Fluorescence microlymphography: diagnostic potential in lymphedema and basis for the measurement of lymphatic pressure and flow velocity

Fluorescence microlymphography (FML) is an almost atraumatic technique used to visualize the superficial skin network of initial lymphatics through the intact skin of man. Visualization was performed with an incident light fluorescence microscope following subepidermal injection of minute amounts of FITC-dextran 150,000 using microneedles. Emanating from the bright dye depot, the surrounding network of microvessels is filled, documentation performed by photography or video film. In congenital Milroy lymphedema, a lack of microlymphatics (aplasia) is typical while in other primary lymphedemas and in secondary lymphedema after mastectomy or irradiation of proximal lymph nodes, the network remains intact but the depicted area is enlarged. Lymphatic microangiopathy characterized by obliterations of capillary meshes or mesh segments develops in phleboedema with trophic skin changes, progressive systemic sclerosis and Fabry's disease. In lipedema, lymphatic microaneurysms are stained. Microlymphatic pressure may also be measured using FML. For this purpose, glass micropipettes are inserted into the capillaries by means of a micromanipulator and pressure is determined by the servo-nulling technique. Normal subjects produced significantly lower pressure (7.9 +/- 3.4 mmHg) compared to patients with primary lymphedema (15.0 +/- 5.1 mmHg, p<0.001). This characteristic lymphatic hypertension may be improved by complex physiotherapy or local application of prostaglandins. Additionally, a modification of the FML procedure can be used to measure lymphatic capillary flow velocity in controls and patients. FML is suited to confirm the clinical diagnosis of lymphedema, contributes to distinguish among various forms of edema, and is useful in clinical research. In addition, FML has also become a tool for experimental animal studies including the depiction of gastric microlymphatics, the measurement of flow velocity in the naked mouse tail, and in evaluation of lymphangiogenesis in a model of Milroy disease.     

Morphometric analysis of elastic fibers in human skin lymphatic capillaries

In contrast to their absence near dermal blood capillaries, elastic fibers are commonly seen adjacent to dermal lymphatic capillaries under light microscopy. Based on morphometric analysis, the elastic fiber network that surrounds these skin lymphatic capillaries is predominantly oriented longitudinally to the lymphatic vessel wall. Quantitative analysis reveals that the density of these pericapillary elastic fibers are almost twice that of the intercapillary elastic fibers but only about one-half as thick. These data suggest that dermal lymph capillaries are surrounded by a specific elastic network of functional significance, morphologically distinct from that seen in the intercapillary dermis. Because lymphatic capillaries are often difficult to identify especially when collapsed, this elastic network may facilitate the positive identification of dermal lymphatic capillaries by light microscopy and thereby help differentiate them from blood capillaries. The possible role of this lymphatic elastic network in the absorptive activity of the dermal lymphatic system is also discussed.     

Microangiopathy of ethylic polyneuropathy.

The authors have studied, by biopsy of the tip of the big toe, the histologic changes in microvessels of patients affected by ethylic polyneuropathy. Patients affected by diabetes, hypertension, peripheral arterial or venous diseases, or dermatologic diseases or vasculitides were excluded. The mean of daily drinks was 310 +/- 105 g of alcohol, chiefly wine. Important pathologic modifications were noted; complete narrowing of the arteriovenous anastomosis, regressive changes of the Vater-Pacini corpuscles, and dilatation of the small veins and the capillaries. The findings were compared with those of a patient affected by ulcerans and mutilans acropathy of Bureau and Barriere, a typical disease of alcoholics. The same characteristics appeared: profound changes of the structure of the smallest vessels, with sclerosis, extended to the interstitium. In a previous study of the authors, 3 other patients affected by Bureau-Barriere disease showed the same pattern. These alterations can be explained as the effect of alcohol on the microvessels.     

Scanning electron microscopy of injection replicas of the cardiac microvessels

The paper concerns three-dimensional study of the organization of the subepicardial network of the blood and lymphatic microvessels of the heart with the aid of scanning electron microscopy of the injected replicas. It is established that the arterioles have regular orientation of the imprints of the nucleus-containing zone of endotheliocytes. The capillaries belonging to the subepicardial network form a thick mesh and are strictly oriented along the muscular bundles. There is no characteristic relief on the surface of the capillaries. The study of the relief of the post-capillary venules made it possible to record different orientation of the imprints of the nuclei-containing zones of endotheliocytes on their surface. The study of structure of "leaks" formed as a result of leaking of resin from the venular segments of the bed enabled one to elicit their passage into the lymphatic capillaries. The latter are represented by flattened tubes, placed over the blood vessels.     

Diameters of lymphatic capillaries in patients with different forms of primary lymphedema

Fluorescence microlymphography was performed near the medial ankle in 12 healthy controls, 12 patients with congenital and 12 with sporadic lymphedema of the lower extremities. Diameters of lymphatic capillaries were determined on the videoscreen by playback of the tape recordings using a morphometric technique. In the patients with congenital disease (Nonne-Milroy) aplasia of microlymphatics was diagnosed in 8 instances, ectasia in 4. Patients with sporadic lymphedema and manifestation after puberty exhibited initial lymphatics of normal caliber. In controls mean capillary diameter was 56.3 +/- 9.0 microns, in congenital disease with ectasia 99.3 +/- 9.0 microns (difference significant at the p less than 0.005 level) and in the sporadic form 49.7 +/- 7.7 microns. Congenital lymphedema may be subdivided into a form with aplastic and ectatic microvessels which possibly show different responses to therapy.     

Differential requirement for ROCK in dendritic cell migration within lymphatic capillaries in steady-state and inflammation

Dendritic cell (DC) migration via lymphatic vessels to draining lymph nodes (dLNs) is crucial for the initiation of adaptive immunity. We imaged this process by intravital microscopy (IVM) in the ear skin of transgenic mice bearing red-fluorescent vasculature and yellow-fluorescent DCs. DCs within lymphatic capillaries were rarely transported by flow, but actively migrated within lymphatics and were significantly faster than in the interstitium. Pharmacologic blockade of the Rho-associated protein kinase (ROCK), which mediates nuclear contraction and de-adhesion from integrin ligands, significantly reduced DC migration from skin to dLNs in steady-state. IVM revealed that ROCK blockade strongly reduced the velocity of interstitial DC migration, but only marginally affected intralymphatic DC migration. By contrast, during tissue inflammation, ROCK blockade profoundly decreased both interstitial and intralymphatic DC migration. Inhibition of intralymphatic migration was paralleled by a strong up-regulation of ICAM-1 in lymphatic endothelium, suggesting that during inflammation ROCK mediates de-adhesion of DC-expressed integrins from lymphatic-expressed ICAM-1. Flow chamber assays confirmed an involvement of lymphatic-expressed ICAM-1 and DC-expressed ROCK in DC crawling on lymphatic endothelium. Overall, our findings further define the role of ROCK in DC migration to dLNs and reveal a differential requirement for ROCK in intralymphatic DC crawling during steady-state and inflammation.     

Dermal lymphatics in myotonic dystrophy.

Myotonic dystrophy is an hereditary disorder of several organ systems. Skeletal muscle is a principal target organ, but abnormalities also occur in the peripheral microcirculation. Because morphological and functional changes in the dermal blood microcirculation may affect interstitial fluid drainage of the skin, we examined dermal lymphatic morphology in adult patients with myotonic dystrophy. Skin biopsies were taken from the big toe from patients with myotonic dystrophy (age 18-50 years) and subjected to light and electron microscopy; five healthy subjects served as controls. The salient findings in myotonic dystrophy were ultrastructural changes of the lymphatic endothelial cells and the fibrillar elements that surround the lymphatic wall. These abnormal lymphatic findings are interpreted in light of changes in the blood microvasculature and loose connective tissue in this disorder.     

Lymphatic microangiopathy of the skin in systemic sclerosis.

METHODS: The cutaneous capillary lymphatic system in patients with systemic sclerosis was investigated using fluorescence microlymphography. The distal upper limbs of 16 healthy controls (mean age 62.3+/-13.1 yr) and 16 patients with systemic sclerosis (mean age 58.9+/-13.6 yr) were examined and the following parameters were evaluated: (a) single lymphatic capillaries; (b) lymphatic capillary network and cutaneous backflow; (c) extension of the stained lymphatics; (d) diameter of single lymphatic capillaries. RESULTS: At the finger level, lymphatic capillaries were lacking in five patients, while they were present in all controls (P < 0.05). Extension of the stained lymphatics was increased in 11 patients (8.1+/-6.0 mm) compared to the 16 healthy controls (2.0+/-1.2 mm) (P < 0.0001). Cutaneous backflow was observed in three patients (P < 0.05). At the hand level, lymphatic network extension was significantly different between patients (3.8+/-2.4 mm) and controls (1.2+/-0.8 mm) (P < 0.01); however, no significant differences were found at the forearm level. CONCLUSION: Lesional skin in patients with systemic sclerosis exhibits evidence of lymphatic microangiopathy.     

Microlymphatic aneurysms in patients with lipedema.

"Lipedema," a special form of obesity syndrome, represents swelling of the legs due to an increase of subcutaneous adipose tissue. In 12 patients with lipedema of the legs and in 12 healthy subjects (controls), fluorescence microlymphography was performed to visualize the lymphatic capillary network at the dorsum of the foot, at the medial ankle, and at the thigh. Microaneurysm of a lymphatic capillary was defined as a segment exceeding at least twice the minimal individual diameter of the lymphatic vessel. In patients with lipedema, the propagation of the fluorescent dye into the superficial lymphatic network of the skin was not different from the control group (p > 0.05). In all 8 patients with lipedema of the thigh, microaneurysms were found at this site (7.9 +/- 4.7 aneurysms per depicted network) and in 10 of the 11 patients with excessive fat involvement of the lower leg, multiple microlymphatic aneurysms were found at the ankle region. Two obese patients showed lymphatic microaneurysms in the unaffected thigh and in only 4 patients were microaneurysms found at the foot. None of the healthy controls exhibited microlymphatic aneurysms at the foot and ankle, but in one control subject a single microaneurysm was detected in the thigh. Multiple microlymphatic aneurysms of lymphatic capillaries are a consistent finding in the affected skin regions of patients with lipedema. Its significance remains to be elucidated although its occurrence appears to be unique to these patients.     

Structure and distribution of lymphatic capillaries and fenestrated blood capillaries in the conduction system of the rabbit heart

Summary The distribution and structure of lymphatic and blood capillaries in the rabbit heart conduction system were investigated by transmission electron microscopy. The sinuatrial node, atrioventricular node, and atrioventricular bundle possessed a rich network of lymphatic capillaries, which were situated not only at the periphery but also in the interior of the conduction system. The fine structure of these lymphatic capillaries was essentially similar to those within the atria and the ventricles. Although blood capillaries within working myocardium were nonfenestrated, the heart conduction system was often supplied by fenestrated blood capillaries. In the atrioventricular node and bundle especially, fenestrated blood capillaries and lymphatic capillaries were topographically associated, forming an extensive microcirculatory system. The presence of fenestrated capillaries suggests that a fast transcapillary passage of metabolites occurs in these regions, while the lymphatic capillaries may play an important role in the removal of macromolecules and excess intercellular fluid.     

Morphology of lymphatics in human venous crural ulcers with lipodermatosclerosis

A morphological evaluation of lymphatic vessels of skin leg ulcers was performed in 39 human subjects with longstanding venous insufficiency and lipodermatosclerosis. Light and electron microscopy demonstrated that the superficial fibrin and inflammatory cell layers and intermediate blood capillary layer of the ulcer bed, which were primarily granulation tissue, did not contain lymphatics. Moreover, lymphatic capillaries were present only sporadically in the transition zone from granulation tissue to the deeper collagenous scar layer of the ulcer. In some instances, in the deepest part of the ulcer bed near the crural fascia, there were one or two thicker lymphatic collectors with valves, which were continuations of collectors from the plantar foot region. Lymphatics were present at the border of the ulcer and in lipodermatosclerotic skin, but the endothelium and muscle lining layer were partially destroyed. Lymphatic capillaries were characterized by open interendothelial junctions in conjunction with subendothelial edema. In lipodermatosclerotic skin, the morphologic changes suggest that absorption of interstitial fluid and lymph is markedly disturbed adjacent to the ulcer bed, which likely contributes to both slow healing and high recurrence of skin ulcers associated with longstanding venous insufficiency.     

Static magnetic fields affect capillary flow of red blood cells in striated skin muscle

Blood flowing in microvessels is one possible site of action of static magnetic fields (SMFs). We evaluated SMF effects on capillary flow of red blood cells (RBCs) in unanesthetized hamsters, using a skinfold chamber technique for intravital fluorescence microscopy. By this approach, capillary RBC velocities (v(RBC)), capillary diameters (D), arteriolar diameters (D(art)), and functional vessel densities (FVD) were measured in striated skin muscle at different magnetic flux densities. Exposure above a threshold level of about 500 mT resulted in a significant (P < 0.001) reduction of v(RBC) in capillaries as compared to the baseline value. At the maximum field strength of 587 mT, v(RBC) was reduced by more than 40%. Flow reduction was reversible when the field strength was decreased below the threshold level. In contrast, mean values determined at different exposure levels for the parameters D, D(art), and FVD did not vary by more than 5%. Blood flow through capillary networks is affected by strong SMFs directed perpendicular to the vessels. Since the influence of SMFs on blood flow in microvessels directed parallel to the field as well as on collateral blood supply could not be studied, our findings should be carefully interpreted with respect to the setting of safety guidelines.     

Molecular differentiation and specialization of vascular beds

Transport in the large and complex bodies of vertebrate organisms is mediated by extensive and highly branched tubular networks that are formed by endothelial cells. Blood vessels are responsible for systemic circulation, while the lymphatic vasculature drains extravasated plasma, proteins, particles, and cells from the interstitium. Endothelial cells of blood vessels and lymphatic vessels can be distinguished by the expression of certain molecular markers, which accompany or even contribute to functional and morphological differences. Even within the blood vessel network, some molecules and pathways selectively mark the endothelium of arteries, veins and capillaries and are thought to contribute to the differentiation of these vessels. Moreover, microvessels can acquire organ-specific specialization in response to local tissue-derived signals. This review summarizes molecular markers and pathways that are specifically expressed in the endothelium of certain vascular beds and vessel types. Special attention will be given to known functional roles in the morphogenesis of these vessels.     

Pulmonary resistance in cardiovascular context

Comparison of the human cardiovascular system with arrangements of circulatory systems found in lower vertebrates and invertebrates allows appreciation of the functional elegance of our double circulation with systemic and pulmonary vascular trees served by a single looped and septated heart. In the pulmonary part of the circulation, consideration of the nature of alveolar microvessels in relation to the system as a whole may throw light on the pathophysiology of pulmonary regurgitation and pulmonary hypertension. Pulmonary microvessels impose remarkably little resistance to flow compared with the systemic. This may be attributed to their delicate, compliant structure, with tissue support on one side only, their respiratory walls remaining relatively free to expand in alveolar air. Low resistance may also depend on the branch pattern of alveolar capillaries, with almost immediate proximity between bifurcations and confluences in a uniquely dense, interconnected network. In the presence of free pulmonary regurgitation, pulmonary microvessels probably play a valve-like role, representing a low-resistance boundary or watershed between pulmonary arteries and veins. This microvascular watershed imposes little resistance to systolic forward flow, but in diastole, with venous pressures being kept low by function of the left heart, there is presumably little or no reversal of gradient to move blood back through the capillaries. The delicacy and potential vulnerability of alveolar capillaries to elevation of flow and pressure is likely, however, to go with a protective feedback circuit which, in abnormal circumstances, could contribute to development of arteriolar medial hypertrophy and pulmonary arterial hypertension.     

Deformation of Erythrocytes in Microvessels and Glass Capillaries: Effects of Erythrocyte Deformability

Objective: The deformation of erythrocytes in microvessels less than 15 μm in inner diameter was analyzed using a microvascular bed isolated from rabbit mesentery. The deformation was compared with that found in glass capillaries. Methods: Human erythrocytes were perfused through two media: first, a microvascular-bed section isolated from rabbit mesentery; and second, a set of glass capillaries. Images of deformed erythrocytes were recorded on videotape under strobe light and analyzed with an image processor. The flow velocity of the erythrocytes was determined from the difference of their positions between video frames or by a dual-spot cross-correlation technique. Erythrocyte deformability was modified with diamide, diazene dicarboxylic acid bis[N,N-dimethylamide], by crosslinking spectrins. Results: Symmetrical (parachute-like or slipper-like) deformation of erythrocytes was observed only in microvessels smaller than 13 μm in inner diameter. Erythrocytes in microvessels were less deformed than those in glass capillaries with corresponding diameters, and the marginal cell-free layer was narrower. The deformation increased by increasing the flow velocity of erythrocytes, and the cell-free layer became wider. Diamide-treated cells in microvessels were less deformed than normal cells and showed slightly narrower cell-free layers. Stronger stress in narrower microvessels induced further deformation of cells. Conclusions: Erythrocyte deformation in microvessels was essentially different from that in glass capillaries, and the effect of erythrocyte deformability on the flow dynamics of erythrocytes in microvessels was properly evaluated using an isolated microvascular bed.     

Effects of ischemia on capillary density and flow velocity in nailfolds of human toes

The purpose of this study was to investigate reactive hyperemia in the capillary network of human skin in terms of the flow per capillary and the density of flow-active capillaries. Seventeen male subjects 20 to 40 years of age were seated with their right foot placed on the stage of a Leitz epi-ilumination microscope such that the nailfold capillary field in their large toes could be viewed. These vessels were video taped while flow velocity in the right posterior tibial artery was recorded via Doppler ultrasound at rest, then following a 45-sec period of arterial occlusion to the foot. Subsequent to experimentation flow velocity in single nailfold capillaries was measured via video densitometry and the number of flow-active capillaries in the field of view were counted. Following the release of arterial occlusion arterial flow velocity increased 142% above rest, the velocity in single capillaries increased by 54%, and the density of flow-active capillaries, as identified by the presence of red cells, decreased by 37%. The fact that capillary flow velocity increased to a lesser degree than arterial velocity during reactive hyperemia vis-a-vis a decrease in the number of flow-active capillaries indicates that ischemia to the foot elicits a smaller dilatory effect in vascular elements controlling blood flow to the superficial cutaneous region of the toe as compared to other regional vascular networks.     

Ultrastructural comparative study on lymphatic capillaries of the subendocardium, myocardium, and subepicardium of the heart left ventricle

A comparative study, with quantitative analysis, was done on the fine structure of the lymphatic capillaries in the subepicardium, myocardium, and subendocardium of the left ventricle from dog hearts. In the three layers, the vesicular system of lymphatic capillaries was quite similar. More than 63% of the plasmalemmal vesicles are in contact with the abluminal or luminal endothelial membrane of cardiac lymphatic capillaries. Volume density measurements showed that plasmalemmal vesicles occupied between 8.0 and 9.3% of the endothelial cytoplasm. Overlapping and interdigitation contacts were frequent, whereas end to end contacts were scarce. The frequency of open junctions wider than 50 nm was inferior to 1%. The intercellular cleft was often obstructed by a focal tight junction (macula occludens). Intercellular spaces without specialized junctions were more frequent with subepicardial and myocardial lymphatic capillaries as compared with subendocardial vessels. Important quantitative variations of the fine structure were encountered on the abluminal side of the endothelial wall. In the subendocardium, the basement membrane covered 33% of the lymphatic endothelium whereas for the subepicardium and myocardium lymphatic capillaries, the values were reduced significantly to 18% (P less than 0.05) and 10% (P less than 0.001), respectively. An inverse relation was observed between the proportion of endothelium covered by basement membrane and the frequency of abluminal endothelial projections associated with the endothelial wall. Endothelial projections (EP) decreased in the following order: myocardium (19 EP/100 micron), subepicardium (15 EP/100 micron), and subendocardium (10 EP/100 micron). Quantitative morphological variations in the fine structure of lymphatic capillaries are believed to result from physiological (muscular movements, metabolic activity) and morphological (thickness of the connective tissue support surrounding lymphatic capillaries) differences existing between the three cardiac layers.     

Skin Capillary Circulation is More Impaired in the Toes of Diabetic Than Non-diabetic Patients with Peripheral Vascular Disease

The aim of the present study was to investigate if diabetes negatively influences the skin microvascular reactivity in the toes of patients with peripheral vascular disease (PVD). Twenty healthy subjects, 20 diabetic, and 20 non-diabetic patients with PVD participated. One foot in each subject was investigated. The patient groups were matched for age, sex, and toe pressure. The capillary blood cell velocity in the nailfold of the great toe was investigated by videophotometric capillaroscopy, and the total skin microcirculation within the same area by laser Doppler fluxmetry. Capillary blood cell velocity and laser Doppler flux were studied during rest, and following a 1 min arterial occlusion at the toe base. The skin microvascular reactivity was impaired in both diabetic and non-diabetic patients. In the diabetic patients the disturbances were mainly seen in the capillaries, and the capillary blood flow was severely reduced during reactive hyperaemia (p<0.01). In contrast, the total skin microcirculation was normal, indicating that sufficient blood reaches the area, but does not come out into the capillaries. The ratio between capillary blood cell velocity and laser Doppler flux, representing the distribution of blood between nutritional and non-nutritional blood compartments, was reduced in the diabetic patients (p<0.05). These findings may contribute to the higher risk for development of chronic foot ulcers in diabetic patients with PVD.     

Microvascular structure of benign and malignant tumors of the colon in humans

Studies of experimental tumors in rodents indicate that there are morphological abnormalities of the tumor microcirculation compared to normal tissues. The aim of this study was to examine the structure of the microvasculature in benign and malignant colonic tumors in humans using microvascular casting techniques. There were 15 adenocarcinomas, four benign sporadic adenomas, and three specimens from patients with familial adenomatous polyposis (FAP). A cast of the microvessels of these tumors was prepared by intraarterial administration of acrylic resin (Mercox) and the cast examined by scanning electron microscopy. Quantitative measures of the microvasculature were obtained from histological sections using stereological techniques in four carcinomas, two sporadic adenomas, and 12 adenomas from patients with FAP. Vascular casts of benign colonic adenomas showed that the microvasculature had a similar organization to normal colon. However, capillaries and venules were elongated and had increased diameters compared to normal. In adenomas greater than 3 mm in diameter, there was an increased density of microvessels in the spaces between tumor cells. Vascular casts of colonic carcinomas were characterized by a disorganized structure and increased density of microvessels. The organization of microvessels within carcinomas had a similar overall pattern to normal colon. However, the increased number and density of microvessels resulted in formation of nodular clusters of capillaries, formation of “sheets” of frequently anastomosing capillaries, or almost complete packing of the interstitial spaces of the tumor by capillaries in places. Most capillaries had a long and tortuous course and numerous capillary sprouts were identified. Tumor microvessels had greater mean diameters than normal. Extravasation of resin from microvessels in carcinomas was frequently seen. The vascular volume of carcinomas (23.1%±12.2), sporadic adenomas (16.3%±3.4), and adenomas >3 mm diameter in patients with FAP (17.7%±3.0) were significantly greater than in normal colon (11.0%±4.2). This study indicates that there is an increased vascular density in benign and malignant tumors of the colon compared to normal colon. The presence of profusely anastomotic microvessels and frequent capillary sprouts is evidence of active neovascularization and suggests control of tumor growth could be achieved by modifiers of angiogenesis.