Photodynamic therapy for cutaneous proliferative vascular tumors in a mouse model

Photodynamic therapy with benzoporphyrin derivative monoacid ring A and red light (PDT-BPD) has been used to treat human choroidal hemangiomas, and may be useful for cutaneous vascular lesions. The potential for PDT-BPD to inhibit selectively vascular tumor growth was tested in a mouse angiosarcoma model, of which the tumor growth mimics the proliferative phase of hemangiomas. Vascular tumors arising after intradermal injection of immortalized murine endothelial cells were exposed to 50 to 150 J per cm2 of 690 nm laser light 15 min after intravenous injection of 1 mg per kg BPD. Tumor volume and gross response were followed after PDT-BPD and compared with control tumors receiving no treatment, light alone, or BPD alone. At 2 wk, hematoxylin-eosin and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling stained tumor sections was performed. There was a selective, fluence-dependent inhibition of tumor growth after PDT-BPD (p< or =0.05), typically with eradication of tumors exposed to higher fluences. A common effect was the replacement of tumor by small scar. Surrounding PDT-BPD exposed normal skin showed no changes. Based on these results, we conclude that PDT-BPD can lead to selective eradication of these tumors. Further studies investigating the efficacy of PDT-BPD for human hemangiomas are warranted.     

Histologic comparison of argon and tunable dye lasers in the treatment of tattoos

Cosmetic benefit from laser therapy of tattoos may simply be the result of thermal injury and host reparative response which remove pigment by a "slough and bury" mechanism. Tattoo pigment of 4 colors (black, white, red, and blue) was introduced into the skin of guinea pigs and studied histologically at 48 h, 7 days, 4 and 6 weeks, and 3 months. Tattoos of each color were treated with argon laser (488 and 514 nm) and tunable dye laser at 3 different wavelengths (505, 577, and 690 nm). Treated tattoos were biopsied immediately and at 48 h, 7 days, and 3 months. Selective laser absorption by the tattoo pigment was suggested by pigment-related differences in threshold doses for histologic damage. Clinical clearing of tattoo pigment correlated well with the extent of immediate epidermal and dermal necrosis and was as well associated histologically with the deposition of parallel bands of collagen fibers (i.e., scar) between the residual pigment and the overlying epidermis. "Lightening" of tattoos probably depends more on widespread necrosis, subsequent tissue sloughing, and resultant dermal fibrosis than on specific changes in tattoo pigment chemistry, morphology, physical properties, or handling by macrophages.     

Low-fluence CO2 laser irradiation: selective epidermal damage to human skin

The interaction of normal human skin with low-fluence CO2 laser irradiation was studied using a three-phase approach. In phase one, freshly excised skin was observed immediately after impact. In phase two, skin irradiated 2 h prior to excision was studied. In phase three, human volunteers were irradiated and biopsied at time zero, 24 h and 48 h. Seventy-five sites were exposed and 60 biopsies were performed. The earliest histologic changes were observed in the 6-10 J/cm2 fluence (radiant exposure) range and these changes included spindle and vacuolar changes in the basal layer of the epidermis. Papillary dermal coagulation was present to a maximum of 0.03 mm. At fluences of 10-25 J/cm2, superficial dermal necrosis (0.06-0.08 mm) was observed. At fluences above 25 J/cm2, transepidermal necrosis was present with increasing papillary dermal necrosis that was in proportion to the energy density delivered. At 2h, basal vacuolar changes were accompanied by diffuse keratinocytic cell death where contact was maintained between the epidermis and dermis, while where separation occurred limited keratinocytic death was observed. The earliest changes occurred at lower threshold fluences (4-6 J/cm2). After 24 h, these doses resulted in extensive epidermal necrosis with focal acute inflammatory infiltrates. At 48 h, the degree of epidermal "slough" was proportional to the energy density delivered and was maximal with a fluence of 5.7 J/cm2 delivered whereas with a fluence of 3.8 J/cm2 thin slough (0.02 mm) was observed. These findings suggest that low-dose CO2 laser irradiation may provide a new approach to selectively damage the epidermis with minimal dermal damage.     

A model approach to laser coagulation of dermal vascular lesions

Summary The quantitative absorption of light by blood and by human skin is discussed in relation to laser coagulation therapy of dermal vascular lesions. The skin is represented by epidermis and dermis. Both layers are assumed to be planparallel. Absorption and scattering of light by turbid materials was accounted for by the Kubelka theory. Reflection and transmission data of blood and of skin were taken from the literature. This model shows that, firstly, argonlaser coagulation at 488/514.5 nm of dermal vascular lesions is a selective method because vascular absorption exceeds non-vascular dermal absorption by a factor of 10. Secondly, a coagulation depth of about 1 mm is predicted in agreement with the literature. Thirdly, the epidermis absorbs the 488/514.5 nm wavelengths about half as strong as blood and is therefore non-transparent. The consequences of this have been discussed in relation to the final cosmetic result. Fourthly, there is theoretical evidence that better cosmetic results may be expected when coagulation is performed at either 420 nm, 540 nm, or 575 nm. This suggests application of the krypton-laser with lines at 407, 413, and 415 nm (violet), at 531 nm, or at 568 nm. The krypton 647 nm line and the ruby 694 nm line are of little value for this purpose.Preliminary draft presented at the 1980 European Conference on Optical Systems and Applications, September 23–25, Utrecht, The Netherlands     

Wavelength effects on contrast observed with reflectance in vivo confocal laser scanning microscopy

Background/purpose: The ability to optically section live biological tissue in vivo with laser light is made possible by confocal laser scanning microscopy (CLSM). In this work, the effects of changing the wavelength of incident light used for CLSM imaging of human skin are reported and analyzed.
Methods: Optical phantoms and the skin of eight human volunteers were imaged using CLSM systems having three different incident light wavelengths (405, 785, and 830 nm).
Results: Qualitative and quantitative differences were observed between images obtained at each wavelength, despite the proximity of the two near infrared 785 and 830 nm wavelengths. Furthermore, the penetration depth achieved with the 405 nm CLSM permitted imaging into the papillary dermis.
Conclusion: The laser wavelength used in CLSM reflectance imaging is important to properly understand and resolve different biological structures within human skin.     

Histological changes produced in skin by equally erythemogenic doses of UV-A, UV-B, UV-C and UV-A with psoralens

The sequential light microscopic histological changes produced in human skin by a single exposure of UV-A, UV-B, UV-C and oral 8-methoxypsoralen plus UV-A (PUVA) causing approximately equal degress of delayed erythema response, have been evaluated. UV-C and UV-B affect the epidermis to a greater degree than UV-A, while UV-A affects the dermis to a greater degree than UV-B and UV-C. PUVA has prominent effects on both epidermis and dermis, differing in degree from those changes induced by UV-A, UV-B, and UV-C and are longer lasting. The sequence of histological changes following UV exposure is completed more rapidly after exposure to shorter UV wavelengths.     

Protoporphyrin IX formation and photobleaching in different layers of normal human skin: Methyl‐ and hexylaminolevulinate and different light sources

Topical photodynamic therapy (PDT) is used for various skin disorders, and selective targeting of specific skin structures is desirable. The objective was to assess accumulation of PpIX fluorescence and photobleaching within skin layers using different photosensitizers and light sources. Normal human skin was tape‐stripped and incubated with 20% methylaminolevulinate (MAL) or 20% hexylaminolevulinate (HAL) for 3 h. Fluorescence microscopy quantified PpIX accumulation in epidermis, superficial, mid and deep dermis, down to 2 mm. PpIX photobleaching by light‐emitting diode (LED, 632 nm, 18 and 37 J/cm²), intense pulsed light (IPL, 500–650 nm, 36 and 72 J/cm²) and long‐pulsed dye laser (LPDL, 595 nm, 7.5 and 15 J/cm²) was measured using fluorescence photography and microscopy. We found higher PpIX fluorescence intensities in epidermis and superficial dermis in HAL‐incubated skin than MAL‐incubated skin (P < 0.001). In mid and deep dermis, fluorescence intensities were higher (37%) in HAL‐treated skin than MAL‐treated skin, although not significant (P = ns). At skin surface, photobleaching was significantly higher (90–98%) after LED illumination (18 and 37 J/cm²) than IPL (29–53%, 36 and 72 J/cm²) and LPDL (43–62%, 7 and 15 J/cm²) (P < 0.001). Within the skin, photobleaching was steady from epidermis to deep dermis by LED illumination (37 J/cm², P = ns), but declined from epidermis to mid and deep dermis for IPL‐treated skin and LPDL‐treated skin (IPL 72 J/cm²: 26–15%; LPDL 15 J/cm²: 37–23%) (P < 0.04). Clinically, erythema correlated linearly with MAL and HAL‐induced photobleaching (r² = 0.175, P < 0.001). In conclusion, selective PpIX accumulation indicates HAL as an alternative to MAL for epidermal‐targeted PDT. In clinically relevant doses, PpIX photobleaching throughout the skin was more profound following LED than LPDL and IPL exposure.     

Comparative histochemistry of port-wine stains after copper vapor laser (578 nm) and argon laser treatment.

The present study compared the histologic changes occurring 15 min after copper vapor laser (CVL; operating at 578 nm) and argon laser (488/514 nm) treatment of port-wine stains (PWS) over a range of energy densities (8-32 J/cm2) with corresponding pulse widths of 50-200 ms. Frozen tissue sections were stained with nitroblue tetrazolium chloride (NBTC). This histochemical method permits an accurate color differentiation between blue-stained viable and unstained thermally damaged cells. At 8, 10, and 12 J/cm2 the argon-laser injury was confined to epidermal cell layers; none to superficial dermal effects were found. Fluences of at least 15 J/cm2 produced a diffuse NBTC-negative coagulation necrosis. Exposure of PWS skin to 8-12 J/cm2 at 578 nm did not alter the integrity of epidermal cells. In the dermis, damage was confined to blood vessels and surrounding collagen, showing a clear demarcation from adjacent viable structures. The maximum penetration depth achieved with these vessel selective energy densities was 0.44 mm. At 15 J/cm2, besides vascular injury, damage to the basal cell layer also occurred. At fluences of 17-20 J/cm2 a diffuse necrosis similar to that induced by the argon laser was found. Vessel selectivity of the 578 nm wave band was achieved with pulse widths from 50-74 ms, exceeding the estimated "ideal" exposure time (0.1-10.0 ms) for a vascular selective laser effect. The NBTC method allowed identification of subtle laser-induced tissue changes providing accurate quantitative data relating to the extent of vascular injury.     

Erythropoietic protoporphyria—submicroscopic events during the acute photosensitivity flare

The immediate response of erythropoietic protoporphyria (EPP) skin to long-wave ultraviolet radiation (UVR) was studied with the electron microscope. The main finding was severe vascular injury. This was confined to the superficial vessels of the dermis and consisted of endothelial cell degeneration and a pronounced leakage of vascular contents. In contrast, the epidermis showed no abnormalities. Short-wave UV irradiation of EPP skin resulted in epidermal changes typical for the usual sunburn reaction and spared the dermal blood vessels. The following conclusions are drawn: (i) Endothelial cells are the primary cellular target for the photodynamic reaction in EPP. (ii) The fibrillar material, characteristic for chronic EPP lesions, originates from the vessels and vascular contents. (iii) The multilayered basement membranes observed in such lesions reflect multiple consecutive reparative processes that follow endothelial injuries.     

Electron microscopic observations of dyskeratosis, apoptosis, colloid bodies and fibrillar degeneration after skin irritation with dithranol

Dying cells undergo coagulative necrosis or apoptosis. In the skin, apoptosis is known to occur in graft-versus-host reactions, in lichen planus, during regression of plane warts and neoplasms, and after physical injury caused by ultraviolet light resulting in sunburn cells. The present study shows that primary skin irritation also causes apoptosis. Mild, or moderate-to-considerable, dithranol irritation of healthy uninvolved human skin caused focally coagulative necrosis of keratinocytes and also apoptosis of scattered keratinocytes, i.e. condensation of chromatin and cytosol, clumping of tonofilaments and budding of membrane-bound cell fragments. These apoptotic cell fragments were engulfed in the epidermis by macrophages. Colloid bodies were detected in the upper dermis and apparently represented nonphagocytosed apoptotic cell fragments that had dropped down from the epidermis. Dithranol also caused fibrillar degeneration of melanocytes and in some cases of Langerhans' cells, indicating that colloid bodies in the upper dermis could partly derive from these cell types. The significance of apoptosis in irritant contact dermatitis could be to maintain homeostasis of epidermis and counteract the hyperplastic response caused by irritant stimuli. Another possibility is that apoptosis was the response to an injury less severe than that causing necrosis.     

Ultraviolet light and dendritic cells.

The ultraviolet (UV) spectrum is divided into UVC (200-280 nm), UVB (280-320 nm) and UVA (320-400 nm). Of these only UVB and UVA are of environmental significance since UVC is effectively absorbed by ozone in the earth's atmosphere. UVB wavelengths penetrate the epidermis and are almost completely absorbed in the upper dermis while UVA penetrates to the deep dermis.     

Vascular changes in human skin after ultraviolet irradiation

Blood-flow changes in human skin after ultraviolet irradiation at 250 and 300 nm have been measured by three separate methods. Those methods which measure overall blood-flow changes in the skin showed increased flow after irradiation at both wavelengths. A method which measured flow only in the superficial vessels showed a slight increase in flow after low doses of both wavelengths, but in contrast, after higher doses, this method showed a marked reduction in flow through the upper dermal vessels. This reduction in flow is probably due to stasis in these superficial vessels, perhaps secondary to vascular damage. Contrary to previous reports, blood-flow changes after irradiation at 250 and 300 nm are similar and may be mediated by identical mechanisms.     

Actinic reticuloid. A clinical, pathologic, and action spectrum study.

A 54-year-old man suffered from a chronic dermatitis that was more severe on light-exposed areas. Skin biopsy specimens revealed histopathologic findings consistent with those seen in mycosis fungoides. Ultrastructural studies demonstrated the existence of lymphoid cells with hyperconvoluted nuclei, typical of the mycosis fungoides cell or Sézary cell, within the epidermis, dermis, and peripheral blood. Light testing with a monochromator showed abnormal photosensitivity to both short- and long-wave ultraviolet light as well as to the violet, blue, and green wavelengths of visible light. The features of this patient's disease indicate actinic reticuloid.     

A study to determine the efficacy of combination LED light therapy (633 nm and 830 nm) in facial skin rejuvenation.

BACKGROUND: The use of visible or near infrared spectral light alone for the purpose of skin rejuvenation has been previously reported. A method of light emitting diode (LED) photo rejuvenation incorporating a combination of these wavelengths and thus compounding their distinct stimulation of cellular components is proposed.Objective. To assess the efficacy and local tolerability of combination light therapy in photo rejuvenation of facial skin. METHODS: Thirty-one subjects with facial rhytids received nine light therapy treatments using the Omnilux LED system. The treatments combined wavelengths of 633 nm and 830 nm with fluences of 126 J/cm(2) and 66 J/cm(2) respectively. Improvements to the skin surface were evaluated at weeks 9 and 12 by profilometry performed on periorbital casts. Additional outcome measures included assessments of clinical photography and patient satisfaction scores. RESULTS: Key profilometry results Sq, Sa, Sp and St showed significant differences at week 12 follow-up; 52% of subjects showed a 25%-50% improvement in photoaging scores by week 12; 81% of subjects reported a significant improvement in periorbital wrinkles on completion of follow-up. CONCLUSION: Omnilux combination red and near infrared LED therapy represents an effective and acceptable method of photo rejuvenation. Further study to optimize the parameters of treatment is required.     

Effect of wavelength on cutaneous pigment using pulsed irradiation

Several reports have been published over the last two decades describing the successful removal of benign cutaneous pigmented lesions such as lentigines, café au lait macules' nevi, nevus of Ota, and lentigo maligna by a variety of lasers such as the excimer (351 nm), argon (488,514 nm), ruby (694 nm), Nd:YAG (1060 nm), and CO2 (10,600 nm). Laser treatment has been applied to lesions with a range of pigment depths from superficial lentigines in the epidermis to the nevus of Ota in the reticular dermis. Widely divergent laser parameters of wavelength, pulse duration, energy density, and spotsizes have been used, but the laser parameters used to treat this range of lesions have been arbitrary, with little effort focused on defining optimal laser parameters for removal of each type. In this study, miniature black pig skin was exposed to five wavelengths (504, 590, 694, 720, and 750 nm) covering the absorption spectrum of melanin. At each wavelength, a range of energy densities was examined. Skin biopsies taken from laser-exposed sites were examined histologically in an attempt to establish whether optimal laser parameters exist for destroying pigment cells in skin. Of the five wavelengths examined, 504 nm produced the most pigment specific injury; this specificity being maintained even at the highest energy density of 7.0 J/cm2. Thus, for the destruction of melanin-containing cells in the epidermal compartment, 504 nm wavelength appears optimal.     

光致皮肤损伤机制研究进展及防护

太阳辐射是指到达地球表面连续电磁辐射,包括部分紫外线和可见光。其中可见光波长为400nm~800nm,紫外线波长为200nm~400nm,紫外线又可分为短波紫外线、中波紫外线和长波紫外线。不同波段紫外线辐射所引起的生物学效应及皮肤疾病不相同,其中UVA引起皮肤晒黑、皮肤光敏反应及皮肤光老化,UVB则主要引起皮肤红斑及皮肤肿瘤。因此,进一步掌握光致皮肤损伤机制及正确的紫外线防护措施,对相关疾病的治疗及预防有着重要的临床指导意义。     

UV irradiation and cutaneous vitamin A: an experimental study in rabbit and human skin

The effect of UV irradiation on the concentration of cutaneous retinoids (retinol and 3-dehydroretinol) in rabbit skin in vivo and in human skin in vitro was investigated. Irradiation with 4 different narrow-wavelength bands produced dose-dependent reductions of retinol in epidermis and dermis. The maximal effect was obtained at 334 nm, a wavelength which coincides with the absorption maximum for retinol in organic solutions. 3-Dehydroretinol was not reduced to the same extent as was retinol. In human skin the photodecomposition of retinol was most extensive in epidermis and progressively less so in dermis, presumably reflecting the extent to which 334 nm radiation penetrates the tissue. The regeneration of cutaneous retinol took over a week in the rabbit. The nutritional and biologic implications of the UV-induced reduction of cutaneous retinol remain to be established.     

The in vivo fluorescence of tryptophan moieties in human skin increases with UV exposure and is a marker for epidermal proliferation

We have investigated the in vivo fluorescence of human skin with UV excitation and the effect of UV irradiation on the UV fluorescence. A particular chromophore was found to be very sensitive to suberythemogenic UV radiation. This chromophore has the spectral characteristics of tryptophan residues in proteins and is characterized by a fluorescence excitation maximum at 295 nm. The fluorescence of this chromophore in human epidermis has an excitation maximum that is coincident with the maximum of the action spectrum of most UV-induced photobiologic responses to human skin. The fluorescence of the chromophore was found to increase with UV exposure. The action spectrum was determined by following the increase of the emission at 345 nm with excitation at 295 nm as a function of skin exposure to a number of wavelengths in the UV region of the spectrum. The results show that irradiation in the UVB (290-320 nm) is more effective in producing the change in the fluorescence of tryptophan. Irradiation in the UVA (320-380 nm) was found to be capable of producing the increase but to a smaller extent. Whereas tryptophan fluorescence is found in both the epidermis and the dermis, it is only the epidermal component that increases with UV exposure. The change in 295 nm fluorescence with UV exposure was determined to be oxygen dependent. The fluorescence of tryptophan moieties measured in situ was found to increase when epidermal proliferation increases. This was verified by inducing epidermal repair after mechanical insult (tape stripping). The results suggest two possible scenarios for the UV-induced increase of the fluorescence: a prompt photooxidation of tryptophan moieties or a fast proliferation response to the insult created by UV irradiation.     

Studies on fibronectin in the skin

Summary Affected and unaffected skin from patients with vulgar psoriasis and normal skin from a control group were investigated for the presence of fibronectin with an indirect immunofluorescence technique. In the control group, fibronectin is missing in the epidermis, but is found in the basement membrane zone of the dermo-epidermal junction area, in the papillary and the reticular dermis, and in the vascular and neural systems of the skin. The same distribution is also found in unaffected psoriatic skin, whereas in affected skin a change in the distribution of fibronectin is found in the dermis and in the basement membranes, together with the presence of fibronectin in the epidermis, mainly in the cornified layers.