Shock absorbtion of the hoof capsule is due to the deformation of the hoof wall, hoof sole and their corresponding laminae. In addition the combination of cartilages, digital cushion, connecting band structures and the frog itself form the important shock absorber of the palmar/plantar part of the hoof. The digital cushion plays a major role, not only because it covers the very sensitive area of the distal sesamoid bone, the deep digital flexor tendon and the coffin joint, but because it has the size and structure to perfectly reduce any force impact. The digital cushion does not consist of elastic fibres or fatty depositions, it is loosly composed of collagenous fibres with mucous depositions. Their hyaluronic acids and glucose-aminoglycans are well hydrated macromolecules and act as jelly puffers between incoming force and sensitive structures.

This study explored the spontaneously hypertensive rat as an animal model of pulmonary hypertension and sought to identify anatomic changes in its pulmonary microvasculature, especially focal constrictions of pulmonary veins (sphincters). The average systemic and pulmonary artery blood pressures were 172/139 (+/- 9/9) and 36/14 (+/- 4/3), respectively, for spontaneously hypertensive Wistar Kyoto rats (SHR), and 134/83 (+/- 8/2) and 20/10 (+/- 2/2) for normotensive Wistar Kyoto rats (WKY) (P < 0.01 for both). Light microscopy of the lungs in SHR showed muscularization of both arteries and veins, but this was more pronounced in the small pulmonary veins. Perivascular edema was also present. There were 20 (+/- 4) leukocytes per 100 microns of capillary length in SHR and 9 (+/- 2) in WKY (P < 0.001). Transmission electron microscopy showed focal venous smooth muscle was greater in SHR than in WKY. Scanning electron microscopy of vascular casts showed the average maximal focal venous contraction (sphincter) was 54% (+/- 10) of its diameter in SHR, but was only 6% (+/- 4) in WKY (P < 0.01). Arterial contraction occurred in the hypertensive rats as bourglass narrowings of the casts, but was less conspicuous than venous constrictions. The mean alveolar capillary diameter was 8.1 microns (+/- 1.6) in SHR, compared with 6.3 microns (+/- 1.0) in WKY (P < 0.01). The central interspace between capillaries was 3.2 microns (+/- 1.6) in SHR and 6.0 microns (+/- 3.6) in WKY (P < 0.01). The venous contraction, capillary size, and capillary interspace distance correlated with the pulmonary blood pressure. The spontaneously hypertensive rat can be a model of pulmonary hypertension with its most notable structural change being increased muscularity in the small pulmonary veins.

BACKGROUND:This study was conducted to investigate the impact of the preservation method of bioprosthetic heart valve materials on calcification rates and biocompatibility of the biologic tissue.METHODS:In subcutaneous rat implants, conventionally preserved bioprosthetic heart valve material was compared with bovine pericardium that was treated with L-glutamic acid to reduce residual glutaraldehyde released from the fixed tissue. Both these methods were compared with bovine pericardium that was stabilized by a dye-mediated photooxidation reaction without glutaraldehyde. Biocompatibility of these biomaterials was tested in vitro using human endothelial cell cultures.RESULTS:Conventionally preserved bovine pericardium with a high amount of glutaraldehyde incorporated into the tissue resulted in severe calcification 63 days after subcutaneous implantation in rats (165.4 +/- 20 mg Ca2+/g dry weight). Postfixation treatment with L-glutamic acid, which reduces free, unbound aldehyde groups, showed a significant decrease in calcification (89.6 +/- 14 mg Ca2+/g dry weight). Glutaraldehyde-free preservation by dye-mediated photooxidation showed no calcification after 63 days of subcutaneous implantation (1.0 +/- 0.4 mg Ca2+/g dry weight). Regular endothelial cell proliferation was observed on photooxidized and L-glutamic acid-treated tissue, whereas conventionally treated tissue caused endothelial cell death.CONCLUSIONS:This study underlines the detrimental role of glutaraldehyde in the calcification process of bioprosthetic heart valve materials and emphasizes alternative preservation methods that reduce or avoid the use of glutaraldehyde.

BACKGROUND:Pulmonary lymphatics are critical to clearing lung fluid. Although their structure can be shown with light and transmission electron microscopy, scanning electron microscopy of their casts can better show their number, size, shape, distribution, and degree of filling. This technique has identified four forms of lung lymphatics, but these forms have not been fully evaluated by tissue microscopy. A most important site of pulmonary edema formation, the pulmonary capillary, is just upstream from small veins which have focal, smooth muscle tufts termed venous sphincters. Because of their constricting potential, these sphincters may control lung perfusion and cause edema.METHODS:With light and transmission electron microscopy of tissue and scanning electron microscopy of casts, the lymphatic forms were explored in relation to the tissue anatomy in rats without pulmonary edema and with mild-to-moderate edema caused by extended vascular rinsing.RESULTS:The edematous lungs had increased sacculo-tubular lymphatics adjacent to the venous sphincters. These lymphatics were in the adventitial connective tissue and were partially endothelialized. As lymphatics became more tubular their endothelium became more complete. Collagen fibers traversed the lumen of these lymphatics even where endothelial cells were present and caused the lines on the surface of the lymphatic casts. Overlapping endothelial cells caused clefts on the casts.CONCLUSIONS:Scanning electron microscopy of lymphatic casts better defines their ultrastructure and shows the spatial relationship of veins and their sphincters to venous lymphatics. Sphincter contraction may influence pulmonary lymph production which could affect other aspects of regional lung perfusion.

BACKGROUND:The microvascular pattern of the duodenal papilla is unknown. Since the duodenal papilla is located in the transition zone between the stomach and duodenum, and because it regulates bile transfer into the duodenum, a particular microangioarchitecture can be expected. Therefore, we examined the microvasculature of the papilla using guinea pigs as a model.METHODS:The microvascularization of the duodenal papilla and common bile duct was studied in 26 adult guinea pigs (Cavia porcellus), using scanning electron microscopy of microvascular corrosion casts and critical point dried specimens, and light microscopy of tissue sections.RESULTS:The duodenal papilla is located in the cranial portion of the duodenum, approximately 5 mm beyond the pyloric valve. At the most luminal aspect of the cast papilla, ring-shaped capillaries, resembling those of the cast gastric mucosa, are present. Deeper parts of the papilla are provided with villi. Subepithelial capillaries of the papilla are 15 microns thick in average. These capillaries have a dual blood supply either via the straight long arterioles arising from the submucosa or by the pericryptal capillaries. The common bile duct comprises numerous mucoid glands with their pits surrounded by ring-shaped capillaries in corresponding casts.CONCLUSIONS:The special arrangement of different capillary patterns, together with their luminal size and the dual blood supply, favor their protective role from the gastric chyme.

The binding of radiolabelled lipoproteins, iodine-123-labelled low-density. lipoprotein (LDL) and indium-111-labelled LDL, to peripheral blood mononuclear cells (MNCs) was compared in normolipaemic subjects and in patients with heterozygous familial hypercholesterolaemia (FH). 123I-LDL and 111In-LDL binding to MNCs exhibited high-affinity, highly specific, time- and temperature-dependent binding reaching saturation at concentrations above 50 nM. The number of LDL binding sites (Bmax) was significantly (P<0.01) lower in FH patients (P<0.001; 123I-LDL: Bmax 279±44 ng protein/108MNCs; 111In-LDL: Bmax 309±43 ng protein/108MNCs) as compared with controls (123I-LDL: Bmax 2874±246 ng protein/108 MNCs; 111In-LDL: Bmax 3145±339 ng protein/108 MNCs). The corresponding dissociation constants (Kd) were 16±8 nM for 123I-LDL and 12±6 nM for 123In-LDL in healthy volunteers (123In-LDL vs 111In-LDL, P<0.05). In FH patients, the Kd values were 20±8 nM for 123I-LDL and 16±6 nM for 123In-LDL (P<0.05 vs controls for both 123I-LDL and 111In-LDL). 111In-LDL binding to MNCs was inhibited (IC50) by 30±8 nM in healthy controls and 38±12 nM in FH patients (P<0.05). 123In-LDL binding to MNCs was inhibited (IC50) by 34±8 nM in healthy controls and 46±10 nM in FH patients (P<0.05). Taken together, these results suggest a reduced number of LDL receptors expressed on MNCs from FH patients. We conclude that 111In-LDL and 123I-LDL are equally well suited as a probe of receptor-mediated binding and uptake of LDL.

Capillaries in the mouse exocrine pancreas were studied by scanning electron microscopy of microvascular corrosion casts, transmission electron microscopy of tissue sections, and high resolution intravital video‐microscopy. Two types of capillaries were discerned by corrosion casting. The first type was rather straight, had a constant diameter of 5–6 μm, and its surface showed multiple circumferential furrows. The frequency of such constrictions was less in the second type, which was more undulated and had a diameter of 7–9 μm. In the second type, these constrictions defined bulged areas of the capillary cast. Corresponding tissue sections also showed two types of capillaries, fenestrated and non‐fenestrated capillaries. Microtubules were abundant in all capillary endothelial cells, whereas bundles of microfilaments were scarce. Microtubules were arranged along the long axis of endothelial cells as well as parallel to endothelial cell border regions. Endothelial cells were joined by intermediate junctions along cell borders running both circumferentially and longitudinally. Flow reversal in capillaries and spontaneous endothelial contractions were documented in vivo. Endothelial cells bulged into the lumen, either at their nuclear region or distant from it. Spontaneous contraction of pericytes was not observed. These results suggest that contraction of capillaries is carried out by endothelial cells, representing an autonomous flow regulatory device. Capillary contraction in exocrine pancreas may be influenced by blood‐borne agents, probably by those released in Langerhans islets. © 1993 Wiley‐Liss Inc.

The rat pulmonary microvasculature was studied using scanning and transmission electron microscopy of vascular corrosion casts and tissue sections. Special emphasis was placed on small pulmonary venous vessels. The shape of vascular casts was analyzed and interpreted concerning the wall composition of corresponding vessels studied in tissue sections. On the casts of pulmonary venules and small pulmonary veins, narrow or wider annular constrictions were regularly observed. Within these constrictions, marks of circularly running grooves were seen as an additional structural detail, which obviously mimic impressions of single or grouped smooth muscle cells. The depth of the constrictions varies; it may be more or less pronounced, occasionally narrowing down the luminal diameter to approximately 50%. These constrictions are caused by muscular sphincters. In tissue sections of small pulmonary veins, sphincter regions were identified as abruptly appearing single or grouped true smooth muscle cells. Smooth muscle cells may be arranged side by side in a group or bundle or even staked in two or three layers. Between the sphincter regions, the venous wall consists merely of endothelium and an accompanying connective tissue layer. The smooth muscle cells of a sphincter are regularly positioned between endothelial layer and elastic lamina. The smooth muscle cells next to the endothelium form myoendothelial junctions. Autonomic nerves near the sphincters were never seen. The venous sphincters described are suggested to be effective devices involved in blood flow regulation. Blood-borne substances or local tissue hormones might govern sphincter function.

The appearance of pulmonary venous sphincters was studied in normotensive and spontaneously hypertensive rats using scanning electron microscopy of microvascular corrosion casts and transmission electron microscopy of tissue sections. Vascular casts were prepared either after lavage with Tyrode solution or after glutaraldehyde prefixation. Pronounced pulmonary venous sphincters were more frequently identified in spontaneously hypertensive rats as compared to corresponding circular indentations in normotensive rats. Tissue sections established venous sphincters in hypertensive animals as consisting of multiple layers of smooth muscle cells in the venous walls. We did not observe any autonomic nerve terminals in close proximity to these bundles of smooth muscle cells. The effect of various casting procedures on the appearance of venous sphincters is discussed. It is concluded that glutaraldehyde prefixation is an appropriate method to demonstrate sphincter functioning, because it causes deepening of sphincter indentations. Pulmonary vascular sphincters are thought to be governed by blood-born substances, vasoactive metabolites, or by tension of oxygen and carbon dioxide. Venous sphincters may influence microvascular flow in general and probably substitute for venous valves in the pulmonary vascular bed where valves are missing.

Bioprosthetic heart valves removed 76 to 150 months after implantation were morphologically investigated to correlate structural alterations with clinical failure modes. Traditional morphologic methods of evaluating valvular heterografts, such as microradiography and electron microscopy, were complemented by undecalcified ground sections, a new technique for analyzing the distribution of mineral deposits. Apart from well-investigated mechanisms that accelerate tissue degeneration, our observations point to additional facts: (1) phagocytosis of collagen fibrils and elastic material by macrophages and foreign body giant cells in areas near tears and perforations and (2) initial calcification indicated by delicate crystals in the intercellular space arranged in close relation to the periodicity of the cross-striation pattern of collagen fibrils. The present report not only calls attention to degenerative changes that are enhanced by mechanical stress but also underlines phagocytosis as an important mechanism in the destruction of bioprosthetic heart valves.

In this in vitro study, the growth properties of cultured endothelial cells on conventionally treated pericardial valve material were measured. These data were compared to endothelial cell proliferation on an alternatively treated valve material. This alternative preservation procedure was developed in order to bind free, residual glutaraldehyde in the valve tissue by reaction with L-glutamic acid. In order to optimize endothelial cell attachment and proliferation, fibronectin and fibrillar collagen type I were tested as surface precoating substances. Cell viability of the seeded cells was evaluated by means of proliferation kinetics, antithrombotic activity, and morphological appearance. Endothelial cell death occurred within the first 2 days after seeding on conventionally treated valve tissue, independent of the type of precoating. On alternatively treated tissue, regular endothelial cell proliferation was observed. Precoating with fibrillar collagen markedly increased endothelial cell attachment and proliferation as compared to fibronectin. Maintenance of antithrombotic activity of the seeded cells was proven by regular release of prostacyclin.

Degenerative alterations of two different glutaraldehyde (GA)-fixed bioprosthetic heart valve materials were investigated in subcutaneous rat implants: Bovine pericardium, prepared according to clinically used bioprosthetic heart valve material (BHV) was compared to alternatively preserved pericardium (APHV), which was fixed in GA and treated with L-glutamic acid. Following 63 days of subcutaneous implantation, calcification of APHV implants was significantly lower as compared to BHV implants (13 +/- 6 versus 158 +/- 18 micrograms Ca/mg dry weight tissue; p less than 0.05). In BHV implants ultrastructural investigations showed nucleation of plate-shaped hydroxyapatite crystals at the surface of collagen fibrils and in remnants of connective tissue cells; no signs of calcification could be detected in APHV implants. The time-course of the inflammatory reaction was determined by quantification of immunohistochemical stained mononuclear host-cells invading the implants. In both preparation groups inflammatory reaction reached maximum 42 days after implantation. However, infiltration rate of inflammatory cells was markedly decreased in APHVs as compared to BHVs (p less than 0.05).

Preliminary in vitro and in vivo studies have shown that endothelialization is improved by a detoxifying postfixation treatment of glutaraldehyde (GA) fixed bovine pericardial patches and grafts. To test whether this is also true for GA tanned human vein (HUV) grafts, patches of commercially available HUV grafts (MHUV), postfixation treated HUV grafts (PTHUV) and GA fixed HUV granfts (GAHUV) were endothelialized in vitro. Eight pairs of MHUV and PTHUV grafts were implanted as femoropopliteal grafts in eight sheep. Endothelial cell adherence was significantly better on PTHUV (11910 +/- 4413 cells/cm2) than on MHUV (6545 +/- 2835 cells/mm2; p = 0.0007) and on GAHUV (3563 +/- 1638; p = 0.0001) one day after cell seeding. After eight days of culture significantly more cells spread on PTHUV material than on MHUV (p = 0.0002), but none of the cultures on GAHUV remained viable. Four PTHUV grafts occluded in the femoropopliteal position, mostly because of kinking, so that only in four pairs of grafts could the thrombus-free surface be compared by planimetry. Again PTHUV material was covered more by endothelial cells than was MHUV material. On PTHUV endothelial cells spread directly on the graft material while on MHUV these cells spread on a layer of fibrin and macrophages. Postfixation treatment of GA-fixed biological graft material by amino-acid solutions improves the biocompatibility of the material and enhances in vitro as well as spontaneous in vivo endothelialization.

Treatment of glutaraldehyde-fixed pericardium with L-glutamic acid and storage in bacteriostatic preservatives (paraben) stably antagonizes free, reactive aldehyde groups within the fixed bioprosthetic heart valve tissue. In 63-day subcutaneous implants in rats, the calcification rate of this treatment (13.3 +/- 2 mg calcium/g wt tissue) was markedly reduced as compared to conventionally treated tissue (169 +/- 24 mg/g; p < 0.05). To test the influence of tissue-released toxic aldehdyes on spontaneous endothelial cell ingrowth in vivo, vascular grafts (8-cm long, 6-mm diameter) from fixed pericardium treated with L-glutamic acid were interposed into the carotid arteries in ten sheep. They were compared to grafts from conventionally treated pericardium implanted at the contralateral side. Following 3 months of implantation, planimetry revealed 49% +/- 20% of the surface of conventionally preserved pericardium to be covered with red thrombus, but only 12% +/- 5% in L-glutamic acid treated pericardium (p < 0.05). The ultrastructural findings of a closed endothelial cell layer on the graft surface reveals the new technique to be a promising approach towards increased biocompatibility of aldehyde-fixed bioprosthetic heart valves.

The rat exocrine pancreas was studied as a model to demonstrate morphological features of different types of capillaries, using scanning electron microscopy of vascular corrosion casts and transmission electron microscopy of tissue sections. Two types of capillaries were discerned. The first type represents less undulated, slender, straight capillaries with numerous, shallow, circular or semilunar furrows on its cast's surface. In tissue sections, this type probably corresponds, to non-fenestrated capillaries. The numerous grooves on its cast correspond to pericyte processes beneath the endothelial lining. The second type comprises capillaries of an undulated course and variable diameter with less numerous furrows. In addition, these casts showed circumscribed, smooth surfaced bulging areas defined by the grooves described. In tissue sections, this type probably corresponds to fenestrated capillaries, the bulging areas on its cast correspond to fenestrated regions of the endothelium. Fenestrated areas of capillary endothelium are less reinforced; pericyte processes are not present beneath these regions in tissue sections. The hypothesis that pericyte processes are responsible for surface indentations on capillary casts was supported by observations on postcapillary venules. Casts of these vascular segments showed also numerous circularly running furrows. Accordingly, the wall of postcapillary venules is provided with pericytes while smooth muscle cells are missing.

The microvasculature of the esophagus was studied by scanning electron microscopy of vascular corrosion casts in human infants and rabbits. In both species, segmental circumferential arteries arise from main longitudinal arteries, the latter giving off numerous perforating arteries. The tunica muscularis is supplied by branches of circumferential and perforating arteries, the submucosa and its glands by branches of perforatings. Terminal arborizations of perforating arteries feed a subepithelial capillary network. These capillaries are drained by a venous plexus in the lamina propria which is connected to a submucosal venous plexus. Perforating veins, running parallel to the corresponding arteries, connect the submucosal plexus with circumferential veins, and finally empty into main longitudinal veins. Valves were not present in any of the veins. Submucosal veins were less numerous in man than in rabbit. The number and caliber of equivalent vessels in human submucosal plexus decreased from the pharyngoesophageal to the gastroesophageal junction, suggesting the latter to be at particular risk in portal hypertension. The subepithelial capillary network reveals a longitudinal arrangement in rabbits, while the same network shows no preferential organization in human infants. The microvascular architecture of the esophagus in humans and rabbits is comparable, especially in the lay-out of the venous plexuses and the absence of venous valves. Therefore the rabbit could serve as an experimental model for studies on portal hypertension. The present results strongly suggest particular significance of the venous plexus in the lamina propria for the genesis of esophageal varices.

The microvasculature of the rat lung was studied by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) of vascular corrosion casts and tissue sections. Particular emphasis was placed on postcapillary venules, pulmonary venules and small pulmonary veins (small interlobular veins). Casts of lung capillaries appeared inconspicuous with smooth surface. On the casts of pulmonary venules and small pulmonary veins, by contrast, series of narrow annular constrictions, present at regular distances of 20-25 microns, were seen. These constrictions may be drastic, narrowing down the caliber of the vessel up to 50%. In the constrictions the marks of circularly running tubular structures were seen and were interpreted as being caused by circular bands of smooth muscle cells. Tissue sections of the corresponding vascular wall showed the presence of single or grouped smooth muscle cells which regularly formed myoendothelial junctions. These smooth muscle cells are interpreted as sphincters, responsible for the constrictions seen on cast preparations. Axon terminals were not found in spatial relationship to these sphincters. It is suggested that the described venous sphincters are governed by blood-borne and/or endothelium-derived substances and may significantly influence the blood flow.