Thermal response of human skin epidermis in different skin types to 595-nm laser irradiation and cryogen spray cooling: an ex-vivo study

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1 Thermal response of human skin epidermis in different skin types to 595-nm laser irradiation and cryogen spray cooling: an ex-vivo study Tianhong Dai a, Brian M. Pikkula a, James W. Tunnell a, David W. Chang b and Bahman Anvari *a a Department of Bioengineering, Rice University, Houston, TX, USA 77251; b Department of Plastic Surgery, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA ABSTRACT Improved laser treatment of port wine stains is expected by utilizing higher incident dosages, longer pulse durations, and longer wavelengths than those currently used in clinical settings. However, higher incident dosages also increase the risk of nonspecific thermal injury to the epidermis. Using ex-vivo human skin samples, we investigated the thermal response of human skin epidermis in different skin types to 595-nm wavelength laser irradiation at high incident dosages (up to 20 J/cm 2 ) and long pulse durations (1.5 to 40 milliseconds) in conjunction with cryogen spray cooling (CSC). Human skin samples (Fitzpatrick types I-VI) from consenting adult females undergoing trans-rectus abdominis myocutaneoues flap procedures were irradiated at the incident dosages D 0 =4, 6, 10, 15, and 20 J/cm 2, pulse durations τ laser =1.5, 10, and 40 milliseconds without and with CSC (Refrigerant-134A, spurt duration τ CSC =100 milliseconds). Thermal injury to the epidermis was evaluated by histological observations. Experimental results showed that thermal injury to the epidermis could not be avoided in skin type VI even at D 0 =4 J/cm 2 in conjunction with CSC. However, CSC allowed utilization of high incident dosages (15-20 J/cm 2 ) in skin types I-IV. Under the same incident dosage, longer pulse durations led to decreased degree of thermal injury to the epidermis. Threshold values for irradiation parameters that resulted in thermal injury to the epidermis for each skin type were obtained. Keywords: Dermatological laser treatment, epidermal protection, port wine stain, pulsed dye laser, selective cooling, selective photothermolysis 1. INTRDUCTION Pulsed dye laser at the wavelength λ= 585 nm, incident dosages D 0 =5-10 J/cm 2, and pulse duration τ laser =0.45 millisecond has provided a superior approach for the treatment of port wine stain (PWS) lesions 1-5. However, clinical studies have shown that, complete blanching of the stains is not commonly achieved, and multiple treatments are required to obtain optimal blanching 1,1,4,5. Moreover, in some cases, patients are unresponsive to the pulsed dye laser treatment 6. These limited therapeutic outcomes are believed to be the insufficient laser-induced heat generation, nonuniform heating, and limited light penetration depth in large-sized PWS blood vessels and blood vessels located in the deep parts of the skin dermis, which result from the sub-optimal irradiation parameters of incident dosage, pulse duration, and wavelength currently used in clinical settings. Improved laser treatment of PWS is expected by utilizing higher incident dosages 7-10, longer pulse durations 10-15, and longer wavelengths Increasing the incident dosage will increase the laser-induced heat generation in the targeted blood vessels, resulting in improved photocoagulation of the blood vessels 7,8,18. The size of the blood vessels coagulated during laser treatment is dependent on the laser pulse duration. Longer pulse durations are more effective in treating larger-sized blood vessels by heating the blood vessels more homogeneously 11,19,20. Longer wavelengths penetrate deeper into tissue and blood, and hence, are able to coagulate larger-sized blood vessels and blood vessels located in the deeper parts of the skin dermis For example, increasing the wavelength from λ=585 nm to 595 nm will increase the light penetration depth in blood from approximately 50 µm (absorption coefficient of blood µ a 19.1 mm -1 ) to 260 µm (µ a 3.8 mm -1 ) 21. * anvari@rice.edu; phone ; fax ; ruf.rice.edu/~banvari Lasers in Surgery: Advanced Characterization, Therapeutics, and Systems XIII, Lawrence S. Bass, et al., Editors, Proceedings of SPIE Vol (2003) 2003 SPIE /03/$

2 However, as epidermal melanin, mainly located at the basal layer of the epidermis (stratum basale), competes with absorption of laser irradiation by subsurface targeted blood vessels, simply increasing the incident dosage will also increase the risk of nonspecific thermal injury to the epidermis. Additionally, a large number of the patients, namely those with high melanin concentration skin types, are still excluded from the laser treatment due to significant light absorption at the basal layer of the epidermis. A novel technique, cryogen spray cooling (CSC), has demonstrated its efficacy in protecting skin epidermis during laser treatment. In this technique, an appropriately short cryogen spurt (on the order of tens of milliseconds) is sprayed onto the skin surface immediately prior to the onset of laser irradiation. Cooling is localized in the epidermis, while leaving the temperature of the PWS blood vessels unchanged Using ex-vivo human skin samples in different skin types, we investigated the thermal response of human skin epidermis to 595 nm wavelength irradiation at high incident dosages (up to 20 J/cm 2 ) and long pulse durations (1.5 to 40 milliseconds) to help identify the irradiation parameters that do not induce thermal injury to the epidermis. 2. METHODOLOGY 2.1 Apparatus The flashlamp-pumped pulsed dye laser V-beam TM (Candela Corporation, Wayland, MA) was used in the study. This laser operates at λ=595 nm and provides user-specified discrete incident dosages between 4 and 20 J/cm 2, macro pulse durations between 1.5 and 40 milliseconds, and a light spot size of 7 mm in diameter. The V-beam TM laser macro pulse is composed of a series of 100-microsecond duration micro pulses with equal time intervals between the pulses. For a 1.5-millisecond macro pulse, three micro pulses are present at the intervals of 0.75 millisecond (τ laser /2). For 10-millisecond and 40-millisecond macro pulses, four micro pulses are present at the intervals of 3.3 (τ laser /3) and 13.3 (τ laser /3) milliseconds, respectively. Hereafter, we term pulse duration as the duration of the macro pulse. A CSC device is incorporated in the hand-piece of the laser system. The cooling medium is Refrigerant-134A (1,1,1,2- terfluoreethane) with boiling point of 26 C at atmospheric pressure. 2.2 Skin samples Normal abdominal skin samples (types I-VI, according to Fitzpatric classification 27 ) were obtained from 28 consenting adult females (age 34 to 66 years) undergoing the trans-rectus abdominis myocutaneoues flap procedures in the Department of Plastic Surgery at University of Texas M.D. Anderson Cancer Center (MDACC). The numbers of samples were 3, 7, 5, 9, 3 and 1 for the respective skin types I to VI. The protocol to obtain the skin samples was approved by the Institutional Review Boards of MDACC and Rice University. 2.3 Study design Each skin sample was irradiated at the incident dosages D 0 =4, 6, 10, 15, and 20 J/cm 2, laser pulse durations τ laser =1.5, 10, and 40 milliseconds, without and with CSC (spurt duration τ CSC =100 milliseconds). The time delay between the termination of cryogen spurt and the onset of laser pulse (τ delay ) was 30 milliseconds throughout the experiments. Biopsy specimens were taken from the irradiated sites by a 6-mm punch and then processed for histological sectioning. Thermal injury to the epidermis was evaluated by histological observations. 3. RESULTS 3.1 Histological observations of skin types V-VI Histological sections of an untreated site (i.e., not irradiated and without CSC) and sites treated at various settings in a type VI skin sample are shown in Figure 1. The settings are denoted by the function form of Φ(D 0, τ laser, τ CSC ) where the units of the parameters are J/cm 2, millisecond, and millisecond, respectively. Fig.1b-1e are sites irradiated at various incident dosages D 0 =4, 6, 10, and 15 J/cm 2, respectively, but the same pulse duration (τ laser =1.5 milliseconds). CSC was not applied to these sites. At D 0 =4 J/cm 2, there was evidence of morphologic 2 Proc. of SPIE Vol. 4949

3 changes as basal cell elongation (Fig. 1b, curve wrapped areas) and cytoplasmic vacuolization (Fig. 1b, arrows). When the incident dosage was increased to D 0 =6 J/cm 2, the cytoplasmic vacuolization enlarged and combined with each other to form multiple basal lacunae (Fig. 1c, ovals); meantime, basal cell elongation evolved to basal cell spindling (Fig. 1c, curve wrapped area). When the incident dosage was further increased to D 0 =10 J/cm 2, the basal lacunae enlarged to affect the entire epidermal papillae, and led to partial basal layer separation (Fig. 1d, arrows). When the incident dosage was D 0 =15 J/cm 2, complete epidermal ablation occurred (Fig. 1e). Fig. 1c and 1f show sites irradiated at the same incident dosage D 0 =6 J/cm 2 but with different pulse durations of 1.5 and 40 milliseconds, respectively, both without CSC. In Fig. 1c, epidermal damage appears in the form of multiple basal lacunae, whereas Fig. 1f shows cytoplamic vacuolization (arrows), a less amount of epidermal damage. These results suggest when the incident dosage remained the same, longer pulse durations helped reduce the thermal injury to the epidermis. The comparison between Fig. 1f and 1g demonstrates the efficacy of CSC in reducing thermal injury to the epidermis. Sites in Fig. 1f and 1g were both irradiated at D 0 =6 J/cm 2, τ laser =40 milliseconds, but the site in Fig. 1f was without CSC and site in Fig. 1g with CSC. When CSC was applied, the degree of epidermal damage was decreased from cytoplasmic vacuolization to basal cell elongation (Fig. 1g, curve wrapped area). This indicates that the application of CSC provided a means to reduce epidermal damage during laser irradiation. Fig. 1h shows the histological section of a site irradiated at the minimum available incident dosage (D 0 =4 J/cm 2 ), and maximum available pulse duration (τ laser =40 milliseconds) of the laser system in conjunction with CSC. Even under this safest setting used, basal cell elongation (curve wrapped area) was present. Histological observations in type V skin samples showed that no evidence of thermal injury to the epidermis was observed at Φ(4, 1.5, 0). Thermal injury to the epidermis initiated when the incident dosage was higher than 4 J/cm 2 (basal cell elongation was observed at Φ(6, 40, 100)). Due to the limited availability of skin types V-VI, only three type V and one type VI skin samples were used in our experiments. Additionally, not all of the designated parameter settings were implemented on each of the skin samples because of the smaller areas on some of them. However, the results on skin types V-VI obtained in this study were in a good agreement with those from an earlier in-vivo study Statistical analyses of histological results in skin types I-IV In contrast to skin types V-VI, skin types I-IV demonstrated much higher threshold for thermal injury to the epidermis. When CSC was not applied and pulse duration was 1.5 milliseconds, no evidence of thermal injury to the epidermis was observed in skin types I-II and III-IV at D 0 15 J/cm 2 and D 0 10 J/cm 2, respectively (Table 1). When CSC was applied and pulse duration was 1.5 milliseconds, no evidence of thermal injury to the epidermis was observed in types I-II skin samples both at D 0 =20 J/cm 2 and 15 J/cm 2. For skin types III-IV, 83% (10 out of 12) of the samples showed no evidence of epidermal damage at Φ(20, 1.5, 100). At Φ(15, 1.5, 100), no thermal injury to the epidermis was observed in skin types III-IV (Table 1). Table1: Statistical results of thermal injury to the epidermis in skin types I-IV, τ laser =1.5 milliseconds. Number of samples exhibiting epidermal damage */ D 0 (J/cm 2 ) τ CSC (millisecond) number of samples treated Skin types I-II Skin types III-IV /6 9/ /7 6/ / /2 2/ /4 0/ * The types of epidermal damage observed in skin types I-IV included basal cell elongation, nuclei shrinkage and cytoplasmic vacuolization, multiple basal lacunae formation, and partial basal layer separation. Proc. of SPIE Vol

4 a b c e f g h Figure 1: Histological sections of an untreated site and sites irradiated at various settings in a type VI skin sample. The settings are denoted by the function form of Φ(D 0, τ laser, τ CSC ), where the units of the parameters are J/cm 2, millisecond and millisecond, respectively. (a) untreated site, Φ(0, 0, 0); (b) Φ(4, 1.5, 0) (arrows indicate cytoplamic vacuolization, curve-wrapped area indicates basal cell elongation); (c) Φ(6, 1.5, 0) (ovals indicate basal lancunae formation, curvewrapped area indicates basal cell spindling); (d) Φ(10, 1.5, 0) (arrows indicate partial basal layer separation; (e) Φ(15, 1.5, 0); (f) Φ(6, 40, 0) (arrows indicate cytoplamic vacuolization); (g) Φ(6, 40, 100) (curve-wrapped area indicates basal cell elongation); (h) Φ(4, 40, 100) (curve-wrapped area indicates basal cell elongation). Bar=100 µm. 4 Proc. of SPIE Vol. 4949

5 For statistical analyses purpose, the degree of thermal injury to the epidermis was scored as the following values: 0-no demonstrable damage, 1-basal cell elongation, 2-nuclei shrinkage and cytoplasmic vacuolization, 3-mutiple basal lacunae formation, 4-partial basal layer separation, and 5-complete epidermal ablation, corresponding to the range from none to the largest amount of epidermal damage observed. Table 2 shows average scores of thermal injury to the epidermis in skin types III-IV at pulse durations of 1.5 and 40 milliseconds and same incident dosages 20 J/cm 2 without CSC. It can be observed from Table 2 that the average score of thermal injury to the epidermis in skin types III-IV decreased from 1.8 to 1.1 as the pulse duration increased from 1.5 milliseconds to 40 milliseconds (the relatively large standard deviations in Fig. 2 are due to the coarse score scales of the epidermal damage and the Fitzpatric classification system for various human skin types). These results are consistent with that obtained from skin types I-IV, i.e., at a given incident dosage, longer pulse durations help reduce the thermal injury to the epidermis. Table 2: Scores of thermal injury to the epidermis for skin types III-IV, τ laser =1.5, 40 milliseconds, D 0 =20 J/cm 2, without CSC. Scores of thermal injury to the epidermis Skin Sample No. Skin type τ laser =1.5 milliseconds τ laser =40 milliseconds 1 III III III III III IV IV IV IV IV IV IV 0 0 Average Score Standard Deviation DISCUSSION Similar ex-vivo 8 and in-vivo studies 7 were performed previously using 585-nm wavelength laser irradiation with the laser pulse duration fixed at 1.5 milliseconds. To our knowledge, the present study is the first investigation using 595-nm laser irradiation at various pulse durations. As mentioned earlier, the V-beam TM laser pulse is composed of a series of 100-microsecond micro pulses with equal intervals between them. Using chick chorioallantoic membrane (CAM), the thermal responses to the irradiation of V- beam TM versus a single pulse laser system (ScleroPlus TM laser, Candela Corporation, Wayland, MA) both at τ laser =1.5 milliseconds were compared in a previous study 20. The sizes of CAM vessels were grouped as µm, µm, and µm. Almost identical average scores of vessel damage were obtained from the irradiation of V-beam TM and ScleroPlus TM for all the vessel sizes. The present study showed that in skin types V-VI, thermal injury to the epidermis could not be avoided when the incident dosage was higher than D 0 =4 J/cm 2 at all laser pulse durations used in the experiments even with a 100- millisecond cryogen spurt. However, clinical studies have demonstrated that the incident dosage of D 0 =8-9 J/cm 2 are required to cause irreversible photocoagulation of PWS blood vessels in patients with skin types V-VI 18,28,29. Therefore, laser treatment of patients with heavy pigmentation remains a challenge. Further studies on optimizing the CSC parameters, such as droplet velocity, mass flux, and cryogen spurt duration, are needed to maximize heat removal from the epidermis in skin types V-VI. For example, enhanced heat transfer between the cryogen spray and skin surface is expected by increasing the droplet velocity 30 to enhance turbulence within the cryogen film. Proc. of SPIE Vol

6 It was found from this study that for a given incident dosage, longer pulse durations led to reduced thermal injury to the epidermis. This result can be explained on the basis that longer pulses allow more heat to diffuse away from the epidermal basal layer during the laser irradiation, and subsequently, reduce the peak temperature within the basal layer. Ideal treatment of PWS requires selective photocoagulation of targeted blood vessels with minimal thermal injury to the overlying epidermis. Therefore, in contrast to the requirement of heat confinement in targeted blood vessels for photocoagulation, heat generated within the epidermis needs to be released from the basal layer of the epidermis during the laser irradiation in order to protect the epidermis from nonspecific thermal injury. Accordingly, it will be beneficial if the laser pulse duration is longer than the thermal relaxation time of the epidermal basal layer, minimizing the peak temperature of the epidermal basal layer. As the thickness of the basal layer of epidermis is much less than the laser spot diameter, the thermal relaxation time of the epidermal basal layer (τ r, b ) can be estimated as 31,32 : 2 δ τ r,b = (1) 4[ k /( ρc)] where δ is the thickness of the epidermal basal layer (typically, δ 20µm 33 ), k is the thermal conductivity of epidermis ( W/m/ o C 34 ), ρ is density (1,110-1,190 kg/m 3 34 ), and C is specific heat (3,530-3,710 J/kg/ o C 34 ). Based on equation (1), τ r,b is in the range of milliseconds. Therefore, the pulse durations used in this study are in the appropriate range in terms of epidermal protection. It was the goal of the present study to help identify the threshold incident dosages for irreversible thermal injury to human skin epidermis, defined as the dosage values above which epidermal damage with a score 2 would be induced. It was considered that an epidermal damage score of 2 possibly could lead to pigmentation change, and an epidermal damage score higher than 2 would induce cell necrosis 7. Based on the analyses of the experimental results, we obtained the threshold incident dosages for various ex-vivo skin types (Table 3). Table 3: Threshold incident dosages (J/cm 2 ) above which thermal injury to the epidermis occurred in various ex-vivo human skin types. Values are provided without and with a 100-milliseond cryogen spurt. τ laser τ CSC Threshold incident dosages (J/cm 2 ) (millisecond) (millisecond) Skin types I-II Skin types III-IV Skin types V-VI < < < < < In skin types I-II, when CSC was applied, no demonstrable thermal injury to the epidermis was observed at Φ(20, 1.5, 100), indicating that the threshold incident dosage in these skin types is very possibly higher than D 0 =20 J/cm 2 when τ laser =1.5 milliseconds and τ CSC =100 milliseconds. Moreover, when the pulse duration is increased, even higher threshold dosage values would be expected. Due to the setting limitation of the incident dosage (maximum D 0 =20 J/cm 2 ) of the laser system used, the exact value could not be identified. This study only used a fixed cryogen spurt duration (τ CSC =100 milliseconds) and a fixed time delay between cryogen spurt termination and onset of the laser pulse (τ delay= 30 milliseconds). Longer spurt durations are expected to allow higher threshold incident dosages than those listed in Table 3 7,35. A previous ex-vivo study 8 using 585-nm wavelength irradiation at pulse duration τ laser =1.5 milliseconds demonstrated that a cryogen spray duration of τ CSC =200 milliseconds could protect the epidermis in skin types II-III from thermal injury at incident dosage as high as D 0 =30 J/cm 2. An in-vivo study 7 also using 585-nm wavelength irradiation at τ laser =1.5 milliseconds showed that no thermal injury to the epidermis was observed in skin types I-IV at D 0 =30 J/cm 2 when τ CSC was 250 milliseconds. Since light absorption by melanin at 585 nm is almost identical to that at 595 nm, similar results would be expected at 595-nm irradiation. However, it has been reported that prolonged spurt duration may induce some epidermal injury due to the tissue freezing 7, and cool the 6 Proc. of SPIE Vol. 4949

7 targeted PWS blood vessels, which would subsequently counteract the photothermal effect of higher incident dosages, decreasing the efficacy of spatial selectivity of cooling. A mathematical modeling study 36 indicated that the optimal cryogen spurt durations are directly related to the irradiation parameters, skin type and location of the targeted blood vessels. Optimum cryogen spurt durations were calculated for different PWS categories (different skin types, different vessel sizes and depths) at incident dosages D 0 =5-10 J/cm 2 and pulse duration τ laser =0.45 milliseconds 36. For example, when incident dosage was 8 J/cm 2, optimal cryogen spurt durations were predicted to be milliseconds for moderate pigmented skin types (types III-IV) with large-sized and deeply located blood vessels, whereas for heavily pigmented skin types (types V-VI) with large-sized and deeply located blood vessels, spurt durations of milliseconds were recommended. In addition, calculated results showed that for lightly pigmented skin types (types I-II), no cooling was need when D 0 10 J/cm 2 ; and for heavily pigmented skin types, when D 0 9 J/cm 2, spatial selectivity of cooling could not be achieved as the spurt durations were so long that the targeted blood vessels were also cooled. Although effects of τ delay on the temperature profiles are not as great as τ CSC, it may also influence the cooling selectivity within human skin, especially when the epidermal basal layer is relatively deep 37. It was suggested that optimal τ delay could be determined by maximizing the temperature difference between the epidermis basal layer and targeted blood vessels 37. Based on this criterion, mathematical modeling predicted that for the commonly used τ CSC =100 milliseconds, optimal τ delay were 5-10 milliseconds and milliseconds for shallow (depth of 60 µm) and deep (depth of 120 µm) epidermal basal layer, respectively 37. We recognize the differences between ex-vivo and in-vivo human skin. Due to the presence of dermal chromophores and higher skin temperature, in-vivo skin would sustain thermal injury at lower incident dosage level than ex-vivo skin 8,38,38. Nevertheless, ex-vivo skin serves as a reasonable model since the structural components are maintained, and light distribution is expected to remain similar to those of the in-vivo skin except for a slight increase in back scattering due to lack of blood vessels and dehydration in ex-vivo skin 38. In summary, the treatment of PWS lesions involves the issues from both the photocoagulation of the targeted blood vessels and the protection of skin epidermis. Optimal set of CSC in conjunction with laser irradiation parameters is required to achieve ideal therapeutic outcome and advance the technology to treat patients of all skin types. 5. CONCULSIONS For a given incident dosage, longer pulse durations help reduce nonspecific thermal injury to the epidermis. When a 100- millisecond cryogen spurt was applied, thermal injury to the epidermis could be prevented in lightly (types I-II) and moderately (types III-IV) pigmented ex-vivo skin when irradiated at higher incident dosages (15-20 J/cm 2 ) than those currently used in clinical settings. Epidermal protection in heavily pigmented skin (skin types V-VI) remains a challenge. Further studies on optimizing the CSC parameters in conjunction with the laser irradiation parameters are needed to protect heavily pigmented skin types from thermal injury to the epidermis. ACKNOWLEDGEMENTS This study was supported in part by the Institute of Arthritis and Musculoskeletal and Skin Disease (IR01-AR47996) at the National Institutes of Health, Texas Higher Education Coordinating Board, and Candela Corporation. We thank Mrs. Carol Johnston from MDACC for preparing the histology slides. REFERENCES 1. Y. Namba, O. Mae, M. Ao, The treatment of port wine stains with a dye laser: a study of 644 patients, Scand. J. Plast. Reconstr. Hand. Surg., 35, , H. Wang, J. Wang, H. Jin, S. Wen, G. Jiang, Flashlamp-pumped pulsed dye laser in treatment of port wine stains, Chin. Med. Sci. J., 16, 56-58, Proc. of SPIE Vol

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