Combined effect of chronic partial occlusion and orthostatic load on the saphenous vein network: A varicosity model in the rat

Abstract

Objectives

We tested the combined effects of chronic flow obstacle and gravitation on the saphenous vein network of rats.

Methods

A narrowing clip (500 µm, partial occlusion) was administered on the saphenous vein main branch for 4, 8 and 12 weeks, either separately or in combination with chronic orthostatic load (tilted tube-cages for four weeks). Resulting network changes were studied on plastic casts, by video-microscopy, histochemistry–immunohistochemistry and image analysis.

Results

A rich collateral venous network developed containing newly formed masses of retrograde conducting small veins. Their walls had less dense elastica, less contractile protein, increased cell division activity and macrophage invasion, and were more sensitive to chronic gravitational load.

Conclusions

Hemodynamic disturbance induces remodeling of the saphenous vein network. Walls of veins being in the process of flow-induced morphological remodeling are weak and more sensitive to gravitational load. Reticular vein conglomerates, veins with local dilations, and convoluted courses were observed.

Keywords

Vein varicosity remodeling gravitational load collateral network venous reflux

Introduction

Lower extremity vein varicosity affects around a quarter of the population.^1–6 Gravitational stress,^4,7–10 hemodynamic disturbance,^7,11–16 local inflammation,^17–19 in combination with genetic factors^18,20–22 are in the background of the human leg varicosity disease. Despite the fact that these initiating factors have been identified, many steps of the pathomechanism have not been revealed yet. Attempts to develop model varicosity in animals have only partially been successful until now.^16,23–26

Previously, we have shown that chronic (four weeks) head-up maintenance of rats in tilted tube-like cages elevates leg venous pressure and induces several morphological and cellular physiological alterations in the saphenous vein wall and network that can be assumed to play an important role in the pathomechanism of the human varicosity disease.^{9,10,24,27,28} Modern clinical observations with ultrasonography confirm an early report by Fegan and Kline⁷ about the significance of flow disturbances^{13–15,29,30} in addition to the elevated venous pressure^2,11,30–34 in initiation and progress of human leg venous varicosity. Recent work by Pfisterer et al.¹⁶ reports on successful induction of a pathologic morphology resembling early phases of the human varicosity disease by occlusion of the auricular vein in the mice. Based upon these observations, we decided to induce flow disturbance in the saphenous vein network of rats by applying a narrowing clip on the main branch and studying the induced changes in network geometry. We have found that not dilation, but involution of the main branch did occur.³⁵ Venous flow has been diverted from the main branch through a newly developed system of collateral veins. The development of these pathologic vascular neoformations with retrograde flow, their morphological appearance, hemodynamics, histology and sensitivity to gravitation will be discussed in this paper. Our question was whether a varicous morphology can be induced by a combined application of venous flow disturbance and gravitational stress in animal experiments.

Materials and methods

Studies were started on young, adult male Wistar rats weighing 190–210 g at initiation of study. In anesthetized animals (Pentobarbital, 50 mg/kg ip.), under sterile conditions, a plastic clip (500 µm) was positioned around the saphenous vein, a few mm below its confluence with the deep femoral vein (Figure 1(a)). It induced only partial occlusion of blood flow. The unclipped right side saphenous vein network served as control. In an additional group of rats, a sham operation was performed. Operation did not induce substantial stress to the animals, no local infection, edema or altered leg movements were observed. Occlusions lasted in separate groups 4, 8 and 12 weeks. Further groups of animals, also with 4,8 and 12 weeks of occlusion, were kept in the last four weeks before sacrifice in tube-like cages tilted at 45° head-up position²⁷ (Figure 1(b)) to elevate gravitational load. Food and water were offered at the top of the tube-cage in which the animals could freely move up and down but they could not turn around. One hour of grooming each day outside the cage was ensured for them. They easily acclimated to their new homes and at the end of grooming time they voluntarily reentered it. All interventions applied during the study conform to the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes, NIH guidelines and local regulations. The program has been accepted by the Animal Care Committee of the Semmelweis University and Hungarian authorities (PEI 001/801-2/2015).

Figure 1.

Saphenous vein stricture and collateral venous network development. (a) The chronic narrowing clip. (b) Chronic tilted cage maintenance to elevate gravitational load. (c) Retrograde conducting collateral venous network, similar to human reticular veins. Four weeks occlusion, immediately after Batson 17 filling. (d) Batson 17 cast of saphenous vein after four weeks of stricture, rich retrograde filling collateral branches at the occluded side (right) while hardly any retrograde conducting side branches at the contralateral nonoccluded side (left). (e) Local dilations, contorted course of collaterals bypassing the site of occlusion (eight weeks of occlusion, Batson 17 cast). (f) Retrograde methylene-blue flow (slow) in collateral network, serial pictures taken at seconds intervals. Fascia was not opened.

In the first group, at termination of study (4, 8 and 12 weeks of occlusion in respective groups, four to nine animals in each group) animals were euthanized (100 mg/kg Pentobarbital). Batson’s 17 plastic fluid³⁶ (Polysciences Inc, PA, USA) was injected into the saphenous vein through a microcannula inserted in a popliteal branch. Retrograde filling in original side branches is blocked by valves while collateral branches which developed in response to occlusion did not have valves (“reflux”, Figure 1(c)). Soft tissues and bone were digested away in 10% KOH. The resulting venous network cast was studied by stereomicroscopy (Figure 1(d)). Collateral networks developing at the side of the occlusion were compared with the contralateral non-occluded side. Typical for varicosity, morphological features (local dilations, contorted courses etc.) were looked for. The frontal projection surface of the network was measured using quantitative image analysis techniques (Leica QWin). A picture of the network was made from the vertical direction. Pixels showing the blue color of the stained Batson17 plastic were counted automatically for each retrograde filling saphenous vein side branch, excluding the saphenous vein main branch itself.

In another series, saphenous vein narrowing clips were applied for 2, 8, and 12 weeks (four rats in each group). Hemodynamics of developing collaterals were investigated in reanesthetized animals by injecting saline stained with methylene blue (50 µl) into one of the microcannulated popliteal end branches of the saphenous vein. Movement of the stain was followed by video-microscopy, making serial pictures (Figure 1(f), slides 1 through 5). Study was terminated by euthanasia as above.

Histological samples were collected from 16 additional animals (8, 4, 4 animals with 4, 8, 12 weeks of occlusion, respectively). Sections, 5µm thick, of thigh tissues were stained with resorcin-fuchsin (RF) for elastic components and by immuno-histochemistry for smooth muscle actin (SMA). Cellular division and macrophage activity were analyzed by immuno-histochemistry for the Ki67 protein and the presence of the CD68 antigen, respectively. Primary rabbit antibodies were obtained from the R&D Systems Inc. Antibody dilutions were 1:100 (Ki67, SMA) and 1:3000 (CD68). Secondary goat antibodies were visualized with the DAB technique. The Ventana Benchmark XT Immune-Automat System was used for the immuno-histochemical staining. This ensured identical staining procedures and made the sections comparable with each other. Control and occlusion side specimens from the same animal were always embedded in the same block and positioned on the same sections, close to each other. Section pictures were digitized (3D Histech Pannoramic250 Scanner, selected high magnification, pixel sizes 0.31 µm at highest capture magnification). From pictures of resorcin-fuchsin stained sections, the walls of small veins were cut, collected on separate picture sheets, and their green intensity was analyzed on histograms (Leica QWin). Careful preliminary analysis has proven that green intensity decreases with increasing purple density of the RF stain, quantitatively marking a denser elastica. Density frequencies on the cross section of small vein walls have been analyzed on statistical histograms. A similar analysis was made for the DAB-stained SMA immuno-histochemical sections.

Data are shown as mean ± standard error of mean. Comparisons were made with one- and two-way ANOVAs. Time-dependence of collateral mass development was analyzed by the Pearson correlation. Pixel frequency was tested by the χ² probe. Statistical significance has been considered at level p < 0.05. Observer blinding could not be assured because of the outstanding morphological differences between strictured and non-strictured networks. However, the quantitative measurements of collateral venous mass and of color intensities ensured objectivity.

Results

Retrograde conducting brush-like masses of bypassing small veins (Figure 1(c), (d) right. and (e)) developed in response to chronic stricture. They appear both on casts (Figure 1(c), (d) right and (e)) and on in vivo methylene-blue marked video-microscopic pictures (Figure 1(f)), but they are missing at the control side (Figure 1(d), left). The extent of this collateral network continuously increased with time (Figure 2(a), p < 0.01), while it was practically missing at control (unclipped Figure 2(a)) and sham-operated sides (Figure 2(b)). Several branches morphologically strengthened (wider lumens) with time while conserving their zigzagging, contorted routes (Figure 1(d) and (e)).

Figure 2.

Diagrams showing amount and quantitative histology of collateral venous mass. (a) Vertical projection of retrograde filling collateral venous mass as a function of time. Note amount of retrograde veins increasing with time. (b) Amount of retrograde venous mass at eight weeks occlusion and eight weeks after sham operation. Sham operation has no effect. (c) Effect of gravitational stress on collateral development after four weeks without and with additional gravitational loading. Gravitational load accelerates collateral vein development. *p < 0.05, **p < 0.01, significantly different with one-way ANOVA, ^#p < 0.05 significance level of the Pearson correlation. (d) Elastica densities in the venous wall cross sections of the femoral triangle connective tissue, normalized for 7-7 animals. Green color intensities are suppressed by intensive purple color of resorcin-fuchsin dye meaning more densely stained elastica. Solid line, normal small veins at control side; dotted line, occluded side. The latter contains newly developed collateral veins with less developed elastica. (e) Smooth muscle actin immuno-histochemical staining comparison of control side (continuous line) and occluded side (dotted line) small vein cross sections. Normalized for 7-7 animals. DAB brown color suppresses green color of pixels marking more intensive actin stain. Note new population of loosely staining (less actin containing) vessels; ***p < 0.001 significantly different with the χ² probe. (f) Number of Ki67 positive nuclei in femoral triangle cross section. Mean values for eight control and seven strictured animals. *p < 0.05, **p < 0.01, significantly different with one-way ANOVA.

Collateral network mass development was accelerated if gravitational overload was applied during the initial phase of their development (stricture and gravitational stress in weeks 1–4, Figure 2(c)). Chronic maintenance in tilted cages as a rule resulted in development of varicosity like venous structures in these collateral veins even if applied in a later phase of collateral development (gravitational stress in the last 4 weeks of 8 and 12 weeks of occlusion). A few convincing samples of local dilations and contorted courses are shown on Figure 3(a) through (f).

Figure 3.

(a–f) Part of the saphenous vein collateral networks developed after partial occlusion for 4, 8 and 12 weeks with four weeks gravitational load before sacrifice. Note convoluted courses, local dilations resembling varicous morphology (encircled). Attached inserts show the time course of the given experiment in weeks.

The wall of these newly formed small collateral veins stained less densely with resorcin-fuchsin, demonstrating the weakness of the elastica in them (Figure 4). Quantitative image analysis (Leica QWin) has demonstrated that they are scant of densely staining elastica (p < 0.001 with the χ² probe at week 4, Figure 2(d)). In many of these small veins at occlusion side, the amount of the contractile protein smooth muscle actin was not less than at control side small veins (Figure 5(b), arrows and left part of the diagram of Figure 2(e)), but large new populations of venules appeared with only limited amount of the contractile protein in their walls (Figure 5(c), arrows, and Figure 2(e), right part of the diagram). Cellular division, as characterized by the number of nuclei in the wall expressing the Ki67 protein, was significantly more frequent at the occluded side (Figure 6(a) and (b), arrows and Figure 2(f)) after 4 and 8 weeks (p < 0.05 and p < 0.01 with one-way anova, respectively). CD68 positive cells, a marker for macrophage activity, were practically missing at control site while frequently observed at the occluded side (Figure 6(c) and (d)).

Figure 4.

Resorcin-fuchsin stained sections. (a) Four weeks occlusion, control side, (b) four weeks occlusion, occluded side, (c) eight weeks occlusion, control side, (e) eight weeks occlusion, occluded side. a′, b′, c′, d′, are enlarged inserts of corresponding a′, b′, c′, d′ figures. Scale bars 50 µm and 10 µm for inserts. Note reduced RF staining in newly formed collaterals. For quantitative analysis, see Figure 2(d).

Figure 5.

Smooth muscle actin (SMA) immuno-histochemistry of thigh connective tissue. Four weeks occlusion. (a) Control small vein cross sections at control side. At occluded side, a large number of venular cross sections with normal (b, arrows) and with limited (c, stars) SMA staining. Scale bars for (a) and (b), 100 µm, for (c), 50 µm. For quantitative analysis, see Figure 2(e).

Figure 6.

Ki67 immuno-histochemistry of thigh connective tissue, four weeks of occlusion. High cell division (Ki67) activity in the wall of newly formed veins at occluded side (b, arrows). Large area without Ki67 positive nuclei at control side (a). Quantitative analysis, see in Figure 2(f). Increased macrophage (CD68) infiltration in the area of venous vasculogenesis (d), missing at control side (c). Scale bars, 100 µm.

Discussion

Reticular vein conglomerates

The morphological appearance of newly developed collateral veins resembles early phases of the human varicosity disease: reticular veins (Figure 1(c), to (f)).³⁷ Several of them seem to originate from the vasa vasorum of the saphenous vein wall (Figure 1(e)).^38–39 Blood flow drifted away from the occluded saphenous vein main branch to find collateral pathways in such networks. These may be constructed of small veins or venous segments either without valves in the reverse direction, or having destructed valves (Figure 2(f)). In the clinical practice, masses of reticular veins are usually supplied by “feeder” veins which can be either superficial branches or incompetent perforantes with reverse flow in them.^{14,15,29,30,33}

Strengthening of branches forming preferred flow routes: similarity with more advanced leg varicosity

Chronic alteration in luminal blood flow in any blood vessel induces morphological remodeling of the lumen as a rule due to endothelial shear stress: larger flow will induce the development of a wider morphological lumen (‘Murphy’s law’).^40–43 Thus, certain routes in the reticular network preferred by blood flow will further develop and gain wider lumina. In the process, however, they will conserve their contorted route (Figure 1(d) and (e)) resembling more advanced forms of the human varicosity disease.^31,37,44

Histological marks of wall weakening during the flow-induced remodeling process

The wall of the newly formed collaterals is scarce of dense elastica (Figures 2(d) and 4). This is an important fact, as weakening of elastic tissue is a central pathological event in the formation of varicous segments.^32,45–48 While cross sections of a high number of SMA staining small veins can be observed at occlusion side (Figures 2(e) and 5(b)), several newly formed small veins do not have that contractile protein expressed in part or in the whole of their walls (Figure 2(e) and 5(c)). Damaged smooth muscle is accepted as an important element in the histopathology of varicosity.^34,49–51 One can theorize that vessel wall cells preparing for frequent mitoses in the process of flow-induced morphological lumen enlargement cannot fulfill their specialized mechanical functions (see Ki67 activity, Figure 6(a) and (b)). Monocytic elements being present close to their walls (CD68 activity, Figure 6(c) and (d)) can damage their outer layer. Inflammatory processes in and around the varicous vein are forming an essential component of the pathomechanism of the disease.^17,19,52

Increased gravitational sensitivity during the flow remodeling process: local dilations, convoluted courses

Earlier we have reported moderate changes in network and wall geometry,^24,28 elevation of innervation density,^9–10 release of endothelial secretory granules,⁵³ reversible elevation of myogenic tone²⁸ and hyperpolarization of vascular smooth muscle cells^27,54 in the saphenous veins of rats kept chronically in tube-like, tilted to 45° cages which doubled saphenous vein pressure.²⁷ In this series of studies, stricture-induced hemodynamic disturbance was combined with elevated gravitational stress. At the occluded side, accelerated development of the collateral small venous mass could be observed if this gravitational load was applied in the first four weeks of chronic stricture (Figure 2(c)). The developing collaterals seemed to be more sensitive to gravitational stress than normal orthograde side branches at control side. Tortuous, convoluted routes, local dilations were seen more frequently and such pathological malformations were more extensive if subjected to gravitational stress. Practically in all networks, the signs of varicous morphology could be identified when developing collaterals were subjected to gravitational stress (Figure 3).

Some comparison with human pathology

Disturbance of flow in the venous network of the leg plays an essential role in the pathological network and segmental wall remodeling. Appearance of venous reflux is a cardinal point in the development of varicous saphenous morphology in the human disease.^{2,14,15,30,33} Weakness of the wall and passive dilation due to elevated venous pressure are thought to be in the background of perforator reflux.^14,55,56 We identified an additional mechanism how retrograde conducting veins can develop: In a complex network of small veins, zigzagging routes will form collateral routes for flow as a row of serially and parallelly connected venous stretches without valves or with valves conducting in the desired direction⁵⁷ (Figure 1(c) to (f)). Our studies support a view according to the so-called “primary” varicosity of the leg, attributed mostly to elevated and lengthy gravitational load alone, in fact can be a “secondary” varicosity.^11,31 We think that sustained isometric contraction of the leg muscles, supporting the standing body position, may occlude the deep venous routes. The development of “recidivas” after phlebological surgical and other type occlusive treatments has long been discussed among specialists. Our studies show that such collateral systems because of their irregular course and histologically weak walls and elevated flows in them are especially prone to develop new varicosities. According to our view, lifestyle changes will be essential to avoid such recidivas. One limitation of our study is that with the techniques at hand, we cannot follow individual vessels or vessel segments in their pathologic development. There is only indirect evidence that the observed venous masses develop from originally existing venular branches without valves. We cannot yet identify those larger branches that form part of the newly developing collateral system by their valve-less stretches or after valvular incompetence induced by the morphological dilation.

Conclusion

Our studies show that the double requirement to adapt to altered flow and to altered pressure at the same time results in varicous remodeling of the venous system of the rat leg. Parallel appearing pressure and flow loads, exceeding genetically inherited adaptation limits seem to be an important factor in the development of the lower extremity varicosity disease. Our studies suggest a new component in the pathomechanism of varicosity: Flow obstacles result in the development of a reticular collateral mass (reticular veins), from which certain zigzagging routes later strengthen (teleangiectasias, spider veins, and “feeder veins”). Such veins are in the continuous state of wall remodeling and as such, they are sensitive to gravitational/pressure loads (local dilations, varicosities, valve incompetence). The suggested mechanism, working in addition to the “classical” pressure distension, is schematically demonstrated on Figure 7.

Figure 7.

Suggested mechanism how flow obstacle, collateral development and flow-induced vein wall remodeling can contribute to development of varicous morphological malformations. (1) Chronic narrowing stricture on main branch. (2) A bypassing collateral system develops from retrograde and orthograde conducting venules connected serially. Many such units then are connected in parallel (“reticular veins”). (3) Flow-induced morphological dilation in reticular vein units forming optimal flow routes. (4) Vein wall weakens while being in the flow induced chronic remodeling process. (a) Less dense inner elastic membrane. (b) Less amount of contractile protein. (c) Monocyte infiltration. (d) Cell divisions. (5) Increased sensitivity of the wall to gravitation (undulations, local dilatations developed).

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The work was supported by Hungarian National grants TO 32019, TO 42670, NVKP 16-1-2016-0004, the Hungarian Kidney Foundation and by a grant from the Dean of the Medical Faculty, Semmelweis University.

Acknowledgements

The authors thank Mrs Ildiko Oravecz and Mrs Erzsebet Hazuma for expert technical assistance.

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