| Part One: Research BackgroundNevus flammeus, also named port-wine stains (PWS), is categorized as a benign capillary vascular malformation which is hard to cure. The incidence of this disease is about 3-5‰, which is just second to strawberry hemangioma. It is a common type of blood vessel disease in superficial skin. Most of the damage is located on exposure parts, such as faces and necks. The color of erythema deepens with aging and with changes of hypertrophic nodularity. It easily bleeds when it is hurt. All of these bring much inconvenience to patients' daily lives. The area of focus enlarges with the growth of the body, with self-vanishing as a rare phenomenon. Nevus flammeus ruins appearance, which causes great psychological obstacles when patients face such important issues as schooling, job-hunting, marriage, and promotion. It affects the living quality throughout one's life. To some patients, this disease may involve other organs and is complicated by general abnormality such as glaucoma and epilepsy, which causes organic functional obstacles. As for treatment of nevus flammeus, the best result that patients desire is keeping the original color and quality of the skin with the total disappearance of erythema, which has remained the pursuit of orthopedics for years. The old-fashioned therapies such as freezing and isotope application, which lead to many complications without desirable curative effects, have been eliminated. Both in the country and abroad, efforts have been made to seek for a new way to damage the capillary vascular of the focus with high selectiveness and at the same time not to hurt the surrounding normal tissues.Different choices of laser, discrepancies of various races, and complication and diversity of blood vessels of nevus flammeus pathological changes are the main factors that affect the curative effects of laser treatment to nevus flammeus. Since the appearance of the treatment of selective photothermolysis in 1985 which has been represented by pulsed dye laser, it has been realized to cure nevus flammeus selectively. No hypertrophic scar is found after the treatment, and effects are especially desirable as to the focus in superficial skin. It has become a widespread therapy on the front line both in the country and abroad. However, it has failed to achieve good results to the focus with a darker color and with hyperplasia of blood vessel dilatation. Photodynamic therapy (PDT) has offset the weakness of laser treatment. Because of the dual selectiveness of the photosensitizer and the laser, PDT has the advantages of better curative effects, less damage and broader applications in comparison with the simple laser treatment. It is especially applicable to the patients with larger areas of nevus flammeus and thickened hyperplasia of the focus.The skin of yellow race belongs to type IV of Fitzpatrick skin. The content of melanin is relatively higher. Such post-treatment complications as hyperpigmentation and hypertrophic scar are likely to occur. Compared with Caucasians, it has its own speciality. Foreign scholars have studied respectively the influences on nevus flammeus of correlative factors such as the colors and locations of the focus, and the ages of patients. Since the collections of clinical case load are different, and most of the cases are under the age of 30, the conclusions vary considerably, or even opposite one another. On the whole, the conclusions are not inherently systematic. Domestic scholars have paid more attention to observing the curative effects of different lasers on nevus flammeus, with a tentative but not insightful morphological research in pathological changes of blood vessels of nevus flammeus with different clinical symptoms. In recent years, based on the ten-odd-year clinical experiences in my faculty of more than 10,000 cases having received laser treatment on nevus flammeus, we have discovered that the curative effects of laser treatment differ greatly from different types of focuses. The reactions to laser are significantly different when nevus flammeus is pink, red, purple or with the focus change of hypertrophic nodularity. With the irradiation of laser with the same wavelength and energy density, the purple focus reacted well, but the red focus might carry complications of hypertrophic scar and hyperpigmentation. It is concluded that for different types of nevus flammeus, the darkness of the color is an important factor affecting the curative effects of laser treatment. Several factors such as the status of malformed blood vessels in the focus and the distribution of the nerves surrounding blood vessels are decisive in forming the color of the focus. It has become a clinical subject deserves insightful research as soon as possible in a view to analyze the correlation between the relative parameters of blood vessels, the nerve distribution, and the curative effects.In clinical practice, doctors and patients generally assess the curative effects by considering the fading rate of the picture color before and after treatment. This method is exposed to subjectivity, with strength of statistic results weakened by obvious errors and low accuracy. It can neither be quantitated for application to exchanges of scientific research nor provide clinicians with timely objective prediction of the curative effects. This breeds the ground for medical disputes. So it is of great realistic significance to establish an objective and accurate method to observe curative effects from the perspective of clinical treatment.Part Two: Research Objectives(1)To explore the correlation between pathologic changes of nevus flammeus and the clinical symptoms through HE staining and immunohistochemistry examination of specimens of nevus flammeus in different colors among Chinese, and through the relative parameters of the distribution of related blood vessels and dominant nerves that are quantificationally measured by computed image assessment;(2)To study the histological and ultrastructure changes of blood vessels after PDT through the observation of the effectiveness that PDT has on capillary vessels in the superficial skin of the dermis of nevus flammeus by resorting to histological examination and transmission electronic microscope;(3)To investigate the effectiveness in assessment of the curative effects of nevus flammeus through quantificational measures by red index of nevus flammeus and normal skin before and after PDT with the Angel-color pigment analysis software;(4) Based on the aforesaid aspects, to analyze the correlation between the curative effects of laser treatment on nevus flammeus by new copper vapor laser according to the colors of pathological changes.Part Three: Research MethodsChapter One: Pathological and histological research in the distribution of skin blood vessels and nerves of nevus flammeus among yellow race.48 cases of nevus flammeus patients who received treatment at our clinic were randomly selected. The patients' age ranged from 9 to 40 with 21 males and 27 females. The 48 patients were grouped by the colors: 12 cases of pink, 12 cases of red, 12 cases of purple and 12 cases of purple with hypertrophic nodularity. All of the pathological changes were located on faces or necks. Those patients with nevus flammeus carrying any type of complications were excluded.After local anesthesia by 3% LiDoCainE, the focus and the corresponding normal tissues were collected by micro-trepan. After regular fixation and embedding, they were sliced up, and the prepared sections were stained with HE and immunohistochemistry. The slices stained with HE were put under light microscopes to observe the pathology of the tissues, especially the distribution of the malformed blood vessels and the surrounding nerve fibers. The slices stained with immunohistochemistry underwent a computed image assessment, and a quantitative assessment of the following parameters was conducted by blood vessels marked with geneⅧ: the Mean Vessels depth, the maximum depth, the Mean ratio of vessel square to skin square (MRVS), the Filling ratio of red blood cells. Moreover, a quantitative assessment was conducted of the average ratio of the nerve to the skin square by the nerve fbers marked with S-100 albumen.Statistic processing: analyzed by the SPSS12.0 statistic software, the experimental data were figured as ((?)±S). The margin of P＜0.05 had statistic significances after the ANOVA, Nonparametric test and T test of materialsChapter Two: The impacts of PDT on histology of blood vessels in nevus flammeus and ultrastructure of skinThe staining with HE and immunohistochemistry and the observation index for PDT to the first 4 groups of nevus flammeus with different colors were the same as those in Chapter One. Some of the specimens were fixed by 2% MDA and were cut into extra-thin slices. Then regular samples were collected for observation by a transmission electronic microscope. After 2 months of PDT, the following analysis was conducted of the specimens of the focus: 1. histological examination of the slices stained with HE; 2. computed image assessment of the slices stained with immunohistochemistry; 3. ultrastructure observation under the transmission electronic microscope. The method of the statistic processing was the same as that in Chapter One.Chapter Three: The measure and assessment of the index of skin color before and after PDT of nevus flammeusBy means of a cross test with contralateral normal skin as the standard, it was measured at multiple points for both the areas of pathological changes and the areas of comparison, and then the average was calculated. Every patient took digital photos before PDT and 2 months after the therapy separately. With the help of the Angel-color pigment analysis software, systematical analysis was conducted to approach the differences of Erythema index of the skin in the pathological area on the normal skin, the focus and the focus after 2 months of therapy. After a comparison with the clinical judgments, assessment was conducted of the effects of Erythema index by the software based on a quantitative analysis of curative effects of nevus flammeus.Chapter Four: The clinical applications of PDT to nevus flammeus with the use of copper vapor laser.HMME was injected intravenously with 3.5-5.0mg/kg. 5 minutes later, the focus was irradiated by copper vapor laser. Irradiation lasted 20 to 40 minutes for each spot. The diameter for the spot was 8 to 9cm. In the group receiving the therapy, 2 spots were irradiated once. If nevus flammeus went beyond the boundary of the spot, it should be irradiated separately, without exposure to light for 1 month after the therapy and with the next therapy to be conducted 2 months later. The results of one single therapy were analyzed to study the impacts of different color types of pathological changes on the effects of PDT.Part Four: Research Results Nevus flammeus, also named port-wine stains (PWS), is categorized as a benign capillary vascular malformation which is hard to cure. The incidence of this disease is about 3-5%0, which is just second to strawberry hemangioma. It is a common type of blood vessel disease in superficial skin. Most of the damage is located on exposure parts, such as faces and necks. The color of erythema deepens with aging and with changes of hypertrophic nodularity. It easily bleeds when it is hurt. All of these bring much inconvenience to patients' daily lives. The area of focus enlarges with the growth of the body, with self-vanishing as a rare phenomenon. Nevus flammeus ruins appearance, which causes great psychological obstacles when patients face such important issues as schooling, job-hunting, marriage, and promotion. It affects the living quality throughout one's life. To some patients, this disease may involve other organs and is complicated by general abnormality such as glaucoma and epilepsy, which causes organic functional obstacles. As for treatment of nevus flammeus, the best result that patients desire is keeping the original color and quality of the skin with the total disappearance of erythema, which has remained the pursuit of orthopedics for years. The old-fashioned therapies such as freezing and isotope application, which lead to many complications without desirable curative effects, have been eliminated. Both in the country and abroad, efforts have been made to seek for a new way to damage the capillary vascular of the focus with high selectiveness and at the same time not to hurt the surrounding normal tissues.Different choices of laser, discrepancies of various races, and complication and diversity of blood vessels of nevus flammeus pathological changes are the main factors that affect the curative effects of laser treatment to nevus flammeus. Since the appearance of the treatment of selective photothermolysis in 1985 which has been represented by pulsed dye laser, it has been realized to cure nevus flammeus selectively. No hypertrophic scar is found after the treatment, and effects are especially desirable as to the focus in superficial skin. It has become a widespread therapy on the front line both in the country and abroad. However, it has failed to achieve good results to the focus with a darker color and with hyperplasia of blood vessel dilatation. Photodynamic therapy (PDT) has offset the weakness of laser treatment. Because of the dual selectiveness of the photosensitizer and the laser, PDT has the advantages of better curative effects, less damage and broader applications in comparison with the simple laser treatment. It is especially applicable to the patients with larger areas of nevus flammeus and thickened hyperplasia of the focus.The skin of yellow race belongs to typeⅣof Fitzpatrick skin. The content of melanin is relatively higher. Such post-treatment complications as hyperpigmentation and hypertrophic scar are likely to occur. Compared with Caucasians, it has its own speciality. Foreign scholars have studied respectively the influences on nevus flammeus of correlative factors such as the colors and locations of the focus, and the ages of patients. Since the collections of clinical case load are different, and most of the cases are under the age of 30, the conclusions vary considerably, or even opposite one another. On the whole, the conclusions are not inherently systematic. Domestic scholars have paid more attention to observing the curative effects of different lasers on nevus flammeus, with a tentative but not insightful morphological research in pathological changes of blood vessels of nevus flammeus with different clinical symptoms. In recent years, based on the ten-odd-year clinical experiences in my faculty of more than 10,000 cases having received laser treatment on nevus flammeus, we have discovered that the curative effects of laser treatment differ greatly from different types of focuses. The reactions to laser are significantly different when nevus flammeus is pink, red, purple or with the focus change of hypertrophic nodularity. With the irradiation of laser with the same wavelength and energy density, the purple focus reacted well, but the red focus might carry complications of hypertrophic scar and hyperpigmentation. It is concluded that for different types of nevus flammeus, the darkness of the color is an important factor affecting the curative effects of laser treatment. Several factors such as the status of malformed blood vessels in the focus and the distribution of the nerves surrounding blood vessels are decisive in forming the color of the focus. It has become a clinical subject deserves insightful research as soon as possible in a view to analyze the correlation between the relative parameters of blood vessels, the nerve distribution, and the curative effects.In clinical practice, doctors and patients generally assess the curative effects by considering the fading rate of the picture color before and after treatment. This method is exposed to subjectivity, with strength of statistic results weakened by obvious errors and low accuracy. It can neither be quantitated for application to exchanges of scientific research nor provide clinicians with timely objective prediction of the curative effects. This breeds the ground for medical disputes. So it is of great realistic significance to establish an objective and accurate method to observe curative effects from the perspective of clinical treatment.(1)To explore the correlation between pathologic changes of nevus flammeus and the clinical symptoms through HE staining and immunohistochemistry examination of specimens of nevus flammeus in different colors among Chinese, and through the relative parameters of the distribution of related blood vessels and dominant nerves that are quantificationally measured by computed image assessment;(2)To study the histological and ultrastructure changes of blood vessels after PDT through the observation of the effectiveness that PDT has on capillary vessels in the superficial skin of the dermis of nevus flammeus by resorting to histological examination and transmission electronic microscope;(3)To investigate the effectiveness in assessment of the curative effects of nevus flammeus through quantificational measures by red index of nevus flammeus and normal skin before and after PDT with the Angel-color pigment analysis software;(4) Based on the aforesaid aspects, to analyze the correlation between the curative effects of laser treatment on nevus flammeus by new copper vapor laser according to the colors of pathological changes.Chapter One: Pathological and histological research in the distribution of skin blood vessels and nerves of nevus flammeus among yellow race.48 cases of nevus flammeus patients who received treatment at our clinic were randomly selected. The patients' age ranged from 9 to 40 with 21 males and 27 females. The 48 patients were grouped by the colors: 12 cases of pink, 12 cases of red, 12 cases of purple and 12 cases of purple with hypertrophic nodularity. All of thethological changes were located on faces or necks. Those patients with nevus flammeus carrying any type of complications were excluded.After local anesthesia by 3% LiDoCainE, the focus and the corresponding normal tissues were collected by micro-trepan. After regular fixation and embedding, they were sliced up, and the prepared sections were stained with HE and immunohistochemistry. The slices stained with HE were put under light microscopes to observe the pathology of the tissues, especially the distribution of the malformed blood vessels and the surrounding nerve fibers. The slices stained with immunohistochemistry underwent a computed image assessment, and a quantitative assessment of the following parameters was conducted by blood vessels marked withgeneⅧ: the Mean Vessels depth, the maximum depth, the Mean ratio of vessel square to skin square (MRVS), the Filling ratio of red blood cells. Moreover, a quantitative assessment was conducted of the average ratio of the nerve to the skin square by the nerve fibers marked with S-100 albumen.Chapter Two: The impacts of PDT on histology of blood vessels in nevus flammeus and ultrastructure of skinThe staining with HE and immunohistochemistry and the observation index for PDT to the first 4 groups of nevus flammeus with different colors were the same as those in Chapter One. Some of the specimens were fixed by 2% MDA and were cut into extra-thin slices. Then regular samples were collected for observation by a transmission electronic microscope. After 2 months of PDT, the following analysis was conducted of the specimens of the focus: 1. histological examination of the slices stained with HE; 2. computed image assessment of the slices stained with immunohistochemistry; 3. ultrastructure observation under the transmission electronic microscope. The method of the statistic processing was the same as that in Chapter One.Chapter Three: The measure and assessment of the index of skin color before and after PDT of nevus flammeusBy means of a cross test with contralateral normal skin as the standard, it was measured at multiple points for both the areas of pathological changes and the areas of comparison, and then the average was calculated. Every patient took digital photos before PDT and 2 months after the therapy separately. With the help of thengel-color pigment analysis software, systematical analysis was conducted to approach the differences of Erythema index of the skin in the pathological area on thenormal skin, the focus and the focus after 2 months of therapy. After a comparisonith the clinical judgments, assessment was conducted of the effects of Erythema index by the software based on a quantitative analysis of curative effects of nevus flammeus.Chapter Four: The clinical applications of PDT to nevus flammeus with the use of copper vapor laser.HMME was injected intravenously with 3.5-5.0mg/kg. 5 minutes later, the focus was irradiated by copper vapor laser. Irradiation lasted 20 to 40 minutes for each spot. The diameter for the spot was 8 to 9cm. In the group receiving the therapy, 2 spots were irradiated once. If nevus flammeus went beyond the boundary of the spot, it should be irradiated separately, without exposure to light for 1 month after the therapy and with the next therapy to be conducted 2 months later. The results of one single therapy were analyzed to study the impacts of different color types of pathological changes on the effects of PDT.Chapter One:1. Histological examination: the cuticle was normal with the stain of HE, and the capillary vascular displayed dilatation with groups in the superficial dermis. With darkening of the color of the pathological changes, part of the dilatation capillary vascular extended to the deep dermis and tissues under the skin. The capillary vascular full of red cells increased. The endothelial cells matured but were not hypertrophic. 2. Immunohistochemical examination: (1) the brown capillary vascular in the superficial and middle dermis were abundant after the blood vessels marked with geneⅧwere stained positively. Most of them were dilated vessels. (2) The brown nerve fibers were scattered in the superficial dermis after the nerves marked with S-100 albumen were stained positively. It was apparently to a less extent stained than normal skin. (3) Computed image assessment: for the focus of the 48 patients, the mean vessels depth was 0.45±0.11mm; the maximum depth was 0.64±0.12mm mm; the filling ratio of red blood cells was 10.63%; and MRVS was 11.39%; the average ratio of the nerve to the skin was 2.07%. Among these, MRVS had statistic differences between groups with different clinical symptoms while the first three had no such differences. The average ratio of the nerve to the skin square of the two groups in the color of pink and red were quite different from those of purple and hypertrophic nodularity. Chapter Two:1.The formation of most of the newly reborn blood vessels and the fibre tissues in the superficial dermis became natural in the immunohistochemical examination two months after PDT. However, part of the dilated vessels in the deep dermis of the groups of purple and hyperthophic nodularity still remained. 2. The antrum of the capillary vascular had apparent dilatation before the therapy by the electronic microscope observation. The endothelial cells were natural. Immediately after the therapy, the endothelial cells showed apparent dilatation. There were sebaceous glands full of grease drops in the epithelia. Two months after the therapy, the micro blood vessels with red cells decreased. New blood vessels formed, and among fibers were full of sebaceous glands. 3. Two months after the therapy, the differences of MRVS among the groups of pink, red and normal carried no statistic significance. The differences between other two groups had such significance. The differences of the average ratio of the nerve to the skin square between the groups of pink and the groups of red carried no statistic significance. The differences between other two groups had such significance.Chapter Three:With the help of the Angel-color pigment analysis software, an examination was conducted on the Erythema index and the square of normal skin, the skin beside the focus and the skin beside the focus after two months of therapy respectively with the 48 patients of nevus flammeus. Before PDT, Erythema index differed significantly from those of the first 4 groups with the normal side. It had statistic significance in comparison with other two groups. After the two-month therapy, the Erythema index of the groups of pink and red dropped significantly, but was still different from that of the normal side. These differences had statistic significance compared with that before the therapy. The post-therapy Erythema index of the groups of purple and hypertrophic nodularity dropped, but was still obviously different from that of the normal side. The differences had statistic significance when compared with those before the therapy. This conclusion accorded with the clinical observation.Chapter Four:According to the standard of GuYin method, the best of the curative effects was that of the pink group, followed by the red group, the purple group and the hypertrophic nodularity group. After one single PDT, the percents of the curative effects on the 4 groups above two degrees were 77.3%, 62.5%, 32.1% and 0 respectively. The differences of the curative effects had statistic significance.The present study is designed to explore the correlation between the pathological changes of nevus flammeus and the clinical symptoms by the study of the pathological histology of the distributions of blood vessels and nerves of nevus flammeus among the yellow race. The conclusion indicates that MRVS is correlated with the types of the clinical pathological changes. The dilating of blood vessels of the focus might be relevant to the loss of flexibility of blood vessels to stretch and shrink which is caused by a lack of nerves surrounding the capillary vascular. PDT targeted to damage endothelial cells of blood vessels leads to highly selective damage to the capillary vascular net in the superficial dermis. The erythema index of the Angel-color pigment analysis software demonstrates a tendency of decrease, which suggests that the index can be used for quantificational assessment of the curative effects of nevus flammeus. PDT sourced by copper vapor laser is safe and effective to cure nevus flammeus, promising a bright future. The curative effects are related to the colors of pathological changes.Five: Research ConclusionThe present study is designed to explore the correlation between the pathological changes of nevus flammeus and the clinical symptoms by the study of the pathological histology of the distributions of blood vessels and nerves of nevus flammeus among the yellow race. The conclusion indicates that MRVS is correlated with the types of the clinical pathological changes. The dilating of blood vessels of the focus might be relevant to the loss of flexibility of blood vessels to stretch and shrink which is caused by a lack of nerves surrounding the capillary vascular. PDT targeted to damage endothelial cells of blood vessels leads to highly selective damage to the capillary vascular net in the superficial dermis. The erythema index of the Angel-color pigment analysis software demonstrates a tendency of decrease, which suggests that the index can be used for quantificational assessment of the curative effects of nevus flammeus. PDT sourced by copper vapor laser is safe and effective to cure nevus flammeus, promising a bright future. The curative effects are related to thecolors of pathological changes.
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