James Nelson

Many commercially available dermatologic lasers utilize cryogen spray cooling for epidermal protection. A previous tissue culture study demonstrated that single cryogen spurts of 80 ms or less were unlikely to cause cryo-injury in light-skinned individuals. More recently, multiple cryogen spurts have been incorporated into commercial devices, but the effects of MCS have not been evaluated. The aim was to study an in vitro tissue culture model and the epidermal and dermal effects of SCS vs patterns of shorter MCS with the same preset total cryogen delivery time and provide an explanation for noted differences. Four different spurt patterns were evaluated: SCS: one 40-ms cryogen spurt; MCS2: two 20-ms cryogen spurts; MCS4: four 10-ms cryogen spurts; MCS8: eight 5-ms cryogen spurts. Actual Δt c and total cooling time were measured for each spurt pattern. RAFT tissue culture specimens were exposed to cryogen spurt patterns and biopsies were taken immediately and at days 3 and 7. Actual Δt c was increased while Δt Total remained relatively constant as the preset Δt c of 40 ms was delivered as shorter MCS. Progressively more epidermal damage was noted with exposure to the MCS patterns. No dermal injury was noted with either SCS or MCS. For a constant preset Δt c of 40 ms, delivering cryogen in patterns of shorter MCS increased the actual Δt c and consequently the observed epidermal cryo-injury as compared to an SCS. © Springer-Verlag London Limited 2005.

Background and Objectives: Previous in vitro studies demonstrated the potential utility of benzoporphyrin derivative monoacid ring A photodynamic therapy for vascular destruction. Moreover, the effects of PDT were enhanced when this intervention was followed immediately by pulsed dye laser irradiation. We further evaluate vascular effects of PDT alone, PDL alone and PDT/PDL in an in vivo rodent dorsal skinfold model. Study Design/Materials and Methods: A dorsal skin-fold window chamber was installed surgically on female Sprague-Dawley rats. One milligram per kilogram of BPD solution was administered intravenously via a jugular venous catheter. Evaluated interventions were: control, PDT alone, PDL alone at 7 J/cm2, PDL alone at 10 J/cm2, PDT/PDL, and PDT/PDL. To assess changes in microvascular blood flow, laser speckle imaging was performed before, immediately after, and 18 hours post-intervention. Results: Epidermal irradiation was accomplished without blistering, scabbing or ulceration. A reduction in perfusion was achieved in all intervention groups. PDT/PDL at 7 J/ cm2 resulted in the greatest reduction in vascular perfusion. Conclusions: BPD PDT can achieve safe and selective vascular flow reduction. PDT/PDL can enhance diminution of microvascular blood flow. Our results suggest that PDT and PDT/PDL should be evaluated as alternative therapeutic options for treatment of hypervascular skin lesions including port wine stain birthmarks. © 2006 Wiley-Liss, Inc.

Port-wine stain birthmarks are congenital, low-flow vascular malformations of the skin. Lasers are the modality of choice for the treatment of PWS birthmarks, and for most patients the pulsed-dye laser in conjunction with epidermal cooling offers the greatest efficacy and safety. Other light devices, including the 532-nm frequency-doubled Nd:YAG laser, intense pulsed light, 1064-nm Nd:YAG laser, and combined 1064/532-nm system, may be useful during a treatment course for resistant PWS. Laser treatment results in blanching of most lesions, although complete resolution may not occur and some resistant PWS birthmarks respond minimally, if at all. Factors limiting laser treatment include variable vascular geometry, inadequate damage of some vessels, and lesional posttreatment recurrence as a result of neovascularization. Alternative or adjunct treatment options that address these limitations should be explored, including noninvasive real-time imaging to optimize the selection of treatment settings, photodynamic therapy, and perioperative use of antiangiogenic compounds.

Background and Objectives: The degree of port wine stain blanching following pulsed dye laser therapy remains variable and unpredictable. Because of the limitations of current PDL therapy, alternative treatment approaches should be explored. The objective was to evaluate a novel methodology for selective vascular damage, combined photodynamic and photothermal treatment, using the in vivo chick chorioallantoic membrane model. Study Design/Materials and Methods: Thirty microliters of benzoporphyrin derivative monoacid ring A solution was administered intraperitoneally into chick embryos at day 12 of development. Study groups were: control ; BPD alone; continuous wave irradiation alone ; CW+PDL; BPD+PDL; PDT ; PDL alone ; and PDT+PDL. Vessels were videotaped prior to, and at 1 hour post-intervention and then assessed for damage based on the following scale: 0, no damage; 1, coagulation; 1.5, vasoconstriction; 2.0, coagulation+vasoconstriction; 2.5, angiostasis; 3.0, hemorrhage. Damage scores were weighted by vessel "order." Results: PDT+PDL resulted in significantly more severe vascular damage than was observed in any other study group: 127% more than PDT, 47% more than PDL alone. Conclusions: PDT+PDL is a novel and promising approach for selective vascular damage and may offer a more effective method for treatment of PWS and other vascular skin lesions. © 2004 Wiley-Liss, Inc.

BACKGROUND AND OBJECTIVE. Vascular ectasia in port wine stain birthmarks might result from reduced innervation with loss of autonomic stimulation. We investigated this theory and evaluated nerve and blood vessel density, and mean blood vessel size in untreated and treated PWS skin. METHODS. Skin biopsy specimens were obtained from uninvolved skin, untreated PWS, PWS with a good response to laser treatment and PWS with a poor response to laser treatment. Confocal microscopy was performed to determine nerve and blood vessel density, and mean blood vessel size. RESULTS. Nerve density was significantly decreased in all PWS sites compared to uninvolved skin. Mean blood vessel diameter was larger in untreated compared to treated PWS. PWS with a good response to treatment had decreased nerve density but blood vessel density and mean diameter was relatively normal. PWS with a poor response to treatment had decreased nerve density but increased blood vessel density and mean blood vessel diameter compared to normal skin. CONCLUSION. Nerve density was decreased in all evaluated PWS sites and this may be a factor in lesion pathogenesis. PWS blood vessel size correlated with pulsed dye laser response and may prove to be a useful prognostic indicator of therapeutic outcome.

Background and Objectives: Cryogen spray cooling is commonly used during dermatologic laser surgery. The epidermal and dermal effects of CSC have not been adequately evaluated. To study the potential for epidermal and dermal injury after CSC using an in vitro model of human skin. Study Design/Materials and Methods: RAFT specimens were exposed to continuous CSC spurt durations of 10, 20, 40, 80, 100, 200, or 500 milliseconds. Biopsies were taken acutely, 3 and 7 days post-CSC exposure. Sections were stained with hematoxylin and eosin for evaluation of possible injury, Ki-67 to determine keratinocyte viability, and Melan-A, to identify and evaluate melanocytes. Results: Minimal, transient epidermal changes were noted in specimens exposed to continuous CSC spurts of 80 milliseconds or less. Keratinocytes and melanocytes remained viable. Continuous CSC spurts of 100, 200, or 500 milliseconds resulted in significant epidermal injury acutely, with partial or full thickness epidermal necrosis at 7 days. Only the 500 milllisecond specimen demonstrated dermal change, decreased fibroblast proliferation at 3 days. Conclusions: Continuous CSC spurts of 80 milliseconds or less induce minimal, if any, epidermal or dermal damage and are unlikely to produce cryo-injury when used during dermatologic laser surgery. © 2004 Wiley-Liss, Inc.

BACKGROUND. The majority of port-wine stain patients treated with the pulsed dye laser do not achieve complete blanching. Safe administration of higher fluences has been proposed as a means of improving treatment efficacy. OBJECTIVE. To determine the safety and efficacy of PWS treatment with the 1.5-msec PDL at high fluences in conjunction with cryogen spray cooling. METHODS. Twenty PWS patients were treated with the PDL in combination with cryogen spray cooling utilizing a 7 or 10 mm spot size and fluences ranging from 6 to 15 J/cm2. Before and after treatment photographs were compared on a blinded basis. RESULTS. No scarring or skin textural changes occurred. Blanching scores were as follows: 20% of patients achieved 75% or greater blanching after an average of 3.3 treatments, 30% achieved 50-74% blanching, 20% achieved 25-49% blanching, and 30% achieved less than 25% blanching. CONCLUSION. In conjunction with cryogen spray cooling, the PDL can be safely used at high fluences. At this time it is not clear that the use of higher fluences improves treatment efficacy; however, as other aspects of PWS laser treatment are optimized, safe administration of higher fluences is likely to be advantageous.

The turbidity of most biologic tissues hinders the use of lasers for diagnostic and therapeutic purposes. Hyperosmotic agents such as glycerol have been used to alter the optical scattering properties of turbid tissues. The mechanism of this effect, "optical clearing", however, remains incompletely understood. Multiphoton microscopy utilizing second harmonic generation can be used to monitor collagen structural changes in the presence of glycerol. This study suggests that the use of glycerol for tissue "optical clearing" screens noncovalent intermolecular interactions between low-order collagen structures, resulting in fiber disassembly. Dissociation of collagen fibers in native and engineered tissues in the presence of glycerol and reassociation with the application of saline are imaged dynamically. Collagen fiber reassembly is governed by the displacement of glycerol by saline in native and engineered skin. These results reveal the assembly process of high-order collagen structures and propose a molecular mechanism for the increase in tissue transparency observed after glycerol application.

Aneuploidy with chromosome instability is a cancer hallmark. We studied chromosome 7 copy number variation in gliomas and in primary cultures derived from them. We found tumor heterogeneity with cells having Chr7-CNV commonly occurs in gliomas, with a higher percentage of cells in high-grade gliomas carrying more than 2 copies of Chr7, as compared to low-grade gliomas. Interestingly, all Chr7-aneuploid cell types in the parental culture of established glioma cell lines reappeared in single-cell-derived subcultures. We then characterized the biology of three syngeneic glioma cultures dominated by different Chr7-aneuploid cell types. We found phenotypic divergence for cells following Chr7 missegregation, which benefited overall tumor growth in vitro and in vivo. Mathematical modeling suggested the involvement of chromosome instability and interactions among cell subpopulations in restoring the optimal equilibrium of tumor cell types. Both our experimental data and mathematical modeling demonstrated that the complexity of tumor heterogeneity could be enhanced by the existence of chromosomes with structural abnormality, in addition to their mis-segregations. Overall, our findings show, for the first time, the involvement of chromosome instability in maintaining tumor heterogeneity, which underlies the enhanced growth, persistence and treatment resistance of cancers. © 2013 Hu et al.

Background and Objectives: The objective of this study was to compare the efficacy and safety of cryogen spray cooled laser treatment at wavelengths of 585 nm vs. 595 nm for port wine stain birthmarks in a large series of patients. Study Design/Materials and Methods: A retrospective review was conducted of 64 patients with PWS treated with the ScleroPLUS® [Candela ] pulsed dye laser over a 3-year period. Subjects' ages ranged between 3 months and 64 years; there were 42 females and 22 males, all of whom were Asian. Number of treatments ranged from 1 to 6. Duration of treatment ranged from 6 months to 2 years 11 months, with a mean of 12 months. Patients received CSC-LT using radiant exposures of 7-10 J/cm2. A second group of patients received CSC-LT using radiant exposures of 7-10 J/cm2. The primary efficacy measurement was the quantitative assessment of blanching response scores for CSC-LT versus CSC-LT. Patients were monitored for adverse effects. Results: Based on chi-squared analysis, there were clinical, and statistically significant, differences in blanching response scores favoring PWS receiving CSC-LT as compared to CSC-LT. Transient hyperpigmentation was noted in 43.7% and 37.5% of patients in the CSC-LT and CSC-LT groups, respectively. In both groups, transient hyperpigmentation resolved in all patients within 1 year. Permanent hypopigmentation or scarring was not observed in either group. Conclusions: CSC-LT resulted in superior blanching as compared to CSC-LT. Further study is required to optimize wavelength selection on an individual patient basis during PWS therapy in order to improve treatment results. © 2002 Wiley-Liss, Inc.

Previous studies suggest that optimal port wine stain laser treatment parameters require knowledge of skin characteristics such as blood vessel size, depth, and distribution. Effective and rapid imaging modalities are not widely available. In the present study, photothermal tomography images of an in vivo hamster window model and human PWS skin were obtained and analyzed. Subtherapeutic laser light pulses at 585 and 600 nm were applied to skin surface and image sequences acquired with an infrared camera. A nonnegatively constrained conjugate gradient algorithm was used to reconstruct a PTT image of the initial temperature distribution immediately following pulsed laser irradiation. Vessel dimensions determined from PTT images of hamster window model skin compared well with those measured directly using video microscopy. PTT images of human PWS skin contained vessels with estimated diameters of 200-250 μm over a 250-320 μm depth range. Use of dual wavelength excitation analysis allowed for imaging of shallow vessels.

BACKGROUND: Despite widespread clinical use of cryogen spray cooling in conjunction with laser dermatologic surgery, in vivo cutaneous effects have not been systematically evaluated. OBJECTIVE: The authors characterize the in vivo cutaneous effects for Fitzpatrick skin types I through VI after CSC exposures of varying spurt durations and spurt delivery patterns. METHODS AND MATERIALS: Twenty-seven normal human subjects were exposed to single cryogen spurts from 10 to 80 milliseconds, and multiple spurt patterns consisting of two 20-millisecond spurts, four 10-millisecond spurts, and eight 5-millisecond spurts. Subjects were evaluated by clinical observation and photography at 1 hour, 1 day, and 1, 4, 8, and 12 weeks after CSC exposure. RESULTS: Acute erythema and urticaria were noted in 14 of 27 and 3 of 27 subjects, respectively. Transient hyperpigmentation occurred in 4 of 27 subjects but resolved spontaneously without medical intervention in all subjects by 8 weeks. No permanent skin changes were noted in any subjects. Skin reactions were more common with longer single-spurt durations and multiple spurt patterns. CONCLUSION: Acute erythema, urticaria, and, less commonly, transient hyperpigmentation were observed after CSC exposure. Permanent skin injury was not observed and is unlikely. © 2006 by the American Society for Dermatologic Surgery, Inc. Published by Blackwell Publishing.

Background and Objective: Although cryogen spray cooling is used to minimize the risk of epidermal damage during laser dermatologic surgery, concern has been expressed that CSC may induce cryo-injury. The objective of this study is to measure temperature variations at the epidermal-dermal junction in ex vivo human skin during three clinically relevant multiple cryogen spurt-laser pulse sequences. Study Design/Materials and Methods: The epidermis of ex vivo human skin was separated from the dermis and a thin-foil thermocouple was inserted between the two layers. Thermocouple depth and epidermal thickness were measured using optical coherence tomography. Skin specimens were preheated to 30°C before the MCS-LPS were initiated. Three MCS-LPS patterns, with total cryogen spray times of 38, 30, and 25 milliseconds respectively, were applied to the specimens in combination with laser fluences of 10 and 14 J/cm2, while the thermocouple recorded the temperature changes at the epidermal-dermal junction. Results: The thermocouple effectively recorded fast temperature changes during three MCS-LPS patterns. The lowest temperatures measured corresponded to the sequences with longer pre-cooling cryogen spurts. No subzero temperatures were measured for any of the MCS-LPS patterns under study. Conclusions: The three clinically relevant MCS-LPS patterns evaluated in this study do not cause sub-zero temperatures in ex vivo human skin at the epidermal-dermal junction and, therefore, are unlikely to cause significant cryogen induced epidermal injury. © 2006 Wiley-Liss, Inc.

Background: Although cryogen spray cooling is used to minimize the risk of epidermal damage during laser dermatologic surgery, concern has been expressed that CSC may induce cryo-injury. In order to address this concern, it is necessary to evaluate the effects of prolonged exposure of human skin phantoms to CSC. Objective: To measure the minimum surface temperature and the time at which it occurs as well as determine the time the sprayed HSP surface remains below 0°C and -26°C during the application of single and multiple cryogen spurts. Two initial HSP substrate temperatures were studied, Ti: 23 and 70°C. Study Design/Materials and Methods: An epoxy-based HSP was constructed to measure Tmin, tTmin, Δts, and Δtr, for 17 spray patterns: 1 SCS with a total cryo-delivery time of 40 milliseconds; 8 MCS patterns with identical Δtc, but with a total cooling time varying from 50 to 280 milliseconds; and 8 SCS patterns that matched the Δttotal of the MCS patterns. Results: For both Ti, our results show that it is possible to distinguish between two different cooling regimes. For Δttotal ≤ 110 milliseconds, the differences between SCS and MCS patterns with the same Δttotal for all variables are negligible. Most importantly, all these variables show a remarkable linear dependence with Δttotal. In the interval 110 milliseconds < Δttotal < 280 milliseconds, T min and tTmin are similar for SCS and MCS, while Δts and Δtr show more pronounced differences between the two spray patterns. In this interval, the values of Tmin and Δts for MCS remain invariant and similar to the corresponding values for Δttotal = 110 milliseconds. Conclusions: These results suggest that: similar epidermal protection may be attained with SCS and MCS for Δttotal ≤ 110 milliseconds; and for 110 milliseconds < Δttotal ≤ 280 milliseconds, MCS help to maintain Δts similar to that of SCS at Δttotal = 100 milliseconds, which may be beneficial to prevent cryo-injury. © 2005 Wiley-Liss, Inc.

Background and Objectives: Quantitative methods to assess port wine stain skin response to laser therapy are needed to improve therapeutic outcome. In this study, PWS skin erythema was analyzed using erythema index difference images before and after treatment to investigate systematically subject-dependent response to laser therapy. Study Design/Materials and Methods: Cross-polarized digital skin color images were acquired from 17 subjects with facial PWS and the associated ΔEI images were computed. Qualitative and quantitative analyses of PWS skin erythema were performed with ΔEI images, in which ranges of 40-6 and 5-0 represented PWS and normal skin, respectively. Results: After laser therapy, we qualitatively observed α reduction in the ΔEI values for all subjects. Regression fitting of ΔEI values before and after PWS laser therapy was associated with strong positive linear correlation. Conclusions: The imaging modality and analysis method allowed systematic analysis of PWS skin erythema in response to laser therapy. PWS skin response was dependent on pretreatment ΔEI values, suggesting that erythema can be utilized as an effective parameter to monitor PWS response to laser therapy. © 2005 Wiley-Liss, Inc.

Background and Objectives: Objective methods to assess quantitatively port wine stain blanching in response to laser therapy are needed to improve laser therapeutic outcome. Previous studies have attempted to assess objectively PWS color based on point measurement devices. To date, these approaches have typically been limited by a number of factors such as small test area and need for contact. To address these issues, a cross-polarized diffuse reflectance imaging system and color image analysis method has been developed to evaluate quantitatively erythema and melanin content in PWS skin. Study Design/Materials and Methods: A cross-polarized diffuse reflectance system has been constructed to acquire high resolution digital images while minimizing artifacts such as glare, shadowing, and nonuniform illumination effects that can compromise image fidelity. Furthermore, an image analysis algorithm has also been developed to analyze normal and PWS skin in terms of CIEL*a*b* color space parameters. Using the algorithm, images of color space intensity, L*, and saturation, a*, indices have been calculated to extract quantitative metrics of melanin and erythema, respectively. Results: Compared to the cross-polarized diffuse reflectance image, the a* index image enhanced the contrast in regions of high and low erythema and seems to have quasi correlation with the L* index image. In the presented PWS patient example, the relative a* index difference image showed less erythema in PWS skin after laser treatment when compared to pre-treatment values. Conclusions: Our cross-polarized imaging system and color image analysis method is a simple, noncontact technique that can indirectly provide quantitative measurement of erythema and melanin content in PWS skin. © 2004 Wiley-Liss, Inc.

Cryogen spray cooling is used to minimize the risk of epidermal damage during various laser dermatologic surgeries. However, as the application of single or multiple cryogen spurts becomes available on some commercial lasers devices, it is necessary to determine the optimal CSC parameters for different laser surgeries. The objective of this study was to measure the time the sprayed surface of a human skin phantom remains below water freezing temperature 0°C, referred to as subzero time, and below the cryogen boiling temperature -26°C, referred to as residence time, as well as the minimum surface temperature and the time at which Tmin occurs for two HSP-initial temperatures during and after the application of single and multiple cryogen spurts. For this propose, a HSP was used to measure ts, tr, Tmin, and tTminn for nine sequences: one SCS of ΔtT = TCT = 40 ms; four MCS sequences, all adding to a ΔtT of 40 ms but with different TCT up to 110 ms and, finally; four SCS that matched the TCT of the four MCS sequences, but lead to different ΔtT. Our results show that the differences between SCS and MCS sequences with the same TCT are negligible for all variables measured. Moreover, in this interval, this variables show a remarkable linear dependence with the TCT. Copyright © 2004 by ASME.