery reperfused for 24 hours, WBC infiltration is similar to normal arteries. Platelets line exposed subendothelium. Thickness of media is significantly reduced. F, Long-term cold-stored artery reperfused for 24 hours, Media width is similar to short-term cold-stored arteries, and WBC infiltration is similar to short-term cold-stored and normal arteries. Extracellular framework is compressed but intact. Journal of Vascular Surgery 1995 22, 769-779DOI: (10.1016/S0741-5214(95)70068-4) Copyright © 1995 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 1 Summary of morphologic changes to rabbit femoral arteries induced by storage at 4° C and by subsequent 24 hours reperfusion. Longitudinal view of rabbit femoral arteries. A, Normal artery, single layer of EC rest on IEL. Typically 15 to 20 layers of SMC occupy rabbit femoral artery media, although only six layers of SMC are drawn in this diagram. B, Short-term cold storage, In arteries cold-stored for short term (24 hours to 1 week), there is patchy cell necrosis and an increase in media thickness. EC are contracted or lost, and subendothelium is exposed. Pycnotic nuclei and mildly damaged cells (as depicted by fibroblast) are visible in media and adventitia. C, Long-term cold storage, After long period of cold storage (4 weeks to 6 months), all cells are necrotic, but extracellular framework remains intact. Media returns to normal thickness. Myelin figures (as seen replacing fibroblast) are occasionally present. D, Normal artery reperfused for 24 hours, Normal artery grafts are infiltrated with WBC, of which neutrophils and macrophages are mostly present. WBC are visible beneath endothelium, in media and in adventitia. E, Short-term cold-stored artery reperfused for 24 hours, WBC infiltration is similar to normal arteries. Platelets line exposed subendothelium. Thickness of media is significantly reduced. F, Long-term cold-stored artery reperfused for 24 hours, Media width is similar to short-term cold-stored arteries, and WBC infiltration is similar to short-term cold-stored and normal arteries. Extracellular framework is compressed but intact. Journal of Vascular Surgery 1995 22, 769-779DOI: (10.1016/S0741-5214(95)70068-4) Copyright © 1995 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 1 Summary of morphologic changes to rabbit femoral arteries induced by storage at 4° C and by subsequent 24 hours reperfusion. Longitudinal view of rabbit femoral arteries. A, Normal artery, single layer of EC rest on IEL. Typically 15 to 20 layers of SMC occupy rabbit femoral artery media, although only six layers of SMC are drawn in this diagram. B, Short-term cold storage, In arteries cold-stored for short term (24 hours to 1 week), there is patchy cell necrosis and an increase in media thickness. EC are contracted or lost, and subendothelium is exposed. Pycnotic nuclei and mildly damaged cells (as depicted by fibroblast) are visible in media and adventitia. C, Long-term cold storage, After long period of cold storage (4 weeks to 6 months), all cells are necrotic, but extracellular framework remains intact. Media returns to normal thickness. Myelin figures (as seen replacing fibroblast) are occasionally present. D, Normal artery reperfused for 24 hours, Normal artery grafts are infiltrated with WBC, of which neutrophils and macrophages are mostly present. WBC are visible beneath endothelium, in media and in adventitia. E, Short-term cold-stored artery reperfused for 24 hours, WBC infiltration is similar to normal arteries. Platelets line exposed subendothelium. Thickness of media is significantly reduced. F, Long-term cold-stored artery reperfused for 24 hours, Media width is similar to short-term cold-stored arteries, and WBC infiltration is similar to short-term cold-stored and normal arteries. Extracellular framework is compressed but intact. Journal of Vascular Surgery 1995 22, 769-779DOI: (10.1016/S0741-5214(95)70068-4) Copyright © 1995 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 1 Summary of morphologic changes to rabbit femoral arteries induced by storage at 4° C and by subsequent 24 hours reperfusion. Longitudinal view of rabbit femoral arteries. A, Normal artery, single layer of EC rest on IEL. Typically 15 to 20 layers of SMC occupy rabbit femoral artery media, although only six layers of SMC are drawn in this diagram. B, Short-term cold storage, In arteries cold-stored for short term (24 hours to 1 week), there is patchy cell necrosis and an increase in media thickness. EC are contracted or lost, and subendothelium is exposed. Pycnotic nuclei and mildly damaged cells (as depicted by fibroblast) are visible in media and adventitia. C, Long-term cold storage, After long period of cold storage (4 weeks to 6 months), all cells are necrotic, but extracellular framework remains intact. Media returns to normal thickness. Myelin figures (as seen replacing fibroblast) are occasionally present. D, Normal artery reperfused for 24 hours, Normal artery grafts are infiltrated with WBC, of which neutrophils and macrophages are mostly present. WBC are visible beneath endothelium, in media and in adventitia. E, Short-term cold-stored artery reperfused for 24 hours, WBC infiltration is similar to normal arteries. Platelets line exposed subendothelium. Thickness of media is significantly reduced. F, Long-term cold-stored artery reperfused for 24 hours, Media width is similar to short-term cold-stored arteries, and WBC infiltration is similar to short-term cold-stored and normal arteries. Extracellular framework is compressed but intact. Journal of Vascular Surgery 1995 22, 769-779DOI: (10.1016/S0741-5214(95)70068-4) Copyright © 1995 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 1 Summary of morphologic changes to rabbit femoral arteries induced by storage at 4° C and by subsequent 24 hours reperfusion. Longitudinal view of rabbit femoral arteries. A, Normal artery, single layer of EC rest on IEL. Typically 15 to 20 layers of SMC occupy rabbit femoral artery media, although only six layers of SMC are drawn in this diagram. B, Short-term cold storage, In arteries cold-stored for short term (24 hours to 1 week), there is patchy cell necrosis and an increase in media thickness. EC are contracted or lost, and subendothelium is exposed. Pycnotic nuclei and mildly damaged cells (as depicted by fibroblast) are visible in media and adventitia. C, Long-term cold storage, After long period of cold storage (4 weeks to 6 months), all cells are necrotic, but extracellular framework remains intact. Media returns to normal thickness. Myelin figures (as seen replacing fibroblast) are occasionally present. D, Normal artery reperfused for 24 hours, Normal artery grafts are infiltrated with WBC, of which neutrophils and macrophages are mostly present. WBC are visible beneath endothelium, in media and in adventitia. E, Short-term cold-stored artery reperfused for 24 hours, WBC infiltration is similar to normal arteries. Platelets line exposed subendothelium. Thickness of media is significantly reduced. F, Long-term cold-stored artery reperfused for 24 hours, Media width is similar to short-term cold-stored arteries, and WBC infiltration is similar to short-term cold-stored and normal arteries. Extracellular framework is compressed but intact. Journal of Vascular Surgery 1995 22, 769-779DOI: (10.1016/S0741-5214(95)70068-4) Copyright © 1995 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 2 Mildly damaged cold-stored rabbit femoral artery. A, Longitudinal section of mid graft of 1-week cold-stored artery. Note karyolytic ECs (*) and spaces (**) in media as result of necrosis of SMCs. 1 indicates lumen; 2 indicates adventitia. (Methylene blue stain; original magnification ~440.) B, SEM of mid graft of 24-hour cold-stored artery. Note general necrosis of most ECs. 1 indicates normal ECs; 2 indicates severely damaged ECs. (Original magnification × 2000.) C, TEM of media in mid graft of 24-hour cold-stored artery. Typical mildly damaged SMCs. Note mildly dilated synthetic organelles (*) and intact plasmalemma (arrow) and myofilaments (**). 1 indicates extracellular framework. (Uranyl acetate and lead citrate staining; original magnification × 4500.) D, TEM of media in mid graft of 24-hour cold-stored artery. Typical mildly damaged SMCs. Note mildly dilated synthetic organelles (*) and intact plasmalemma (arrow), basal lamina (double arrows), and myofilaments (**). (Uranyl acetate and lead citrate staining; original magnification × 40,000.) Journal of Vascular Surgery 1995 22, 769-779DOI: (10.1016/S0741-5214(95)70068-4) Copyright © 1995 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 2 Mildly damaged cold-stored rabbit femoral artery. A, Longitudinal section of mid graft of 1-week cold-stored artery. Note karyolytic ECs (*) and spaces (**) in media as result of necrosis of SMCs. 1 indicates lumen; 2 indicates adventitia. (Methylene blue stain; original magnification ~440.) B, SEM of mid graft of 24-hour cold-stored artery. Note general necrosis of most ECs. 1 indicates normal ECs; 2 indicates severely damaged ECs. (Original magnification × 2000.) C, TEM of media in mid graft of 24-hour cold-stored artery. Typical mildly damaged SMCs. Note mildly dilated synthetic organelles (*) and intact plasmalemma (arrow) and myofilaments (**). 1 indicates extracellular framework. (Uranyl acetate and lead citrate staining; original magnification × 4500.) D, TEM of media in mid graft of 24-hour cold-stored artery. Typical mildly damaged SMCs. Note mildly dilated synthetic organelles (*) and intact plasmalemma (arrow), basal lamina (double arrows), and myofilaments (**). (Uranyl acetate and lead citrate staining; original magnification × 40,000.) Journal of Vascular Surgery 1995 22, 769-779DOI: (10.1016/S0741-5214(95)70068-4) Copyright © 1995 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 2 Mildly damaged cold-stored rabbit femoral artery. A, Longitudinal section of mid graft of 1-week cold-stored artery. Note karyolytic ECs (*) and spaces (**) in media as result of necrosis of SMCs. 1 indicates lumen; 2 indicates adventitia. (Methylene blue stain; original magnification ~440.) B, SEM of mid graft of 24-hour cold-stored artery. Note general necrosis of most ECs. 1 indicates normal ECs; 2 indicates severely damaged ECs. (Original magnification × 2000.) C, TEM of media in mid graft of 24-hour cold-stored artery. Typical mildly damaged SMCs. Note mildly dilated synthetic organelles (*) and intact plasmalemma (arrow) and myofilaments (**). 1 indicates extracellular framework. (Uranyl acetate and lead citrate staining; original magnification × 4500.) D, TEM of media in mid graft of 24-hour cold-stored artery. Typical mildly damaged SMCs. Note mildly dilated synthetic organelles (*) and intact plasmalemma (arrow), basal lamina (double arrows), and myofilaments (**). (Uranyl acetate and lead citrate staining; original magnification × 40,000.) Journal of Vascular Surgery 1995 22, 769-779DOI: (10.1016/S0741-5214(95)70068-4) Copyright © 1995 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 3 Severely damaged cold-stored rabbit femoral artery. A, Longitudinal section of mid graft of 6-month cold-stored artery. Note total karyolytic ECs (*) and pycnotic SMCs (**). 1 indicates lumen; arrow indicates IEL; double arrows indicate EEL. (Methylene blue staining; original magnification × 460.) B, SEM of mid graft of 1-week cold-stored artery. Note loss of most ECs and exposed IEL. 1 indicates exposed IEL; 2 indicates severely damaged ECs. (Original magnification × 2500.) C, TEM of media in mid graft of 1-week cold-stored artery. Typical severely damaged SMCs. Note grossly dilated synthetic organelles (*), ruptured nucleus with myelin figure (arrow), and loose myofilaments (double arrows). (Uranyl acetate and lead citrate staining; original magnification × 5400.) D, TEM of EC in mid graft of 1-week cold-stored artery. Unusual severely damaged cell. Note that despite lack of plasmalemma, there is necrotic nucleus (1), mitochondria with intact outer membranes (*) and intact extracellular framework (2). (Uranyl acetate and lead citrate staining; original magnification × 23,500.) Journal of Vascular Surgery 1995 22, 769-779DOI: (10.1016/S0741-5214(95)70068-4) Copyright © 1995 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 3 Severely damaged cold-stored rabbit femoral artery. A, Longitudinal section of mid graft of 6-month cold-stored artery. Note total karyolytic ECs (*) and pycnotic SMCs (**). 1 indicates lumen; arrow indicates IEL; double arrows indicate EEL. (Methylene blue staining; original magnification × 460.) B, SEM of mid graft of 1-week cold-stored artery. Note loss of most ECs and exposed IEL. 1 indicates exposed IEL; 2 indicates severely damaged ECs. (Original magnification × 2500.) C, TEM of media in mid graft of 1-week cold-stored artery. Typical severely damaged SMCs. Note grossly dilated synthetic organelles (*), ruptured nucleus with myelin figure (arrow), and loose myofilaments (double arrows). (Uranyl acetate and lead citrate staining; original magnification × 5400.) D, TEM of EC in mid graft of 1-week cold-stored artery. Unusual severely damaged cell. Note that despite lack of plasmalemma, there is necrotic nucleus (1), mitochondria with intact outer membranes (*) and intact extracellular framework (2). (Uranyl acetate and lead citrate staining; original magnification × 23,500.) Journal of Vascular Surgery 1995 22, 769-779DOI: (10.1016/S0741-5214(95)70068-4) Copyright © 1995 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 3 Severely damaged cold-stored rabbit femoral artery. A, Longitudinal section of mid graft of 6-month cold-stored artery. Note total karyolytic ECs (*) and pycnotic SMCs (**). 1 indicates lumen; arrow indicates IEL; double arrows indicate EEL. (Methylene blue staining; original magnification × 460.) B, SEM of mid graft of 1-week cold-stored artery. Note loss of most ECs and exposed IEL. 1 indicates exposed IEL; 2 indicates severely damaged ECs. (Original magnification × 2500.) C, TEM of media in mid graft of 1-week cold-stored artery. Typical severely damaged SMCs. Note grossly dilated synthetic organelles (*), ruptured nucleus with myelin figure (arrow), and loose myofilaments (double arrows). (Uranyl acetate and lead citrate staining; original magnification × 5400.) D, TEM of EC in mid graft of 1-week cold-stored artery. Unusual severely damaged cell. Note that despite lack of plasmalemma, there is necrotic nucleus (1), mitochondria with intact outer membranes (*) and intact extracellular framework (2). (Uranyl acetate and lead citrate staining; original magnification × 23,500.) Journal of Vascular Surgery 1995 22, 769-779DOI: (10.1016/S0741-5214(95)70068-4) Copyright © 1995 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 4 Severely damaged cold-stored rabbit femoral artery after 24 hours reperfusion in contralateral femoral artery. A, Longitudinal section of mid graft of 4-week cold-stored artery. Note total loss of endothelium and karyolytic SMCs (arrow). Neutrophils (double arrows) lined luminal surface and infiltrate into media. 1 indicates IEL; 2 indicates EEL. (Methylene blue staining; original magnification × 380.) B, SEM of mid graft of 6-month cold-stored artery. Note total loss of endothelium. Exposed IEL is covered by platelets and WBCs. 1 indicates WBCs; 2 indicates platelets. (Original magnification × 4100.) C, TEM of media in mid graft of 1-week cold-stored artery. Typical severely damaged SMCs (1) with WBC infiltration (2) and an intact extracellular framework. (Uranyl acetate and lead citrate staining; original magnification × 4200.) Journal of Vascular Surgery 1995 22, 769-779DOI: (10.1016/S0741-5214(95)70068-4) Copyright © 1995 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 4 Severely damaged cold-stored rabbit femoral artery after 24 hours reperfusion in contralateral femoral artery. A, Longitudinal section of mid graft of 4-week cold-stored artery. Note total loss of endothelium and karyolytic SMCs (arrow). Neutrophils (double arrows) lined luminal surface and infiltrate into media. 1 indicates IEL; 2 indicates EEL. (Methylene blue staining; original magnification × 380.) B, SEM of mid graft of 6-month cold-stored artery. Note total loss of endothelium. Exposed IEL is covered by platelets and WBCs. 1 indicates WBCs; 2 indicates platelets. (Original magnification × 4100.) C, TEM of media in mid graft of 1-week cold-stored artery. Typical severely damaged SMCs (1) with WBC infiltration (2) and an intact extracellular framework. (Uranyl acetate and lead citrate staining; original magnification × 4200.) Journal of Vascular Surgery 1995 22, 769-779DOI: (10.1016/S0741-5214(95)70068-4) Copyright © 1995 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions