| Part One:Research Background.Port-wine stains (PWS) or micro-venous malformations (venular malformation),commonly known as "red birthmark", are the congenital dilation of blood vessels in the superficial dermis which are presented as abnormal skin erythema at birth. PWS incidence rate is 0.3 to 0.5%. There is no significant gender differences and 90%occur in the face and neck areas. Its mechanism is not clear and the most accepted hypothesis is that the postcapillary venules are lack of neurons controlling blood flow and blood vessels cannot contract properly therefore perpetually stay in dilated state.Port wine stains do not subside on their own and can become dark with age. In addition to affect the feeling, adjusting the temperature and skin barrier function,more importantly, port wine stains-related facial defects can affect patient psychologically. Some PWS are part of a variety of serious syndrome, such as Sturge-Webers syndrome, KT syndrome and vascular pigmented hamartoma fibromatosis etc.Prior to Professor Andersen’s landmark theory of selective photothermolysis and the birth of pulsed dye laser (PDL) therapy in 1983, PWS treatment had experienced the traditional non-selective and destructive stages. PDL can selectively interact with the hemoglobins within the abnormal vascular network and induce coagulative necrosis, vascular embolism, and achieve the destruction of the vascular malformation. Its efficacy and safety have improved significantly. Photodynamic therapy (PDT) of PWS was first reported in China in 1991. The mechanism of action of PDT includes the photosensitizer absorption of laser energy,production of reactive oxygen species, and direct damage of vascular endothelial cells to achieve therapeutic goals. Although this vascular targeting treatment has improved the efficacy of PWS treatment, the PWS treatment is still facing the following problems:1.The relationship of PWS lesion types and their structure is uncertain.To date, PWS classification is based on lesion color and thickness. Lesion is broadly divided into pink, purple, and nodular hyperplasia. Currently, it is believed that PWS color depends mainly on the extent of the papillary layer of dilated abnormal blood vessels. It is unclear whether there are other factors to be considered,such as vessel thickness, depth, perfusion and circulation status. What parameter affect the color, single or multiple? Is the color determined by the extent of PWS pathogenesis? It has been found that the clinical type of lesion is closely related to the efficacy. Therapeutic outcomes of red type is better than that of purple type. Thicken lesion is the worst. However, due to the lack of in vivo non-destructive observation techniques for lesion structure, the pathological examination is subjected to many limitations (ethical, not real-time, not fully reflect the lesion nature, etc.). Due to the lack of full understanding of the diseased structure, based solely on the color of lesions, it is difficult for doctor to choose a personalized treatment option and the number of treatments and to predict the best discoloration. Therefore,it is necessary to establish the relationship between lesion type and structure.2.Lack of objective standard of lesion classification and outcome evaluation.Currently,lesion classification and outcome evaluation are based on doctor’s subjective observation which is lack of objective base. Since individual’s color perception can be affected by many factors, such as environmental factors (light, the color of surrounding objects), physiological factors (visual sensitivity, resolution of color), and psychological factors, there is a big variation in lesion classification. It is difficult to accurately identify small color differences. Certain errors can happen in evaluating the efficacy. Current criteria used for subjective visual evaluation of efficacy are: 1 (cure): lesion color fading, close to the normal skin; 2 (significant):most of the lesion color fading, small amount of residual light color, or thicken lesion becomes flattened; 3 (effective): pathological changes color significantly fading,thicken lesion flattened slightly; 4 (minor): color fading slightly, thicken lesion flattened slightly; 5 (invalid): lesion color unchanged. This non-quantitative subjective assessment method is associated with some errors so that the comparability of treatment outcomes is affected. There is no objective criteria when doctor and patient disagree with each other on the clinical efficacy. Furthermore,the residual deep vascular malformation cannot be identified by the color. Therefore,it is urgently needed to establish objective and standardized criteria for lesion classification and efficacy assessment.3.Lack of objective basis in selecting optimized treatment options.At present, PDL and PDT are the mainstream of PWS treatment in China. PDL requires multiple treatment and its color fading effect is not ideal. Except for superficial lesions of small areas, the cure rate (completely discoloration, the skin returns to normal) is less than 20%. Uneven piebald and scaring can happen after treatment. There are 20% of PWS are resistant to PDL. For vascular malformation either less than 12 μm or large size, thermal coagulation of hemoglobin could be inadequate and not be able to completely close the vascular malformation. Because of it vascular targeting effect, PDT can solve some of these problem. However, the mechanisms of PDT are complex and its efficacy could be influenced by many factors,such as the structure, circulation status and red blood cell flow path. But one cannot select treatment parameters based on the structural characteristics of the vascular malformation so that treatment parameters can not be individualized. Real-time monitoring of photodynamic treatment is impossible and the dose-effect relationship is still not clear. Excessive treatment or inadequate dosage is still existing.It is still lack of objective basis in selecting these two therapies. Doctors usually select the treatment method based on their own terms, technology and experience.This might affect the cure rate. Therefore, it is necessary to compare the efficacy of both treatments, use optical detection methods to understand the structural characteristics of PWS, establish the relationship between structural characteristics and the efficacy of treatments, and objectively select the best treatment for different types of lesions.Part Two:Research Objcetives1 .By analyzing differences in color parameters between the different types of PWS and in discoloration of the various types of PWS before and after treatment and quantitatively calculating the percentage of discoloration before and after treatment,to establish the objective classification method for PWS color and the quantitative standard for assessing the efficacy.2 .Establish the relationship between lesion type and tissue structure.Understanding the tissue structure of PWS is the key to solve the treatment problem.The tissue structure of PWS were examined using non-invasive optical technology.Photoacoustic imaging technology developed in recent years were sued to image different types of PWS lesion and obtain their morphological structures (e.g.epidermal thickness at the top of the malformation vascular, melanin content, dermal thickness,the diameter,density,thickness and depth of vascular malformation, etc.).We compared the geometric parameters of pathological structure with photoacoustic image, such as the mean depth of blood vessels, the largest depth of blood vessels, the mean area ratio between the blood vessels and skin,the filling ratio of blood,etc. "We studied the relationship between photoacoustic imaging and structural characteristics,pathology and color type. We demonstrated the authenticity of the photoacoustic imaging for PWS blood vessels and laid the foundation for clinical application.3 .By comparing the efficacy of both treatments and objectively assess the efficacy of the two mainstream treatments, to establish the relationship between PWS pathological vascular parameters and corresponding color parameters and between structural characteristics and efficacy in order to provide an objective basis for selecting the treatment method,optimize the treatment parameters and predict prognosis for port wine stains.Part Three:Research MethodsChapter One:chromatism of port wine stains before and after laser therapyA total of 15 subjects with various types of PWS were recruited. Before treatment and at 2, 4 and 6 months follow-up color parameters in each experimental site were measured - A: PWS blank control, B: PDT treatment, C: PDL treatment, and D: contralateral normal skin. Using MINOLTA CM-2600d Spectrophotometer and normal skin as the standard sample, the color parameter L, *, a*, b. * values of normal skin, PWS lesion before and after treatment were measured. The average value was obtained from 3 consecutive measurements. The color differences between pre-treatment of PWS lesions and normal skin and between post-treatment of PWS lesions and normal skin were compared, the percentage of color fading calculated,and the meanings of color parameter changes analyzed.Chapter Two: Photoacoustic imaging for blood vessels of port wine stains in vivoImages of different types of PWS lesions were acquired with photoacoustic technology. The morphological characteristics and parameters of blood vessels of PWS were obtained from the photoacoustic images, such as epidermal thickness at the top of the malformed vascular, melanin content, dermal thickness, the diameter of vascular malformation, density, thickness and depth, etc. Pathological examination was carried out for the corresponding locations and the parameters of blood vessels were calculated with the image processing software to obtain following parameters:the mean depth of blood vessels, the largest depth of blood vessels, the mean area ratio between the blood vessels and skin, the filling ratio of blood, etc. We studied the relationship between the pathological parameters and photoacoustic parameters,demonstrated the authenticity of the photoacoustic imaging for PWS blood vessels and laid the foundation for clinical application.Chapter Three: The research of the relationship with structure of port wine stains and treatment effectThis perspective self-control study assessed the efficacy of both treatments on the same type of PWS lesion. In previous studies, the comparative assessment of two therapies were often carried out on different PWS types, anatomic location and different individuals. This study was a self control study which excluded a variety of differences and improved the reliability. A total of 32 PWS subjects, including 11 red,14 purple and 7 thickening, underwent PDL and PDT treatment, Follow-up observations were carried out at 2, 4 and 6 months after treatment. Spectrophotometer was used to obtain the Lab color parameters of the experimental sites. The percentages of color difference and color fading were calculated and the efficacy of the two treatment areas was objectively and quantitatively analyzed.Part Four:Research ResultsStudy of Lab values in color changes before and after treatmentIn the Lab color coordinate system developed by the International Illuminating Engineering Society, the △ a value gradually increases when the lesion color gradually darkenes, but the statistical analysis of △ a value of proliferative lesions shows no significant differences between the various types. So the value alone cannot be used as the parameter for PWS classification. This agrees with that the color coordinates of a single color value cannot determine the coordinate of the color position. Along with the gradual increase in lesion color, the blood vessels of the dermal layer become more dilated,and the levels of hemoglobin higher,which leads to more absorption of complementary shade and more reflection of the red spectrum,therefore causes a corresponding increase in △ a. However, for the proliferative lesions, skin melanin content usually will increase,the spectrometry color measurement instrument cannot fully distinguish between the contribution of the reflectance spectra of melanin and hemoglobin. The increase of melanin can decrease △ a. and gradually increase△ E values in various types significantly. △ E value reflects the colors of the three variables, namely, hue, brightness and color saturation. It is the sum of squares of all values and even this change is negative it will increase the total △ E values. Therefore,there is a good correlation between △E values and PWS lesion grading. △ E values are valuable in PWS classification.This color evaluation system for the assessment of efficacy showed that 2, 4 and 6 months after treatment the changes in three color variables were significant: i.e.the increase of L value, the decrease of red and green values or a value, and the increase in yellow-blue values. This indicated that the red color faded after treatment while the skin brightness increased. However,it was unclear why the yellow values increased. It might be related to the increase of yellow reflection ratio after treatment.Efficacy analysis of follow-up results of different time points showed that after treatment improvement was presented as a reduction in the color difference between lesion color and normal skin color lesion. When △ E decreased to 0 after treatment,the efficacy was close to 100%, i.e. the best therapeutic effect. For cases with no treatment,△ E values were equal before and after treatment,efficacy value is 0.Therefore, △ E can be considered as an objective index for the evaluation of the efficacy.In situ photoacoustic imaging of PWS vesselsThe parameters of blood vessels of cock comb were quantitatively analyzed. The mean diameter, depth range and blood vessel density were calculated. The mean diameter,depth range and blood vessel density were 29.7±5.2 μm,38.5-268.3 μm and 49.2%, respectively.Photoacoustic imaging was carried out for two PWS subjects with red color lesion and the parameters including the mean diameter, max diameter, mean depth,max depth and blood vessel density were calculated. They were 38.6±5.7μm, 62.1μm,309.1±32.9 μm,351.3 μm,3.96±0.29%,and 30.5±6.8 μm,47.1 μm,280.2±63.1μm,335.1μm,and 3.1 ±0.61%,respectively.Photoacoustic imaging was also carried out on the normal skin and PWS sites for two PWS subjects with purple color lesion and the parameters were calculated.The mean diameter, max diameter, mean depth, max depth and blood vessel density of PWS lesion of the first patient were 74.1 ± 10.9 μm,93.3 μm,499.2±76.2 μm,581.3μm,and 6.56±0.28%,respectively. That of normal skin were 51.2±8.8 μm,58 μm,370.6±32.5μm, 405.8 μm, and 1.91±0.13%, respectively. The parameters of arm PWS lesion were ～1.44,～1.61,～1.35,～1.43 and ～3.43 times higher than that of normal arm skin.The mean diameter, max diameter, mean depth, max depth and blood vessel density of PWS lesion of the second patient were 62.6± 13.2 μm,87.3 μm,374.6±63.1μm,428.8 μm,5.16±0.31%,respectively. That of normal skin were 49.3±6.3 μm,56.1μm,336.7±26.3 μm,363.5 μm, and 0.85±0.12%,respectively. The parameters of arm PWS lesion were ～1.27,～1.55,～1.11,～1.18 and ～6.07 times higher than that of normal skin.The port wine stains were imaged before and after PDL treatment. The shape and size of blood vessels was decreased after treatment.Study the relationship between PWS structure and treatment efficacyPWS color depends on the extent of the papillary layer telangiectasia. In this study, the vascular parameters of three types of PWS were analyzed histopathologically. The blood vessel depth of red type PWS was 460 ± 210 μm,the depth of the largest blood vessel 580 ± 220 μm, the ratio of vascular filling 8.30%,and the area ratio of vessels and dermis 11.45%. The blood vessel depth of purple type was 570 ± 190 μm,the depth of the largest blood vessel and the largest blood vessel depth of 730 ± 220 μm,the ratio of vascular filling 8.70%,and the area ratio of vessels and dermis 18.45%. The blood vessel depth of thickened lesion was 860 ±210 μm, the depth of the largest blood vessel and the largest blood vessel depth of 1060 ± 220 μm,the ratio of vascular filling 11.30%,and the area ratio of vessels and dermis 50.45%. Among four vascular parameters, the statistical analysis showed a significant difference in the ratio of vessel and dermis area between the groups which was also correlated with △ E.This self-control study evaluated the efficacy of two experimental sties at 2, 4 and 6 months after treatment. Results of 11 subjects of red type showed that at 2-month follow up PDT is superior to PDL. Pigmentation was seen in PDL group which became light but uneven at 4-month follow up. The last follow-up at 6-month showed a return of red color. For 14 cases of purple type, the efficacy of PDT was superior to that of PDL at all three follow-up time points. Analysis of the vascular structure parameters showed that the maximal vessel depth was 730 μm. Some studies conclude that PDL is only effect for vascular lesions located 830 μm below the epidermis and less effective for lesions located greater than 1000 μm. Reported tissue penetration depth of PDL is 0.5 mm. With the increase of pulse width, the effective depth can be increased to 1.5 mm. PDL is less effective for lesion located deeper than the penetration depth. Since the mechanism of PDT is the damage of vascular endothelial cells mediated by singlet oxygen produced by photodynamic effect, which is not affected by the blood vessel depth, therefore PDT was superior to PDL for all three follow-up points. For the 7 cases of thickening lesion, PDT was also superior to PDL for all three follow-up points.Part Five:Research ConclusionThis project utilized photoacoustic imaging and spectroscopy color measurement instrument for in situ examination of PWS non-invasively. We analyzed the consistency in vascular parameters between photoacoustic imaging and pathological examination of various types of PWS lesions and photoacoustic images before and after treatment. We confirmed the reliability of the photoacoustic imaging and the advantages of in vivo non-invasive real-time multi-dimensional imaging and laid the foundation for clinical application and clinical research. Color parameters of objective quantitative analysis. To assess the efficacy of the two mainstream treatments and three types of PWS,the Lab color space system was utilized in a self-control study. For red lesions, the efficacy of two treatments were equal, but PDL group showed visible spot and treated area looked uneven at 4-month follow up. At 6-month follow up, red color reappeared. Hyperpigmentation was seen 2 months after treatment and gradually subsided after 4 months. For purple lesion,the efficacy of PDT was superior to that of PDL. Thicken lesions of vascular malformation showed significant expansion, the increase of blood vessel density, and lesion thickening. The blood vessel depth was greater than 1000 microns so that the selective photothermolysis effect of PDL and its effective death could not reach and destroy the vascular malformations, therefore the treatment effect was not obvious. Whereas,PDT could directly affect vascular endothelial cells which was not affected by the vascular structure and thus partially effective in color bleaching. PDT is more effective for purple and thicken PWS lesions. |
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