Microscopic and ultrastructural evaluation of the saphenous vein endothelium for CABG prepared by the no touch technique.

OBJECTIVE: Saphenous vein grafts (SV) used in coronary artery bypass grafting have a limited life and vein occlusion may be the final adverse effect. Efforts to develop new techniques to harvest the saphenous vein may improve the viability of the graft. METHODS: Twenty patients were randomly divided into two groups with the objective of evaluating the vascular endothelium. The No Touch (NT) technique consists in removing the saphenous vein with perivascular tissue. The conventional technique consists in harvesting with "in situ" removal of the perivascular tissue. The standard saphenous vein harvesting procedure used bridged incisions. Characteristics of the vein were considered. Evaluation of the endothelium was achieved by electron microscopy and histologic analysis using hematoxylin eosin staining. The Picrosirius and Masson Trichrome methods were used to analyze subendothelial collagen. RESULTS: Electron microscopy demonstrated that the NT Group had larger non-denudated endothelial areas as well as a smaller number of degraded cells. Histological analysis showed the form and integrity of the saphenous vein layers. A larger amount of collagen fibers were identified in the NT Group. CONCLUSIONS: The NT technique better preserves the saphenous vein endothelium suggesting a more viable graft in the long term.

Glycol methacrylate-embedding medium to study morphological alterations of saphenous vein under brief and crescent pressurizations.

PURPOSE: This study sought to evaluate the efficiency of glycol methacrylate-embedding medium to detect morphological alterations of human saphenous vein submitted to brief and crescent pressurizations. METHODS: Saphenous veins of 20 CABG patients were randomly distributed into four experimental groups (control, 100, 200 and 300 mmHg pressures during 15 seconds). To quantify the percentage of endothelium spread over vein surface a microscope magnification of 100x was used for measurements. Morphometric analysis was performed using videomicroscopy with the Leica Qwin software in conjunction with a Leica microscope, videocamera, and an on-line computer. RESULTS: A slight tendency of quantitative increase was observed in all parameters including percentage of endothelium spread over vein surface and thickness of saphenous vein walls (intima and media layers). CONCLUSIONS: The glycol methacrylate-embedding allowed sections with adequate resolution of structural details and revealed to be an extremely useful method to study pressurized human saphenous veins.

Human saphenous vein organ culture under controlled hemodynamic conditions.

INTRODUCTION: Saphenous vein grafting is still widely used to revascularize ischemic myocardium. The effectiveness of this procedure is limited by neointima formation and accelerated atherosclerosis, which frequently leads to graft occlusion. A better understanding of this process is important to clarify the mechanisms of vein graft disease and to aid in the formulation of strategies for prevention and/or therapeutics. OBJECTIVE: To develop an ex vivo flow system that allows for controlled hemodynamics in order to mimic arterial and venous conditions. METHODS: Human saphenous veins were cultured either under venous (flow: 5 ml/min) or arterial hemodynamic conditions (flow: 50 ml/min, pressure: 80 mmHg) for 1-, 2- and 4-day periods. Cell viability, cell density and apoptosis were compared before and after these intervals using MTT, Hoeschst 33258 stain, and TUNEL assays, respectively. RESULTS: Fresh excised tissue segments were well preserved prior to the study. Hoechst 33258 and MTT stains showed progressive losses in cell density and cell viability in veins cultured under arterial hemodynamic conditions from 1 to 4 days, while no alterations were observed in veins cultured under venous conditions. Although the cell density from 1-day cultured veins under arterial conditions was similar to that of freshly excised veins, the TUNEL assay indicated that most of these cells were undergoing apoptosis. CONCLUSION: The results observed resemble the events taking place during early in vivo arterial-vein grafting and provide evidence that an ex vivo perfusion system may be useful for the identification of new therapeutic targets that ameliorate vein graft remodeling and increase graft patency over time.

Avaliação microscópica e ultra-estrutural do endotélio de veia safena preparada pela técnica "no touch" / Microscopic and ultrastructural evaluation of the saphenous vein endothelium for CABG prepared by the no touch technique

OBJETIVO: O enxerto de veia safena (VS) utilizado em revascularização miocárdica possui uma vida útil, sendo o estágio final a oclusão do vaso. Esforços em adquirir novas técnicas de coleta da VS podem possibilitar uma viabilidade maior do enxerto. MÉTODOS: Vinte pacientes foram randomizados e divididos em dois grupos com o objetivo de avaliação do endotélio vascular. A técnica "no touch" (NT) consiste em retirar o segmento de VS com o tecido perivascular. A técnica convencional consiste em retirar a VS, com remoção "in situ" do tecido perivascular e conseqüente vasoespasmo. Houve um padrão de retirada das VS com incisões longitudinais escalonadas. Características da VS foram consideradas. A avaliação do endotélio das VS foi realizada usando microscópio eletrônico (ME) pelo método de varredura e de transmissão. Cortes histológicos das VS foram corados em Hematoxilina-Eosina (HE). O colágeno subendotelial foi analisado pelos métodos de Picro-Sirius e Tricrômio de Masson. RESULTADOS: A ME evidenciou que o Grupo NT possui maiores áreas endoteliais não desnudadas, além de um menor número de células degradadas. A coloração em HE nos permitiu verificar a forma e a integridade das camadas das VS. Há um predomínio maior de fibras colágenas coradas no Grupo NT. CONCLUSÕES: A técnica NT permite uma melhor preservação endotelial da VS, sugerindo um enxerto mais viável em longo prazo.

OBJECTIVE: Saphenous vein grafts (SV) used in coronary artery bypass grafting have a limited life and vein occlusion may be the final adverse effect. Efforts to develop new techniques to harvest the saphenous vein may improve the viability of the graft. METHODS: Twenty patients were randomly divided into two groups with the objective of evaluating the vascular endothelium. The No Touch (NT) technique consists in removing the saphenous vein with perivascular tissue. The conventional technique consists in harvesting with "in situ" removal of the perivascular tissue. The standard saphenous vein harvesting procedure used bridged incisions. Characteristics of the vein were considered. Evaluation of the endothelium was achieved by electron microscopy and histologic analysis using hematoxylin eosin staining. The Picrosirius and Masson Trichrome methods were used to analyze subendothelial collagen. RESULTS: Electron microscopy demonstrated that the NT Group had larger non-denudated endothelial areas as well as a smaller number of degraded cells. Histological analysis showed the form and integrity of the saphenous vein layers. A larger amount of collagen fibers were identified in the NT Group. CONCLUSIONS: The NT technique better preserves the saphenous vein endothelium suggesting a more viable graft in the long term.

Glycol methacrylate-embedding medium to study morphological alterations of saphenous vein under brief and crescent pressurizations / Inclusão em glicol metacrilato para estudar alterações morfológicas de veias safenas humanas submetidas a pressurizações breves e crescentes

PURPOSE: This study sought to evaluate the efficiency of glycol methacrylate-embedding medium to detect morphological alterations of human saphenous vein submitted to brief and crescent pressurizations. METHODS: Saphenous veins of 20 CABG patients were randomly distributed into four experimental groups (control, 100, 200 and 300 mmHg pressures during 15 seconds). To quantify the percentage of endothelium spread over vein surface a microscope magnification of 100x was used for measurements. Morphometric analysis was performed using videomicroscopy with the Leica Qwin software in conjunction with a Leica microscope, videocamera, and an on-line computer. RESULTS: A slight tendency of quantitative increase was observed in all parameters including percentage of endothelium spread over vein surface and thickness of saphenous vein walls (intima and media layers). CONCLUSIONS: The glycol methacrylate-embedding allowed sections with adequate resolution of structural details and revealed to be an extremely useful method to study pressurized human saphenous veins.(AU)

OBJETIVO: Avaliar a inclusão em glicol metacrilato para estudar alterações morfológicas de veias safenas humanas submetidas a pressurizações breves e crescentes. MÉTODOS: Veias safena de 20 pacientes submetidos a cirurgia de revascularização do miocárdio foram distribuídas ao acaso em quatro grupos experimentais (controle, pressões de 100, 200 e 300 mmHg durante 15 segundos). Para quantificar a percentagem da superfície venosa recoberta por endotélio utilizou-se o aumento de 100x. A análise morfométrica foi realizada utilizando-se videomicroscopia com auxílio do software Leica Qwin em conjunto com um microscópio Leica e videocâmera, acoplados a um computador. RESULTADOS: Observou-se uma leve tendência de aumento quantitativo de todos os parâmetros avaliados, incluindo a percentagem de superfície recoberta por endotélio e a espessura das paredes das veias safenas. CONCLUSÕES: A inclusão em glicol metracrilato permitiu secções com adequada resolução dos detalhes estruturais, revelando-se um método extremamente útil para o estudo de veias safenas humanas pressurizadas.(AU)

Human saphenous vein organ culture under controlled hemodynamic conditions

INTRODUCTION: Saphenous vein grafting is still widely used to revascularize ischemic myocardium. The effectiveness of this procedure is limited by neointima formation and accelerated atherosclerosis, which frequently leads to graft occlusion. A better understanding of this process is important to clarify the mechanisms of vein graft disease and to aid in the formulation of strategies for prevention and/or therapeutics. OBJECTIVE: To develop an ex vivo flow system that allows for controlled hemodynamics in order to mimic arterial and venous conditions. METHODS: Human saphenous veins were cultured either under venous (flow: 5 ml/min) or arterial hemodynamic conditions (flow: 50 ml/min, pressure: 80 mmHg) for 1-, 2- and 4-day periods. Cell viability, cell density and apoptosis were compared before and after these intervals using MTT, Hoeschst 33258 stain, and TUNEL assays, respectively. RESULTS: Fresh excised tissue segments were well preserved prior to the study. Hoechst 33258 and MTT stains showed progressive losses in cell density and cell viability in veins cultured under arterial hemodynamic conditions from 1 to 4 days, while no alterations were observed in veins cultured under venous conditions. Although the cell density from 1-day cultured veins under arterial conditions was similar to that of freshly excised veins, the TUNEL assay indicated that most of these cells were undergoing apoptosis. CONCLUSION: The results observed resemble the events taking place during early in vivo arterial-vein grafting and provide evidence that an ex vivo perfusion system may be useful for the identification of new therapeutic targets that ameliorate vein graft remodeling and increase graft patency over time.

Glycol methacrylate-embedding medium to study morphological alterations of saphenous vein under brief and crescent pressurizations / Inclusão em glicol metacrilato para estudar alterações morfológicas de veias safenas humanas submetidas a pressurizações breves e crescentes

PURPOSE: This study sought to evaluate the efficiency of glycol methacrylate-embedding medium to detect morphological alterations of human saphenous vein submitted to brief and crescent pressurizations. METHODS: Saphenous veins of 20 CABG patients were randomly distributed into four experimental groups (control, 100, 200 and 300 mmHg pressures during 15 seconds). To quantify the percentage of endothelium spread over vein surface a microscope magnification of 100x was used for measurements. Morphometric analysis was performed using videomicroscopy with the Leica Qwin software in conjunction with a Leica microscope, videocamera, and an on-line computer. RESULTS: A slight tendency of quantitative increase was observed in all parameters including percentage of endothelium spread over vein surface and thickness of saphenous vein walls (intima and media layers). CONCLUSIONS: The glycol methacrylate-embedding allowed sections with adequate resolution of structural details and revealed to be an extremely useful method to study pressurized human saphenous veins.

OBJETIVO: Avaliar a inclusão em glicol metacrilato para estudar alterações morfológicas de veias safenas humanas submetidas a pressurizações breves e crescentes. MÉTODOS: Veias safena de 20 pacientes submetidos a cirurgia de revascularização do miocárdio foram distribuídas ao acaso em quatro grupos experimentais (controle, pressões de 100, 200 e 300 mmHg durante 15 segundos). Para quantificar a percentagem da superfície venosa recoberta por endotélio utilizou-se o aumento de 100x. A análise morfométrica foi realizada utilizando-se videomicroscopia com auxílio do software Leica Qwin em conjunto com um microscópio Leica e videocâmera, acoplados a um computador. RESULTADOS: Observou-se uma leve tendência de aumento quantitativo de todos os parâmetros avaliados, incluindo a percentagem de superfície recoberta por endotélio e a espessura das paredes das veias safenas. CONCLUSÕES: A inclusão em glicol metracrilato permitiu secções com adequada resolução dos detalhes estruturais, revelando-se um método extremamente útil para o estudo de veias safenas humanas pressurizadas.

Elastic fibers in saphenous varicose veins.

The purpose of this study is to describe the elastic fibers of varicose collateral saphenous veins. Sections were obtained from venous segments of patients with essential varices and stained with resorcin-fuchsin for elastic system fibers and analyzed with laser scanning microscopy after Evans blue staining. Vein portions (270 microm) were classified as without thickening, with a cushion, or with a diffuse thickening. The elastic material density of the intima (Dei) and media (Dem) were tested for differences by the Kruskal-Wallis test. Diffuse thickening (87.1+/-8.6 microm) represents 54.5% of the segment. Cushion occupies 23.5% with 42.4+/-4.66 microm. The elastic network present in the cushion is formed by elastic fibers of different diameters that branch into delicate oxytalan fibers in association with the smooth muscle cells. The diffuse thickening elastic network varies from elastic lamellae and delicate oxytalan fibers related to the intima smooth muscle cell bundles to fragmented elastic fibers in the collagenous areas. Dei increases as the intima enlarges (10.19, 14.63, and 16.01, respectively to without thickening, cushion, diffuse thickening). In the media, the elastic network encircles the circular muscle bundles connecting them to the elastic internal lamina and to elastic fibers in the adventitia. Smooth muscle cells were coiled by numerous oxytalan fibers and the elastic fibers are irregular and fragmented. In the sclerotic portions, the elastic fibers are sparse. No correlation was found between Dei and Dem. The important thickening of varicose vein intima shows increasing quantities of elastic material formerly associated with smooth muscle cells. In the media, the elastic network around smooth muscle cell bundles is disrupted and the Dem diminishes as the media becomes sclerotic.

Immunolabeling of type IV collagen, laminin, and alpha-smooth muscle actin cells in the intima of normal and varicose saphenous veins.

Smooth muscle cells (SMC) of normal and varicose human saphenous intima were studied on cryostat sections by immunohistochemistry with alpha-smooth muscle actin (ASMA), type IV collagen, and laminin antibodies and also by transmission electron microscopy. The findings suggest two structurally distinct subtypes of smooth muscle cells with thin and thicker external lamina. Thin external lamina SMC were characterized by laminin, type IV collagen, weaker external lamina reactivity, and intense cytoplasmic alpha-smooth muscle actin immunoreactivity. Ultrastructurally, they exhibited abundant cytoplasmic microfilaments and thin external lamina. These cells were found isolated or, more frequently, clustered in fascicles close to the subendothelium in focal or zonal cushions, or in diffuse enlargement of the intima. In contrast, thicker external lamina smooth muscle cells were intensely immunolabeled for laminin and collagen IV, showing irregular cytoplasmic ASMA reaction. Single or clustered thicker external lamina SMC were seen predominantly in zonal cushions and in intima diffuse enlargement. It is very likely that these cells secrete these matrices in a nonpolarized fashion. The thicker external lamina of these SMCs showed a fine granular amorphous aspect sometimes intermingled with microfibrils. These external lamina were interposed between neighboring cells and exposed to collagen fibrils and elastic fibers. The cells also exhibited rarefaction of the cytoplasmic filaments. Intermediary cells exhibiting both features were rarely seen. Thicker external lamina SMC should be discussed in the context of an adaptive/proliferative response leading to dysfunction of the fibroelastic properties of the vein wall.

Microscopic observations on the great saphenous vein at the upper third level of the thigh and at the level of discharge into the femoral vein

For the microscopical study of the great saphenous vein wall, the venous segments from six male necropsiedcadavers: were obtained three young individuals between 16 and 35 years old and three aged individuals between 50 and 83 years old. The tunica media of the great saphenous vein at the upper third level of the thigh belonging to both young and aged individuals is made up of two layers: an internal formed by fascicles of smooth longitudinal muscular fibers and the external layer made up by fascicles of smooth circular muscular fibers. At the level of the discharge in the femoral, the tunica media of the great saphenous vein wall of both, young and aged individuals, is made up of one layer, whose fascicles of smooth muscular fibers are circularly orientated in relation to the vascular axis. The tunica adventitia of the great saphenous vein wall at the upper third level of the young individuals thigh, presents elastic fibers whereas it the aged individuals it is made up of fascicles of smooth longitudinal fibers; at the level of the femoral vein disdarge in both young and aged individuals, the tunica adventitia presents a fiber elastic constitution. The young and aged individuals' parietal venous valve is elastic fiber in nature. The young individuals' ostial valve is also elastic and that of aged individuals is muscular fiber in nature.

Estudo estrutural da veia safena magna normal e varicosa / Structural study of the normal long saphenous vein and varicose

O uso da veia safena magna como implante em cirurgias de revascularizaçäo do miocárdio e de extremidades, ao lado do seu acometimento pela doença varicosa, tem despertado o interesse dos pesquisadores quanto aos aspectos de sua estrutura microscópica. Este trabalho teve por objetivo descrever as variaçöes que ocorrem nas túnicas da veia safena magna normal e da safena magna varicosa, comparar os resultados obtidos e correlacionar as anormalidades encontradas com o mecanismo patogênicodas varizes. Para isso, os fragmentos foram submetidos a técnicas histológicas para estudo ao microscópio óptico de sua morfologia geral, para determinaçöes morfométricas e para análise histoquímica e imuno-histoquímica de componentes da matriz extracelular das túnicas parietais. Os aspectos mais relevantes destes achados foram levados a um estudo ao microscópio eletrônico, complementando-se com um teste para avaliaçäo da reatividade motora frente ao K e à noradrenalina. Os resultados permitiram concluir que o espessamento da túnica íntima é uma eventualidade que pode acometer a veia, independente do processo varicoso, embora seja encontrado mais intensamente nesta condiçäo. Na túnica média, a interposiçäo das fibras do sistema elástico associadas aos feixes musculares constituem as modificaçöes mais importantes nas veias safenas varicosas. Com o estabelecimento de uma sistemática no estudo da parede das safenas magnas, deverá ser possível aprofundar os estudos sobre a adequaçäo do seu uso como enxerto, assim como aqueles que visam tornar mais claro o mecanismo do seu acometimento pela doença varicosa e pela hiperplasia intimal

[Chronic hexosaminidase A deficiency associated with pure sensory peripheral neuropathy]. / Deficiencia crónica de hexosaminidasa A asociada a neuropatía periférica sensitiva pura.

One patient with hexosaminidase A (Hx A) deficiency, which produces GM2 gangliosidosis, developed a complex progressive neurological syndrome, starting when he was 10 years old, which encompassed intellectual impairment, cerebellar involvement, features of upper and lower motoneurones compromise and sensory neuropathy without signs of motor fibre damage within the peripheral nerves. Sural nerve biopsy demonstrated loss of myelinated fibres, mainly of those of large and small diameters, clusters of small diameter fibres, fibres with abnormal thin myelin sheaths related to their axonal diameters, axonal degeneration, segmental and paranodal demyelination and remyelination. Electronmicroscopic examination showed small electrondense, non specific, bodies and concentric lamellar inclusions within the cytoplasm of the Schwann cells. These findings demonstrate that pure sensory peripheral neuropathy should be considered as part of the spectrum which may result from Hx A deficiency.