The cytoarchitecture of the wall and the innervation pattern of the microvessels in the rat mammary gland: a scanning electron microscopic observation

This report describes the morphology of the smooth muscle cells, pericytes, and the perivascular autonomic nerve plexus of blood vessels in the rat mammary gland as visualized by scanning electron microscopy after removal of connective-tissue components. From the differences in cellular morphology, eight vascular segments were identified: 1) terminal arterioles (10-30 microns in outer diameter), with a compact layer of spindle-shaped and circularly oriented smooth muscle cells; 2) precapillary arterioles (6-12 microns), with a less compact layer of branched smooth muscle cells having circular processes; 3) arterial capillaries (4-7 microns), with " spidery " pericytes having mostly circularly oriented processes; 4) true capillaries (3-5 microns), with widely scattered pericytes having longitudinal and several circular processes; 5) venous capillaries (5-8 microns), with spidery pericytes having ramifying processes; 6) postcapillary venules (10-40 microns), with clustered spidery pericytes; 7) collecting venules (30-60 microns), with a discontinuous layer of circularly oriented and elongated stellate or branched spindle-shaped cells which may represent primitive smooth muscle cells; and 8) muscular venules (over 60 microns), with a discontinuous layer of ribbon-like smooth muscle cells having a series of small lateral projections. No focal precapillary sphincters were found. The nerve plexus appears to innervate terminal arterioles densely and precapillary arterioles less densely. Fine nerve fibers are only occasionally associated with arterial capillaries. Venous microvessels in the rat mammary gland seemingly lack innervation.     

Hints of a functional connection between the neuropeptidergic innervation of arteriovenous anastomoses and the appearance of epithelioid cells in the rabbit ear

Peripheral blood flow can be regulated by specialized vessel segments, the arteriovenous anastomoses. Their wall consists of a relatively thick layer of smooth muscle cells and so-called epithelioid cells. The epithelioid cell is a specialized myogenic cell phenotype expressing nitric oxide synthase. We studied the innervation of the different segments of arteriovenous anastomoses in the rabbit ear using antisera against neuropeptide Y, tyrosine hydroxylase, calcitonin gene-related peptide and substance P, as well as neuron-specific enolase, calbindin D and neurotubulin. The participation was especially examined of neuropeptidergic innervation and a possible morphological connection to the occurrence of epithelioid cells and a paracrine function. The NADPH diaphorase reaction and -smooth muscle actin immunoelectron microscopy served to distinguish epithelioid cells from smooth muscle cells. Using conventional fluorescence microscopy and confocal laser scanning microscopy, we found the most dense innervation pattern of pan-neuronal markers (neurotubulin, neuron-specific enolase), tyrosine hydroxylase-immunor eactive nerve fibres and neuro-peptidergic nerve fibres (neuropeptide Y, calcitonin gene-related peptide, substance P) around the intermediate segment in arteriovenous anastomoses, whereas the venous segment was barely marked. Single nerve fibres penetrated into the medial layer and reached the epithelioid cells. Using immunoelectron microscopy, we found intercellular contacts between epithelioid cells, but not the gap junction protein connexin 43. Here, we report for the first time a correlation of the innervation pattern with epithelioid cell type in arteriovenous anastomoses. Our findings suggest that epithelioid cells of the arteriovenous anastomoses are controlled by a dense network of neuropeptidergic nerve fibres in functional connection to their paracrine role as a nitric oxide producer. © 1998 Chapman & Hall     

Morphometry of the amount of smooth muscle cells in the media of various rabbit arteries

Our objective in this study was to evaluate the relative amount of smooth muscle cells in the medial layer of various rabbit arteries. The fixation of smooth muscle cells in the arterial wall is difficult and the differential effect of glutaraldehyde (GA) and fixative vehicle on cell ultrastructure in different tissues is controversial. We compared the effect of various concentrations of the vehicle and glutaraldehyde (osmolarity ranges for total fixative, 350-1030 mOsm) on the arterial wall ultrastructure. We found that a 600 mOsm GA solution (isotonic vehicle; 2.5% GA) adequately preserves arterial wall structures. The relative amount of smooth muscle cells in the media differed in various segments along the arterial tree. It ranged from 35% (thoracic aorta) to 74% (tibial artery). The importance of weighting the contractile response of different arteries in vitro to their relative smooth muscle cell content is discussed.     

Arterial wall neovascularization induced by glycerol

An intense and significant neovascularization, with numerous capillaries growing into the media layer of the rat femoral artery, was demonstrated when glycerol was administered into the interstitium between the femoral vein and the femoral artery. The maximum microvascularization was observed at days 7 and 9 after glycerol administration. Afterwards, involution of the majority of the newly-formed microvessels in the arterial wall occurred. Other substances containing glycerol in their molecules, such as triacetyl-glycerol and tributyril-glycerol, failed to produce significant neovascularization in the media layer of the femoral artery. Neovascularization of the arterial wall was preceded by a considerable decrease in the number of the smooth muscle cells, which experienced apoptosis and necrobiosis, disappearing in extense areas of the arterial segment affected by glycerol. Coinciding with neovascularization and microvascular involution, repopulation of the media layer by smooth muscle cells was observed.     

Regeneration of the arterial wall in microporous,compliant, biodegradable vascular grafts after implantation into the rat abdominal aorta

Summary The ultrastructure of a new type of vascular graft, prepared from a mixture of polyurethane (95 weight %) and poly-L-lactic acid (5 weight %), was examined six weeks after implantation into the abdominal aorta of rats. These microporous, compliant, biodegradable, vascular grafts function as temporary scaffolds for the regeneration of the arterial wall.Smooth muscle cells, covering the grafts, regenerated a neo-media underneath an almost completely regenerated endothelial layer (neo-intima). These smooth muscle cells varied in morphology from normal smooth muscle cells to myofibroblasts. They were surrounded by elastic laminae and collagen fibers.Macrophages, epithelioid cells, multinucleated giant cells, fibroblasts and capillaries were present in the disintegrating graft lattices. The epithelioid cells and multinucleated giant cells engulfed polymer particles of the disintegrating grafts.The regeneration of the endothelial and smooth muscle cells is similar to the natural response of arterial tissue upon injury. The presence of macrophages, epithelioid cells, multinucleated giant cells, fibroblasts and capillaries in the graft lattices resembles the natural response of tissue against foreign body implants. Both of these responses result in the formation of a neo-artery that possesses sufficient strength, compliance and thromboresistance to function as a small caliber arterial substitute.Supported by Grant nr. 82.042 from the Dutch Heart Foundation     

Morphology of smooth muscle cells in the rat thoracic duct

Summary The three-dimensional cytoarchitecture and ultrastructure of the smooth muscle cells in the wall of the rat thoracic duct were investigated by scanning and transmission electron microscopy. The muscle layer basically consists of a single layer of circularly arranged cells. The smooth muscle cell is fusiform or ribbon-like in shape, as in veins or venules with a similar or smaller diameter. Connections by spinous processes are observed between adjacent muscle cells along their length. Spot-like membrane contacts frequently occur in areas where facing membranes are closely apposed. These are thought to be gap junctions and may be responsible for electrical coupling and mechanical attachment. Large invaginations arranged regularly in rows on the surface of the smooth muscle cells can be observed. These invaginations are closely associated with a flattened sarcoplasmic reticulum, and caveolae tend to open into the invaginations.     

Study of neuropeptide Y-containing nerve fibers in the human penis

Summary Neuropeptide Y 1–36 (IR-NPY) immunoreactive nerve-fiber processes have been observed in tunicae of veins and arteries and in smooth muscles of the human penis taken at autopsy or during surgery by use of light-and electron-microscopic immunohistochemical techniques. Numerous IR-NPY nerve fibers were mostly concentrated in the inner part of the adventitia close to the media of the arterial and venous vessels and among the intracavernous smooth muscle cells. IR-NPY nerve fibers were less abundant in veins than in arteries. Positive somata were not observed in the penises. At the ultrastructural level, IR-NPY were localized exclusively in large, dense granules of nerve terminals by means of the postembedding immunogold technique. In the deep dorsal vein, IR-NPY nerve fibers were also located in the media formed by an outer circular and an inner longitudinal layer. In the intracavernous and dorsal arteries, they showed the highest density in the inner part of the adventitia. In the corpora cavernosa and in the corpus spongiosum, IR-NPY nerve processes were intermingled between the smooth-muscle fibers around the sinusoid spaces. IR-NPY nerve fibers were present in the cavernous nerves close to the central arteries. The urethra did not show any IR-NPY-positive nerve fibers. This peculiar distribution of IR-NPY nerve fibers suggested that they could participate in regulating arterial and venous blood flow and intracavernous smooth-muscle tone. NPY may therefore be of importance in some of the mechanisms of penile erection especially during detumescence.     

Pathophysiology of motility dysfunction in bowel obstruction: role of stretch-induced COX-2

In gastrointestinal conditions such as bowel obstruction, pseudo-obstruction, and idiopathic megacolon, the lumen of affected bowel segments is distended and its motility function impaired. Our hypothesis is that mechanical stretch of the distended segments alters gene expression of cyclooxygenase-2 (COX-2), which impairs motility function. Partial obstruction was induced with a silicon band in the distal colon of rats for up to 7 days, and wild-type and COX-2 gene-deficient mice for 4 days. Mechanical stretch was mimicked in vitro in colonic circular muscle strips and in primary culture of colonic circular smooth muscle cells (SMC) with a Flexercell system. The rat colonic circular muscle contractility was significantly decreased in the distended segment oral to obstruction, but not in the aboral segment. This change started as early as day 1 and persisted for at least 7 days after obstruction. The expression of COX-2 mRNA and protein increased dramatically also in the oral, but not aboral, segment. The upregulation of COX-2 expression started at 12 h and the effect persisted for 7 days. At 24 h after obstruction, the COX-2 mRNA level in the oral segment increased 26-fold compared with controls. This was not accompanied by any significant increase of myeloperoxidase or inflammatory cytokines. Immunohistochemical studies showed that COX-2 was selectively induced in the colonic SMC. In vitro stretch of colonic muscle strips or cultured SMC drastically induced COX-2 expression. Incubation of circular muscle strips from obstructed segment with COX-2 inhibitor NS-398 restored the contractility. The impairment of muscle contractility in obstructed colon was attenuated in the COX-2 gene-deficient mice. In conclusion, mechanical stretch in obstruction induces marked expression of COX-2 in the colonic SMC, and stretch-induced COX-2 plays a critical role in the suppression of smooth muscle contractility in bowel obstruction.     

18β-甘草次酸对Wistar大鼠脑微动脉平滑肌细胞间缝隙连接的影响

目的：探讨18β-甘草次酸对Wistar大鼠脑微动脉平滑肌细胞间缝隙连接的影响,为寻求强效和可逆的缝隙连接阻断剂提供实验依据。方法：去除脑微动脉段外层结缔组织后,应用全细胞膜片钳技术,观察不同种类的缝隙连接阻断剂对Wistar大鼠脑微动脉段上平滑肌细胞膜电容（Cinput）、膜电导（Ginput）和膜电阻（Rinput）的影响。结果：（1）Wistar大鼠脑微动脉段上平滑肌细胞Cinput高于消化分离的单个平滑肌细胞。（2）18β-甘草次酸（18pGA）能浓度依赖性的抑制Wistar大鼠脑动脉平滑肌细胞间的缝隙连接,IC50分别为2．0μM。当18βGA100μM时,Wistar大鼠脑微动脉段上平滑肌细胞的Cinput、Ginput或Rinput与单个平滑肌细胞十分接近。结论：1813GA可以浓度依赖性的抑制Wistar大鼠脑微动脉平滑肌细胞间缝隙连接。     

Predictive Factors of Late Venous Aortocoronary Graft Failure: Ultrastructural Studies

Background

Venous aortocoronary graft arterialization may precede a preterm occlusion in some coronary artery bypass grafting (CABG) patients. The aim of the present study was to identify ultrastructural variations in the saphenous vein wall that may have an impact on the development of venous graft disease in CABG patients.

Methods

The study involved 365 consecutive patients with a mean age of 62.9±9.4 years who underwent isolated CABG. The thickness and area of the whole venous wall, the tunica intima, the tunica media and the adventitia and the number and shape (length, thickness and length/thickness ratio) of the nuclei in the medial smooth muscle cells nuclei in the distal saphenous vein segments were evaluated by ultrastructural studies. Patients were followed up for 41 to 50 months (mean 45.1±5.1). Saphenous vein graft patency was assessed by follow-up coronary angiography. Logistic regression models were used to identify independent risk factors for late graft failure.

Results

In 71 patients significant lesions in the saphenous vein grafts were observed. The whole venous wall thickness (437.5 µm vs. 405.5 µm), tunica media thickness (257.2 µm vs. 211.5 µm), whole venous wall area (2.23 mm² vs. 2.02 mm²) and tunica media area (1.09 mm² vs. 0.93 mm²) were significantly larger for this group of patients than for those without graft disease. In the latter group more elongated smooth muscle cell nuclei (higher length/thickness ratio) were found in the tunica media of the saphenous vein segments. Thickening of the saphenous vein tunica media and chunky smooth muscle cell nuclei were identified as independent risk factors for graft disease development.

Conclusions

Saphenous vein tunica media hypertrophy (resulting in wall thickening) and chunky smooth muscle cell nuclei might predict the development of venous graft disease.     

Close relation of arterial ICC-like cells to the contractile phenotype of vascular smooth muscle cell

This work aimed to establish the lineage of cells similar to the interstitial cells of Cajal (ICC), the arterial ICC-like (AIL) cells, which have recently been described in resistance arteries, and to study their location in the artery wall. Segments of guinea-pig mesenteric arteries and single AIL cells freshly isolated from them were used. Confocal imaging of immunostained cells or segments and electron microscopy of artery segments were used to test for the presence and cellular localization of selected markers, and to localize AIL cells in intact artery segments. AIL cells were negative for PGP9.5, a neural marker, and for von Willebrand factor (vWF), an endothelial cell marker. They were positive for smooth muscle alpha-actin and smooth muscle myosin heavy chain (SM-MHC), but expressed only a small amount of smoothelin, a marker of contractile smooth muscle cells (SMC), and of myosin light chain kinase (MLCK), a critical enzyme in the regulation of smooth muscle contraction. Cell isolation in the presence of latrunculin B, an actin polymerization inhibitor, did not cause the disappearance of AIL cells from cell suspension. The fluorescence of basal lamina protein collagen IV was comparable between the AIL cells and the vascular SMCs and the fluorescence of laminin was higher in AIL cells compared to vascular SMCs. Moreover, cells with thin processes were found in the tunica media of small resistance arteries using transmission electron microscopy. The results suggest that AIL cells are immature or phenotypically modulated vascular SMCs constitutively present in resistance arteries.     

Phenotypic conversion leads to structural and functional changes of smooth muscle sarcolemma

Continuous changes in the length of smooth muscles require a highly organized sarcolemmal structure. Yet, smooth muscle cells also adapt rapidly to altered environmental cues. Their sarcolemmal plasticity must lead to profound changes which affect transmembrane signal transduction as well as contractility. We have established porcine vascular and human visceral smooth muscle cultures of epithelioid and spindle-shaped morphology and determined their plasma membrane properties. Epithelioid cells from both sources contain a higher ratio of cholesterol to glycerophospholipids, and express a less diverse range of lipid-associated annexins. These findings point to a reduction in efficiency of membrane segregation in epithelioid cells. Moreover, compared to spindle-shaped cells, cholesterol is more readily extracted from epithelioid cells with methyl-beta-cyclodextrin and its synthesis is more susceptible to inhibition with lovastatin. The inability of epithelioid cells to process vasoactive metabolites, such as angiotensin or nucleotides further indicates that contractile properties are impaired. Phenotypic plasticity extends beyond the loss of smooth muscle cell marker genes. The plasma membrane has undergone profound functional changes which are incompatible with cyclic foreshortening, but might be important in the development of vascular disease.     

Enzymatic heterogeneity of the capillary bed of rat skeletal muscles

This study of the capillaries in rat skeletal muscle involved the use of a histochemical method that allows one to distinguish between arterial and venous portions of capillaries. Under controlled staining conditions, the arterial portion of the capillary bed reacts positively for alkaline phosphatase (AP) activity, and the venous portion is positive for dipeptidyl peptidase IV (DPP IV) activity. A short transitional capillary segment is positive for the activity of both enzymes. Capillaries of the normal soleus muscle and the red and white portions of the sternomastoid muscle have been quantitatively analyzed. Quantitative data demonstrated differences in capillary dimensions among the muscles studied. Capillaries of the white part of the sternomastoid were the longest, and they had the shortest DPP IV-positive segment (8% of the total capillary length). Capillaries of the soleus muscle were the shortest, and they also had short DPP IV-positive segments (16%). In contrast, the DPP IV-positive segments of the red part of the sternomastoid occupied 60% of the total capillary length. Survey cross sections reveal a mosaic distribution of patches of capillaries stained for AP and DPP IV activity. This study reveals that within given bundles of muscle fibers, the capillaries that run parallel to the muscle fibers are aligned relative to one another in such a manner that their arterial and venous segments are in register.     

The fine structure of smooth muscle cells and their relationship to connective tissue in the rabbit portal vein

Summary The smooth muscle of rabbit portal vein was studied by electron microscopy with particular emphasis on the mechanical linkage between the muscle cells and on the distribution of connective tissue.The media of this vein is composed of inner circular and outer longitudinal muscle layers which are orientated almost perpendicularly to each other. The muscle of the inner circular layer shows very irregular contours with much branching and anastomosing of the cytoplasmic processes, which often make membrane contacts with neighbouring cells to form an extensive network of cytoplasmic processes. The muscle cells of the outer longitudinal layer are arranged in densely packed bundles and are spindle-shaped, with no branching processes. Opposing dense areas from neighbouring cells, with variable gap distances (30–100 nm) and close membrane contacts (intermediate junctions) with a gap of 11 nm were observed in both circular and longitudinal muscle layers.In the terminal regions of muscle cells in both circular and longitudinal layers a specialized anchoring structure was present which was closely related to extracellular elastic tissue. Muscle cells in the longitudinal layer showed the most elaborate structure, the tapering end of the muscle cell showing a honeycomb-like structure penetrated by columns of connective tissue compounds. The functional implications of these structures are discussed.     

Trophoblastic invasion and the development of uteroplacental arteries in the macaque: immunohistochemical localization of cytokeratins,desmin, type IV collagen,laminin, and fibronectin

The processes by which trophoblast cells invade and modify the walls of the uteroplacental arteries of macaques during the course of gestation were examined. Antibodies to cytokeratins were employed to identify trophoblast, anti-desmin antibody to identify smooth muscle, and antibodies to type IV collagen, laminin, and fibronectin to examine changes in extracellular matrix distribution in the arterial wall. During early gestation, endovascular trophoblast adhered to the arterial wall, often in an asymmetrical distribution. As trophoblast cells moved outwardly into the tunica media, the basement membrane underlying the endothelium was lost, as indicated by gaps in the layer when stained for type IV collagen and laminin. Trophoblast cells became sequestered in the vessel wall where they hypertrophied and became surrounded by a capsule containing type IV collagen and laminin. As the trophoblast cells became established in the vessel wall, the muscular layer of the artery became discontinuous. Throughout gestation it was common for trophoblast cells to invade the vessel intimal layer and share the lining of the artery with typical endothelial cells. This general disposition of endovascular and intramural trophoblast persisted into late gestation. In addition, and contrary to the results of earlier studies of macaques, we identified trophoblastic invasion and modification of myometrial segments of the uteroplacental arteries in later gestation. We also found evidence of interstitial trophoblast cells among the stromal cells of the endometrium, especially during early gestation.     

Vascular anatomy of the lateral musculature of the flathead,Platycephalus bassensis (Teleostei: Perciformes)

Summary The vascular anatomy of the lateral musculature of the flatheadPlatycephalus bassensis, was studied by scanning electron microscopy of corrosion casts. Arteries and veins showed an alternating pattern in neighbouring vertebral segments. The red muscle was supplied by five major branches of the intermuscular artery, and the white muscle by infrequent branches of the intermuscular artery, dorsal segmental artery and ventral segmental artery. Venous drainage of the red and white muscles broadly mimicked the arterial supply. The functional unit of the trunk vasculature can be considered as an artery, a vein and connecting fine blood vessels. There appear to be 2 over-lapping types leading to alternating clockwise and counter-clockwise flows of blood. Small satellite vessels were observed running parallel to most of the larger blood vessels. No anatomical A-V shunt vessels, or series vascular connections between the red and white muscle, were observed. The irregular, alternating adult system is postulated to have developed from an earlier system showing strict bilateral symmetry and equal arterial and venous development in each vertebral segment.     

Co-expression of cytokeratins and vimentin by highly invasive trophoblast in the white-winged vampire bat, Diaemus youngi, and the black mastiff bat, Molossus ater, with observations on intermediate filament proteins in the decidua and intraplacental trophoblast

Histological and immunocytochemical studies of gravid reproductive tracts obtained from the white-winged vampire bat (Diaemus youngi) and the black mastiff bat (Molossus ater) have established that both species develop unusually invasive trophoblast. This is released by the developing discoidal haemochorial placenta, expresses both cytokeratins and vimentin, and invades the myometrium and adjacent tissues (including the ovaries) via interstitial migration within the walls of maternal blood vessels. Hence, this trophoblast is noteworthy for the extent to which it undergoes an epithelial-mesenchymal transformation. In Molossus, it originates from the cytotrophoblastic shell running along the base of the placenta, is mononuclear, and preferentially invades maternal arterial vessels serving the discoidal placenta. This trophoblast may have a role in dilatation of these vessels when the discoidal placenta becomes functional. In Diaemus, the highly invasive trophoblast appears to originate instead from a layer of syncytiotrophoblast on the periphery of the placenta is multinucleated, and vigorously invades both arterial and venous vessels. During late pregnancy, it becomes extensively branched and sends attenuated processes around many of the myometrial smooth muscle fibres. In view of its distribution, this trophoblast could have important influences upon myometrial contractility and the function of blood vessels serving the gravid tract. Other aspects of intermediate filament expression in the uteri and placentae of these bats are also noteworthy. Many of the decidual giant cells in Molossus co-express cytokeratins and vimentin, while the syncytiotrophoblast lining the placental labyrinth in Diaemus late in pregnancy expresses little cytokeratin.     

Morphological and functional changes of rabbit mesenteric artery cultured with fetal bovine serum

The aim of this study was to examine the morphological and functional changes in rabbit mesenteric arterial tissue cultured with fetal bovine serum. In the endothelium-denuded arteries cultured under a serum-free condition for one week (serum-free arteries), morphology of the smooth muscle layer was intact. In the serum-free arteries, high K+ -induced contraction did not change but norepinephrine-induced contraction slightly decreased compared with that in the freshly isolated arteries, whereas the sensitivity to these stimulants was significantly augmented. In the medial layer of the arteries cultured with 10% fetal bovine serum for one week (serum-treated arteries), proliferation, disorientation and death of smooth muscle cells were observed. In the serum-treated arteries, both the amplitude of contractions induced by high K+ and norepinephrine and the sensitivity to these stimulants were significantly reduced compared with those of the serum-free arteries. The reduced norepinephrine-induced contraction in the serum-treated arteries was partially recovered by adding NG-monomethyl-L-arginine (L-NMMA), a nitric oxide (NO) synthase inhibitor, to the assay medium. In alpha-toxin permeabilized arteries, the amplitude of Ca2+ -induced contraction and the sensitivity of the contractile apparatus to Ca2+ were significantly reduced after serum-treatment. These results suggest that chronic serum-treatment of rabbit mesenteric arteries impairs muscle contractility by the morphological and phenotypic changes in smooth muscle cells. NO production in smooth muscle cells is also responsible for the decreased contractility after the serum-treatment.     

Neuronal systems immunoreactive with antiserum to lamprey gonadotropin-releasing hormone in the brain of Petromyzon marinus

Summary The wall structure of arteriovenous anastomoses in the rabbit ear was investigated. (1) Clusters of epithelioid smooth muscle cells form 3–4 longitudinally oriented plicae. The channel shows a single, irregularly outlined lumen, and its wall is very thin between adjacent plicae. (2) Endothelial cells covering the plicae protrude into the lumen, thus suggesting active contraction or shortening of the plicae. (3) The tunica adventitia is composed of 4–6 sheaths of flat fibroblasts, which may serve as a barrier to prevent loss of neurotransmitters. Processes of some of the fibroblasts also extend into the tunica media. (4) The tunica media is composed of an outer circular layer of typical smooth muscle cells, and an inner longitudinally running plica of ramified smooth muscle cells. Wide intercellular spaces between these ramified cells are filled with collagen fibrils, microfibrils, amorphous intercellular substances, and fibroblasts. Fibroblasts form close membrane contacts with each other, and with the smooth muscle cells. (5) Fibroblasts and other connective tissue components may function as an elastic support during active motility of the anastomotic channel.