The particularities of connective fibers from the wall of varicose veins extirpated by cryostripping

Abstract

Introduction: The varicose vein affects more than 30% of the general population. Significantly increased rates were noticed in women and older population. From the histopathological point of view, venous arterialization, smooth muscle cell hypertrophy, and hyperplasia are the main changes noticed in varicose vein disease. Some of the main therapeutic methods used in the management of varicose disease are injection sclerotherapy, conservative, surgical, saphenous vein inversion and removal, high saphenous ligation, ambulatory phlebectomy, transilluminated powered phlebectomy, endovascular management, cryostripping. Aim: The aim of this study was to evaluate the morphology of connective fibers from the wall of the varicose veins extirpated by cryostripping. Patients, Materials and Methods: The study included 109 samples taken by cryostripping method. Hematoxylin–Eosin, Masson’s trichrome, Silver and Orcein staining were applied. The assessment of fibers was made according to score values between 0 and 3. Results: It was found no major structural differences in terms of alterations of collagen fibers induced by the applied surgical procedure. It was noticed duplications and multiplications of the internal elastic lamina, as diffuse and nodular forms. Depletion of elastic fibers at the media was a lesion identified in most of the specimens. The depletion of reticulin fibers correlates with the accumulation of collagen fibers, which partially or completely replace the network in the media and intima. No correlation was found between changes in the reticulin network and the time between prelevation and buffered formalin fixation, the maximum time investigated being five days. Conclusions: The Orcein staining in the venous vessel evaluation panel may be a useful investigation

Introduction

Varicose veins disease is a chronic venopathy characterized by the permanent and tortuous dilation of the veins, especially those in the lower limbs where blood stasis and alteration of the venous walls are found. Varicose veins represent the local manifestation of varicose disease, a general condition characterized by a degenerative process of the venous wall. Varicose veins include telangiectasias, reticular veins, and true varicose veins. It is generally more common in women and older adults. Varicose veins affect 22 million women and 11 million men between the ages of 40 and 80 [1]. Of these, 2 million men and women will develop symptoms and signs of chronic venous insufficiency, including venous ulceration. Worldwide, the incidence of varicose veins varies between 10% and 60%. This incidence is up to 30% higher in the Asian region, compared to the Western world [2, 3].

The cryostripping is a surgical minimally invasive method used for the ablation of varicose veins. It is also an effective and safe method of treatment [4]. The treatment of varicose veins by cryostripping was practiced for the first time in 1987, and since 1990 it has been successfully used in some phlebological centers. Even if the technique of removing veins using a cryosonde is simple, the method is not widely used [5]. Some data launched the hypothesis that patients prefer endovenous laser ablation because it is less painful, and it has a lower postoperative morbidity. Besides the fact that it offers a faster return to normal activity [6], cryostripping is a less expensive method and seems to be more effective [7]. The clinical results of cryostripping are not inferior to those obtained by endovenous laser ablation. Thus, considering its profitability, cryostripping is a safe and feasible treatment method for chronic venous disease [8]. Among the advantages of this procedure are the high success rate, the low number of complications, the short execution time and low costs, including the possibility to practice this procedure as a usual surgical intervention.

The particular aspects of the connective fibers in the walls of varicose veins are very little studied in the literature. Wali et al. [9] demonstrated that all sections of varicose veins showed a marked hypertrophy of the intima due to fibrous tissue infiltration and a localized thinning of the muscle layer compared to normal veins. The same study showed that elastic fibers are deficient and dispersed with the loss of the normal network at the level of varicose veins and noticed a decrease in the elastin/collagen ratio. Another, more recent study found an intensive development of collagen fibers in varicose veins. The direction of the collagen fibers was parallel to the endothelium and in some cases, they were arranged at an angle to it. The elastic fibers in the media were largely unchanged. Occasionally, their fragmentation was noticed [9, 10].

Aim

At the moment, there is no conclusive data in the literature regarding the particularities of connective fibers in the wall of varicose veins. The aim of this study was to analyze the structural features of collagen, elastic and reticulin fibers present in the wall of varicose veins collected by cryostripping.

Patients, Materials and Methods

Patients and biopsies

The study included 109 samples taken by cryostripping method. The principle of this procedure consists of venous catheterization with a special probe which is cooled to -85°C. Due to these very low temperatures, the venous intimal layer strongly adheres to the probe, thus enabling the stripping of the insufficient vein. The probe is made of smooth metal materials, and it can be easily inserted in the venous lumen even if the vein path is tortuous. By connecting the probe to the ERBOKRYO device (Erbe USA, Inc., Marietta, GA, USA) using liquid nitrogen the cooling is subsequently carried out. When the insufficient vein adheres to the probe (this phenomenon usually occurs in about five seconds), it is removed by repeated traction [11, 12]. Signed consents forms were obtained from each patient, the principles of the Declaration of Helsinki were respected, and the study was approved by the Ethics Committee of Victor Babeş University of Medicine and Pharmacy, Timişoara, Romania (Approval No. 66_22/22.03.2023). The specimens were directly fixed in formalin or maintained in saline for two, four, 12, 24, 48, 72 hours, four and five days and after that fixed. The fragments were kept for a variable time in saline in order to follow the persistence of the viability of the graft. After that variable time, they were fixed in buffered formalin.

Histochemical stainings

For the microscopic analysis of the specimens, common stains including Hematoxylin–Eosin, Masson’s trichrome, Silver and Orcein staining were applied according to standard recommended protocols. The following kits were used: Artisan Masson’s Trichrome Stain kit, Artisan Reticulin–Nuclear Fast Red Stain kit, Artisan Orcein stain kit. All of the kits were from Agilent Technologies Denmark ApS (Glostrup, Denmark).

Microscopic evaluation

Microscopic evaluation of the fibers was made according to the followings scores. In the case of the collagen fibers, the quantification was done as follows: score 0 (rare thin collagen fibers disposed between muscle cells, in the media); score 1 (minor disorganization of the media, with thick and thin collagen fibers in equal proportions); score 2 (major disorganization of the media, with thick collagen fibers, forming bundles and a marked decreased of smooth muscle cells number); score 3 (massive/complete collagenization of the media). The elastic fibers were evaluated as: score 0 (normal distribution of elastic fibers in all three layers of the vein); score 1 (partial depletion of elastic fibers in the intima and media, but present without changes in the adventitia); score 2 (total depletion of elastic fibers in the media +/- intima, but present in the adventitia); score 3 (absence of elastic fibers in all three layers of the vein). A similar score was used for the reticular fibers’ evaluation. The microscopically assessed score with values between 0 to 3 reflects the disposition and density of connective fibers by comparison with their appearance in the normal vein.

Results

The analysis of the arrangement of collagen fibers on the vein specimens collected by cryostripping was performed on slides stained with the Masson’s trichrome method. This staining highlights isolated or bundled collagen fibers in deep blue. The purpose of applying this staining was to identify the degree of collagenization/fibrosis/sclerosis of the vascular wall. The quantification of collagen fibers under these conditions was based on an original score, previously mentioned (Figure 1A, 1B, 1C).

Figure 1.

The normal distribution and main changes of collagen, elastic and reticulin fibers in the venous wall: (A) Venous wall with rare and thin collagen fibers in media, not organized in bundles, score 0; (B) Moderate collagenization of the media in which smooth muscle cells predominate, score 1; (C) Numerous collagen fibers in media, which predominate compared to smooth muscle cells, score 2; (D) Fibrosis in the wall of varicose veins removed by cryostripping; extensive fibrosis of the media; (E) Fibrosis of the media and subendothelial space; (F) Disorganization of the fibrous wall; (G) Venous vessel with normal microscopic structure; the organization in the three distinct layers; (H) Detail with the internal elastic lamina; the apparently fragmented elastic fibers in the middle and the inner part of the adventitia; (I) Thick elastic fibers in the adventitia; (J) Thickening with doubling of the internal elastic lamina; (K) Detail with the multiplication of the internal elastic lamina, diffuse plane pattern; (L) Nodular multiplication of the internal elastic lamina; (M) Normal distribution of reticulin fibers in all three layers of the vein wall; (N) Detail of the reticulin fibers from the media, the most numerous, with multiple branches; (O) Focal depletion of reticulin fibers in media; (P) Severe depletion of reticular fibers in media and intima; (Q) Complete depletion in all three layers of the vein; (R) Complete depletion in the media and rare reticular fibers in the intima; (S) Thrombus which partially obstructed the vascular lumen; (T) Recanalization with extensive development of collagen fibers from intima; (U) Thrombotic and recanalized venous vessels; the internal elastic lamina is distinct, the elastic fibers are present in the media; (V) Detail, with weak orceinophilic fibrillar material in the area of the organized thrombus; (W) Massive accumulation of collagen fibers especially in the media, in the case with severe depletion of reticulin fibers; (X) In the same case, severe depletion of reticulin fibers. Masson’s trichrome staining: (A–F and W) ×200; (S and T) ×100. Orcein staining: (G) ×100; (H–L and U) ×200; (V) ×400. Silver staining: (M–R) ×200; (X) ×100

From the evaluated cases, four specimens presented normal histological structure. Thus, the three layers of the venous wall were clearly identified, the media presenting a predominance of smooth muscle cells, the intima was thin, without accumulation of collagen fibers. In all these cases, in the adventitia were noticed thick collagen fibers, arranged relatively orderly and homogeneously, without extension in the media.

We further followed the distribution and severity of lesions noticed in harvested veins that showed pathological changes. In the evaluation of the lesions, it was considered the vascular architecture, the presence or absence of collagen material in the intima and media, but we excluded from the interpretation the collagen fibers of the adventitia. This last aspect was invariably present in all cases. In most of the cases, the venous lumen was permeable without major changes, except for cases that showed thrombus or thrombus recanalization.

Regarding the collagen fibers distribution and score it was noted the rarity of cases with minor or absent changes (five cases). The majority presented fibrosis lesions signaled by the hyperplasia of collagen fibers. In approximately 25% of cases, the fibrosis was extended to the entire venous wall (Figure 1D, 1E, 1F). A particular aspect was the diffuse collagenization of the subintimal space, occupied in the cases evaluated with value 3 of score mostly by the presence of an amorphous collagen-type material that greatly modifies the architecture of the venous wall.

Significant changes in collagen fibers dependent on the period of harvesting, fixation and histological processing were not found. Our data argue for the existence of changes with onset during the evolution of the varicose disease and not induced by the cryostripping procedure.

We did not notice conclusive aspects related to the duration and method of prelevation in terms of the disposition of elastic fibers. The lesions described below, according to our observations, precede the time of prelevation. These aspects are correlated with the resistant character of elastic fibers that have a very long-life span.

From the 109 specimens analyzed, we found four with a normal microscopic structure in terms of the disposition of the elastic fibers. In these cases, we identified classical aspects at the level of the three layers of the venous wall (Figure 1G). The internal elastic lamina was present in all cases of this group, continuous, thick and sinuous, without ramifications. In the media, the elastic fibers were short, preferentially arranged among the smooth muscle cells and created the impression of fragmentation (Figure 1H). It may be considered that these are short elastic fibers, the product of smooth muscle cells. These fibers of the media are disposed in an orderly manner at relatively equal distances from each other and sustain the existence of a network occupying the media. In the adventitia, the fibers were not organized into bundles (Figure 1I). They had a plexiform distribution, forming the outer elastic lamina. The internal elastic lamina was the most constant structure we have identified, both in normal and depleted cases.

Also, the normal structure of the elastic fibers in all the vasa vasorum vessels was noticed in the excised varicose veins.

The lesions of the elastic fibers noticed most often in cases with varicose veins were in the intima and media. In the intima of 36 specimens, was found an architectural change of the network of elastic fibers which was not reported in the specialized data. This aspect consists in doubling and multiplication of the internal elastic lamina (Figure 1J). The elastic fibers from the intima–media interface are multiple, sinuous, without anastomoses, slightly paler stained with Orcein than in veins with a normal structure. Multiplication of the internal elastic lamina has at least two patterns, respectively plane (Figure 1K) and nodular type. The plane variant occupies relatively large areas on the circumference of the vein and may occupy up to half the thickness of the venous wall. This aspect underlines the hypothesis of the synthesis activation of elastic fiber precursors during the pathological process. The second aspect in the same category is represented by the duplication in its nodular form (Figure 1L), which affects limited areas of the venous circumference, but which protrudes towards the venous lumen and which, at least theoretically, could induce favorable changes in the process of thrombosis.

In six of the specimens, the multiplication of the internal elastic lamina was associated with the deposition of an amorphous orceinophilic material at the border with the media. The practical significance of this material is still unknown.

We mention that we identified such duplications and multiplications also in specimens that did not show any other change in the architecture of the venous elastic system. It was found a particular aspect to the cases in which thrombi were present (eight specimens) in different stages of recanalization. In these cases, surprisingly, the internal elastic lamina was present and continuous, it did not present duplications, instead, in area of organized thrombus, we identified fibrillar substrate positive for Orcein, like elastic fibers, but much shorter and pale stained. Under these conditions, it was considered that they represent fibrin elements, without meaning for the interpretation of the reaction.

Elastic fiber depletion was a lesion reported in 56 specimens and is a common feature in varicose disease. It was noticed the lack of elastic fibers on more or less stretched areas. The evaluation of the specimens according to the previously indicated score revealed the following results: four cases marked with normal; 36 specimens evaluated with 1; 55 specimens marked with 2 and nine specimens marked with 3. We maintain this type of scoring for the purpose of making a viability protocol of excised veins. It was noticed that the depletion of elastic fibers in the average in many cases, varying from their numerical reduction to their absence (Table 1).

Table 1.

Scorification of the collagen, elastic and reticular component in the venous wall

Score	0	1	2	3
Collagen fibers	5	44	41	19
Elastic fibers	4	36	55	9
Reticular fibers	9	48	42	10

From the perspective of the score we propose, apparently elastic fibers have a higher significance than collagen fibers.

In samples with normal morphological appearance, reticulin fibers were present in all layers of the vascular wall. It was noticed the highest density in these cases in media, a parameter that did not change significantly depending on the duration of preservation in saline and the moment of fixation. The mentioned aspects were kept under the same aspect for the entire investigated period, including the specimens fixed after five days in saline. In nine of the examined specimens, we noted a normal appearance from the point of view of the distribution of reticulin fibers, being the most constant fibrillar elements during the period evaluated by us. Normally disposed reticulin fibers (Figure 1M, 1N) show thick elements in media and significantly thinner in intima, without the obvious distinction between the two networks.

In most of the cases was found more or less extensive areas of depletion in reticulin fibers. If in normal conditions, the reticulin fibers are homogeneously and orderly distributed in the three layers of the vein, in the case of partial depletion, we noticed irregular areas devoid of fibers, of variable sizes. Thus, we identified 48 samples with mild depletion, scored 1, compatible with the viability of the structure. In 55 samples, we identified depletion on extended areas, which initially affects the subendothelial layer, and includes extended media and adventitia areas, giving these layers a heterogeneous appearance from the point of view of the distribution of reticulin fibers (Figure 1O). Severe partial depletion, noted with score 2, was characterized by the rarefaction of the network of reticulin fibers. In these cases, the arrangement in the form of a network of fibers is no longer noticed, but only isolated elements, present especially in the media (Figure 1P).

Severe depletion in reticulin fibers was characterized by the absence of the network of reticulin fibers (Figure 1Q, 1R). In some cases of this group, thin and short fibrillar elements may persist, which we consider remnants of the degraded fibrillar network. Most of the samples in this group marked with a score of 3 do not show a positive histochemical reaction for fibers and, like the previous group, do not ensure the viability of a possible venous graft.

As can be seen from Table 1, the reticulin fibers seem to be the most resistant to the changes that occur in varicose disease, because we observed normal appearance in nine of the samples. Severe depletion was observed in 10 cases, significantly less than the similar ones noted for elastic and collagen fibers. In cases scored 1 and 2, we did not observe significant differences between the samples included in this study.

In the cases with thrombi or recanalization, Masson’s trichrome staining revealed some particular aspects. In the area of the initial lumen, it was noticed the proliferation of a tissue with numerous thin collagen fibers, with variable numbers of fixed cells, without inflammatory elements. In all these cases, numerous neoformation vessels, small in size, like blood capillaries were present. The collagen fibers showed variable thicknesses from one area to another, with a heterogeneous disposition, forming a true network (Figure 1S, 1T).

A particular aspect was noticed to the cases in which thrombi were present (eight specimens) in different stages of recanalization. In these cases, the inner elastic limiting membrane was present and continuous, it did not show duplications. In the area of organized thrombus, we identified an Orcein-positive fibrillar substrate, similar to elastic fibers, but much shorter and paler stained (Figure 1U, 1V).

It was found that the inverse proportional ratio between reticulin fibers and collagen fibers between the two fibrillar forms, there being an inversely proportional relationship. Respectively, in cases with partial or complete depletion of reticulin fibers, we noticed a massive accumulation of collagen fibers, especially at the media level (Figure 1W, 1X).

Discussions

Most of the veins are small or medium size veins, with diameters of 10 mm or even less. Such veins are usually located near the corresponding muscular arteries. Histologically, the vein wall consists of three layers: intima, media and adventitia, which are not as distinct as they are in arteries. The intima usually has a thin subendothelial layer, the media consists smooth muscle cells mixed with reticulin fibers and a delicate network of elastic fibers. The adventitia is well developed, consisting of connective tissue containing nerve fibers and vasa vasorum [13].

In patients with varicose veins, the venous wall shows a series of morphological changes: hypertrophy of the structural elements of the wall, initial phenomena of sclerosis of the venous wall on the background of hypertrophy, as well as initial phenomena of atrophy of the venous wall on the base of severe sclerosis. At the level of the intima of the varicose veins, a hypertrophy of the endothelium associated with small areas of desquamation and hypertrophy of the subendothelial layer may be noticed. Other microscopic changes that can appear are collagenosis and the fragmentation of individual collagen fibers. The smooth muscle cells of the media are hypertrophied, and interfascicular sclerosis is noticeable among them. The muscular tunic is made up of two well-differentiated layers of muscle fibers. The external layer, the adventitia, is thin and sometimes not visible microscopically [10].

Even if is highly prevalent, chronic venous disease remained underestimated global concern situation [14]. For the chronic venous disease classification, the Clinical–Etiology–Anatomy–Pathophysiology (CEAP) system was used [15]. Varicose veins are the most common clinical sign of chronic venous disease. Starting from this stage, corresponding to the C2 classification, it may be noticed a progression to the C3 (edema), C4 (cutaneous manifestations) and C5/C6 (healed and active ulcerations) [16].

Data showed some differences in the number of elastic fibers depending on the presence or absence of venous reflux and the age of the patients. Thus, it was noticed that the R (pathological reflux) patients had a significant decrease in the number of elastic fibers in the tunica media of the vein wall. Considering the age factor, it was found that the NR (absence of pathological reflux) ≥50 and R<50 have the highest number of elastic fibers that were statistically significant. Similar aspects were found in the intimal and adventitial tunics [17, 18, 19].

In our study was noticed that the cryostripping procedure and the histological techniques of fixation and inclusion do not specifically alter the architecture and structure of the elastic fibers in the maximal range studied. Elastic fibers have a specific distribution in the three layers of morphologically normal veins. We described for the first-time duplications and multiplications of the internal elastic limiting membrane, in a diffuse and nodular pattern. In some of these cases, at the interface between the intima and the media, it was found the accumulation of an orceinophilic material of unknown significance. Depletion of elastic fibers at the media level is a lesion that was identified in most specimens.

Some studies have shown differences in the expression of precursors of the elastic fibers. Thus, tropoelastin was significantly upregulated in patients with venous reflux in comparison to patients without reflux [20]. The differences were independently by age. Tropoelastin is synthetized by cells, especially smooth muscle cells and secreted to extracellular matrix (ECM). The same cells are involved in the synthesis of fibrilin-1. It was shown an increase of fibrilin-1 expression in the veins and skin of patients with chronic venous disease [21]. It is well known the role of fibrillin-1 in the modulation of transforming growth factor-beta (TGF-β).

Different therapeutic methods for the varicose disease were described, such as: conventional stripping, invaginated stripping, cryostripping, extraluminal stripping, phlebectomy. But no significant advantages were described for either method [22, 23, 24]. Not so much data is found regarding the collagen and reticular fiber of the vein wall after this intervention.

Compared with the normal control of the saphenous vein, varicose vein sections showed increased diameter of the lumen and hypertrophy of the wall, mainly of the intima, due to increased amounts of collagen fibers. Collagen fibers also lost their normal pattern and showed abnormal forms. Elastic fibers lost their regular laminar arrangement and formed clumps or scattered fragments [9, 25]. In our study, in some cases, the fibrosis was extended to the entire venous wall. A particular aspect was the diffuse collagenization of the subintimal space, occupied in the cases evaluated with value 3 of score mostly by the presence of an amorphous collagen-type material that greatly modifies the architecture of the venous wall. The accumulation of collagen fibers was correlated with depletion of reticulin fibers. The collagen fibers partially or completely replace the network in the media and intima.

Reticulin fibers are present in all three layers of the vascular wall in specimens with normal morphology. We report changes such as partial or total depletion of reticulin fibers in most of the evaluated samples. We believe that these changes are the result of the long-term effects of varicose disease. We found no correlation between changes in the reticulin network and the time between harvest and buffered formalin fixation, the maximum time investigated being five days. The cases marked with scores 2 and 3 do not present important elements that ensure the viability of the investigated venous segment.

Conclusions

With Masson’s trichrome staining, we highlighted the changes in the collagen fibers in the structure of the varicose vein wall. Based on the morphology and scorification of this fibrillar element, we found no major structural differences in terms of alterations of these fibers induced by the applied surgical procedure. An interrelation between the accumulation of collagen fibers and depletion of reticulin fibers was found. Based on the results obtained, we consider the introduction of Orcein staining in the venous vessel investigation panel may be useful.

Conflict of interests

The authors declare that they have no conflict of interests.