Background. Nevus flammeus (NF) is a congenital vascular malformation.
Objectives. To investigate the acute effect of a variable pulse width Nd:YAG laser combined with hematoporphyrin monomethyl ether (HMME)-mediated photodynamic therapy (PDT) on a cockscomb model of NF.
Material and methods. Forty-two leghorn roosters were randomly divided into the following 7 groups: group A1 (treated with HMME-mediated PDT; energy density of 75 J/cm2), group A2 (treated with HMME-mediated PDT; 125 J/cm2), group A3 (treated with HMME-mediated PDT; 150 J/cm2), group A4 (treated with HMME-mediated PDT; 175 J/cm2), group B (treated with a variable pulse width Nd:YAG laser), group C (treated with a variable pulse width Nd:YAG laser and HMME-mediated PDT), and group D (the control group). Changes in the cockscomb tissues were observed visually and microscopically on days 1, 3, 7, and 14 after treatment. The capillary reduction and the ratio of collagen type I to type III were examined.
Results. The response rate was higher in groups A3 and A4 than in group B. In group A, a higher energy density resulted in a higher response rate and a greater capillary reduction (p < 0.05 for all). However, we concluded that PDT at an energy density of 175 J/cm2 is not suitable for treating NF, as severe tissue damage, markedly lower capillary numbers, and markedly higher collagen type I:III ratios were observed in the cockscombs treated at this energy density; instead, 150 J/cm2 may be a more appropriate energy density. Moreover, HMME-mediated PDT at 150 J/cm2 combined with a variable pulse width Nd:YAG laser achieved better treatment outcomes than PDT or a variable pulse width Nd:YAG laser alone (p < 0.05 for both).
Conclusions. Compared to PDT or a variable pulse width Nd:YAG laser alone, the combination of the 2 therapies achieved a better acute effect in treating a cockscomb model of NF, and 150 J/cm2 may be a suitable energy density for PDT.
Key words: photodynamic therapy, laser, nevus flammeus, hematoporphyrin monomethyl ether
Nevus flammeus (NF), also known as port-wine stain, is a congenital vascular malformation characterized by ectasia of capillaries and venules; it often occurs in dermal papillary and reticular layers.1, 2 The disease does not resolve without treatment, and the incidence rate is about 0.4% in children.3, 4 Treatment options for NF include laser therapy, cryotherapy and surgery.5, 6, 7 A pulsed dye laser, which is the most widely used therapy for NF, can achieve good results but may cause irreversible vessel injury, thereby increasing the risk of purpura. Moreover, there may be a recurrence after pulsed dye laser therapy, and it is ineffective in some NF cases.7, 8 Therefore, it is essential to find a more effective method for the treatment of NF.
In recent years, photodynamic therapy (PDT) has been considered a good method for treating NF.9 In this therapy, photosensitizers can accumulate selectively in the vascular endothelial cells, and the recurrence rate is low. The greatest advantage of PDT is that it has high selectivity in the target tissue. Some studies have demonstrated that hematoporphyrin monomethyl ether (HMME) displays characteristic absorption peaks in multiple wavelengths, and a 532-nm laser is often used in PDT for treating NF.10, 11 Therefore, a 532-nm continuous laser was chosen for our experiment. During PDT, the capillary malformation is targeted by substances generated from the reaction between the laser and photosensitizers. This therapy can be applied in large-area skin damage and offers a low recurrence rate.12 However, most of the studies to date investigated only the effect of PDT on patients in clinical practice and no effective animal experiments were carried out; moreover, the energy densities applied in those studies were empirical doses. The effectiveness of PDT in the treatment of NF has been demonstrated in many studies.5, 9, 11, 13 Different energy densities (75 J/cm2, 100 J/cm2 and 150 J/cm2) were investigated in some of these studies, but the effects of different energy densities on the treatment outcomes were not thoroughly compared. Therefore, in order to find a suitable energy density for treating NF, we compared 4 energy densities in our experiments. It has been revealed that a variable pulse width Nd:YAG laser can achieve good efficacy in treating some skin diseases.14 In this therapy, a 532-nm wavelength is often used to treat NF, as a laser at this wavelength can deeply penetrate the skin, cause a large area of thermal damage and achieve good coagulation; the effect of a variable pulse width Nd:YAG laser at a wavelength of 532 nm is especially noteworthy in the management of deep vascular malformation.15, 16 However, no other study has been performed on the effect of combined variable pulse width Nd:YAG laser and PDT. Therefore, in this study, we investigated the effect of the combined use of the 2 methods and compared the efficacy of such use to the efficacy of a variable pulse width Nd:YAG laser or PDT alone.
Material and methods
Forty-two leghorn roosters (age: 8–9 months; weight: approx. 3 kg) were chosen as subjects. The cockscombs (thickness: 7–9 mm) were ruddy and without damage, necrosis or ulcers. The subjects were divided into the following 7 groups: group A1 (treated with HMME-mediated PDT; an energy density of 75 J/cm2), group A2 (treated with HMME-mediated PDT; 125 J/cm2), group A3 (treated with HMME-mediated PDT; 150 J/cm2), group A4 (treated with HMME-mediated PDT; 175 J/cm2), group B (treated with variable pulse width Nd:YAG laser), group C (treated with variable pulse width Nd:YAG laser and HMME-mediated PDT), and group D (the control group). The study was approved by the Animal Ethics Committee of our hospital.
The cockscombs of the leghorn roosters met the criteria for the disease model of NF.17
The HMME is the most commonly used photosensitizer in PDT. For the treatment in our study, HMME (Fudan-Zhangjiang Bio-Pharmaceutical, Shanghai, China) was diluted with normal saline solution (10 mg/mL/kg). The subjects received chloral hydrate (2 mL/kg) orally for anesthesia, and the experimental area on the left side of the comb was marked in a circle with a diameter of 1.5 cm. The HMME was then injected intravenously into the root of the chicken wings in group A, and laser radiation with a power density of 150 mw/cm2 was applied to the experimental areas. During the radiation, both the experimental and non-experimental areas were covered. The energy densities in groups A1, A2, A3, and A4 were 75 J/cm2, 125 J/cm2, 150 J/cm2, and 175 J/cm2, respectively.
Variable pulse width Nd:YAG laser
Before applying the variable-pulse frequency-doubled Nd:YAG (532 nm) laser, the subjects received chloral hydrate (2 mL/kg) orally for anesthesia, and the experimental areas on the combs were marked in the same way as that in the PDT groups. The energy density was 20 J/cm2, the pulse-width was 10–50 ms and the spot diameter was 8 cm.
After comparing the effects in the 4 PDT groups, we chose 150 J/cm2 as the most suitable energy density and applied it with the variable pulse width Nd:YAG laser for the combination therapy. The PDT was first conducted, followed by variable pulse width Nd:YAG laser treatment 10 min later. The 2 procedures were carried out in the same way as described above.
Main outcome measures
On days 1, 3, 7, and 14 after treatment, samples from the experimental areas of the combs were collected in each group for visual and microscopic inspection, and the non-experimental area was used as a control. The blood capillaries in the combs were observed under a microscope, and the percentage decrease of the capillary number was calculated with the following formula: percentage decrease of the capillary number = (the number of capillaries before treatment – the number of capillaries after treatment)/the number of capillaries before treatment × 100%.
Secondary outcome measures
On day 14 after treatment, 5 fields in the experiment area of each comb were randomly picked and drilled for immunohistochemistry staining to examine the levels of collagen types I and III. The samples were fixed with 10% formaldehyde, sectioned after paraffin embedment, and then dewaxed and hydrated. After antigen retrieval, the samples were blocked with serum and incubated with primary antibodies to collagen types I and III. Next, the samples were incubated with the secondary antibodies after rewarming. Then, they were stained with DAB followed by counterstaining with hematoxylin. The sections were then sealed for observation, and the ratio of collagen type I to type III were calculated.
The response rate in each group was observed and classified into 5 levels as listed in Table 1.18 The total response rate = the total number of samples with response rate in levels 1, 2 and 3/the total number of samples × 100%.
Adverse reactions in each group such as pigmentation, blistering and scar formation were recorded 14 days after treatment.
SPSS v. 19.0 software (IBM Corp., Armonk, USA) was used for the statistical analysis. The measurement data is expressed as means ± standard deviation (SD). Comparison between 2 groups was conducted using a t-test for independent samples. Measurement data at different time points between the 2 groups were compared with repeated-measures analysis of variance (ANOVA) and a Bonferroni post hoc test. A p-value <0.05 was considered to indicate a statistically significant difference.
Results of visual inspection
After treatment, the color and morphology remained the same in the combs of group D and on the right side of the combs (opposite to the experimental side) in other groups. In contrast, changes were observed in the experimental areas of combs in groups A, B and C. In groups A1–4, the higher energy density resulted in a better clearance of NF. However, the energy density in group A4 was not optimal, as scars were formed on the combs in this group 14 days after treatment. Therefore, we chose the energy density from group A3 for PDT in the combination therapy. The results showed that the outcome in group C was much better than that of the other groups. In group C, 14 days after treatment, marked blanching was observed in some areas and no noticeable scars had formed (Table 2).
Results of microscopic observation
On days 1, 3, 7, and 14 after treatment, the combs in each group were observed under a microscope. Prior to the treatment, the structure of the stratum corneum and the dermis was intact in each group; however, changes were observed after treatment in the blood vessels in the experimental areas of groups A, B and C, whereas the combs in group D and the right side of the combs in other groups remained unchanged. As with the results of the visual inspection, good clearance of NF was achieved in group A4, but proliferative collagen fibers, which could lead to scars, were observed in this group. Thus, the energy density from group A3 was chosen for the combination therapy. Fourteen days after treatment in group C, the capillaries had almost disappeared, the epidermis had thickened, no blisters had formed, and the results of the treatment were much better than that of other groups (Table 3).
Capillary reduction in each group
Compared to group D at each time point, the other groups had a higher percentage of decrease in capillary number (p < 0.05 for all), suggesting that the treatment in these groups may have damaged the blood vessels. In groups A1–4, we found that a higher energy density resulted in a greater percentage of decrease in capillary number. Moreover, compared to groups B and A3, group C had a greater percentage decrease in capillary number (p < 0.05 for both; Table 4).
Collagen type I:III ratio in each group
The collagen type I:III ratios in groups A1, A2, A3, B, and C were similar to that in group D, while group A4 had a higher ratio than group D (p < 0.05). This result indicates that the energy density in group A4 was too high and that it may cause a risk of fibrosis in the combs (Figure 1).
Response rate and adverse reactions in each group
The results from groups A1–4 demonstrated that the response rate increased with the increase of energy density. In fact, the energy density of 175 J/cm2 used in group A4 could easily cause blisters and scarring. Considering that NF usually occurs on the facial and neck area, any therapy that may lead to blisters and scarring in these areas is not suitable for clinical treatment. Thus, we chose an energy density of 150 J/cm2 for further experiments. The results showed that group C achieved a better response rate than groups A3 and B (p < 0.05 for both), and had a similar incidence rate of adverse reactions in comparison to groups A3 and B (29.2% vs 29.2%, 29.2% vs 25.0%; p > 0.05 for both; Table 5, Table 6).
Methods for treating NF include surgical incision, cryotherapy with liquid nitrogen and skin grafting if the wound is large.19, 20 However, these methods cannot achieve excellent results and are likely to cause scarring or pigmentation. Therefore, it is essential to find a safer and more effective method for treating NF. In recent years, PDT and variable pulse width Nd:YAG lasers have often been employed in clinical practice. The PDT can achieve good results for treating superficial vascular lesions, but not for deep vascular malformations, due to its weak laser penetration. In contrast, variable pulse width Nd:YAG lasers, with stronger laser penetration, can treat the capillary malformations in deep tissues.15, 21 Therefore, in order to effectively treat both superficial and deep vascular malformations in NF, we investigated the effect of a combination therapy of PDT and a variable pulse width Nd:YAG laser.
We found that in the PDT groups, the clearance improved with increased energy density, and that PDT at an energy density of 175 J/cm2 achieved the highest clearance rate. However, the group with this energy density had the most adverse reactions, including severe blistering and scarring, and had markedly higher collagen type I:III ratios, suggesting the potential risk, or even the presence, of fibrosis in the combs.22 Therefore, an energy density of 175 J/cm2 cannot be employed clinically; instead, 150 J/cm2 may be considered a suitable energy density. The PDT at 150 J/cm2 in group A3 was found to achieve a good clearance rate and a similar incidence rate of adverse reactions as found in groups B and C; likewise, PDT at this energy density did not cause a noticeable elevation in the collagen type I:III ratio, indicating a low risk of fibrosis.
In this study, we found that the combination of PDT and a variable pulse width Nd:YAG laser was able to achieve a better outcome than PDT or variable pulse width Nd:YAG laser alone. The combination therapy achieved a similar response rate with a lower incidence of adverse reactions in comparison with the PDT therapy at 175 J/cm2. The combined use of PDT and a variable pulse width Nd:YAG laser can treat both deep and superficial capillary malformations. So far, many scholars have explored the combination of 2 or more therapies for treating NF, including cryotherapy combined with laser therapy, surgery combined with laser therapy, microneedle therapy combined with PDT, and pulsed dye laser therapy combined with long-pulse 1064-nm Nd:YAG laser therapy. However, many of these methods can easily cause adverse reactions, including scarring and pigmentation. The combination of microneedle therapy and PDT, even though it can achieve good outcomes, requires a more complicated operation than the combination of PDT and a variable pulse width Nd:YAG laser. Therefore, the combined use of PDT and variable pulse width Nd:YAG laser therapy can be recommended for further clinical trials.
Due to time constraints, there were some limitations in our study. Firstly, there are still some differences between cockscomb skin and human skin, even though the cockscomb can be used as a disease model of NF. Secondly, since human facial skin is more delicate, whether PDT at 150 J/cm2 can be used to treat faces needs to be investigated further. In this study, we found that the combination of 2 therapies, even though it did not increase the incidence of adverse reactions, could still lead to blisters and scars on the subjects. Therefore, we do not recommend its clinical application on facial areas to avoid scarring or other adverse reactions. Lastly, the study only demonstrated the short-term effect within 14 days of treatment, and more studies need to be carried out in the future to investigate the adverse reactions and recurrence rate after a longer course of treatment.
In conclusion, 150 J/cm2 can serve as a suitable energy density in PDT for the treatment of NF, and the combination of HMME-mediated PDT and variable pulse width Nd:YAG laser therapy can achieve a better outcome than PDT or such a laser alone, so it can be recommended for clinical application.