# Preclinical Demonstration of In-Vivo Vein Occlusion by Mechanical HIFU Without Contrast Agent Injection: Paving the Way for the Non-Invasive Treatment of Varicose Veins

A. Ganeau, T. Charrel, O. Pichot, C. Lafon

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A. Ganeau, T. Charrel, O. Pichot, C. Lafon. Preclinical Demonstration of In-Vivo Vein Occlusion by Mechanical HIFU Without Contrast Agent Injection: Paving the Way for the Non-Invasive Treatment of Varicose Veins. Ultrasound in Medicine & Biology, 2025, 51 (8), pp.1210-1216. 10.1016/j.ultrasmedbio.2025.04.004. hal-05235646

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Original Contribution

# Preclinical Demonstration of In-Vivo Vein Occlusion by Mechanical HIFU Without Contrast Agent Injection: Paving the Way for the Non-Invasive Treatment of Varicose Veins

A. Ganeau<sup>a,*</sup>, T. Charrel<sup>a</sup>, O. Pichot<sup>b</sup>, C. Lafon<sup>c</sup>

<figure><img src="image_4.png" /></figure>

ARTICLE INFO

Keywords:

Pulsed Ultrasound

Ultrasonic cavitation

Non-thermal HIFU

Vein occlusion

ABSTRACT

**Objective:** High-intensity focused ultrasound (HIFU) is an emerging non-invasive treatment for various pathologies, including in the field of vascular medicine. Clinical studies have demonstrated its efficacy for vascular occlusion through thermal effects. An interesting yet early-stage alternative is mechanical HIFU, where occlusion is achieved by cavitation initiated by the injection of microbubble contrast agents. Our study aims to demonstrate the feasibility of an innovative ultrasound-guided mechanical HIFU device for non-invasive in-vivo vein occlusion by cavitation without microbubble contrast agents.

**Methods:** A four piezoelectric ceramic device was developed and acoustically characterized. Erosion efficiency by cavitation was assessed on agar phantom models with cylindrical channels created to mimic veins. A preclinical feasibility demonstration was carried out in-vivo on a sheep model, targeting a collateral saphenous vein in the hind limb. Vein occlusion was investigated using ultrasound imaging during a 7-day follow-up and at the cellular level by histological analysis.

**Results:** A maximum negative pressure of -23 MPa was measured at the focal point of dimension 1.27 mm³ at -6 dB. In agar phantom models, a centrally applied HIFU treatment was sufficient to erode small veins, while an application at multiple points was needed for larger veins. In vivo, cavitation was triggered in a small-diameter vein, causing occlusion and preventing blood flow. Histology confirmed vein wall damage and occlusion.

**Conclusion:** Vein occlusion was successfully achieved in-vivo by cavitation using mechanical HIFU without micro-bubble contrast agents. This approach holds real potential for the non-invasive treatment of varicose veins, without the limitations of current techniques.

Introduction

High-intensity focused ultrasound (HIFU) is an emerging therapeutic approach for a large and growing number of clinical applications [1]. Non-invasive and non-ionizing, HIFU enables localized tissue ablation through two main mechanisms: thermal or mechanical. Thermal HIFU involves continuous HIFU pulses, emitted for several seconds, with acoustic intensity at the focus ranging from 1300 to 2200 W.cm⁻². The result is a localized rapid temperature increase to 85–90°C inducing tissue necrosis at the target. Applications include the treatment of prostate cancer, uterine fibroids and essential tremors [2,3]. In comparison, mechanical HIFU, involves short HIFU pulses (2–100 µs) resulting in high acoustic intensity at the focus (>10 kW.cm⁻²) [4] that corresponds to peak negative pressures of -12 to -25 MPa [5]. A pulse repetition frequency (PRF) of 100 Hz – 1 kHz triggers the phenomenon of cavitation

which is characterized by the formation of a dense bubble cloud inside the tissue [6]. The subsequent collapse of these cavities causes the discharge of intense micro-jets that mechanically damage the targeted tissue, a method referred to as histotripsy [7]. There are a number of promising applications for mechanical HIFU including the treatment of a number of cancers [8], in particular liver cancer [9], as well as deep vein thrombosis [10]. Real-time monitoring is possible due to the visibility of the cavitation cloud in ultrasound imaging and its characterization by the frequency content of the scattered acoustical noise [11,12]. An additional advantage of mechanical compared to thermal HIFU treatment concerns the better preservation of the targeted and surrounding tissue from thermal damage due to the low duty cycle.

An increasing number of applications are emerging for both thermal and mechanical HIFU in the field of vascular medicine, such as thrombolysis, where cavitation has been shown to dissolve blood clots in

**Objective:** High-intensity focused ultrasound (HIFU) is an emerging non-invasive treatment for various pathologies, including in the field of vascular medicine. Clinical studies have demonstrated its efficacy for vascular occlusion through thermal effects. An interesting yet early-stage alternative is mechanical HIFU, where occlusion is achieved by cavitation initiated by the injection of microbubble contrast agents. Our study aims to demonstrate the feasibility of an innovative ultrasound-guided mechanical HIFU device for non-invasive in-vivo vein occlusion by cavitation without microbubble contrast agents.

**Methods:** A four piezoelectric ceramic device was developed and acoustically characterized. Erosion efficiency by cavitation was assessed on agar phantom models with cylindrical channels created to mimic veins. A preclinical feasibility demonstration was carried out in-vivo on a sheep model, targeting a collateral saphenous vein in the hind limb. Vein occlusion was investigated using ultrasound imaging during a 7-day follow-up and at the cellular level by histological analysis.

**Results:** A maximum negative pressure of -23 MPa was measured at the focal point of dimension 1.27 mm³ at -6 dB. In agar phantom models, a centrally applied HIFU treatment was sufficient to erode small veins, while an application at multiple points was needed for larger veins. In vivo, cavitation was triggered in a small-diameter vein, causing occlusion and preventing blood flow. Histology confirmed vein wall damage and occlusion.

**Conclusion:** Vein occlusion was successfully achieved in-vivo by cavitation using mechanical HIFU without micro-bubble contrast agents. This approach holds real potential for the non-invasive treatment of varicose veins, without the limitations of current techniques.

<footnote><sup>a</sup> R&D Department, Veinsound, Lyon, France</footnote>

<footnote><sup>b</sup> Département of Vascular Medicine, Groupement Hospitalier Mutualiste, Grenoble, France</footnote>

<footnote><sup>c</sup> LabTAU, INSERM, Centre Léon Bérard, Université Claude Bernard Lyon 1, Lyon, France</footnote>

<footnote>* Corresponding author. R&D Department, Veinsound SAS, 60 avenue Rockefeller, Bâtiment Quanta, 69008, Lyon, France.</footnote>

<footnote>E-mail address: aliceg@veinsound.com (A. Ganeau).</footnote>
preclinical studies [10,13,14], and vascular occlusion, typically achieved by coagulation necrosis induced by thermal effects [15–19]. Vascular occlusion by HIFU shows significant promise for the treatment of varicose veins [20–22] and also tumors by cutting off the blood supply [23]. A number of different mechanisms have been identified as contributing to vascular occlusion by HIFU: vein spasm, damage to the endothelial layer, the acute inflammatory response triggered by platelet aggregation and the formation of intraluminal occlusion [24,25]. In the case of thermal HIFU, collagen shrinkage can further narrow the vein, increasing the inflammatory response and contributing to fibrosis and vascular occlusion. Though vein occlusion by the effect of ultrasonic cavitation on the vein wall has been demonstrated [26], the minimally invasive nature of this study should be noted as it requires the injection of microbubbles to initiate the cavitation process.

The objective of our study is to demonstrate the feasibility of non-invasive in-vivo vein occlusion by cavitation using mechanical HIFU and without microbubble contrast agents. An innovative device – composed of an ultrasound-guided HIFU probe – has been designed for the treatment of superficial veins. Characterization of the device was achieved through numerical simulations and measurements. The efficiency of mechanical HIFU to erode vein walls by cavitation was assessed using agar phantom models. A preclinical in-vivo demonstration of the feasibility of the method for non-invasive vein occlusion was carried out using an animal model, with comprehensive evaluation during a 7-day follow-up with ultrasound imaging, and tissue examination via histological analysis.

## Materials and Methods

### Innovative ultrasound-guided HIFU device

The ultrasound-guided HIFU device is designed to treat superficial veins located 5-20 mm below the skin surface. The HIFU probe is composed of four spherical bowl-shaped piezoelectric ceramic elements (radius of curvature: 50 mm, outer diameter: 50 mm), with a central frequency of 1.1 MHz (PZ28, Fuji ceramics, Shizuoka, Japan), manufactured to optimize the total active surface area (67.8cm²). The four elements are assembled such that their geometric focal points overlap (Fig. 1a). This confocal set-up [27] has several advantages compared to a single-element set-up, including reduced linear distortion, lower acoustic power requirements, and minimized thermal effects [27], in addition to a more stable focal point and an interference pattern that can help trap cavitation bubbles. The HIFU elements are powered by a 2 kW power amplifier (Sinaptec, Lezennes, France) with electrical power

measured using a wattmeter (Rhode&Schwarz, Meudon-la-Forêt, France).

Ultrasound-guidance of the HIFU treatment enabling real-time visualization of the vein is achieved by an ultrasound imaging system (Evo-Touch+, Quantel Medical, Cournon d'Auvergne, France) using a 10 MHz linear transducer (LC1038V), positioned at the center of the HIFU probe and aligned with the focal point of the HIFU beams.

When in use, a latex membrane (EDM Imaging, Domont, France) is attached to the probe and filled with degassed, demineralized water. The water balloon formed improves ultrasound propagation and enables the focal depth to be controlled by adjustment of the water volume.

### Acoustic characterization of the ultrasound-guided HIFU device

The volume of the focal area was estimated by simulating HIFU propagation from the four piezoelectric ceramic elements, using the Rayleigh-Sommerfeld integral method and assuming a homogeneous medium close to water (speed of sound: 1482 m/s, density: 1000 kg/m³) with a 0.1 mm pitch spatial grid. Peak pressure measurements were carried out in a water tank using a 3D positioning bench with 10 µm displacement precision (Precision Acoustics, Dorchester, UK). A fiber optic sensor with a 100 µm active surface was mounted on the bench and powered by an 810 nm laser diode (200 nW), a photodetector and an amplifier (FOPH 2000, RP Acoustics, Leutenbach, Germany). The HIFU probe was fixed to the bench frame, powered by a function generator (33220A Agilent, Santa Clara CA, USA) and amplified with a power amplifier (E&I-1kW, Rochester NY, USA). The acoustic power and electro-mechanical efficiency were measured using a radiation force balance system (RFB-2000, Onda Corp., Sunnyvale CA, USA).

### Assessment of erosion efficiency by cavitation in agar phantom models

Agar phantom models were prepared by mixing degassed water with 0.7% agar powder, heated to 90°C and set in a rectangular mold. The acoustic properties are approximated as: ultrasound velocity 1500 m/s [28] and attenuation of 0.09 dB/MHz-cm [29]. Once firm, 2 and 5 mm cylindrical channels were hollowed out 1 cm below the surface. The 5 mm diameter channel represents the typical saphenous veins encountered in clinical practice, while the 2 mm diameter channel represents a contracted vein, commonly observed during thermal HIFU treatment [18].

Erosion of the channel wall by cavitation was characterized for 3 different treatment protocols. For the 5 mm channels, the focal point was either (1) positioned at the center of the channel with HIFU applied for

<figure><img src="image_5.png" /><figcaption>**Figure 1.** Acoustic characterization of the ultrasound-guided HIFU device. (a) A 3D representation of the device. (b) -20 dB pressure field simulated at the focal area. (c) Dimensions of the focal area for different pressure level thresholds. (d) Non-linear pressure measurements at the focal point using an optic fiber.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Figure 2.** Assessment of erosion efficiency by cavitation in agar phantom models. (a) Transverse view of the ultrasound-guided HIFU device, positioned on an agar phantom model. The x symbols represent the position of the HIFU focal point for each treatment protocol. Adjustment of the height of the water balloon allow control of the focal depth. (b) Ultrasound images acquired during HIFU treatment of channel 2. The focal spot is positioned at the lower point of the channel wall. The cavitation cloud triggered inside the channel is visible as a hyperechoic area. (c) A longitudinal view of the cylindrical channels after treatment. Channel 1: no visible erosion; channel 2: lateral and proximal but not distal erosion, illustrated in a zoom of one treatment point; channel 3: near-homogeneous erosion in all directions except distal. (d) Image of erosion at one treatment point of channel 3 with no erosion visible distally, acquired with the microscope. (e) Average erosion dimensions per channel.</figcaption></figure>

50 s, or (2) positioned at the proximal, right, left and distal points of the channel wall with HIFU applied for 12.5 s at each of the four points. (3) For the 2 mm channel, the focal point was positioned at the center of the channel with HIFU applied for 50 s (see Fig. 2a). For all protocols, HIFU pulses were of 100 µs duration at a PRF of 150 Hz. The peak electrical power transmitted to the probe was fixed at 416 W, corresponding to an equivalent negative pressure of -13MPa. Each protocol was repeated four times, shifted 1 cm along the channel each time. An additional untreated 5 mm channel served as a control. The experiment was carried out in a tank of water at room temperature on three different agar phantom samples. The resultant erosion was observed using a microscope camera with 0.5X magnification (OCS-SK2-HDMI-1080PU, Omano Scientific, China).

## Preclinical feasibility demonstration in-vivo

Sheep are recommended (ISO 10993-6) for the evaluation of vascular treatments and are a proven model for venous occlusion. For our study, the sheep were acclimatized for one week with daily health checks. Our protocol, approved under APAFIS #50841-2024061815097801v3 by the VetAgro Sup Ethics Committee (CEEA No.18 Project No.2447), is as follows. Macroscopic examinations were conducted before and after treatment on

the awake animal to assess changes to the skin. On D0, the sheep was anesthetized with morphine and propofol injected intravenously and maintained by inhalation. The animal was positioned laterally to target the veins of its hind limb, which was carefully shaved with a clipper and depilatory cream (Klorane, Pierre-Fabre, France) to optimize ultrasound coupling. The position of the lateral saphenous and collateral veins, 5-20 mm below the skin surface, was located by ultrasound (L3-12T probe, Alpinion, Seoul, Republic of Korea). The HIFU probe was then positioned to achieve a cross-sectional visualization of the vein to be treated (Fig. 3a–b and Fig. 3c–top). A custom mineral oil-based coupling medium was applied to the skin and medical-grade latex membrane to ensure effective coupling between the device and the skin. HIFU was applied, with the focal point positioned at the center of the collateral vein of 3mm diameter whose blood flow is similar to varirose human veins [30], for 50 s with pulses of 100 µs duration and a PRF of 150 Hz. The electrical power was gradually increased from 900 W to 2000 W to assess the power required to trigger cavitation, as monitored by ultrasound images. The HIFU treatment was repeated at 3 different points, spaced 1 cm apart, along the targeted vein.

After HIFU treatment, ultrasound imaging was carried out using B-mode and Color Doppler to investigate vein shrinkage or occlusion. This

<figure><img src="image_7.png" /><figcaption>**Figure 3.** Preclinical feasibility demonstration in-vivo. (a) Probe positioning on the sheep hind limb. (b) Transverse view of the ultrasound-guided HIFU device positioned on the hind limb. The height of the water balloon was adjusted to target the collateral lateral saphenous vein with the help of ultrasound-guidance. (c) Transversal view of the treatment monitored in real-time by ultrasound imaging: the cavitation cloud triggered in the vein appears as a hyperechoic area. The vertical line corresponds to the HIFU beam artifacts.</figcaption></figure>
ultrasound imaging was repeated on D3 without anesthesia and again on D7 under anesthesia.

After euthanasia, treated and untreated veins were marked with green anatomical paint and fixed using 10% buffered formaldehyde. Veins were cut into 5-8 mm sections with treatment points indicated by yellow anatomical paint. Each section was included in a single Formalin-Fixed Paraffin-Embedded (FFPE) block in a pathological cassette, sliced at 3 µm intervals and stained using hematoxylin-eosin (H&E). Histology slides were digitized at 20X magnification using a slide scanner (VS200, Olympus, Japan) and analyzed to assess the impact of HIFU treatment on the vein.

## Results

### Acoustic characterization of the ultrasound-guided HIFU device

Numerical simulation of the focal zone of the HIFU device is shown in Figure 1b. The focal area consists of a central spot corresponding to the 6 dB pressure level, of dimensions 0.8 mm x 0.9 mm x 2.9 mm. Secondary spots, located around the central spot, are due to interference from the different ultrasound beams, as observed in [27] for a dual-element confocal probe. Figure 1c details the dimensions of the focal area for different pressure level thresholds. Pressure measurements at the focal point (Fig. 1d) using an optical fiber indicate a maximum positive pressure of 48 MPa and a maximum negative pressure of -23 MPa, with a measurement uncertainty of 5%, for an acoustic intensity at the probe surface of 10 W.cm⁻². It should be noted, however, that at high pressures, this approach has limitations due to the radiation force and cavitation-induced phenomena, which can damage the optic fiber. Finally, an average electro-mechanical efficiency of 68% was measured using a radiation force balance, with a measurement uncertainty of 5%.

### Assessment of erosion efficiency by cavitation in agar phantom models

Figure 2b shows ultrasound images acquired during HIFU treatment of a 5 mm diameter channel, with the focal point positioned at the distal point of the channel wall. It can be seen that the cavitation cloud appears as a hyperechoic area in the image aligned with the position of the focal point. Cavitation-induced erosion is visible macroscopically (Fig. 2c). The erosion shape was investigated using a microscope camera for each treatment protocol: (1) the 5 mm channel with HIFU applied at the center of the channel (channel 1 in Fig. 2c), presents no evidence of erosion, similar to the control (channel 0 in Fig. 2c); (2) the 5 mm channel with HIFU applied at 4 points (channel 2 in Fig. 2c) shows lateral and proximal erosion (see zoom of Fig. 2c) but not distal; (3) the 2 mm channel (channel 3 in Fig. 2c) presents near-homogeneous erosion in all directions except distal (see Fig. 2d). Erosion dimensions were consistent within each channel and across agar phantom samples (see Fig. 2e).

### Preclinical feasibility demonstration in-vivo

The HIFU treatment was monitored in real time using ultrasound imaging, as presented in Figure 3c. A cavitation cloud, appearing as a hyperechoic area, can be observed inside the vein lumen (Fig. 3c center and right image). In our study, cavitation inside the vein was triggered at an electrical power of 1800 W, equivalent to an acoustic power of 1224 W. This corresponds to an equivalent negative pressure at the focal point of -23 MPa, which is within the intrinsic pressure threshold range for inducing inertial cavitation inside soft tissue (-12 to -25 MPa) [5].

Ultrasound images of the lateral saphenous and collateral veins before treatment are shown in Figure 4a, while Figure 4b shows the collateral vein lumen was observed, which could be explained either by venous spasm and/or immediate lumen occlusion. After application of

<figure><img src="image_8.png" /><figcaption>**Figure 4.** Ultrasound images of the lateral saphenous (LSV) and collateral (Col. V) veins before and after treatment. (a) D0 pre-treatment: A longitudinal view of the LSV and Col. V (diameter: 3 mm). (b) D0 post-treatment: Same view showing occlusion of the Col. V (diameter: 2.9 mm). Arrows approximately indicate the 3 treatment points. (c) D3: B-mode imaging: Longitudinal view of the LSV and occluded Col. V (diameter: 3.1 mm). (d) D3: Color Doppler imaging: Longitudinal view showing continued occlusion of the Col. V with blood flow in the LSV and a surrounding artery. (e) D7: B-mode imaging: Longitudinal view of the LSV and occluded Col. V (diameter: 4.3 mm). (f) D7: Color Doppler imaging: Longitudinal view showing continued occlusion of the Col. V with blood flow in the LSV.</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>**Figure 5.** Histological analysis of portions of the treated and untreated veins. The \* symbol indicates the vein wall closest to the HIFU device. (a), (b) Untreated vein: the lumen of the vein was unobstructed. The 3 layers of the wall vein - intima, media and adventitia – are all intact. (c), (d) Treated collateral vein: the lumen was completely occluded. The intima layer was no longer present, and the media layer presented advanced sclerification, with differentiation from the adventitia layer difficult.</figcaption></figure>

HIFU at the 3 treatment points, the treated collateral vein appeared occluded, with a 2.9 mm diameter and complete hyperechogenic filling of the lumen. No damage to the skin was observed after HIFU treatment. Ultrasound imaging on D3 and D7 showed continued occlusion of the collateral vein with a diameter of 3.1 and 4.3 mm respectively (Fig. 4c and 4e). Color Doppler imaging on D7 showed no blood flow in the treated collateral vein, unlike the untreated lateral saphenous vein (Fig. 4d and 4f).

Occlusion of the collateral vein lumen in histology can be seen in Figure 5c. Histological analysis showed significant changes in all three layers (intima, media and adventitia) of the treated collateral vein (Fig. 5c-d) compared to the untreated lateral saphenous vein (Fig. 5a-b). In detail, the endothelial layer (intima) was absent or melted in the treated collateral vein and the second (media) and third (adventitia) layers showed advanced sclerosis, hindering their differentiation (Fig. 5d). This advanced sclerosis is responsible for vein occlusion and the absence of blood flow.

## Discussions

This study aimed to demonstrate the feasibility of non-invasive vein occlusion by cavitation using a novel and innovative ultrasound-guided HIFU device and without microbubble contrast agents. Vein occlusion was successfully achieved in-vivo on a sheep model using a probe composed of four piezoelectric ceramic elements coupled with imaging to successfully treat a lateral saphenous collateral vein in the hind limb. The HIFU focal point was positioned at the center of the 3 mm diameter vein, triggering the formation of a cavitation cloud inside the vein lumen that was observed in real-time via ultrasound imaging. The HIFU treatment was applied for 50 s and repeated at 3 different points along the vein, with the resultant occlusion of the collateral vein observed on the day of treatment and again 3 and 7 days post-treatment. Histological analysis confirmed the occlusion of the treated vein.

Focal pressure measurements showed a maximum negative pressure of -23MPa which is within the intrinsic threshold pressure range needed to trigger cavitation inside soft tissue [5]. Assessment of erosion efficiency by cavitation in agar phantom models demonstrated the capability of the device to trigger cavitation inside cylindrical channels inducing local erosion. In a 5 mm diameter channel, no visible erosion was observed when HIFU was applied at the center of the channel. Erosion was, however, observed on the proximal and lateral points of the

channel walls when the focal point was positioned at those points. In a 2 mm diameter channel, erosion in all directions except the distal was achieved by cavitation. The lack of erosion distally may be due to the bubble cloud acting as a barrier, reflecting ultrasound waves and reducing cavitation activity. Between agar phantom samples, erosion was similar with dimensions varying with a maximum standard deviation of 1 mm. These differences could be attributed to the manufacturing process of the agar phantom models, targeting errors during ultrasound guidance, or potential inaccuracies in the measurement process, which could stem from the limitations of the microscopic imaging technique.

In general, the erosion dimension is approximately spherical with a diameter of 4 mm. This is larger than the - 6dB focal zone of the HIFU beam and closer in size to the -12 dB focal zone (see Fig. 1c). It should be noted, however, that numerical simulations were carried out at low acoustic intensity and do not take into account non-linear ultrasound propagation and the resulting focal zone consequences. Additional non-linear numerical simulations could be of interest to assess the pressure distribution, in particular the negative pressure field over the focal zone. Furthermore, although cavitation is triggered locally at the negative pressure threshold, bubble activity extends to nearby areas increasing erosion. The phantom models studied here do not take into account the physical properties of biological tissues, such as absorption (0.09 dB/MHz.cm in 0.7% agar compared to 1.45-0.17 dB/MHz.cm for venous and perivascular tissue [18]), elasticity or temperature, which can affect when cavitation is triggered, cavitation activity and erosion [31,32]. In conclusion, these model results indicate that for 2 mm veins in-vivo, occlusion could be achieved by positioning the HIFU focal point at the center of the lumen, whereas for 5 mm veins in-vivo, the focal point should be positioned at several points on the vein wall.

The pre-clinical study carried out on a sheep model demonstrates the feasibility of non-invasive in-vivo vein occlusion by cavitation induced by mechanical HIFU without microbubble contrast agents. Though in-vivo vein occlusion by cavitation has been previously described in rabbit ears, it should be noted that this study required the injection of a micro-bubble contrast agent into the circulatory system before cavitation was triggered at -9 MPa by a single-element device [26]. Our study demonstrated occlusion in a vein with an inner diameter of 3 mm before treatment with the result almost instantaneously observed with ultrasound imaging on D0 and confirmed on D3 and 7 with no blood flow recurrence on D7. Ultrasonic cavitation was triggered by a negative pressure of -23 MPa, which exceeds the soft tissue intrinsic threshold [5]. Vein
occlusion by cavitation may be due to erosion of the vein wall, achieved by targeting the center of the lumen in small-diameter veins, as demonstrated by model experiments.

Vein wall shrinkage in sheep, with a mean diameter constriction of 53%, has also been achieved using thermal HIFU [18], with 3400 J (85 W for 8 s, repeated 5 times) applied with a 30 s pause between applications to allow heat dissipation in surrounding tissue and skin. In our study, 2754 J was applied in our study (1224 W with a 1.5% duty cycle for 50 s, representing 918 J, repeated 3 times). Though optimization of the HIFU treatment parameters must now be carried out, our method, employing mechanical HIFU with short pulses, avoids the disadvantage of thermal diffusion into surrounding tissue, which can occur during thermal HIFU.

Finally, histological analysis showed a major transformation of the vein wall in the treated compared to the untreated vein. The biological effect observed was similar to previous studies that showed that non-thermal HIFU induces the formation of a fibrous lesion inside the vein wall whose expansion results in vein occlusion [26,33]. It should be noted, however, that unlike our study, pro-inflammatory agents were injected to increase the efficiency and long-term persistency of the biological effect in [33]. Though we demonstrated continued vein occlusion without blood flow recurrence 7 days post-treatment, a longer follow-up is necessary to evaluate long-term efficacy.

Considering the similar blood flow between varicose veins [30] and the treated collateral saphenous vein in our preclinical study, this work highlights the real potential of mechanical HIFU for the non-invasive treatment of varicose veins by cavitation-induced occlusion without microbubble contrast agents. Interventional varicose vein treatment has now largely shifted from surgery to thermal or non-thermal endovenous techniques [34]. Thermal endovenous ablation is mainly achieved by laser and radiofrequency energy that damages the first layers of the vein wall, inducing an inflammatory response that can be observed macroscopically by a sharp reduction in diameter and microscopically by the formation of sclerosis [35]. Though less invasive than surgery, this first-line procedure requires catheterization of the vein and tumescent anæsthesia within an operating environment. Non-thermal endovenous ablation techniques include ultrasound-guided foam sclerotherapy, which involves the intravenous injection of a pharmacological agent that induces an inflammatory response and the formation of sclerosis [36]; physico-chemical techniques that require catheterization of the vein and the intravenous injection of a pharmacological agent; and glue embolization that requires catheterization of the vein and the intravenous injection of cyanoacrylate.

Our proposed method for vein occlusion by cavitation using mechanical HIFU and without microbubble contrast agents achieves similar pathophysiological effects but with all the inherent advantages and reduced risk of complications associated with non-invasive procedures. The absence of thermal effects and contrast agents distinguishes our method from current treatments and their associated disadvantages [20,37]. With no skin damage observed following treatment, our proposed method appears highly promising for the non-invasive treatment of varicose veins.

Further preclinical studies will now refine treatment parameters, in order to optimize the ultrasound dose delivered and demonstrate the occlusion of veins at a depth of 5–20 mm. The reproduction of these encouraging initial results will pave the way for clinical trials and enable patients worldwide to benefit from vein occlusion by cavitation using mechanical HIFU without microbubble contrast agents for the non-invasive treatment of varicose veins in the near future.

## Conclusion

This study demonstrates the feasibility of in-vivo vein occlusion by cavitation using mechanical HIFU. Non-invasive and without requiring the injection of any microbubble contrast agent, our innovative method

holds great potential for the non-invasive treatment of varicose veins without the disadvantages of current treatments.

## Data availability statement

The data that support the findings of this study are available from the corresponding author, AG, upon reasonable request.

## Ethical procedure

All in-vivo experiments were conducted in the Biovivo laboratory, accredited by AAALAC International. The personnel who were in contact with the animals, to ensure observation, care and performance of technical procedures, were qualified and trained according to the latest recommendations concerning animal welfare in experimentation. The protocol was approved under APAFIS #50841-2024061815097801 v3 by the VetAgro Sup Ethics Committee (CEEA No.18 Project No.2447).

## Conflict of interest

A.Ganeau and T.Charrel are employees of Veinsound. Olivier Pichot and Cyril Lafon own shares of the company Veinsound.

## Acknowledgments

We thank the BioVivo laboratory where in-vivo experiments were performed. We thank also the CGFL laboratory (U1231, INSERM, Dijon) for the histopathology analysis and especially Dr. Valentin Derangere and Pr. François Ghiringhelli. We thank the Focused Ultrasound Foundation (FUS) for their financial support (FUSF-RAP-S-18-00022).

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