Introduction

Despite of progress in the development of great saphenous vein (GSV) trunk obliteration endovascular methods the problem of varicose veins radical treatment still stay in the focus of specialists attention today [1—6].

In purpose to increase the varicose veins thermal obliteration efficiency during the past 10 years, the method of its implementation has been significantly improved. There were changes in the optical fibers features design and the laser radiation wave length. But the thermal obliteration methods usage in cause of ectasia of the GSV main trunk is still the subject of discussion [7, 8]. Many authors in their daily varicose veins treatment practice continue to use non-thermal methods of obliteration (injection and catheter sclerosis of veins). Using this methods without taking into account the severity of ectasia can completely discredit one [7]. Nowdays the effectiveness of varicose veins non-thermal obliteration new method with usage a cyanoacrylate adhesive base is being discussed in the world [9].Due to the small number of observations, it is not yet possible to make final conclusions about the new chemical obliteration method [10]. One of the unresolved issues remains the determination of the maximum allowable diameter of the ectated vein for glue obliteration [11].

On the other hand there are studies in which authors try to get away from traditional varicose veins treatment methods and prove the theory validity of the possible ectated GSV trunk preserved remodeling after isolated varicose veins tributary elimination. This technique is called "ASVAL" [12]. However specialists, tried to promote usage the "ASVAL" method in phlebological practice, are not able to determine the maximum permissible ectated GSV diameter, in which the regression of the initiated changes can be expected with the help of this method [13].

In order to study the recrudescence causes when using various obliteration methods, the traditional histological studies at different time intervals with an analysis of the vein lumen obliteration degree and the recanalization degree was conducted. One significant drawback such histological analysis is the lack of morphological structural changes understanding of the initially present in the ectated vein and the severity degree which could be different.The vein lumen obliteration onset timing may depend on the structural morphological changes present initially in its wall. There are some studies on this topic [14—16]. However, it should be mentioned that in all these studies, considering particular issues of morphological GSV wall remodeling, two extreme forms were taken — a normal variant and its varicose transformation. The dynamics of transient changes in the vein wall from normal to its gradual ectasia was not analyzed.

Thus, the histological studies aim was to establish, on the great saphenous vein example, the general points of the its wall morphological rearrangement in the ectasia cases.

Materials and methods

In accordance with research aim, 43 patients with the GSV main trunk ectasia presence and the absence of its varicose transformation were sequentially included in the study group. All patients met the clinical class C2 according to the International classification of Chronic venous diseases CEAR.

GSV main trunk ectasia was understood as the vessel lumen expansion, determined by preoperative ultrasound performed in the patient vertical position, based on the generally accepted criteria for the GSV diameters in the norm [17]. Ectasion of GSV was considered at the size of its lumen more than 5—6 mm with the presence of retrograde blood flow along its main trunk. Ultrasound examination was performed on PHILIPS-EPIQ 5G and 7G devices in B-mode with color Doppler mapping and spectral analysis. All studies were conducted at the morning.

The criteria for exclusion from the general cohort of subjects were: patients with a history of surgery for varicose veins or sclerosis, in the presence of thrombotic or post-thrombotic venous lesions, patients with congenital malformations, arterial or lymphatic vasculopathy, collagenosis and myopathy, traumatic injuries with or without concomitant fractures, patients with lower extremities obliterating atherosclerosisand diabetes mellitus. The patients age was limited to 20 and 40 years.

The histological material removal for 1 cm was performed on the operating-table before endovasal vein obliteration by one of the thermal or non-thermal methods using local miniflebectomy using Varadi hooks in the projection of the most ectatized area, the marking of which was carried out during the preliminary ultrasound scan. In some cases, depending on the situation, 2 or 3 different GSV ectasia sections were excised.

A total of 58 GSV fragments vein were removed for histological analysis.

All histological material was fixed in a 10% solution of neutral formalin. After the standard wiring, paraffin blocks were prepared. A total of 138 blocks were prepared. Sections with a thickness of 6—7 microns were stained with hemotoxylin-eosin, fuchselin-picrofuxin, and hematoxylin-picrofuxin according to Van Gieson.

The obtained histological material was studied using a Levenhuk-Zoom microscope with a magnification of PL 4x / 0.10, PL10x / 0.25, and PL40x / 0.65. Photoprotocoling was performed using a TOUPCAM-UCMOS 14000 KPA video camera with the possibility of video and digital photo processing in the program“TaupView”.

Study results

The comparative GSV wall structural changes analysis was based on the previously obtained and already published information about the human GSV morphological structure in normal conditions [18].

Despite the polymorphism of the GSV morphological rearrangement during its ectasia (Fig. 1), a detailed analysis revealed the f smooth muscle proliferation degree and connective tissue structures characteristic of this stage in all three GSV wall membranes.

Fig. 1. Large saphenous vein. Cross section. Staining with hematoxylin and eosin. PL 4×/0.10.

a — clear visualization of all three membranes of the wall of the great saphenous vein in normal conditions; b — a decrease in the degree of differentiation of the inner and middle membranes of veins while still without signs of hyperplastic processes in the intima.

Structural changes in the GSV wall smooth muscle elements during the GSV ectasia development.

A smooth muscle fibers morphological rearrangement at the level of the GSV inner shell (Tunica Intima-Ti) is shown in Fig. 2. Directly morphological changes in the longitudinal smooth muscle fibers were detected already at the subendothelial layer level. There was a distinct hyperplasia tendency of these fibers with a transition to their hypertrophy. Several typical variants of structural changes in the longitudinal smooth muscle fibers in Ti were identified, which are shown in Fig. 3. Thus in 39 (28.3%) cases, single longitudinal smooth muscle fibers in the subendothelial layer of Ti showed no signs of hyperplasia, although the overall hyperplastic activity of Ti in the GSV wall exposed to ectasia was already noted (Fig. 3A). In 15(10.8%) of the studied preparations, the morphological rearrangement of Ti was characterized by incipient hyperplasia of the longitudinal smooth muscle fibers of Ti, against the background of increased activity of hyperplastic processes in it (Fig. 3B). In another variant, which was the most specific, in 84 (60.9%) cases, there was a distinct hypertrophy of previously only hyperplastic longitudinal smooth muscle fibers (Fig. 3C).

Fig. 2. Characteristics of hyperplastic processes occurring in the intima (Ti) with the gradual development of ectasia of the great trunk of the great saphenous vein without varicose transformation.

Staining with hematoxylin and eosin. PL 10×/0.25. a — type I ectasia (local Ti hyperplasia); b — type II ectasia (progressive Ti hyperplasia); c — type III ectasia (widespread Ti hyperplasia with the formation of an intimate ridge).

Fig. 3. Characterization of smooth muscle longitudinal fibers (SML) of the Ti sub-endothelial layer at different stages of the hyperplastic process during the development of GSV ectasia.

Staining with hematoxylin and eosin. PL 40×/0.65. a — type I Ti ectasia; b — type II Ti ectasia; c — type III Ti ectasia.

A smooth muscle fibers morphological rearrangement at the level of the GSV middle shell (Tunica Media-TM) is shown in Fig. 4. In 25(18.1%) cases with morphological rearrangement, the venous wall middle shell circulatory and longitudinal smooth muscle fibers with the hyperplastic processes general activity retained their orientation and sequence (Fig. 4A). In 86(62.3%) cases, against the background of increased general hyperplastic activity of connective tissue elements and intimal hyperplasia, active hyperplasia and hypertrophy of circular smooth muscle fibers were observed in the middle shell (Fig. 4B). In 27(19.6%) of the studied segments the GSV wall morphological rearrangement was characterized by decreasing in the middle vein sheath circular smooth muscle fibers hypertrophic activity and the circular muscle fibers reorientation process beginning to the oblique-longitudinal orientation (Fig. 4C).

Fig. 4. Characterization of longitudinal (SML) and circular (SMC) smooth muscle fibers of the Ti sub-endothelial layer at various stages of the hyperplastic process during the development of GSV ectasia.

Staining with hematoxylin and eosin. PL 40×/0.65. a — type I TM ectasia; b — type II TM ectasia; c — type III TM ectasia.

A smooth muscle fibers morphological rearrangement at the level of the GSV outer shell (Tunica Adventicia-TA) is shown in Fig. 5. In 18(13%) cases, despite the presence of incipient general hyperplastic processes in other ectated GSV wall membranes, authors did not observe morphological rearrangement of smooth muscle fibers at the adventitia level. In all cases, the smooth muscle fibers had a longitudinal direction and were localized in the area of the inner periadventitial layer, that is, at the border with TM (Fig. 5A). In 34(24.6%) cases, with a distinct increase in the hyperplastic activity of connective tissue elements at the Ti level and hypertrophic processes affecting the circular smooth muscle fibers TM, the adventitia also showed hyperplasia of longitudinal smooth muscle elements in the periadventitial layer (Fig. 5B). In 72(52.2%) of the ectazed GSV studied segments the morphological rearrangement of smooth muscle elements in the TA was characterized by a pronounced hypertrophic process of longitudinal smooth muscle cells smooth muscle fibers (Fig. 5C). This hypertrophic smooth muscle process occurred against the background of a decrease in the vein wall middle shell smooth muscle fibers hypertrophic activity.

Fig. 5. Characteristics of the morphological rearrangement of smooth muscle longitudinal fibers (SML) of the outer layer of TA at different stages of the hyperplastic process during the development of ectasia of GSV.

Staining with hematoxylin and eosin. PL 40×/0.65. a — type I ectasia TA; b — type II TA ectasia; c — type III ectasia TA.

The GSV wall connective tissue elements structural changes during the development of GSV ectasia

A GSV wall inner layer carcase connective tissue morphological rearrangement during the GSV ectasia development is shown in Fig. 6. In 58 (42%) cases of Ti morphological rearrangement against the general hyperplastic processes background, the beginning subendothelial level connective tissue fibers growth towards the middle shell, acquirly forming of unidirectional thickened intermuscular partitions(Fig. 6A) were possible to mention. In 52(37.7%) cases, connective tissue fibers further growth at the middle shell border was observed by the type of individual sectors(Fig. 6B). In 28(20.3%) cases in the studied material, further connective tissue elements proliferation at the background of general hyperplastic processes led to the sectoral growths formation at the border of the inner and middle shells of connective tissue rollers in Ti (Fig. 6).

Fig. 6. Characteristics of morphological rearrangement of the connective tissue skeleton of the GSV wall at the level of the inner membrane at various stages of the hyperplastic process during the development of GSV ectasia.

Staining with hematoxylin and eosin. PL 10×/0.25. a — type I ectasia; b — type II ectasia; c — type III ectasia.

In the middle shell connective tissue hyperplasia had two ways of development. The first variant — the connective tissue elements growth was a continuation of their linear ingrowth on the side of the intima (Fig. 7A). Connective tissue hyperplasia occurred mainly between bundles of smooth muscle fibers. This type of connective tissue rearrangement was observed in 113 (81.9%) cases.In 25(18.1%) cases, connective tissue growths lost their linear slenderness, becoming increasingly diffuse (Fig. 7B).

In the outer shell, changes in the connective tissue skeleton occurred in 84 (60.9%) cases almost immediately with a clear tendency to increase the mass of the main intercellular substance, which indicated a high activity of proliferative processes in the TA for the formation of the secondary connective tissue framework of the venous wall (Fig. 7C). In a more detailed analysis, in 54 (39.1%) cases in the study material, it was possible to note that the formation of the secondary connective tissue carcasses at the adventitia level was primarily due to hypertrophied collagen fibers (Fig. 7D).

Fig. 7. Characteristics of proliferation of connective tissue elements in the wall of the GSV during the development of its ectasia.

Coloring according to Van Gieson. PL 40×/0.65.

a — linear connective tissue ingrowths into the middle shell from the TI side; b — diffuse growths of connective tissue (pink) in TM; c — diffuse increase in the mass of the main intercellular substance (BS) at the border of TM and TA; d — formation of the secondary connective tissue frame of the GSV wall due to hypertrophy of collagen fibers (CF) in TA.

Research results discussion

The first attempts to describe the morphological changes in the lower extremities veins of the occurred in varicose veins refer to the late 60s and early 70s. It is necessary to recall the fundamental work of the Bulgarian histologist and pathologist Vankov V. N. [19]. The author noted the advancing proliferative changes in various membranes of the venous wall in varicose veins. His concept of the saphenous vein wall general connective tissue proliferation occurred in varicose veins,was further developed in the studies of E. P. Dumpe, Yu. I. Ukhov, and P. Schwalb.G. [20]. The irrefutable merit of Dumpe E. P. et al., was the first attempt in the world to describe the structural changes in theGSV wall by the "vasa-vasorum" and to give these microcircular changes occurring in the vein wall one of the main roles in the aging of any venous vessel. However, the main research drawback at that time was the generally accepted gradual development theory of age-related "phlebosclerosis". As a result, the concept of age-related "phlebosclerosis" and the existing morphological changes in the GSV wall during its varicose transformation were practically identified. Subsequently, from the same point of view, in their numerous studies, Vedensky A. N. and Gladkikh V. G. also explained the phenomenon of the appearance of relative valvular insufficiency of deep veins as the cause of not only the development of varicose veins, but also the progression of chronic venous insufficiency [21, 22].

In the early 2000s, studies began to appear abroad, in which the authors increasingly used a new theory of endothelial dysfunction as the primary cause of the lower extremities chronic venous insufficiency development[23, 24]. According to a number of morphological studies, changes in the intima of the venous wall with the subsequent development of endothelial cell dysfunction triggered a reactions cascade which resulted in progressive vein wall endothelium dystrophy and desquamation [25, 26].Nowadays more than 20 molecular and biochemical endogenous substances are considered as endothelium-dependent triggers in the development of venous endothelial dysfunction. It has been proven that with a high degree of probability, this endothelium-dependent cascade is important in the development of venous thrombosis. But the endothelial dysfunction factors influence on the lower extremities varicose veins development is still under active discussion [27].

That is why pathologists have again turned to studying the restructuring of the venous wall structure [14—16]. However, the published studies were conducted exclusively on a varicose vein.

The presented research data is devoted to the venous wall structure morphological rearrangement study in the venous ectasia stage. The great saphenous vein, as the most frequently affected by varicose transformation, was taken as the object of study.Based on previous studies, the vein wall features morphological rearrangement depending on the limb level in different time periods of normal human life were studied on the GSV example [18]. Based on this, morphological material, so necessary for conducting a comparative analysis was always in present.

The results of present study not only confirmed or clarified the data of other authors [23, 28, 29], but also allowed to take a different look at the dynamics of changes occurring in the GSV wall, corresponding to the initial manifestations of its ectasia, which is the main scientific novelty. Based on the analysis of the identified GSV wall morphological rearrangements features with its gradually developing ectasia. So it could be established not only general patterns, but also to understand a certain stage-by-stage nature of the structural changes occurring.

The initial ectasia stage (30.4%). Against the GSV ectasia beginning development background there is an increasing activity of hyperplastic processes in the intima due to the connective tissue elements gradual growth at the border of the subendothelial layer of the Ti and TM by the type of linear eccentric invasion (42%). As a result of this process, the boundary between the inner and middle shells is gradually erased, with the complete loss of the previously formed inner elastic membrane. At the vein ectasia initial stage the first radially arranged collagen fibers begin to appear from the subendothelial layer, which are interwoven into the TM circular smooth muscle fibers slender lines, which in structure do not differ from the age norm. There were no changes in the smooth muscle fibers TM and TA.

Progressive stage of ectasia (44.9%).As the proliferative process progresses, the connective tissue elements gradual growth at the border of Ti and TM from linear takes the form of separate sectors in 79.7%. Radially directed individual collagen fibers of the Ti subendothelial layer acquire a decorated and hypertrophied appearance. There is hyperplasia and hypertrophy of circular smooth muscle fibers (62.3%), which retain their orientation. Individual longitudinal smooth muscle fibers with signs of hypertrophy (24.6%) at the TM level . Collagen fibers at the TA level without signs of hypertrophy.

Diffuse stage of ectasia (24.7%).At this ectasia stage, the connective tissue hyperplasia process becomes diffuse and spreads throughout the entire border of the inner and middle shells. In some cases, these growths have the form of pronounced connective tissue rollers (20.3%). At the middle layer level circular smooth muscle fibers part begins to change their orientation to the longitudinal direction with a gradual shift from the center to the periphery. The longitudinal smooth muscle fibers of TA have a hypertrophied appearance (52.2%). In adventitia hypertrophied (39.1%) longitudinally oriented collagen fibers are powerful supporting structures that are the ectated vein connective tissue carcasses basis.

For a better GSV wall structural changes understanding occurring during its ectasia developing ,in present study new morphofunctional characteristics were introduced. Taking into account the described connective tissue hyperplastic processes specific direction from Ti to TM), article authors gave them the name — eccentric connective tissue hyperplasia. The GSV wall smooth muscle elements morphological remodeling main feature as its ectasia progresses is an eccentric longitudinal smooth muscle hypertrophy. The one's nub is increasing in the longitudinally directed smooth muscle fibers number at the level of all three venous wall membranes (subendothelial layer, middle shell, periadventitial layer). Thus, circularly oriented smooth muscle fibers are pushed away from the central parts of the TM to the periphery, at the border with the TA. Normally presented, well-defined circular smooth muscle layers with the development of vein ectasia take the form of separate and hypertrophied fibers. The presence of such hypertrophied circular smooth muscle fibers can stand for the ectated vein ability to actively restore its outer diameter and inner lumen.

Conclusion

Based on the achieved data of morphological patterns of GSV wall restructuring in cause of its ectasia, it seems appropriate to try to answer the main pathophysiological question — is the described morphological changes the GSV wall structure in course of its ectasia compensatory-adaptive or pathological?

Considering that the GSV wall ectasia always begins segmentally and most often affects only one of its main trunk branches, it seems that the local hemodynamic factors that should play a major role in the ectasia appearance. For several decades, a local increase in intravenous pressure has been considered as the main factor of GSV wall ectasia [30]. However, as a result of many years of discussion, most pathophysiologists have not confirmed the phenomenon of increased intravenous pressure, not only in varicose veins or post-thrombotic disease, but also in people with normal veins, for example, with a change in body position or prolonged orthostasis [31].

We fully support the point of view of Tsukanov Yu. T. that one of the main factors precipitating local ectasia of the great saphenous vein is segmental venous hypervolemia [32]. The presence of locally venous hypervolemia causes a change in the transmural pressure, not the total intravenous one [30].

A further increase in transmural pressure associated with the deposition of additional blood volumes begins to cause initial vein ectasia [33]. Based on the classical biophysical and hydrodynamic blood flow principles [34], structures located outside the venous wall can resist the increase in transmural pressure in the venous wall. In the lower limbs’ deep veins, such strength is possessed, for example, by the muscles of the lower leg, in the cases of which these venous trunks pass [35]. For subcutaneous veins, covered from the outside only with sheets of superficial fascia and layers of adipose tissue, there are no anatomical structures that can actively resist further increases in transmural pressure.

It is logical to presuppose that such sources should be located in the vein wall. In the outer shell there are collagen fibers, which protect the vein connective tissue basement. This fibers can not actively play role in the vein' work, since collagen is more of a support than a contractile element. This also applies to the single smooth muscles fibers located in the periadventitial layer, runs longitudinally. The saphenous vein wall structure is characterized by the presence of the well-developed smooth muscle fibers circular layer in the middle shell. These structure is the mechanism of the short-term adaptation in the event of a vessel lumen expansion (in research case — vein ectasia).

If there is venous ectasia for a long time period due to persistent or increasing segmental hypervolemia, mechanisms of long-term adaptation should be involved in the vein work to normalize transmural pressure. Based on our studies, such mechanisms with the corresponding morphological rearrangement of the GSV wall in long-term ectasia are:

— the inner shell hyperplasia with the significant elastogenesis;

— hyperplasia and hypertrophy of smooth muscle fibers;

— changing the structure of the primary collagen framework and creating a secondary one .

As it is shown in our researches the degree of participation of these mechanisms in the overall compensatory-adaptive response of the venous wall is directly dependent on the GSV segmental ectasia development stage.

At the initial stages, this primarily concerns the elastic-contractile elements of Ti. The hyperplastic process primarily concerns elastic fibers, we have given this morphological phenomenon the name compensatory-optimal elastosis. The hyperplasia of collagen fibers is single and linear.

At the next stage, with persistent stable or progressive ectasia of the vein segment, compensatory hypertrophy of the circular smooth muscle fibers occurs. As it follows from the biomechanics principles, following the increase in vein diameter (ectasia) begins its relative elongation, which in itself is compensatory-adaptive mechanism whose purpose is to reduce segmental transmural pressure by distributing the (elimination) hypervolemia with increasing segment length. As a result, from our point of view, hyperplasia is observed in all three shells, and then compensatory hypertrophy of the longitudinal smooth muscle fibers. In addition, as our studies have shown, as a result of an increase in the diameter of the vein with its existing ectasia, some of the smooth muscle fibers located circularly begin to take first a radial, and then a completely longitudinal direction.

The first signal of the coming overload of the elastic-contractile structures of the venous wall is the activation of collagenogenesis, which is observed mainly in the outer layers of the TA. The task of this process is to strengthen the framework of the GSV wall at a given diameter and prevent its ectasia. Given that the main carrier of the connective tissue backbone of the wall is collagen, it is the collagen proliferative activity at the TA level that is one of the main compensatory and adaptive mechanisms involved, along with other supporting elements, in the overall work of the venous wall to stabilize the increasingly rapidly changing transmural pressure. The presence of longitudinal hypertrophied muscle fibers in the periadventitial layer in developing ectasia allows us to conclude not only about the passive, but also about the active participation of TA in this regulatory process. In addition, the ongoing reactive hyperplasia of the intima in some areas forms sufficiently voluminous connective tissue rollers, which indicates the intensity of proliferative processes in Ti and may be indirectly related to and characterize the severity and rate of increasing endothelial dysfunction.

But the reserves are not unlimited. And as we will show in our next work, there is not much time left before the first signs of varicose transformation appear from the point of view of the formal genesis of the morphological changes taking place in the GSV wall. Obtained data on the GSV morphological rearrangement in humans during the development of its ectasia coincide, with the results of experimental studies on modeling the pathology of superficial veins in rats conducted in 2020 by Gabriella Dörnyei, Judit Hetthéssy, Bettina Pataietal [36].

Taking into account not only the purely morphological, but also the applied nature of our work, it should be noted that it is at the stage of the onset of ectasia, while the structural changes in the GSV wall have not yet acquired an irreversible character, the treatment of developing venous ectasia can be conservative, and if necessary, invasive manipulation using, for example, the ASVAL method can be performed not only minimally traumatic, but also with the most radical effect.

The participation of the authors:

Concept and design of the study — A.B. Sannikov, E.V. Shajdakov, V.M. Emelyanenko

Data collection and processing — A.B. Sannikov, E.V. Shajdakov

Statistical processing of the data — A.B. Sannikov, V.M. Emelyanenko

Text writing — A.B. Sannikov

Editing — A.B. Sannikov, E.V. Shajdakov, V.M. Emelyanenko

The authors declare no conflicts of interest.