Removing subcutaneous microvessels using photo-mediated ultrasound therapy

Mingyang Wang; Yu Qin; Tao Wang; Jeffrey S Orringer; Yannis M Paulus; Xinmai Yang; Xueding Wang

doi:10.1002/lsm.23260

. Author manuscript; available in PMC: 2021 Dec 1.

Published in final edited form as: Lasers Surg Med. 2020 May 11;52(10):984–992. doi: 10.1002/lsm.23260

Removing subcutaneous microvessels using photo-mediated ultrasound therapy

Mingyang Wang ¹, Yu Qin ^1,², Tao Wang ^1,³, Jeffrey S Orringer ⁴, Yannis M Paulus ^1,⁵, Xinmai Yang ⁶, Xueding Wang ¹

PMCID: PMC7655656 NIHMSID: NIHMS1592056 PMID: 32394475

Abstract

Objectives

We have developed a novel anti-vascular technique, termed photo-mediated ultrasound therapy (PUT), which utilizes nanosecond duration laser pulses synchronized with ultrasound bursts to remove microvasculature through cavitation. The objective of the current study is to explore the potential of PUT in removing subcutaneous microvessels.

Methods

The auricular blood vessels of two New Zealand white rabbits were treated by PUT with a peak negative ultrasound pressure of 0.45 MPa at 0.5 MHz, and a laser fluence of 0.056 J/cm² at 1064 nm for 10 minutes. Blood perfusion in the treated area was measured by a commercial laser speckle imaging (LSI) system before and immediately after treatment, as well as at one hour, three days, two weeks, and four weeks post treatment. Perfusion rates of 38 individual vessels from 4 rabbit ears were tracked during this time period for longitudinal assessment.

Results

The measured perfusion rates of the vessels in the treated areas, as quantified by the relative change in perfusion rate (RCPR), showed a statistically significant decrease for all time points post treatment (p<0.001). The mean decrease in perfusion is 50.79% immediately after treatment and is 32.14% at four weeks post treatment. Immediately after treatment, the perfusion rate decreased rapidly. Following this, there was a partial recovery in perfusion rate up to 3 days post treatment, then followed by a plateau in the perfusion from 3 days to 4 weeks.

Conclusions

This study demonstrated that a single PUT treatment could significantly reduce blood perfusion by 32.14% in the skin for up to 4 weeks. With unique advantages such as low laser fluence as compared with photothermolysis and agent-free treatment as compared with PDT, PUT holds potential to be developed into a new tool for the treatment of cutaneous vascular lesions.

Keywords: laser, ultrasound, anti-vascular treatment, cutaneous vascular lesions, photo-mediated ultrasound therapy

Introduction

Cutaneous vascular lesions are often a cause of great concern to patients for both medical and cosmetic reasons. For example, port-wine stains (PWS) are congenital vascular malformations in the skin ranging in color from pink to dark purple. Histopathological analysis of PWS shows a normal epidermis overlying an abnormal plexus of dilated blood vessels, which appear as a layer in the upper dermis.^1–3 PWS biopsy has shown that 50% of PWS blood vessels have a circumference ranging from 50 to 200 μm⁴. Due to the abnormal aggregation of these blood vessels in the dermis, PWS can cause hypertrophy and result in disfigurement in adults⁵, leading to a profound psychological impact on the quality of life of patients and other sequelae such as bleeding.

Laser treatment through photothermolysis to remove the abnormal blood vessels is the most popular choice for the clinical management of cutaneous vascular lesions including PWS³. Pulsed dye laser (PDL) therapy with epidermal cooling is widely considered as the gold standard treatment option⁶. When yellow light (585–595 nm) emitted by a PDL is preferentially absorbed by hemoglobin, heat will transfer into the vessel wall and induce thermal damage. This procedure will cause blood vessels to occlude and be removed after a few weeks, leading to shrinkage, fading, or possible removal of vascular lesions⁷. In addition to PDL, Nd:YAG and frequency-doubled Nd:YAG laser have also been used as alternative laser devices for the treatment of resistant vascular lesions^8,9. Nevertheless, vascular lesions such as PWS can recur after laser therapy due to the reformation and reperfusion of blood vessels⁸. Consequently, laser treatment generally needs to be repeated multiple times in order to achieve the desired outcome and clinical results. To reduce the recurrence and persistence of vascular lesions, angiogenesis inhibitors, which can modulate biological repair processes, may also be used¹⁰.

Photodynamic therapy (PDT) is another light-based treatment method for vascular lesions. Early studies have shown that photosensitizing agents exposed to light at a specific wavelength can cause selective destruction of vessels in the dermis without damage to the normal overlying epidermis^6,9,11. This technique requires the systemic injection of photosensitizers, which necessitates the avoidance of sun exposure during a certain time period after treatment. With all these technological developments, however, current clinical treatment of cutaneous vascular lesions is still suboptimal. For example, it was reported that less than 20% PWS can be completely cleared, while up to 10% of PWS patients do not respond to treatment at all¹². Therefore, there is still a strong need for alternative and better treatment methods for cutaneous vascular lesions.

We have recently developed a novel technology termed photo-mediated ultrasound therapy (PUT). PUT is a noninvasive and agent-free anti-vascular therapy that applies synchronized laser pulses and ultrasound bursts to the target blood vessels. The technique is based on controlled induction and promotion of micro-cavitation activity in blood vessels¹³. When a laser pulse is absorbed by hemoglobin, the produced photoacoustic wave can possess a strong rarefactional phase, which can induce micro-cavitations. The concurrently applied ultrasound bursts can further increase the likelihood of micro-cavitations in the vessels. The generated micro-cavitations in the vessels are further driven by ultrasound bursts to induce microjets and shear stress which can cause vascular responses such as disruption and occlusion, depending on the ultrasound and laser parameters used.

In our previous studies, PUT has been proven to be a practical anti-angiogenic therapy method, and its potential application to the treatment of eye diseases associated with pathological microvessels has been demonstrated^13,14. In the current work, the potential of PUT for clinical management of cutaneous vascular lesions was explored. The performance of PUT in removing skin microvessels was examined by studying a rabbit ear model. In order to objectively evaluate the treatment effects, a commercial laser speckle imaging (LSI) system was used to measure the perfusion rates of individual blood vessels before and after treatment longitudinally.

Materials and Methods

PUT system

Figure 1 shows the schematic of our PUT system. The system included a standard Nd:YAG laser (Continuum Powerlite DLS 8010, Santa Clara, CA) which was employed to produce 1064-nm laser pulses with 5-ns pulse duration and 10-Hz pulse repetition rate. The laser beam was delivered to the sample with its energy measured by a Nova PE25BB-SH-V2 pyroelectric head (Ophir Optronics Ltd., Jerusalem, Israel). The laser spot size was adjusted to 3 mm through an iris diaphragm. When working in the treatment mode, the switch in Figure 1 stayed on position 2. The ultrasound bursts were originally generated by a function generator (DS345, Stanford Research System, Sunnyvale, CA) which, triggered by a delay generator (DG535, Stanford Research System, Sunnyvale, CA), produced 0.5-MHz bursts with 0.2% duty cycle at a pulse repetition rate of 10 Hz. The signals from the function generator were then amplified by a 50-dB radio frequency amplifier (2100L, Electronics and Innovation, Rochester, NY) before being sent to a 0.5-MHz center-frequency high intensity focused ultrasound (HIFU) transducer (H107, Sonic Concepts, Bothell, WA). The HIFU transducer had a geometric focal distance of 63.2 mm, a focal depth of 21.42 mm, and a focal width of 3.02 mm. A custom-built, 3D printed cone was designed and attached to the ultrasound transducer, and filled with agar-gelatin based couplant to provide acoustic coupling. A middle hole was reserved inside the cone for light propagation. In order to ensure ultrasonic transmission, the hole was sealed from the side close to the sample and then water was used to fill the gap between the couplant and the seal film. Care was taken to ensure that the ultrasound focal point was concentric with the laser beam. The detailed geometry of this 3D printed cone is shown in Figure S1 in the Supplementary Materials.

Synchronization between laser pulses and ultrasonic bursts

Since the laser and the function generator were both triggered by the delay generator, the time difference between a laser pulse and an ultrasound burst reaching the target was primarily dependent on the traveling time of an ultrasound wave propagating from the HIFU transducer to the target. This traveling time was measured by detecting the photoacoustic signals emitted from the target using the HIFU transducer. To measure the traveling time, the switch in Figure 1 was put on position 1. The photoacoustic signal generated from the target tissue was detected by the HIFU transducer, and then, after amplification, displayed on the oscilloscope together with the trigger signal. The traveling time was determined by the arrival time of the photoacoustic wave. The delay between the function generator and the Q-switch of the Nd:YAG laser was then adjusted according to the measurement result to ensure that each laser pulse was delivered to the target tissue to overlay the negative phase of an ultrasound cycle¹⁵.

In vivo rabbit ear model

Two New Zealand white rabbits (3.5 and 4.0 kg, 4 months old, both genders) were acquired from the Center for Advanced Models for Translational Sciences and Therapeutics at the University of Michigan Medical School. All the animal handling procedures were carried out according to NIH guidelines and an animal protocol approved by the Institutional Animal Care and Use Committee at the University of Michigan (protocol number PRO00008567; PI: Paulus). Briefly, each rabbit was anesthetized using a combination of xylazine (5 mg/kg) and ketamine hydrochloride (40 mg/kg) via intramuscular injection. Repeated injections of ketamine were administered, as required, to keep the rabbits under anesthesia. Respiratory rate, heart rate, response to stimuli, and temperature were monitored. Under anesthesia, the hairs on the ear were removed using depilatory cream. To facilitate better ultrasound coupling, ultrasound gel was applied to the skin of the rabbit ear. Then, the rabbit was placed on a 3-D motion platform. The ear under treatment was placed on a block of agar gel which was mounted to a custom-built holder enabling flexible and precise positioning of the ear.

Rabbit auricular blood vessels were treated by PUT with a peak negative ultrasound pressure of 0.45 MPa and a surface laser fluence of 56 mJ/cm² at 1064 nm. Determined by the ultrasound focal point (focal width: 3.02 mm) and the laser spot size (3 mm), each treatment covered a spot size of 3 mm. To cover a larger area, the treatment spot was moved to an adjacent location after each treatment. The treatment at each spot took 10 minutes. Therefore, the treatment over 4–5 spots on each rabbit ear took about 40–50 minutes. In the future, the treatment speed can be significantly enhanced by using lasers with higher pulse repetition rates, or stronger lasers and more powerful HIFU transducers that can cover a larger area. After the treatment, the rabbits recovered from anesthesia and were returned to the housing room. To study the treatment effect longitudinally, the treated vessels in the rabbit ears were monitored using a commercial LSI system at different time points after the treatment. One rabbit was monitored over a total period of 2 weeks, and the other one was monitored over a total period of 4 weeks.

Perfusion analysis

LSI was used in this study for evaluating the treatment effects because it is a flexible, wide-field optical imaging approach capable of measuring blood perfusion in the upper layer of the dermis in real time¹⁶. By detecting the speckle pattern and changes produced from the blood vascular network, LSI can map the velocity distribution in the entire field on the photograph^17,18. The LSI system employed was a commercial device (PeriCam PSI System, Jarfalla, Sweden), namely laser speckle contrast analysis (LASCA). Each blood perfusion map was an average of 5 images acquired continuously with a 12-second interval. The blood perfusion maps measured by this device were analyzed with “PIMSoft”, a built-in software of the imager, to quantify the perfusion rate of each vessel.

Figure 2(A) shows an example of the blood perfusion maps acquired on a rabbit ear in vivo. Figure 2(B) shows the same image presented in another color scale. To get Figure 2(B), the average speckle flow index (SFI) of Figure 2(A) was subtracted from the value of each pixel, and the color scale was changed to highlight the vascular structure. The sensitivity and resolution of the LSI system satisfied the quantitative evaluation of the perfusion in each vessel. In our study on each rabbit ear, all the vessels within the treatment area were identified in the perfusion map, like those marked in Figure 2(B). The perfusion of each identified vessel and its change after the treatment were assessed. Hence, by working on the four ears of the two rabbits, a total of 38 independent vessels treated by PUT were quantitatively evaluated.

Besides the treated vessels, a randomly selected vessel in the untreated area was also studied for its perfusion before and after the treatment procedure. The perfusion in this untreated vessel served as a reference. It was noticed that the perfusion rate quantified from the LSI system was sensitive to the skin’s condition. For example, the depilatory cream for removing the hairs can irritate the skin, leading to an acute increase in blood perfusion. To compensate all these variables and improve the repeatability in the quantitative assessment of perfusion, we quantified the relative change in perfusion rate (RCPR) of each treated vessel by using the following equation:

RCPR = \frac{\frac{Treated vessel perfusion rate after}{Untreated vessel perfusion rate after} - \frac{Treated vessel perfusion rate before}{Untreated vessel perfusion rate before}}{\frac{Treated vessel perfusion rate before}{Untreated vessel perfusion rate before}} \times %

The percentile RCPR of each vessel was evaluated at different time points after the treatment, including immediately after, 1 hour, 3 days, 2 weeks, and 4 weeks following treatment.

Results

LSI imaging results

In Figure 3(A)–(D), the perfusion maps of a rabbit ear before and after treatment are shown. Compared to the image before the treatment, the image acquired immediately after the treatment, as shown in Figure 3(B), shows an obvious reduction in perfusion in the treated area. This result demonstrated the short-term treatment effects from PUT. At three days after the treatment, as shown in Figure 3(C), the acquired LSI image shows strongly blurred perfusion in the treated area, and the vascular structure is difficult to recognize. This may suggest that, when originally perfused vessels in the treated area were largely blocked, the surrounding small microvessels started to dilate in order to compensate for the deficiency in blood flow. At 2 weeks after the treatment, as shown in Figure 3(D), the treated area became stable, and no significant changes were noted after that. Some of the blood vessels (typically those with diameters less than 100 μm) in the treated area were no longer visible on the perfusion maps, while almost all the treated blood vessels were smaller in size and showed reduced perfusion rates. Figures 3(E)–(H) show the same perfusion maps as in Figures 3(A)–(D) but in a different color scale which benefits the tracking of individual vessels. In this ear, a total of 12 vessels, including 11 treated vessels and 1 untreated vessel as a reference, were monitored longitudinally.

Quantitative analysis

Table 1 shows the quantified perfusion rates and RCPR at different time points of the 12 tracked vessels shown in Figure 3. The first column shows the vessel ID, where 1.1–1.11 are the 11 treated vessels and 1.0 is the untreated vessel. The perfusion rate of each vessel at each time points (immediately before, immediately after, and at 1 hour, 3 days, 2 weeks, and 4 weeks post-treatment), as quantified by the built-in software of LASCA, are listed in the columns 3–8, respectively. The second numbers in the column 4–8 show the quantified relative changes in perfusion rate (i.e. RCPR) for each of treated vessels. In this rabbit ear, all of the 11 tracked vessels show decreased perfusion after the treatment.

Table 1.

Diameter and perfusion rate change of each tracked vessel.

Vessel ID	Diameter of vessel (mm)	Perfusion rate and RCPR
Vessel ID	Diameter of vessel (mm)	Immediately before	Immediately after	1 hour	3 days	2 weeks	4 weeks
1.0	0.57	268.56	492.43	310.2	385.6	345.22	321.64
1.1	0.57	251	161.75 −64.85%	116.97 −59.65%	195.33 −45.80%	201.37 −37.59%	144.56 −51.91%
1.2	0.51	262.38	152.03 −68.40%	171.54 −43.40%	126.24 −66.49%	81.8 −75.75%	187.95 −40.19%
1.3	0.47	301.36	165.5 −70.05%	156.03 −55.17%	180.51 −58.28%	117.52 −69.66%	204.24 −43.41%
1.4	0.45	194.97	134.58 −62.35%	167.06 −25.82%	144.48 −48.39%	68.86 −72.52%	163.59 −29.94%
1.5	0.39	219.52	134.31 −66.63%	217.25 −14.32%	173.85 −44.84%	118.57 −57.98%	177.05 −32.66%
1.6	0.30	184.42	165.86 −50.95%	126.36 −40.68%	126.09 −52.38%	173.72 −26.72%	121.87 −44.82%
1.7	0.22	186.15	91.7 −73.13%	99.18 −53.87%	152.13 −43.08%	114.33 −52.22%	119.49 −46.40%
1.8	0.22	140.06	111.7 −56.51%	84.94 −47.50%	99.45 −50.55%	88.32 −50.94%	90.95 −45.78%
1.9	0.20	168.32	170.21 −44.85%	117.63 −39.50%	145.2 −39.92%	137.49 −36.46%	150.4 −25.39%
1.10	0.17	117.52	102.61 −52.38%	119.09 −12.27%	112.35 −33.42%	53.77 −64.41%	100.51 −28.59%
1.11	0.14	138.2	145.6 −42.54%	114.93 −28.00%	140.58 −29.15%	67.3 −62.12%	58.83 −64.46%

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In this study, a total of 38 vessels within the treated areas of the four rabbit ears were tracked, as the results shown in Table S1 in the Supplementary Materials. Figure 4 shows a histogram of RCPR of all the 38 treated vessels at two time points, including immediately after and at 2 weeks after the treatment. The result shows that, when immediately after treatment, 97.37% of the treated vessels showed RCPR reduction larger than 5%. At two weeks post-treatment, 81.58% of the treated vessels showed RCPR reduction larger than 5%. To account for the possible inaccuracy in quantifying perfusion by the LASCA system, only the changes in RCPR larger than the ±5% range are considered obvious. Among the 38 treated vessels, 3 vessels (7.89%) showed increased RCPR larger than 5%, which may be due to the revascularization.

Figure 4. — The histogram showing the RCPR of the 38 treated blood vessels at two time points, including immediately after the treatment and at 2 weeks after the treatment. Each column shows the number of vessels with an RCPR within the ±5% range. For example, four vessels show RCPR in the range of −40±5% immediately after the treatment, while nine vessels show RCPR in the range of −40±5% at 2 weeks after the treatment.

Figure 5 presents the longitudinal study and statistical analysis of the quantified perfusion measurements from all the 38 rabbit ear vessels after PUT treatment. The average and the standard deviation of the quantified RCPR of all the treated vessels at each time point are shown. When comparing to the perfusion before the treatment, the paired t-test studying the perfusion at each of the time points post-treatment, including immediate after, and at 1 hour, 2 weeks, and 4 weeks after treatment, leads to p<0.001, indicating that PUT caused a significant reduction in perfusion. Based on the results quantified from the LSI data, we found that, when immediately after the treatment, the perfusion rate descended drastically, with a 50.79% reduction in average RCPR from the baseline. Following that, there was a steady recovery in perfusion until 3 days after treatment when the average reduction in RCPR was 29.10% from the baseline. The paired t-test comparing the RCPR immediately after treatment and the RCPR at 3 days after treatment shows statistically significant difference with p<0.001. During the time period from 3 days to 4 weeks after treatment, the average RCPR of all the treated vessels was stable, and the paired t-tests between different time points after 3 days did not show any statistically significant difference.

Conclusion and Discussion

In this work, via the experiments in a rabbit ear model in vivo, the feasibility of PUT in the treatment of cutaneous microvessels was explored. Both the short-term effects and the long-term effects up to 4 weeks post-treatment were quantitatively assessed by measuring the perfusion rates of the vessels after treatment. Immediately after treatment, the perfusion map demonstrated significantly reduced blood flow in the treated microvessels with blurring of the vascular network. The decrease in the perfusion rate may be explained by the vascular damage caused by PUT. Damage to endothelial cells and the vascular basement membrane leads to the establishment of thrombogenic sites within the vessel lumen, initiating a physiological cascade of responses including platelet aggregation, release of vasoactive molecules, leukocyte adhesion, increase in vascular permeability, and blood vessel constriction^19,20. The rapid decrease in perfusion noticed during PUT is different from PDL therapy where the perfusion rate would rapidly increase during treatment. This may be due to the difference in treatment mechanism. PDL depends on the photothermal effect, where local warming of the skin causes direct and substantial vasodilation in the site being warmed, leading to perfusion rate increase. In PUT, the 5-ns laser pulses used was not only low in total energy (within the ANSI safety limit) but also within the thermal confinement when compared to the thermal relaxation time of the target vessels being on the order of milliseconds²¹. Moreover, the very small 0.2% duty cycle of the HIFU bursts concurrently applied also hardly raises the local temperature during PUT²². Therefore, although not yet measured, the photothermal effect during PUT is considered minimal.

The perfusion rates of the treated vessels at 4 weeks after PUT, although partially recovered compared to the results immediately after the treatment, were also significantly reduced from the baseline. Some vessels, especially those with smaller sizes, became completely invisible on the perfusion maps. While the perfusion through some relatively large vessels was not completely stopped, the perfusion rates were much less than the baseline. Although vessel sizes and depths might vary in different individuals and even from site to site in the same ear, this trend was consistently demonstrated by the statistically analyzed perfusion rates. The majority of treated vessels did not recover to pre-treatment perfusion rates. The long-term treatment effect can be attributed to endothelial cell injury and the release of coagulation factors that eventually lead to vascular blockage. While vascular channels might occlude, the endothelial cells that form them would be expected to be the conduits if the vessels were to reopen. In this study, the assessments of the treatment effect were up to one month. Vascular perfusion was similar at 3 days, 2 weeks, and 4 weeks after treatment, indicating that the vascular changes were stable and likely chronic after day 3. A similar finding has been reported in other anti-vascular treatment research, suggesting that vascular changes at 1 month are likely permanent²³. It is also worth noting that our current study used only a single treatment. We expect that the therapeutic outcome may be improved further if multiple treatment sessions are conducted. Multiple treatment sessions are commonly used in PDL and PDT treatments of cutaneous vascular lesions in the clinical setting.

We expect that PUT, by synergistically combining laser pulses and ultrasound bursts, has some unique advantages over the existing technologies for the treatment of cutaneous vascular lesions such as PWS. First and foremost, PUT removes blood vessels through the mechanical effect of cavitation, completely avoiding unwanted damage due to thermal diffusion. Second, PUT requires much lower laser fluence than PDL and produces treatment effects at a very low level of ultrasonic energy. In this work, PUT utilizing a laser fluence of 0.056 J/cm² was effective in reducing the perfusion of treated vessels, while photothermolysis with PDL generally requires laser fluence larger than 1 J/cm². Working with lower laser fluence, PUT is safer with lower potential of thermally-mediated side effects. The applied peak negative ultrasound pressure in this work was 0.45 MPa at 0.5 MHz, which, when used alone without involving laser, would not induce the cavitation effect and damage blood vessels. This was confirmed by the finding from a former study where 0.7 MPa ultrasound at 0.5 MHz, even applied as a continuously wave, did not induce any detectable cavitation in blood vessels²⁴. In our previous study¹³, the laser-only and ultrasound-only controls have been examined on the same animal model, and no photocoagulation effect has been observed. Third, PUT is highly precise and selective. Similarly as PDL, the high precision and selectivity of PUT are achieved by the high optical absorption contrast between blood and other tissues in the visible to near-infrared spectrum. Based on the high optical contrast, PUT can treat vessels selectively without causing strong collateral damage to the surrounding skin tissue. Furthermore, in comparison with PDT, PUT does not need systemic administration of photosensitizing agents. All these advantages would be highly valuable for future translation of PUT to the clinical management of cutaneous vascular lesions.

Different from our previous PUT experiments using 532-nm laser light¹³, the laser wavelength used in this study was 1064 nm which was highly effective in reducing vascular perfusion. Despite the fact that 1064-nm wavelength is relatively poorly absorbed by oxygenated hemoglobin²⁵, it can penetrate deeper in the skin, and can completely cover the cross-section of a blood vessel, thereby removing the vessels in the skin effectively. This finding is consistent with some experimental results from photothermolysis studies which found that the 1064-nm Nd:YAG laser was highly effective in the treatment of hypertrophic PWS^26–28. Longer wavelengths, such as 2940 nm, may also be used for PUT. However, because of the high optical absorption of water at longer wavelengths, blood vessels may not be treated in a selective manner, and significant collateral damage to the surrounding tissue could be produced.

Although the study described here has for the first time validated the feasibility of PUT in the treatment of cutaneous microvessels, there are some limitations in this work. In our experiments, no damage to the surrounding skin tissue after PUT has been visually noticed (see Figure S2 in the Supplementary Materials). The laser fluence applied was 0.056 J/cm² which was lower than the ANSI safety limit of 0.1 J/cm² for 5-ns laser pulses at 1064-nm wavelength. However, as the first limitation, the safety of PUT in treatment of cutaneous microvessels still needs to be further validated via histopathological assessment of the skin after treatment. To demonstrate the clinical value of PUT, the safety study should include both short-term and long-term assessments, and the results should be compared to those from alternative treatment modalities including PDL and PDT. Second, enabled by the LSI measurements, a total of 42 individual vessels (including 38 treated and 4 untreated) were tracked longitudinally after the treatment, which enabled statistical analyses of the treatment outcome showing statistical and clinical significance. However, the number of animals involved in this study was limited, and thus could not elucidate treatment effects as a function of variables such as vessel size and depth as well as skin thickness and color. With the measurements from the 38 treated vessels, we have conducted an initial statistical study to explore possible correlations between the vessel size and the treatment effects, as the result shown in Figure S3 in the Supplementary Materials. Although no statistically significant correlation was found, this initial study does suggest that the vessels with smaller sizes may have a larger variance in treatment response. Third, as the microvessels in the rabbit ears were normal vessels, the rabbit auricular model involved in this work did not allow for the study of the efficacy and safety of PUT in treating pathological microvessels. Future studies in clinically relevant models of cutaneous vascular lesions are necessary for understanding the potential of PUT as a practical tool for dermatology clinic. Despite these limitations, however, this proof of principle study has successfully indicated that PUT, as a novel anti-vascular therapeutic modality, can treat cutaneous microvasculature safely and effectively.

Supplementary Material

Figure S1

Figure S1. Design of the 3D printed cone.

NIHMS1592056-supplement-Figure_S1.png^{(214KB, png)}

Figure S2

Figure S2. Color photos of a rabbit ear taken at the selected time points before and after PUT treatment. No skin damage after the treatment was noticed. Microhemorrhage secondary to increased vessel permeability by inflammation was noticed in the short term (within 3 days) post treatment; while in the long term (after one week) post treatment, microhemorrhage disappeared and shrinkage of the treated vessels could be seen.

NIHMS1592056-supplement-Figure_S2.jpg^{(165KB, jpg)}

Figure S3

Figure S3. Statistical analysis to explore the possible correlation between the vessel size and the treatment effects quantified by the relative changes in perfusion rate (i.e. RCPR). Based on their sizes, all treated vessels were divided into three groups (Group 1: 0.01–0.1 mm; Group 2: 0.1–0.34 mm; Group 3: 0.34–0.57 mm). The boxplots show the RCPR measured at two different time points (i.e. immediately after treatment and 2 weeks after treatment) for the three vessel groups. The unpaired t-test comparing any two of the three groups could not lead to statistically significant difference (i.e. p>0.05). However, the larger interquartile (Q1-Q3) ranges from Group 1 suggest that the vessels with smaller sizes may have a larger variance in treatment response.

NIHMS1592056-supplement-Figure_S3.jpg^{(102.4KB, jpg)}

NIHMS1592056-supplement-1.pdf^{(158.7KB, pdf)}

Acknowledgments and conflict of interest statement:

This work was supported in part by a grant from the National Eye Institute R01EY029489 and 1K08EY027458, unrestricted departmental support from Research to Prevent Blindness, and the Alliance for Vision Research. The authors would like to thank the University of Michigan Medical School Center for Advanced Models for Translational Sciences and Therapeutics for the generous donation of the rabbits used in these experiments.

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16.Yang B, Yang O, Guzman J, et al. Intraoperative, real-time monitoring of blood flow dynamics associated with laser surgery of port wine stain birthmarks. Lasers in Surgery and Medicine. 2015;47(6):469–475. [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Chen D, Ren J, Wang Y, Zhao H, Li B, Gu Y. Relationship between the blood perfusion values determined by laser speckle imaging and laser Doppler imaging in normal skin and port wine stains. PHOTODIAGNOSIS PHOTODYN THER. 2015;13:1–9. [DOI] [PubMed] [Google Scholar]
18.Choi B, Wenbin T, Wangcun J, et al. The Role of Laser Speckle Imaging in Port-Wine Stain Research: Recent Advances and Opportunities. JSTQE. 2016;22(3):307–318. [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Fingar VH. Vascular Effects of Photodynamic Therapy. Journal of Clinical Laser Medicine & Surgery. 1996;14(5):323–328. [DOI] [PubMed] [Google Scholar]
20.Sanovic R, Verwanger T, Hartl A, Krammer B. Low dose hypericin-PDT induces complete tumor regression in BALB/c mice bearing CT26 colon carcinoma. PHOTODIAGNOSIS PHOTODYN THER. 2011;8(4):291–296. [DOI] [PubMed] [Google Scholar]
21.Stuart Nelson J, Milner TE, Svaasand LO, Kimel S. Laser pulse duration must match the estimated thermal relaxation time for successful photothermolysis of blood vessels. Lasers Med Sci. 1995;10(1):9–12. [Google Scholar]
22.Tu J, Ha Hwang J, Chen T, et al. Controllable in vivo hyperthermia effect induced by pulsed high intensity focused ultrasound with low duty cycles. APPL PHYS LETT. 2012;101(12):124102. [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Paulus YM, Jain A, Gariano RF, et al. Healing of retinal photocoagulation lesions. Invest Ophthalmol Vis Sci. 2008;49(12):5540–5545. [DOI] [PubMed] [Google Scholar]
24.Gross DR, Miller DL, Williams AR. A search for ultrasonic cavitation within the canine cardiovascular system. Ultrasound in Medicine & Biology. 1985;11(1):85–97. [DOI] [PubMed] [Google Scholar]
25.Kono T, Sakurai H, Takeuchi M, et al. Treatment of Resistant Port-Wine Stains with a Variable-Pulse Pulsed Dye Laser. DERMATOL SURG. 2007;33(8):951–956. [DOI] [PubMed] [Google Scholar]
26.Verkruysse W, Gemert MJCv, Smithies DJ, Nelson JS. Modelling multiple laser pulses for port wine stain treatment. Physics in Medicine and Biology. 2000;45(12):N197–N203. [DOI] [PubMed] [Google Scholar]
27.Jia W, Choi B, Franco W, et al. Treatment of cutaneous vascular lesions using multiple-intermittent cryogen spurts and two-wavelength laser pulses: Numerical and animal studies. Lasers in Surgery and Medicine. 2007;39(6):494–503. [DOI] [PubMed] [Google Scholar]
28.van oge AM, Bosveld B, van der Veen JPW, de Rie MA, Wolkerstorfer A. Long-pulsed 1064 nm Nd:YAG laser improves hypertrophic port-wine stains. J EUR ACAD DERMATOL VENEREOL. 2013;27(11):1381–1386. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1

Figure S1. Design of the 3D printed cone.

NIHMS1592056-supplement-Figure_S1.png^{(214KB, png)}

Figure S2

NIHMS1592056-supplement-Figure_S2.jpg^{(165KB, jpg)}

Figure S3

NIHMS1592056-supplement-Figure_S3.jpg^{(102.4KB, jpg)}

NIHMS1592056-supplement-1.pdf^{(158.7KB, pdf)}

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[R13] 13.Hu Z, Zhang H, Mordovanakis A, et al. High-precision, non-invasive anti-microvascular approach via concurrent ultrasound and laser irradiation. SCI REP. 2017;7(1):40243–40243. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R14] 14.Zhang H, Xie X, Li J, et al. Removal of choroidal vasculature using concurrently applied ultrasound bursts and nanosecond laser pulses. SCI REP. 2018;8(1):12848. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R15] 15.Qin Y, Yu Y, Xie X, et al. The effect of laser and ultrasound synchronization in photo-mediated ultrasound therapy. TBME. 2020:1–1. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R16] 16.Yang B, Yang O, Guzman J, et al. Intraoperative, real-time monitoring of blood flow dynamics associated with laser surgery of port wine stain birthmarks. Lasers in Surgery and Medicine. 2015;47(6):469–475. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R17] 17.Chen D, Ren J, Wang Y, Zhao H, Li B, Gu Y. Relationship between the blood perfusion values determined by laser speckle imaging and laser Doppler imaging in normal skin and port wine stains. PHOTODIAGNOSIS PHOTODYN THER. 2015;13:1–9. [DOI] [PubMed] [Google Scholar]

[R18] 18.Choi B, Wenbin T, Wangcun J, et al. The Role of Laser Speckle Imaging in Port-Wine Stain Research: Recent Advances and Opportunities. JSTQE. 2016;22(3):307–318. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R19] 19.Fingar VH. Vascular Effects of Photodynamic Therapy. Journal of Clinical Laser Medicine & Surgery. 1996;14(5):323–328. [DOI] [PubMed] [Google Scholar]

[R20] 20.Sanovic R, Verwanger T, Hartl A, Krammer B. Low dose hypericin-PDT induces complete tumor regression in BALB/c mice bearing CT26 colon carcinoma. PHOTODIAGNOSIS PHOTODYN THER. 2011;8(4):291–296. [DOI] [PubMed] [Google Scholar]

[R21] 21.Stuart Nelson J, Milner TE, Svaasand LO, Kimel S. Laser pulse duration must match the estimated thermal relaxation time for successful photothermolysis of blood vessels. Lasers Med Sci. 1995;10(1):9–12. [Google Scholar]

[R22] 22.Tu J, Ha Hwang J, Chen T, et al. Controllable in vivo hyperthermia effect induced by pulsed high intensity focused ultrasound with low duty cycles. APPL PHYS LETT. 2012;101(12):124102. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R23] 23.Paulus YM, Jain A, Gariano RF, et al. Healing of retinal photocoagulation lesions. Invest Ophthalmol Vis Sci. 2008;49(12):5540–5545. [DOI] [PubMed] [Google Scholar]

[R24] 24.Gross DR, Miller DL, Williams AR. A search for ultrasonic cavitation within the canine cardiovascular system. Ultrasound in Medicine & Biology. 1985;11(1):85–97. [DOI] [PubMed] [Google Scholar]

[R25] 25.Kono T, Sakurai H, Takeuchi M, et al. Treatment of Resistant Port-Wine Stains with a Variable-Pulse Pulsed Dye Laser. DERMATOL SURG. 2007;33(8):951–956. [DOI] [PubMed] [Google Scholar]

[R26] 26.Verkruysse W, Gemert MJCv, Smithies DJ, Nelson JS. Modelling multiple laser pulses for port wine stain treatment. Physics in Medicine and Biology. 2000;45(12):N197–N203. [DOI] [PubMed] [Google Scholar]

[R27] 27.Jia W, Choi B, Franco W, et al. Treatment of cutaneous vascular lesions using multiple-intermittent cryogen spurts and two-wavelength laser pulses: Numerical and animal studies. Lasers in Surgery and Medicine. 2007;39(6):494–503. [DOI] [PubMed] [Google Scholar]

[R28] 28.van oge AM, Bosveld B, van der Veen JPW, de Rie MA, Wolkerstorfer A. Long-pulsed 1064 nm Nd:YAG laser improves hypertrophic port-wine stains. J EUR ACAD DERMATOL VENEREOL. 2013;27(11):1381–1386. [DOI] [PubMed] [Google Scholar]

PERMALINK

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