Backscattered Scanning Electron Microscopy Approach for Assessment of Microvessels under Conditions of Normal Microanatomy and Pathological Neovascularization.

We evaluated the efficiency of an original method for studying of the microvascular bed under conditions of normal microanatomy and pathological neovascularization. The blood vessels, tissues surrounding the stent in the pulmonary artery and subcutaneously implanted titanium nickelide plate, atherosclerotic plaque, and vascular stent with restenosis were examined. The specimens were fixed in formalin and stained in OsO4, embedded into fresh epoxy resin, grinded, polished, and counterstained with uranyl acetate and lead citrate. Numerous vasa vasorum were found in all native vessels. Around the pulmonary artery stent and metal plates, numerous newly formed vessels of small diameter were seen. The intensity of neovascularization in atherosclerosis and carotid stent restenosis differed significantly. Our technique can be successfully used for evaluation of the microvascular bed.

Antigen removal process preserves function of small diameter venous valved conduits, whereas SDS-decellularization results in significant valvular insufficiency.

Chronic venous disease (CVD) is the most common reported chronic condition in the United States, affecting more than 25 million Americans. Regardless of its high occurrence, current therapeutic options are far from ideal due to their palliative nature. For best treatment outcomes, challenging cases of chronic venous insufficiency (CVI) are treated by repair or replacement of venous valves. Regrettably, the success of venous valve transplant is dependent on the availability of autologous venous valves and hindered by the possibility of donor site complications and increased patient morbidity. Therefore, the use of alternative tissue sources to provide off-the-shelf venous valve replacements has potential to be extremely beneficial to the field of CVI. This manuscript demonstrates the capability of producing off-the-shelf fully functional venous valved extracellular matrix (ECM) scaffold conduits from bovine saphenous vein (SV), using an antigen removal (AR) method. AR ECM scaffolds maintained native SV structure-function relationships and associated venous valves function. Conversely, SDS decellularization caused significant changes to the collagen and elastin macromolecular structures, resulting in collagen fibril merging, elimination of fibril crimp, amalgaming collagen fibers and fragmentation of the inner elastic lamina. ECM changes induced by SDS decellularization resulted in significant venous valve dysfunction. Venous valved conduits generated using the AR approach have potential to serve as off-the-shelf venous valve replacements for CVI. STATEMENT OF SIGNIFICANCE: Retention of the structure and composition of extracellular matrix (ECM) proteins within xenogeneic scaffolds for tissue engineering is of crucial importance, due to the undeniable effect ECM proteins can impose on repopulating cells and function of the resultant biomaterial. This manuscript demonstrates that alteration or elimination of ECM proteins via commonly utilized decellularization approach results in complete disruption of venous valve function. Conversely, retention of the delicate ECM structure and composition of native venous tissue, using an antigen removal tissue processing method, results in preservation of native venous valve function.

Comparison of Different Thawing Protocols in Human Cryopreserved Venous Grafts.

BACKGROUND: The aim of our study was to assess the impact of different thawing protocols on morphological changes arising in cryopreserved human saphenous vein grafts. METHODS: The study was performed in 12 saphenous vein grafts harvested in brain death donors. Storage in the vapor phase of liquid nitrogen for 3 or 5 years followed. Two thawing protocols were tested: slow thawing in a refrigerator at temperature +4°C for 2 hr and rapid thawing-in a water bath at +37°C. Grafts were processed for scanning electron microscopy. Comparisons of continuous parameters under study between experimental groups were performed using the t-test (age, cold ischemia time, exposure to cryoprotectant, time of storage, total thawing time, mean thawing rate, morphology scoring of thawed HSVG) and the median test (HSVG length). Categorical parameters (sex and blood group) were formally tested using the chi-square test. RESULTS: All samples were evaluated according to morphological changes and scored in terms of morphologically intact endothelium, confluent endothelium with structural inhomogeneity, disruption of the intercellular contacts, separation of the endothelial cells, complete loss of the endothelium, and damage of the subendothelial layers. There is no statistically significant difference between the sample sets at the significance level of 0.05. There was no association with donors' age, sex, and time of storage. CONCLUSIONS: Human cryopreserved saphenous vein grafts in our experimental work showed no difference in terms of structural deterioration of the endothelial surface and basal membrane depending on different thawing protocols used.

The influence of age on valve disease in patients with varicose veins analysed by transmission electron microscopy and stereology.

BACKGROUND: The aim of this study was to investigate the influence of age on the ultrastructure of venous valve morphology in patients with C2 classified chronic venous disorders according to the CEAP classification. PATIENTS AND METHODS: The study population consisted of 16 consecutive patients with varicose veins (C2). The mean age was 49.8 years (30-66). The (pre-) terminal valve including the vessel wall was harvested within the proximal 2 centimetres of the great saphenous vein. The mean thickness (volume-to-surface ratio = V/S ratio) of elastin, collagen, endothelium and of the entire valve was determined. A blinded morphologist performed the examination by transmission electron microscopy and stereology. Analyses by Pearson's product moment correlation, Kendall's tau and Spearman's rank correlation were performed to investigate whether there is a correlation between age and the ultrastructural morphology. RESULTS: Stereological analysis of the valves demonstrated a mean V/S ratio (signifying a thickness estimation) for elastin of 0.87 µm3/µm2, for collagen of 18.0 µm3/µm2, for endothelium of 0.65 µm3/µm2, and for the entire valve of 25.2 µm³/µm². Statistical analyses showed no statistically significant correlation between age and the ultrastructural morphology in this patient group. CONCLUSIONS: The ultrastructural morphology of the venous valves in chronic venous disorders may not depend on age in patients presenting with C2 disease. This conclusion may or may not apply to all C classes as we investigated a homogenous group of patients with C2 limbs.

Preservation of Endothelial and Smooth Muscle Function of Human Saphenous Vein Transplants.

OBJECTIVES: Methods for conservation and preservation of vascular grafts are often controversially discussed. Furthermore, immunologic monitoring or immunotherapy for allogeneic graft is not considered necessary in many cases. The present study was initiated to examine the cellular vitality and functional efficiency of vein transplant during preservation. MATERIALS AND METHODS: Twenty-seven human vein segments (vena saphena magna) were stored after explant in University of Wisconsin solution or histidine-tryptophan-ketoglutarate solution at 4 °C. After 3, 24, 48, 72, and 96 hours, vein functionality was tested. Ring segments were fixed by triangles in Krebs-Henseleit buffer. Contractile function was measured after addition of potassium chloride solution (80 mM) and phenylephrine (0.2, 2, or 20 µM). To investigate endothelium-dependent vasorelaxation, 1 µM acetylcholine was added. RESULTS: Of 27 segments, 5 showed endothelium-dependent relaxation. Vasorelaxation continued for up to 48 hours after administration of acetylcholine in University of Wisconsin solution and for up to 24 hours in histidine-tryptophane-ketoglutarate solution. At 48 hours, potassium chloride solution-induced vasocontraction was 17% more effective than phenylephrine in University of Wisconsin solution. University of Wisconsin solution was significantly more effective than histidine-tryptophane-ketoglutarate solution in terms of preservation of phenylephrine (0.2, 2 µM)-induced vasocontraction. Phenylephrine (2 µM)-induced contraction was retained in University of Wisconsin solution after 24 hours by 81% and after 48 hours by 55%, with comparable results in histidine-tryptophane-ketoglutarate solution of only 62% and 34% after 24 and 48 hours. CONCLUSIONS: At 48 hours, human saphenous vein transplants had better endothelium and smooth muscle function when preserved in University of Wisconsin solution versus histidine-tryptophane-ketoglutarate solution.

Dysregulated apoptosis of the venous wall in chronic venous disease and portal hypertension.

Introduction The etiology of varicose veins remains elusive. We hypothesized that abnormal cell cycle events in the vein wall may contribute to changes in the structural integrity, thus predisposing to the development of varicosities. The present study was designed to determine whether or not the same molecular apoptotic pathway exists between great saphenous and splenic veins. Methods Thirty-six samples of diseased splenic veins and varicose great saphenous veins were collected. Twenty-five samples of control splenic and great saphenous veins were also collected. The apoptotic cell proteins expression was immunohistochemically stained with antibodies (anti-Bax and anti-Bcl-xl). Apoptosis was evaluated by the terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) assay and immunofluorescence staining. The morphology of apoptotic cells was observed with an electron microscope. Results The apoptotic ratio in walls (intima and media) of diseased splenic vein and varicose great saphenous vein groups were significantly lower than the corresponding regions in the splenic vein and great saphenous vein groups ( p < 0.01), respectively. A significant difference was not noted in the ratio change of apoptotic cells between the diseased splenic vein and varicose great saphenous vein groups ( p > 0.05). The high positive expression of Bcl-xl proteins was detected in the diseased splenic vein and varicose great saphenous vein groups, respectively. While the high positive expression of Bax proteins was also observed in the splenic vein and great saphenous vein groups, respectively. Electron microscopic observations confirmed that endothelial and smooth muscle cells in diseased splenic vein, varicose great saphenous vein, splenic vein, and great saphenous vein walls exhibited apoptotic morphologic features, such as fuzzy mitochondrial cristae, medullary changes, and margination of the nuclear chromatin. Conclusions Our results showed the same dysregulation of apoptosis via the intrinsic pathway in diseased splenic veins and varicose great saphenous veins. This observational study suggests that apoptotic down-regulation in the veins wall is a cause of diseased splenic veins and varicose great saphenous veins, but does not exclude the possibility that other mechanisms are involved.

Myelinated mouse nerves studied by X-ray phase contrast zoom tomography.

We have used X-ray phase contrast tomography to resolve the structure of uncut, entire myelinated optic, saphenous and sciatic mouse nerves. Intrinsic electron density contrast suffices to identify axonal structures. Specific myelin labeling by an osmium tetroxide stain enables distinction between axon and surrounding myelin sheath. Utilization of spherical wave illumination enables zooming capabilities which enable imaging of entire sciatic internodes as well as identification of sub-structures such as nodes of Ranvier and Schmidt-Lanterman incisures.

Morphological, immunohistochemical and biochemical effects of non-pulsatile ex vivo perfusion with crescent pressures in human saphenous veins.

AIM: On the average, 15% to 25% of peripheral grafts and 10% to 30% of coronary grafts fail within 5 years. Changes in mechanical forces to which the vein is subjected could be an explanation for this phenomenon. We submitted human saphenous vein segments to non-pulsatile ex vivo perfusion with crescent pressures and evaluated morphology, nitric oxide synthase immunohistochemical expression; tissue levels of nitrite/nitrate and oxidative stress products. METHODS: Intact segments of human saphenous veins were obtained from 30 patients submitted to elective coronary artery bypass graft surgery. Ex vivo perfusion was performed during 3 hours, using oxygenated Krebs solution, flow of 100 mL/min and pressures of 0, 50, 100, 200 and 300 mmHg, defining five groups. RESULTS: Optical microscopy showed that veins of groups perfused with 200 and 300 mmHg presented increased luminal area and endothelial denuding. Electron microscopy transmission showed alterations in veins perfused with 200 and 300 mmHg. Immunohistochemical expression of the three nitric oxide synthase isoforms was observed in all vein layers, without significant difference among groups. Tissue levels of nitrite/nitrate were not significantly different among distinctive perfusion. Nitrotyrosine was not immunohistochemically expressed in all veins and malondialdehyde tissue levels were not different among groups. CONCLUSION: Non-pulsatile ex vivo perfusion during 3h caused morphological alterations in human saphenous veins (HSVs), which were not accompanied by immunohistochemical and biochemical alterations. Even with mechanical lesions, HSVs maintained the ability of express nitric oxide synthase (NOS) and release nitric oxide.

Characterization of the vasa vasorum in the human great saphenous vein: a scanning electron microscopy and 3D-morphometry study using vascular corrosion casts.

The vasa vasorum (VV) of explanted segments of the human great saphenous vein (Vena saphena magna; HGSV), harvested during dissection for coronary bypass grafts or diseased vein segments from the "Salzburger Landesklinikum," were studied by scanning electron microscopy and three-dimensional morphometry of microvascular corrosion casts. The main objective of this study was to examine the VV's structural arrangement in order to find the most vital segments of the HGSV and in turn to improve the results of coronary bypass surgeries. The study presents a meticulous analysis of the whole microvascular system of the VV of the HGSV and its three-dimensional arrangement. It is one of the first studies yielding detailed quantitative data on geometry of the VV of the HGSV. A detailed insight into different vascular parameters such as vessel diameter, interbranching, intervascular distances, and branching angles at different levels of the VV's angioarchitecture and in different parts of the HGSV in health and disease is given. Further, the geometry of bifurcations was examined in order to compute the physiological optimality principles of this delicate vascular system based on its construction, maintenance, and function.

Effects of high hemodynamics upon the morphology of the walls of the great saphenous vein and splenic vein.

AIM: Studies have shown that the incidence and development of pathological changes in the walls of the great saphenous vein and splenic vein are closely related to high venous pressure. Such changes are referred to as "vascular adaptive remodeling responses under high venous pressure". The proposition of the concept of vascular remodeling contributes to our knowledge of pathological changes in the venous wall (dilation of the venous lumen and thickening of the venous wall). In the present study, we compared the histomorphology and cytomorphology of the walls of varicose great saphenous veins (GSVs) and diseased splenic veins (SVs) to investigate the remodeling of the venous wall under high hemodynamic pressure. METHODS: We collected 34 samples of varicose great saphenous veins and diseased splenic veins. Thirty-four samples of normal great saphenous veins and splenic veins were also collected (control group). Samples were made into slices and observed under light microscopy and electron microscopy. The thickness of the tunica intima and tunica media as well as the inner diameter of the venous lumen were measured. RESULTS: Under light microscopy, the walls of varicose veins stained with H&E were unevenly thickened, and those of diseased splenic veins were evenly thickened; mucoid degeneration of the tunica intima of varicose veins was not obvious by Masson staining (2/20 cases). The boundary between the tunica intima and tunica media was clearly defined. Uneven hyperplasia of muscular connective tissues was observed. For the diseased splenic-vein group, mucoid degeneration of the tunica intima was obvious (8/14 cases), with an unclearly defined boundary between the tunica intima and tunica extima. Uneven hyperplasia of muscular connective tissues was also observed. Differences in the thickness and inner diameter of the tunica intima and tunica media between the great saphenous vein and the splenic vein were significantly different. Under electron microscopy, mitochondrial degeneration in endothelial cells was observed in both groups. Increased numbers of rough endoplasmic reticula in the cytoplasm of smooth muscle cells, ribosomes and mitochondria and decreased numbers of myofilaments were also observed. CONCLUSION: High hemodynamics affected the remodeling of varicose great saphenous veins and diseased splenic veins. The histomorphology of visceral veins showed more significant pathological changes than that of peripheral veins. Similar cytomorphological changes were observed in both groups.

Morphological characteristics of the walls of thrombophlebitic saphenous vein.

INTRODUCTION: To investigate the morphological changes in the walls of thrombophlebitic saphenous veins. METHODS: Fifty-four specimens were made into slices for haematoxylin and eosin and Masson trichrome staining; ultrathin slices were also created. Slices were observed under light microscopy and electron microscopy. RESULTS: Under light microscopy, the tunica intima of venous wall in the thrombophlebitic saphenous veins group was obviously thickened and incomplete; intravascular papillary endothelial hyperplasia was observed. Hyperplasia of collagenous fibres in the tunica media was observed. Elastic fibres in the tunica externa became fewer, whereas nourishing vessels were significantly increased in number. Infiltration of many inflammatory cells was observed. Under electron microscopy, Auer bodies with high electron density and round granules could be seen in endothelial cells in the thrombophlebitic saphenous veins group. Smooth muscle cells had an irregular karyotype, with blurred cristae in some mitochondria. Myofilaments basically disappeared. CONCLUSION: Thrombus formation might aggravate re-modelling of the walls of varicose veins.

Valve disease in chronic venous disorders: a quantitative ultrastructural analysis by transmission electron microscopy and stereology.

INTRODUCTION: The ultrastructure of venous valves and walls in chronic venous disease was investigated. METHODS: Consecutive patients were categorised into one of three groups (group A: patients with C1 venous disease in accordance with CEAP (Clinical severity, Etiology, Anatomy, Pathophysiology); group B: C2 and C3; group C: C4, C5 and C6). The terminal or preterminal valve and adjacent vessel wall was harvested from the great saphenous vein. Sections were examined with a transmission electron microscope. The volumes of elastin and of collagen per unit surface area of valve were assessed, as well as the surface endothelium of valve and vessel wall. RESULTS: The study population consisted of 17 patients. The elastin ratio was analysed by means of stereology. Mean values were: in group A, 0.45 µm3/m2; in group B, 0.67 µm3/m2; in group C, 0.97 µm3/m2. The ratio was similar for collagen (A, 15.7 µm3/m2; B, 26.8 µm3/m2; C, 30.1 µm3/m2). Surface analysis of the valve endothelium and the adjacent vessel wall endothelium showed a trend towards increasing damage with more severe disease. CONCLUSIONS: With progression of venous disease, the valve elastin content, assessed morphologically, seems to increase, and the endothelium of the venous valve and the vein wall tend to show more damage.

Pressure applied during surgery alters the biomechanical properties of human saphenous vein graft.

Pressure applied during harvesting of the saphenous vein (SV) graft in coronary artery bypass surgery might change its mechanical properties and thereby decrease the patency. This study was performed to assess the mechanical properties of the SV graft distended manually with different levels of pressure and to determine the pressure level that induces changes in its structure and mechanics. Saphenous vein graft segments, collected from 36 patients undergoing coronary artery bypass surgery, were distended with pressures of either 50-60, 75-100, or 130-150 mmHg. Grafts were tested for the stress-strain relationship; the Young's moduli at the low- and high-strain regions were calculated, and their structures were examined by light and electron microscopy. Pressures of 50-60 mmHg did not influence the mechanics of the vein graft, whereas pressures of 75-100 mmHg elevated the elastic modulus of the vein at the low-strain region while pressures above 130 mmHg increased the elastic moduli at both low- and high-strain regions. There was a prominent loss of microfibrils at all distending pressure levels. The mechanical results suggest that distending pressures above 75 mmHg might play a role in graft failure. Furthermore, the absence of microfibrils surrounding elastin suggests that application of distending pressures, even as low as 50 mmHg, can cause degeneration of the elastic fibers following implantation, increasing the stiffness of the graft and thus impairing the graft's function under its new hemodynamic conditions.

The effects of aging on the intimal region of the human saphenous vein: insights from multimodal microscopy and quantitative image analysis.

We hypothesized that structural remodeling associated with advancing age occurs in human saphenous veins. To address this hypothesis, we have identified structural remodeling in human saphenous veins by applying histochemistry, fluorescence staining and quantitative image analysis to specifically assess intimal area, intimal cellularity and intimal collagen content and organization. Saphenous veins were collected from patients undergoing coronary artery bypass graft surgery. Area measurements and cellularity were quantified using the image analysis software Stereo Investigator, employing planimetry and counting frames, respectively. Collagen content and organization were quantified in MetaMorph image analysis software based on measurements of color (hue, saturation, and intensity) from polarized light images. Intimal area and cellularity showed no statistically significant increases with age; in contrast, total collagen content showed a significant decrease with advancing age. Furthermore, collagen fiber types also demonstrated a statistically significant alteration with age; increases in age resulted in decreases in larger collagen fibers. No significant changes in small collagen fibers were identified. These results raise the possibility that age-associated structural alterations in total collagen content, specifically collagen fiber size, could be a factor in the etiology of age-associated venous diseases.

Giant saphenous vein graft pseudoaneurysm causing tricuspid valve stenosis.

We present the case of a 72-year-old male who was diagnosed with a saphenous vein graft pseudoaneurysm, detected on routine chest echocardiogram 13 years after undergoing coronary artery bypass graft surgery. Intraoperative transesophageal echocardiography revealed the pseudoaneurysm to be causing functional tricuspid stenosis, which was relieved after surgical excision of the mass.

The "no-touch" harvesting technique for vein grafts in coronary artery bypass surgery preserves an intact vasa vasorum.

OBJECTIVES: Our objective was to evaluate the impact of vein graft harvesting technique on structure and function of vasa vasorum. METHODS: Paired segments of great saphenous veins harvested either with conventional harvesting technique or no-touch technique were obtained from 9 consecutive patients undergoing coronary artery bypass grafting. Quantitative measurements, using immunohistochemistry and morphometry, were performed. Ultrastructural analyses of vasa vasorum were performed with electron microscopy. Video footage of superficial vasa vasorum in an implanted saphenous vein graft harvested with the no-touch technique was captured during a coronary bypass operation and is presented for online viewing. RESULTS: The total area of vasa vasorum in vein grafts harvested with the conventional technique was significantly reduced both in the media (P = .007) and in the adventitia (P = .014) compared with vein grafts harvested with the no-touch technique. Ultrastructural findings indicated that the no-touch technique preserved an intact vasa vasorum whereas the conventional technique did not. Video footage showed retrograde flow in the vasa vasorum in vein graft harvested with the no-touch technique. CONCLUSIONS: These findings show that the no-touch technique for saphenous vein graft harvesting for coronary bypass grafting preserves an intact vasa vasorum. This could represent one of the mechanisms underlying the improved patency of saphenous vein grafts harvested with this technique.

Pathogenic mechanisms in varicose vein disease: the role of hypoxia and inflammation.

AIMS: Although the aetiology of varicose veins remains unknown, recent studies have focused on endothelial cell integrity and function. Among the regulatory factors of vessel tone, synthesises, pro- and anti-inflammatory, adhesion molecules and the transcription factor hypoxia inducible factor-1 alpha (HIF-1alpha), which are responsible for recruiting leukocytes, are very important. METHODS: Investigation in this study focused on the expression of ICAM-1, E-selectin and HIF-1alpha on endothelial cells using immunostaining and RT-PCR in varicose vein specimens compared with controls. RESULTS: Findings of this study showed alterations of the intima, such as focal intimal discontinuity and denudation of endothelium in varicose veins. Based on data derived from immunostaining and RT-PCR, no major differences were identified between ICAM-1 and E-selectin expression in varicose vein specimens compared with controls. In contrast, immunostaining results identified HIF-1alpha expression in five (5/20) varicose vein specimens, whereas no control saphenous vein specimens expressed HIF-1alpha. CONCLUSIONS: These findings could explain other evidence of hypoxia in varicose veins. Finally, results already obtained in this investigation suggest that the process of pathogenesis of varicose veins is not restricted to the role of adhesion molecules.