Intraoperative monitoring of blood perfusion in port wine stains by laser Doppler imaging during vascular targeted photodynamic therapy: A preliminary study

Photodiagnosis and Photodynamic Therapy 14 (2016) 142–151

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Intraoperative monitoring of blood perfusion in port wine stains by laser Doppler imaging during vascular targeted photodynamic therapy: A preliminary study Defu Chen a , Jie Ren b , Ying Wang b , Buhong Li c , Ying Gu a,b,∗ a

School of Information and Electronics, Beijing Institute of Technology, Beijing 100081, China Department of Laser Medicine, Chinese People’s Liberation Army General Hospital, Beijing 100853, China c Key Laboratory of OptoElectronic Science and Technology for Medicine of Ministry of Education, Fujian Provincial Key Laboratory for Photonics Technology, Fujian Normal University, Fujian 350007, China b

a r t i c l e

i n f o

Article history: Received 15 January 2016 Received in revised form 11 March 2016 Accepted 5 April 2016 Available online 9 April 2016 Keywords: Blood perfusion Laser Doppler imaging Port wine stains Vascular targeted photodynamic therapy Microvascular response

a b s t r a c t Objective: The objective of this study was to monitor blood perfusion dynamics of port wine stains (PWS) during vascular targeted photodynamic therapy (V-PDT) with laser Doppler imaging (LDI). Methods: The PWS lesions of 30 facial PWS patients received V-PDT, while the normal skins on the forearm of 5 healthy subjects were treated as light-only controls for comparison. Furthermore, two different PWS lesions in the same individual from each of 3 PWS patients successively received laser irradiation only and V-PDT, respectively. LDI was used to monitor intraoperative blood perfusion dynamics. Results: During V-PDT, the blood perfusion (278 ± 96 PU) in PWS lesions for 31 of 33 PWS patients significantly increased after the initiation of V-PDT treatment, then reached a peak (638 ± 105 PU) within 10 min, followed by a slow decrease to a relatively lower level (515 ± 100 PU). Furthermore, the time for reaching peak and the subsequent magnitude of decrease in blood perfusion varied with different patients. For light-only controls, an initial perfusion peak at 3 min followed by a nadir and a secondary increase were found not only in normal skin, but also in PWS lesions. Conclusion: The preliminary results showed that the LDI permits non-invasive monitoring blood perfusion changes of PWS lesions during V-PDT. There was a clear trend in blood perfusion responses during V-PDT and laser irradiation. The blood perfusion changes during treatment were due to V-PDT effects as well as local temperature increase induced by laser irradiation. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Port wine stains (PWS) birthmarks are congenital vascular malformations histologically characterized by ectatic capillaries predominantly in the upper dermis [1,2]. The occurrence rate of PWS is estimated to be 0.3–0.5% [3]. PWS are mainly located on the face and neck area, and appear red-purple [3]. Without treatment, the PWS lesions may deepen in color with age, and about twothirds of the lesions develop nodular components and can produce facial deformity [4]. The pulsed dye laser (PDL) therapy, based on the principle of selective photothermolysis, is the current standard treatment modality for PWS; however, only about 10% of patients achieve complete blanching of the PWS [3,5]. Alternatively, vascular targeted photodynamic therapy (V-PDT) is also known to be

∗ Corresponding author. E-mail address: [email protected] (Y. Gu). http://dx.doi.org/10.1016/j.pdpdt.2016.04.002 1572-1000/© 2016 Elsevier B.V. All rights reserved.

a safe and effective therapeutic modality for PWS, and has been successfully used for PWS treatment in China for more than two decades [6,7]. The basic principle of V-PDT for the treatment of PWS is as follows [8–11]: a photosensitizer is administered intravenously and accumulated in the ectatic capillaries. Shortly after administration, the PWS lesion is irradiated with laser light of specific wavelength, and the light-activated photosensitizer initiates a cascade of photochemical reactions to generate reactive oxygen species (e.g. singlet oxygen) that selectively damage the dilated and malformed blood vessels and cause vessel closure in the papillary layer without the destruction of surrounding skin tissue. A recent side-by-side comparison study has demonstrated that V-PDT for the treatment of PWS is at least as effective as PDL and, in some cases, superior [11]. Although V-PDT is effective for the treatment of PWS, the treatment outcomes still remain variable not only between different PWS patients but also within the same lesion [8–10]. The PDT efficacy is highly dependent on the oxygen concentration and pho-

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tosensitizer concentration, which in turn can be affected by blood perfusion especially for systematic administration of photosensitizers [12]. The skin blood perfusion may change due to the blood vessel damage during V-PDT, and the changes maybe a useful surrogate marker for monitoring the microvascular responses to V-PDT [12]. Therefore, monitoring blood perfusion in PWS during V-PDT may lead to a better understanding of the microcirculation changes in PWS lesions and its response to V-PDT, and hold promise for guiding clinical V-PDT treatment [8,9]. In fact, several studies have investigated the blood perfusion responses to the topical 5aminolevulinic acid (ALA)-PDT for treating tumors, and the results showed that monitoring blood perfusion parameter may be useful for assessing ALA-PDT response [12–15]. However, the blood perfusion responses during V-PDT for PWS have not been fully investigated. Many noninvasive optical technologies have been proposed for monitoring skin blood perfusion, such as laser Doppler flowmetry, laser Doppler imaging, capillary microscopy, laser speckle imaging, tissue viability imaging and diffuse correlation spectroscopy [12,16–19]. Among these, laser Doppler imaging, which can provide wide-field imaging of skin blood perfusion, is widely used in the studies of microvascular skin characterization and clinical applications [8–10,12,16–18]. LDI can provide a “digital photograph” of PWS surface under examination as well as the corresponding “Doppler photograph” that reflects skin perfusion. In the present study, we intended to use LDI to monitor the blood perfusion changes that occur in PWS lesions during V-PDT with systematic administration of photosensitizer HiPorfin. Furthermore, the blood perfusion changes of the control group, which received V-PDT laser irradiation only without applied photosensitizers, have been also comparatively monitored.

2. Materials and method 2.1. Subjects 5 healthy non-smoking volunteers and 33 patients aged from 6 to 38 years old and clinically diagnosed with PWS were included in this study at the Department of Laser Medicine, Chinese PLA General Hospital. (Tables 1–3 show the information of each subject). This study was approved by the Ethics Committee of the Chinese PLA General Hospital, and informed consent was obtained from all 38 subjects.

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2.3. Measurement protocol 33 PWS patients and 5 healthy subjects were recruited. The PWS lesions of 30 PWS patients received V-PDT, while the normal skins on the forearm of 5 healthy subjects were treated as light-only controls for comparative studies. Furthermore, two different PWS lesions in the same individual for each of 3 PWS patients also successively received laser irradiation only and V-PDT, respectively. LDI was used to record the skin perfusion immediately before, and at the time points of 1, 3, 5, 7, 10, 15, and 20 min during laser irradiation and V-PDT. All the measurements were performed on the skin to be measured with the subject supine in a temperature-controlled room (24 ± 1 ◦ C). A diode-pumped allsolid-state frequency-doubled Nd: YAG laser with a wavelength of 532 nm (Beijing Newraysing Laser Tech Co., Ltd., Beijing, China) transformed by a flat cut fiber combined with an optics system was used as a light source during laser irradiation and V-PDT. For light-only controls, the normal skins on the forearm were irradiated vertically with respect to the surface of skin with the 532 nm laser light at a power density of 100 mW/cm2 , and the irradiation time was set to 20 min. In the V-PDT group, the PWS lesions and contralateral healthy skin were measured by the LDI before VPDT, and then the skin not receiving treatment was covered with an opaque black cloth to avoid expose to treatment laser light during V-PDT. Afterwards, the patients were intravenously injected with a domestically produced photosensitizer HiPorfin (Chongqing Huading Modern Biopharmaceutics Co., Ltd., Chongqing, China) with a dosage of 2–3 mg/kg of body weight. Immediately after the injection, the PWS lesions were vertically irradiated with the 532 nm laser light source at a power density of 80–100 mW/cm2 , and the irradiation time was set to 20 min. The temperature was measured by an infrared thermometer (ST631, Sentry Optronics Corp., Taipei) at each time point. The typical output laser power of treatment light was 5 W and the diameter of the laser spot was 8 cm, and the power density was determined to be about 100 mW/cm2 . The intensity of the periphery of the light spot may be about 85% of the light spot center, and the central part of the light spot was used for irradiation in order to avoid the large intensity variation in the fluence rate of treatment light. 2.4. Evaluation of color bleaching rates The digital photographs of all the 33 PWS patients taken before and at follow-up were evaluated by an independent experienced clinician using a 5-level grading scale [9]: 0-no significant change, 25%-minimal lightening, 50%-obvious lightening, 75%-slight residual color, 100%-appears as normal skin.

2.2. Laser Doppler imaging system

2.5. Data analysis

A LDI system (moorLDLS2-IR, Moor instrument Ltd., Cambridge, UK) was adopted in the study. The LDI system sweeps a low power laser line with a wavelength of 785 ± 10 nm across the skin surface. The Doppler shifted light from moving blood cells and the nonshifted light from stationary tissue is directed by the same scanning mirror and other optics onto a linear photo detector array, to produce signals that are proportional to the beat frequency between the Doppler shifted and non-shifted light, and thereby proportional to tissue blood perfusion [20]. The desired 2-dimension color coded blood perfusion image is built up line by line. The maximum accessible power within the provided aperture is limited to 2.5 mW for the infrared working beam, which has no significant impact on the blood perfusion. The processing frequency bandwidth was from 30 Hz to 15 kHz. The LDI scanner head was placed 15 cm above the skin. The image size was 15 × 12 cm2 with the resolution of 256 × 256 points.

Data were digitized and stored in a computer, and analyzed offline with signal processing software (moorLDLS laser Doppler line scanner research version 2.2, Moor Instruments Ltd., Cambridge, UK). All the measured data were processed and analyzed using OriginPro 9.1 software (OriginLab Corporation, MA, USA). Data are presented as mean ± SD. Statistical analysis was performed using SPSS 13.0 for Windows (SPSS Inc., Chicago, IL, USA). 3. Results 3.1. Blood perfusion in PWS before V-PDT Before V-PDT treatment, 30 PWS patients were imaged by LDI. For the patient in Fig. 1(A, B), the average perfusion value of the PWS lesion was higher than the contralateral healthy control (478 ± 101 versus 295 ± 51 PU), while the average perfusion value of the PWS

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Table 1 Details of the PWS patients for monitoring blood perfusion. V-PDT: vascular targeted photodynamic therapy, PDL: pulsed dye laser. Case

Gender

Age (years)

PWS type

Location of scanning

Treatment history

Rate of color bleaching (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

M F M F F F M M F M F M F M M F M F F M M M M F M M F F F F

31 18 29 29 28 28 27 25 25 24 23 14 14 13 21 10 18 16 24 16 15 13 13 12 12 12 11 10 20 25

Purple with proliferation Purple Purple Purple with proliferation Purple Purple Purple Purple with proliferation Purple Purple Purple with proliferation Purple Purple Purple Purple Purple Purple Purple Purple Purple Purple Purple Purple Purple Purple Purple with proliferation Purple Purple Purple Purple with proliferation

Cheek Cheek Lower jaw Temple Temple Lateral cheek Lateral cheek Cheek Cheek Neck Cheek Temple Lateral cheek Cheek Cheek Lateral cheek Lateral cheek Cheek Cheek Lateral cheek Neck Upper lip Neck Lower jaw Lateral cheek Lateral cheek Lateral cheek Lateral cheek Cheek Cheek

PDL None None PDL None V-PDT, PDL V-PDT Isotope therapy PDL V-PDT PDL V-PDT None V-PDT V-PDT None V-PDT, PDL V-PDT None PDL V-PDT V-PDT V-PDT, PDL V-PDT V-PDT V-PDT, PDL V-PDT, PDL V-PDT PDL V-PDT, PDL

25 25 50 50 50 50 25 50 25 25 50 50 75 50 25 75 50 25 50 25 75 25 25 25 25 25 25 25 50 25

Blood perfusion (PU)

Beginning

Peak

End

388 468 447 152 316 194 417 223 276 107 259 194 238 364 417 122 253 342 272 323 229 316 237 178 302 178 163 190 239 260

684 779 670 504 564 594 749 537 618 499 756 544 609 464 645 459 616 752 659 579 714 717 740 656 709 606 741 754 514 633

558 674 454 478 534 435 608 373 339 485 588 414 518 421 504 401 419 662 567 547 646 484 594 565 648 499 768 590 379 517

Table 2 Details of the 5 healthy subjects for monitoring blood perfusion. Case

Gender

1 2 3 4 5

Age (years)

M M M M M

24 27 28 25 32

Location of scanning

forearm forearm forearm forearm forearm

Blood perfusion (PU) Beginning

Peak

Valley

End

54 54 51 123 73

317 210 480 253 225

182 112 441 239 173

363 369 488 340 365

Table 3 Details of the 3 PWS patients scanned by LDI during laser irradiation and V-PDT, respectively. PDL: pulsed dye laser. Case Gender Age (years) PWS type Location of scanning Treatment history Rate of color bleaching (%) Laser irradiation (PU)

V-PDT (PU)

Beginning Peak Valley End Beginning Peak End 1 2 3

M F F

22 38 38

Purple Purple Purple

Cheek Cheek Cheek

PDL PDL PDL

lesion was approximately equivalent to the contralateral healthy control (297 ± 34 versus 287 ± 60 PU), as for the patient in Fig. 1(C, D). Furthermore, the perfusion were found to be highly heterogeneous even in the same PWS lesion in Fig. 1. The substantial inter- and intra-patient perfusion heterogeneity in PWS lesion were clearly observed for all the 30 subjects. The different microcirculation in PWS may lead to different response to the V-PDT treatment. For all the subjects, the comparison of skin perfusion in PWS lesions and contralateral healthy controls indicated that PWS skin perfusion could be higher than, or occasionally equivalent to that of control healthy skin before V-PDT.

75 50 75

314 195 124

722 411 368

407 318 273

619 449 335 147 327 227

903 485 555

725 388 452

3.2. Blood perfusion changes in PWS during V-PDT As shown in Fig. 2(A) and (B), the skin perfusion of PWS lesion was heterogeneous and were relatively low at the beginning of V-PDT, and then the overall level of perfusion was dramatically increasing within 3 min, followed by a slow decrease. Furthermore, the perfusion changes in three region of interests (ROIs) within the same lesion shows that the magnitude of increase (or decrease) in PWS skin perfusion differed from site to site, but the change tendency is consistent. The perfusion changes in the contralateral healthy controls show a slight fluctuation during V-PDT (data not shown). For 28 of the 30 PWS patients, during V-PDT treatment,

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Fig. 1. The typical (A, C) white light photograph and (B, D) the corresponding PWS perfusion image of (A, B) a 28-year-old female and (C, D) a 23-year-old female, respectively. The average perfusion values were taken from the black dotted bordered circle.

the perfusion in PWS significantly increased after the initiation of V-PDT treatment, then reached a peak within 10 min, followed by a slow decrease to a relatively lower level, which was still higher than, or occasionally equivalent to the perfusion value before VPDT. Furthermore, the time for reaching peak and the subsequent magnitude of decrease in perfusion varied with different patients, as well as different PWS lesion locations. For only 2 cases, the blood perfusion in PWS lesions was dramatically increased and then reached a plateau (Case no. 10 and 27 in Table 1).

summarizes all the 5 normal skin perfusion changes during laser irradiation. The change tendency of perfusion values are consistent, and an initial perfusion peak at 3 min and subsequent nadir followed by a secondary increase were observed for all the subjects. However, the peak and following valley perfusion values differ from person to person.

3.3. Blood perfusion changes in normal skin during laser irradiation only

As shown in Fig. 4(A) and (B), the blood perfusion changes in two different PWS lesions that received laser irradiation only and V-PDT in the same individual were consistent with the changes mentioned above. Fig. 4(C) showed that the blood perfusion rapidly increased to an initial peak for both laser irradiation (722 ± 191 PU) and V-PDT (903 ± 126 PU) for the first 3 min. And then, the blood perfusions continue to decrease to a relatively lower level during VPDT, while the blood perfusion increased again after a significant

Normal skin on the forearms of 5 healthy subjects received laser irradiation and were scanned by LDI. As shown in Fig. 3(A) and (B), the blood perfusion in normal skin rapidly increased to an initial peak at 3 min after the initiation of laser irradiation, then significantly decreased, followed by a secondary progressive rise. Table 2

3.4. Blood perfusion changes in PWS during laser irradiation only and V-PDT in the same individual

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Fig. 2. (A)The typical perfusion dynamics of a 23-year-old female with PWS lesion on the right cheek for V-PDT treatment. (B) The skin perfusion changes in three different ROIs of PWS lesion during V-PDT. The average perfusion values were taken from the marked ROI1, ROI2, and ROI3.

decrease during laser irradiation. Table 3 summarizes the blood perfusion changes during laser irradiation and V-PDT in the same individual for each of 3 PWS patients. For V-PDT groups, the blood perfusion (278 ± 96 PU) in PWS lesions for 31 of all the 33 PWS patients significantly increased after the initiation of V-PDT treatment, then reached a peak (638 ± 105 PU) within 10 min, followed by a slow decrease to a relatively lower level (515 ± 100 PU) during V-PDT. For light-only controls, the blood perfusion (211 ± 96 PU) in the PWS lesions for the 3 PWS patients increased and reached an initial perfusion peak (500 ± 193 PU) at 3 min, followed by a nadir (332 ± 68 PU) and a secondary increase to higher level (427 ± 166 PU) during laser irradiation. The blood perfusion dynamics during V-PDT and laser irradiation were different.

3.5. Response of PWS lesions to V-PDT During V-PDT, the color of the PWS lesions became darker or purple, but there was no blistering, erythema, or scabbing. At the end of V-PDT, mild edema in the treated PWS sites were observed for all of the patients. At the follow-up after V-PDT, the results showed that all the PWS patients had varying degrees of fading without scar formation (Tables 1 and 3 show the overall rate of color bleaching for each PWS lesion scanned by LDI during V-PDT). The color bleaching rates varied with different patients as well as different PWS lesion locations. There was not a significant correlation between the degree of blood perfusion change during V-PDT and PWS color bleaching rates.

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Fig. 3. (A) The skin perfusion images take from the marked red rectangular box in normal skin on the forearm of a 26-year-old male immediately before; and 1, 3, 5, 7, 10, 15 and 20 min during laser irradiation. (B) The corresponding average perfusion values varied over time. The average perfusion taken from the black dotted bordered rectangle. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

4. Discussion LDI is a well-established technique which utilizes the optical Doppler effect to measure superficial blood perfusion in biological tissue, which could provide a better knowledge of microcirculation [17,18]. Previous studies have shown that LDI is capable of assessing the skin perfusion in PWS lesions before and after treatment with PDL [21–25]. In the present study, a new generation line-scanning LDI technique, which rasters a line of pixels in parallel at a single time step, was used. Compared to traditional LDI based on single-point laser scanning (imaging time: on the order of minute), the imaging time for the LDI used in this study is reduced significantly due to the line scanning of laser light, and the typical scanning time for a 15 × 12 cm2 perfusion image was estimated to be 13 s in the fast imaging mode. Furthermore, Svedman et al. has

investigated the effect of measuring with LDI on an inclined skin surface, and the results showed that the obtained mean LDI values almost remained unchanged until the inclination of the skin surface exceeded 38◦ [26]. Therefore, LDI is applicable for perfusion imaging in the uneven surface of PWS lesion, which are often large area and not flat due to being mainly located on the face. The cutaneous perfusion values may be affected by several factors, such as ambient temperature [25] and physical activity [27]. Therefore, all the measurements were performed on the skin to be measured with the subject supine in a temperature-controlled room, and all the subjects had an acclimatization period before measurement and were asked to keep still during measurements. In order to minimize the influence of LDI measurement on the V-PDT treatment, the skin perfusion were measured at only 6 time points (1, 3, 5, 7, 10, 15 min) during V-PDT. The measurements were performed

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Fig. 4. The skin perfusion image of a 22-year-old male with a PWS lesion located on (A) the lower jaw during the laser irradiation only and (B) on the cheek during V-PDT immediately before; and 1, 3, 5, 7, 10, 15 and 20 min. (C) The average perfusion values change over time during laser irradiation and V-PDT, respectively.

in a short time interval within the first 10 min in order to record the significant perfusion changes after initiation of V-PDT or laser irradiation.

Before V-PDT, it was found that most of the PWS lesions have a higher level perfusion than the contralateral healthy controls, and the differences between perfusion values in PWS lesions and con-

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tralateral healthy controls may vary from patient to patient or even from lesion to lesion. Since PWS contains an abnormal density of blood vessels, the higher level perfusion in PWS lesions may be ascribed to the increase in blood content of the hyper-dilated blood vessels [18]. However, in the clinical diagnosis, the color (red-topurple) and thickness of PWS lesions varied from patient to patient or even from lesion to lesion [8,9], and approximately two-thirds of the lesions without treatments may develop nodular components and can produce facial deformity [4]. The large variations in skin characterizations of PWS as well as contralateral healthy controls may lead to varying skin optical properties (e.g., absorption coefficient (␮a ), and scattering coefficient (␮s )), and the optical properties of PWS lesions will be significantly different from the contralateral healthy controls [28,29]. The measurement of LDI is affected by skin optical properties [28]. Accordingly, the differences between perfusion values in the PWS lesions and control areas may vary significantly. Even in the PWS lesions with same skin color of the same patient, there was considerable spatial heterogeneity of blood perfusion across the skin surface. This may be attributed to the heterogeneous nature of PWS. The three-dimensional histological reconstruction of PWS vascular anatomy revealed that the spatial distribution of blood vessels is non-uniform and multiple connected clusters of small diameter vessels may be distinguished in PWS lesion [30]. As a result, there was large inter- and intrapatient perfusion heterogeneity in PWS lesion. During laser irradiation, an initial peak at 3 min, followed by a nadir and a secondary increase of blood perfusion was observed not only in normal skin of 5 healthy subjects but also in the PWS lesions of 3 PWS patients. The blood perfusion changes during laser irradiation in human skin was different from the results obtained from mouse animal models, in which the light-only controls showed rather constant blood flow through ALA-PDT treatment [12,15]. The reason for these blood perfusion changes in human skin during laser irradiation are not well understood [12]. However, it’s found that the blood perfusion changes during laser irradiation were very similar to the perfusion changes during local thermal hyperemia. Local warming of the skin causes a direct and substantial vasodilation in the site being warmed, and local thermal hyperemia achieves a maximal vasodilation between 42 ◦ C and 44 ◦ C [31,32]. Local thermal hyperemia is characterized by an initial peak in skin perfusion within the first 5 min, a subsequent nadir followed by a sustained plateau in a healthy subjects [33]. The initial rapid-phase vasodilation relies predominantly on local sensory nerves and is mediated by an axon reflex thought to be dependent on calcitonin-gene-related peptide and substance P, and the plateau that follows, mediated by nitric oxide [31–33]. In the study, the infrared thermometer showed that the temperature increased to around 39–42 ◦ C in the skin treated by laser irradiation. As a result, the blood perfusion changes during laser irradiation may be mainly due to the local thermal hyperemia caused by the laser irradiation. In order to verify that the initial peak in perfusion was caused by the laser-induced local thermal hyperemia, a suction cup was used to induce local hypobaric pressure on the skin, and to cause vasodilation in the skin before laser irradiation [4]. When suction is applied to a localized region on the skin surface, it is believed that the endothelial cells exposed to the mechanical strain and stress release nitric oxide that in turn triggers the vasodilation [4]. The vacuum cupping-treated skin and surrounding non-treated skin were simultaneously irradiated with 532 nm laser with power density of 100 mW/cm2 . As shown in Fig. 5, the blood perfusion decreased rather than increased in the vacuum-treated normal skin at the early time points during laser irradiation, while the blood perfusion still increased in the non-treated skin. The results further confirmed that the skin blood perfusion changes during laser irra-

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diation may be attributed to the local thermal hyperemia induced by laser irradiation. During V-PDT treatment, the perfusion in PWS significantly increased after the initiation of therapy, then reached a peak within 10 min, followed by a slow decrease to a relatively lower level. The trend was consistent with our previous study of investigating blood perfusion responses to V-PDT by using laser speckle imaging in a pilot study of seven PWS patients [8]. The reasons for these blood perfusion changes during V-PDT are not well understood. However, in this study, the initial increase in perfusion during V-PDT was found to correspond well with initial increase during laser irradiation. The infrared thermometer showed that the temperature increased to around 39–42 ◦ C in the PWS lesion treated by V-PDT without cooling. Therefore, during V-PDT, the increase in skin temperature caused by the laser irradiation plays an important role in the initial increase in perfusion. After reaching the peak, the skin perfusion slowly decreased to a relatively lower level, which was significant different from the perfusion changes during only laser irradiation. This decrease may be mainly due to the malformation vascular damage caused by photodynamic reaction. PDT-induced endothelial cell damage leads to the establishment of thrombogenic sites within the vessel lumen and this initiates a physiological cascade of responses including platelet aggregation, the release of vasoactive molecules, leukocyte adhesion, increases in vascular permeability, and vessel constriction [34,35]. In addition, the sequence of vascular events may last for some time after the treatment [8,9]. As a result, the perfusion in PWS immediately after V-PDT was still higher than, or occasionally equivalent to the perfusion value before V-PDT. Finally, the large error bar of the blood perfusion changes also showed that there exist considerable variations in the blood perfusion response to V-PDT treatment from area to area within the same treated lesion. However, the changes and trend of the evaluated blood perfusion response were largely consistent not only between affected individuals but also within the same lesion. The variations in blood perfusion may be due to the highly heterogeneous nature of PWS. This might also explain why the treatment outcomes differ not only between affected individuals with same clinical PWS characterization but also within the same lesion after given the same V-PDT treatment. Blood perfusion responses to PDT in animal models have been investigated in several studies, and the results showed great variability [12,15,36–39]. Mesquita et al. have shown that there are clear effects of mouse strain on tumor hemodynamics with consequences to PDT [37]. The variability could also be due to differences in animal models, applied photosensitizers [39], treatment protocols and dosimetry. Furthermore, different patterns of blood perfusion response to PDT were found in animal models and in humans [12,39]. The effects of PDT on malignant tissue in humans are more pronounced than would be predicated by some animal studies [39]. As mentioned above, studies have also shown that the blood perfusion changes for light-only controls in human normal and PWS skins were significantly different from the results in animal models, and thus the blood perfusion responses to V-PDT in the study were difficult to directly compare with the animal studies. Several studies have investigated the blood perfusion responses to the topical ALA-PDT for treating basal cell carcinomas (BCCs) patients [12–14]. Consistent with our results, increase in microvascular blood flow in the BCCs immediately after ALA-PDT were also observed using LDI by Wang et al. [13] and by Enejder et al. [14]. Becker et al. have investigated blood flow responses in BCCs during topical ALA-PDT using diffuse correlation spectroscopy, and the results showed that the elevated blood perfusion was persistent throughout treatment [12]. They also found that ALA-PDT induced early blood flow changes and these changes were irradiance dependent. These studies further confirmed that the PDT effects may last

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Fig. 5. The skin perfusion changes in vacuum-treated skin and non-treated skin during laser irradiation. (A) Photograph and (B) the corresponding perfusion image, and (C) the perfusion changes during laser irradiation. The average perfusion taken from the black dotted bordered circles.

for some time after the treatment, and the targeted blood vessels could not be completely shut down immediately after PDT. No obvious correlation between the degree of blood perfusion changes during V-PDT and the PWS color bleaching rates was observed in this study. However, we cannot conclude that there is no correlation between them. First, we made no attempt to control the factors such as: age, gender, types of PWS and baseline skin temperature at this time. Second, the LDI data may be affected by the changes in tissue optical properties during V-PDT treatments. The manifestation of these changes is that the color of the PWS area may became darker or purple, and edema gradually appear during surgery, and thus the tissue optical properties may change in course of time [8,9]. Previous studies have shown that the overall response and the depth sensitivity of the LDI method and eventually the LDI perfusion data are influenced by speckle size changes that occur with changes of tissue optical properties [28]. Nevertheless, further work is required to investigate the influence on the final measured LDI data when both the optical properties and blood perfusion changes within the same tissue volume in time.

blood perfusion were found during both laser irradiation only and V-PDT, which implies that the initial increase in perfusion during V-PDT maybe due to the local thermal hyperemia induced by laser irradiation. The correlation between the blood perfusion changes and V-PDT response for PWS treatment is still under investigation. Conflict of interest The authors declare no conflict of interest in any form with respect to this article. Acknowledgements This study was supported by the National Natural Science Foundation of China (61527827; 61036014; 61108078), the Fujian Provincial Natural Science Foundation (2014J07008), and the Program for Changjiang Scholars and Innovative Research Team in University (IRT-15R10). References

5. Conclusion For light-only controls, an initial perfusion peak at 3 min followed by a nadir and a secondary increase were found not only in normal skin but also in PWS lesions. The blood perfusion in PWS for most patients significantly increased after the initiation of V-PDT treatment, then reached a peak within 10 min, followed by a slow decrease to a relatively lower level. The blood perfusion changes during treatment were due to V-PDT effects as well as local temperature increase induced by laser irradiation. Initial increases in skin

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