Comparison of the effects of endovenous laser ablation at 1470 nm versus 1940 nm and different energy densities

Abstract

Objectives

To evaluate histological and immunohistochemical changes in the great saphenous vein after endovenous laser ablation at two different wavelengths (1470 vs. 1940 nm) and linear endovenous energy density values (50 vs. 100 J/cm).

Method

Segments were obtained from the conventional eversion removal of great saphenous vein and divided into a control group and four groups for ex vivo irradiation (control group; A: 1470 nm, 50 J/cm; B: 1470 nm, 100 J/cm; C: 1940 nm, 50 J/cm; D: 1940 nm, 100 J/cm). Fifty venous segments (n = 10/group) were analyzed. Changes were classified into low-temperature changes, moderate-temperature changes, high-temperature changes, and very high-temperature changes.

Results

In the intima, low-temperature changes + moderate-temperature changes were significantly more prevalent in group A (65.4%) than in D (p = 0.001). In the media, low-temperature changes + moderate-temperature changes were achieved mostly in groups A and C (77.4% and 75.0%, respectively). In adventitia fragments, 100% of changes in group A were low-temperature changes + moderate-temperature changes.

Conclusions

The 1940-nm laser wavelength with linear endovenous energy density of 100 J/cm was excessively destructive to the intima and media causing a high rate of high-grade thermal damage. These findings corroborate the possibility of using lower linear endovenous energy densities with 1940-nm devices to achieve effective occlusion with less high grade thermal damage to the intima and media, as well as to prevent damages to the adventitia and perivenous tissues, including venous perforation and its attendant clinical consequences.

Keywords

Varicose veins endovenous thermal ablation

Introduction

The objective of endovenous laser ablation (EVLA) is to abolish pathologic retrograde blood flow by persistently obliterating the venous lumen. Its mechanism of action is based on inflicting thermal injury to cause endothelial and media damage, thus leading to thickening and fibrosis of the vessel wall and, ultimately, non-thrombotic occlusion of the incompetent vein.¹ The intensity of vessel wall contraction appears to play an important role, as the residual lumen remaining after laser ablation is subject to clot formation and thrombotic occlusion. These vessels may later recanalize. In such cases, laser irradiation is not sufficient to heat the vessel wall, because the light energy is almost entirely absorbed by the blood; thus, the initial success rate is mainly due to a thrombotic effect and, after thrombus dissolution, leads to recanalization of the vessel.²

Some EVLA parameters are known to affect the outcomes of patients treated with this technique. These parameters include power, wavelength, linear endovenous energy density (LEED), and type of laser fiber.³

The energy of 810-nm, 940-nm, and 980-nm diode lasers is absorbed by hemoglobin, and that of 980-nm laser, by water as well, while 1320-nm, 1470-nm, and 1940-nm lasers are water specific.⁴ Whether these water-specific systems act more selectively on the vessel wall and less through the indirect effects of intraluminal vapor bubbles, as demonstrated in 810- to 980-nm lasers, is still a matter of debate.²

Optimal wavelength and linear LEED settings would be able to provide the best balance between a high anatomical success rate and a low procedure-related complication rate.

Within this context, the present study sought to evaluate histological and immunohistochemical changes in the great saphenous vein (GSV) after ex vivo EVLA at different wavelengths (1470 nm and 1940 nm) and with different LEED values.

Materials and methods

This study is part of a line of research in early detection methods and evaluation of prognostic factors in surgical conditions of the Graduate Program in Clinical Surgery at Universidade Federal do Paraná, Brazil. The research project was approved on 1 June 2015 by the Research Ethics Committee of Hospital Angelina Caron (project approval no. 45209015.9.0000.5226) and conducted in accordance with Brazilian Ministry of Health guidelines.

The inclusion criteria were patients of both sexes, aged 18–70 years, with indications for eversion stripping of the GSV and a GSV diameter ranging from 3 to 10 mm, as determined on Doppler ultrasound study. The exclusion criteria were history of GSV thrombophlebitis, GSV diameter < 3 mm, or GSV diameter > 10 mm.

To obtain venous specimens for the present study, five patients who met the inclusion criteria and were scheduled to undergo conventional eversion stripping of the GSV were selected. All signed an informed consent form authorizing experimental use of their veins after the surgical procedure and collection of a 20-mL blood sample for later use in the ex vivo model. Ten GSV segments of measuring approximately 23-cm and 3-cm segments were sectioned to be used as a control group for the analysis of venous lesions after being removed by stripping. Subsequently, segments of approximately 20 cm long were prepared for experimental thermal ablation.

For ex vivo thermal ablation of the GSV, we used an experimental model previously described by Araujo et al., which involves the use of glass tubes and introducer sheaths and is able to reproduce the physiological conditions of in vivo thermal ablation, such as tumescence and blood flow.⁵

In brief, the 20-cm GSV segments were placed in physiological position, stretched, and tied to an introducer sheath at either end. The glass tube was then filled with normal saline solution at room temperature. A radial laser fibre was introduced into the vein through one of the introducer sheaths, and the previously collected human blood (containing ethylenediaminetetraacetic acid [EDTA] as anticoagulant) was injected through the introducer sheath infusion port and simultaneously aspirated through the other sheath, to simulate continuous blood flow. At this point, thermal ablation started.

Experiments were performed using 1470-nm and 1940-nm diode laser emitters (DMC, Brazil), coupled to a radial fibre (DMC, Brazil) and an automatic pullback device (CoolTouch Corporation, USA). Thermal ablation was performed in 10 distinct GSV segments. Each segment was irradiated with four different parameter settings, which were applied to distinct 3-cm long segments within the vein. A 2-cm interval was left between segments to create a transition zone free of the effects of thermal ablation (Figure 1).

Figure 1.

Experimental model. Thermal ablation parameter groups (a–d).

Immediately after irradiation, each GSV segment was removed from the system. The venous segments were then divided at the transition zones, yielding the 3-cm subsegments irradiated with each set of parameters. The 50 resulting specimens were fixed in 10% formalin and constituted um control group CG (CG1, CG2, CG3, CG4, CG5, CG6, CG7, CG8, CG9, and CG10) and four experimental groups of the study: group A (A1, A2, A3, A4, A5, A6, A7, A8, A9, and A10); group B (B1, B2, B3, B4, B5, B6, B7, B8, B9, and B10); group C (C1, C2, C3, C4, C5, C6, C7, C8, C9, and C10); and group D (D1, D2, D3, D4, D5, D6, D7, D8, D9, and D10).

The pathologist in charge of histological and immunohistochemical examination was blinded to group allocation and to irradiation parameters, except for the control group. At the pathology laboratory, specimens were cut into 1-mm cross sections for histological processing and paraffin embedding; The resulting slides from each specimen were stained with hematoxylin and eosin (H&E), histochemical staining for elastic fibers and Gömöri's trichrome, and immunohistochemical staining for smooth-muscle actin. Fragments from the control group were analyzed first and changes in venous wall resulting from stripping were ruled out. Fragments that underwent stripping totalled 208 fragments. Changes were classified into four categories: low-temperature changes (LTCs), defined as smooth-muscle or elastic fibre oedema; moderate-temperature changes (MTCs), including merging or vacuolization of elastic fibres; high-temperature changes (HTCs), including tissue carbonization; and very high-temperature changes (VHTCs), characterized by frank tissue loss (Figure 2).

Figure 2.

Changes induced by endovenous laser ablation (a–j). (a) Histochemical staining for elastic fibres in a GSV segment not subjected to thermal ablation. The fibers (stained dark brown) are regular, continuous, and organized. (b) Low-temperature changes (LTCs) in a GSV segment subjected to thermal ablation, showing disorganization, oedema, and greater spacing between elastic fibres. (c) Histological appearance of a central segment of GSV not subjected to thermal ablation. (d) Representative image of moderate-temperature changes (MTCs), including eosinophil infiltration of the media and vacuolization of the luminal portions of the intima and media (haematoxylin and eosin staining). (e) Gömöri's trichrome staining of a central segment of GSV not subjected to thermal ablation. (f) MTCs in an ablated segment, including changes in stain affinity (red) and vacuolization. Evidence of high-temperature changes (HTCs) on (g) H&E staining and (h) Gömöri's trichrome staining, including carbonization of the intima, vacuolization of the media, marked eosinophilia, and changes in stain affinity as a result of protein denaturation. Evidence of very high-temperature changed (VHTCs) on (i) H&E staining and (j) Gömöri's trichrome staining (b), including full-tissue tissue loss affecting the intima, media, and adventitia (perforation), on a background of HTCs and MTCs.

The four categories of change used for histological and immunohistochemical analysis (LTCs, MTCs, HTCs, and VHTCs) were subsequently pooled into two categories for statistical analysis: LTCs + MTCs and HTCs + VHTCs, taking into account the severity of tissue injury and the fact that any temperature high enough to cause tissue carbonization or ulceration is excessive.

In each vessel layer, groups were compared two by two to test the null hypothesis of equal likelihood of high-grade thermal damage versus the alternative hypothesis of different likelihood. Fischer's exact test was used for comparison with Bonferroni's correction for p values. Overall significance level in each vessel layer was set at 0.05 and, due to Bonferroni's correction, p < 0.008 was considered statistically significant. Data were analyzed using IBM SPSS Statistics, version 20.

Results

Intima

All 208 fragments (100%) exhibited some change in the intima: 52 in group A (25.00%), 50 in group B (24.0%), 52 in group C (25.0%), and 54 in group D (26.0%). Changes in the tunica intima, stratified by intensity of thermal injury into LTCs + MTCs vs. HTCs + VHTCs in each experimental group, are described in Table 1 and Figure 3.

Table 1.

Changes in the intima, media, and adventitia, stratified by intensity of thermal injury into LTCs + MTCs vs. HTCs + VHTCs, in each experimental group.

Group	No. of fragments	LTCs + MTCs	HTCs + VHTCs
Intima
A	52	34 (65.4%)	18 (34.6%)
B	50	24 (48.0%)	26 (52.0%)
C	52	22 (42.3%)	30 (57.7%)
D	54	17 (31.5%)	37 (68.5%)
Media
A	53	41 (77.4%)	12 (22.6%)
B	49	28 (57.1%)	21 (42.9%)
C	52	39 (75.0%)	13 (25.0%)
D	53	23 (43.4%)	30 (56.6%)
Adventitia
A	14	14 (100%)	0 (0%)
B	11	6 (54.5%)	5 (45.5%)
C	10	3 (30.0%)	7 (70.0%)
D	30	22 (73.3%)	8 (26.7%)

LTCs: low-temperature changes, MTCs: moderate-temperature changes, HTCs: high-temperature changes, and VHTCs: very high-temperature changes

Figure 3.

Representative graphs of the degree of changes in the intima, stratified by intensity of thermal injury into low grade (LTCs + MTCs) vs. high grade (HTCs + VHTCs) changes, in each experimental group.

The majority of LTCs and MTCs in the intima occurred in group A (65.4%), with significant differences A vs. D (p = 0.001) comparisons. The majority of HTCs and VHTCs occurred in group D (68.5%), with significant differences on comparison with group A (p = 0.001). Table 2 lists the p values for all comparisons.

Table 2.

P values for pairwise statistical comparison of groups regarding changes in the intima, media, and adventitia.

Group comparison	P value*
Intima
A vs. B	0.109
A vs. C	0.030
A vs. D	<0.001
B vs. C	0.691
B vs. D	0.109
C vs. D	0.314
Media
A vs. B	0.035
A vs. C	0.822
A vs. D	<0.001
B vs. C	0.063
B vs. D	0.234
C vs. D	0.001
Adventitia
A vs. B	0.009
A vs. C	<0.001
A vs. D	0.041
B vs. C	0.387
B vs. D	0.280
C vs. D	0.024

Fisher's exact test; Bonferroni-corrected p value < 0.008.

Boldface indicates statistical significance.

Media

A total of 207 fragments (99.51%) exhibited some change in the media: 53 in group A (25.6%), 49 in group B (23.7%), 52 in group C (25.1%), and 53 in group D (25.6%). These changes are described in Table 1 and Figure 4.

Figure 4.

Representative graphs of the degree of changes in the media, stratified by intensity of thermal injury into low grade (LTCs + MTCs) vs. high grade (HTCs + VHTCs) changes, in each experimental group.

The majority of LTCs and MTCs in the tunica media occurred in groups A and C (77.4% and 75.0%, respectively), with no significant difference between them; significant differences were found on A vs. D (p < 0.001), and C vs. D comparisons (p = 0.001). Conversely, most HTCs and VHTCs in the media occurred in group D (55.6%). Differences were significant on comparison with groups A (p = 0.001) and C (p = 0.001). Table 2 lists p values for all comparisons.

Adventitia

A total of 65 fragments (31.25%) exhibited some change in the adventitia: 14 in group A (21.5%), 11 in group B (16.9%), 10 in group C (15.4%), and 30 in group D (46.2%). These changes are described in Table 1 and Figure 5.

Figure 5.

Representative graphs of the degree of changes in adventitia, stratified by intensity of thermal injury into low grade (LTCs + MTCs) vs. high grade (HTCs + VHTCs) changes, in each experimental group.

In group A, 100% of changes were LTCs and MTCs. The majority of HTCs and VHTCs changes occurred in group C, with significant differences on comparison with group A (p < 0.001). Again, Table 2 lists the p values for all comparisons.

Discussion

The purpose of EVLA is to cause irreversible circumferential damage to the vessel wall, triggering an inflammatory response which includes cell infiltration and fibrogenic reactions. These inflammatory events lead to permanent occlusion of the incompetent vein. However, any temperature high enough to cause tissue carbonization or ulceration is excessive. Carbonization of the vessel wall increases the likelihood of perforation and the risk of clinical complications, such as postoperative pain, bruising, and recanalization.⁶ Corcos et al.⁷ assessed histological changes in the vessel wall after laser irradiation and described such processes as necrosis, vacuolization, delamination, coagulation, tissue loss, and perforation. Each of these changes reflects a specific temperature reached during the procedure.⁷

Yamamoto and Sakata⁸ devised a simplified classification of histological changes into LTCs, MTCs, HTCs, and VHTCs. The cell response to heating follows a well-established sequence of events. Prolonged heating to 42–45 ℃ leads to reversible, sublethal damage not visible on microscopic examination. Above 50–60 ℃, proteins lose their structure, leading to irreversible necrosis, denaturation, and coagulation. Microscopically, swelling of elastic and muscle fibres (LTCs) is visible. At 90–100 ℃, cells melt, causing merging of numerous fibers and loss of intracellular components. Tissue water is vaporized, leading to steam formation and desiccation. Shrinkage of desiccated tissue leads to vacuolization, i.e., the formation of small gaps between fibers (MTCs). When heated to 200–300 ℃ under oxygen-poor conditions, organic compounds release nitrogen, hydrogen, and other elements, and begin to dissociate (HTCs). Direct contact between an excessively hot fiber tip and the vessel wall leads to ulceration and perforation (VHTCs).⁸

Taking into account that temperatures high enough to cause tissue carbonization or ulceration (HTCs and VHTCs) during endovascular thermal ablation must be considered excessive, we decided to stratify changed into two pooled categories—LTCs + MTCs and HTCs + VHTCs—for statistical analysis.

Laser devices which target water appear to be more efficient in terms of light energy absorption than those which operate with hemoglobin as the chromophore target.⁹ Patients treated with higher-wavelength diode lasers have been shown to experience greater postoperative comfort, higher satisfaction and acceptance, less postoperative pain (and, consequently, lower analgesia requirements), and less bruising.¹⁰

Aktas et al.¹¹ compared the efficacy of 980-nm and 1470-nm lasers in terms of output power, complications, recanalization rates, and treatment response, and concluded that 1470-nm radial-tip laser devices are safe and effective for endovascular laser ablation. The authors also found that a lower energy density (LEED 50–60 J/cm) was effective for GSV occlusion, and that treatment response, as assessed by improvement in clinical severity score, was superior with the 1470-nm laser.¹¹

Regarding histological changes, the use of higher-wavelength lasers (1320, 1470, 1500 nm) leads to greater vacuolization of the muscle layer of the vessel wall, with marked thermal destruction.¹² Vuylsteke et al. compared histological destruction of the vessel wall induced by 980-nm and 1500-nm diode lasers during endovenous laser treatment (EVLT) of the saphenous vein in a goat model. They observed that energy issued from the 980-nm laser is absorbed to a lesser extent by cells in the vessel wall and induces greater local convection, which explains the deeper ulcerations and more severe carbonization seen at sites of direct contact with the laser tip. Conversely, the 1500-nm laser induced less eccentric destruction of the vessel wall and shallower ulcerations; from a clinical standpoint, this might correlate with less hazardous tissue destruction and less postoperative pain.¹³

Sroka et al.¹⁴ studied the early histological effects of laser in the ex vivo blood-filled ox-foot model. With a LEED of 30 J/cm, the authors found circular destruction of the intima and vacuolization and delamination of the media on H&E-stained sections. A later prospective observational study used a 1940 nm laser diode, radial fibre, and automatic pullback device with a standard speed of 1 mm/s. At six-month follow-up, complete resolution of venous reflux had been achieved in 100% of insufficient saphenous veins, as had a significant reduction in treated vein diameter. Risk profile correlated with other endovenous treatment options and with low postoperative pain and analgesic utilization rates, suggesting a high level of patient comfort.^14,15

In a mathematical modelling study, Poluektova et al. demonstrated that, at the same amount of delivered energy, the vessel wall would reach temperatures at near-1950 nm wavelengths only in vessel diameters < 1 mm. Otherwise, one would theoretically need to deliver a higher energy output to reach the same temperatures achieved with a 1470-nm laser. This was justified by the fact that high absorption of near-1950 nm wavelengths leads to reduced heat penetration capacity. In other words, the generated heat would be “retained” near the site of emission.¹⁶

In the present study, EVLA at a wavelength of 1470 nm and LEED of 50 J/cm resulted in a higher rate of low grade thermal injury (LTCs and MTCs) in all vessel layers (intima, media, and adventitia). Conversely, ablation at a wavelength of 1940 nm and LEED of 100 J/cm resulted in a higher rate of high grade thermal injury (HTCs and VHTCs) in the intima and media. This was also the group with most fragments (46.2%) showing changes in the adventitia. These findings suggest that use of lower LEEDs with 1940-nm devices may lead to effective occlusion with less destruction of the adventitia and perivenous tissues, possibly preventing venous perforation and its attendant clinical consequences.

Comparison of the effect of the two tested wavelengths (1470 nm vs. 1940 nm) at the same LEED (50 J/cm) on the adventitia demonstrated superiority of the 1470-nm wavelength in terms of inducing LTCs and MTCs. Although comparison of the effects of 1470 vs. 1940 nm at the same LEED level (50 J/cm or 100 J/cm) in the tunica intima and media revealed no significant differences, contradicting the mathematical model of Poluektova et al.,¹⁶ these findings are consistent with the notion and hypothesis that the 1940-nm wavelength with high LEED (100 J/cm) could thus lead to greater vacuolization and thermal destruction of the venous wall.

Limitations

Although these ex vivo experiments look promising, they have some limitations, such as: the fact that the vein segment may undergo changes during harvesting; only the immediate effects of treatment can be analysed; and study of perivascular tissue is impossible.

In the present study, all treated veins had a diameter of 3–10 mm, thus preventing generalization of results to vessels <3 mm or >10 mm.

The LEEDs used in this study (50 J/cm and 100 J/cm) precluded assessment of the specific effects of the 1940 nm wavelength in relation to its predecessors, particularly considering the use of LEEDs < 50 J/cm.

We conclude that EVLA at a wavelength of 1470 nm and LEED of 50 J/cm produced the highest rates of low grade thermal injury (LTCs and MTCs) in all vessel layers (intima, media, and adventitia). The 1940-nm laser wavelength with LEED of 100 J/cm was excessively destructive to the intima and media, causing a high rate of high grade thermal damage (HTCs and VHTCs). These findings corroborate the possibility of using lower LEEDs with 1940-nm devices to achieve effective occlusion with less high-grade thermal damage to the intima and media, as well as to prevent damages to the adventitia and perivenous tissues. Although these ex vivo experiments appear promising, several limitations must be considered. Randomized clinical trials and a more in-depth analysis of the effects of different wavelengths and LEEDs, especially in large-diameter veins, are warranted.

Footnotes

Acknowledgements

We thank DMC and Cardio and Endo Luminal for their gracious loan of the laser devices used in this study and Professor Roberto Kasuo Miyake for his loan of a CoolTouch automatic pullback device (CoolTouch Corporation, Roseville, CA, USA).

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: this study was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

Ethical approval

The research project was approved on 1 June 2015 by the Research Ethics Committee of Hospital Angelina Caron (project approval no. 45209015.9.0000.5226) and conducted in accordance with Brazilian Ministry of Health guidelines.

Guarantor

WJBA

Contributorship

WJBA and JRRT conceived and designed the study. WJBA, LRK, and CRVC were involved on data analysis and interpretation. WJBA wrote the first draft of the article. JRRT made the critical revision of the article. WJBA and LRK were responsible for statistical analysis. WJBA took overall responsibility for the study. All authors reviewed and edited the manuscript and approved the final version of the manuscript.

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