Immunohistochemical evaluation of the heat shock response to nonablative fractional resurfacing.

Despite the emergence of nonablative fractional resurfacing (NFR) as a new therapeutic modality for skin photoaging, little is known about the molecular events that underlie the heat shock response to different treatment parameters. Human subjects are treated with a scanned 1550-nm fractional laser at pulse energies spanning 6 to 40 mJ and a 140-µm spot size. The heat shock response is assessed immunohistochemically immediately through 7 days posttreatment. At the immediately posttreatment time point, we observe subepidermal clefting in most sections. The basal epidermis and dermal zones of sparing are both found to express HSP47, but not HSP72. By day 1, expression of HSP72 is detected throughout the epidermis, while that of HSP47 remains restricted to the basal layer. Both proteins are detected surrounding the dermal portion of the microscopic treatment zone (MTZ). This pattern of expression persists through day 7 post-NFR, although neither protein is found within the MTZ. Immediately posttreatment, the mean collagen denaturation zone width is 50 µm at 6 mJ, increasing to 202 µm at 40 mJ. The zone of cell death exceeds the denaturation zone by 19 to 55% over this pulse energy range. The two zones converge by day 7 posttreatment.

In vivo confocal imaging of epidermal cell migration and dermal changes post nonablative fractional resurfacing: study of the wound healing process with corroborated histopathologic evidence.

In vivo wound healing response post nonablative fractional laser treatment is evaluated. Seven healthy subjects receive treatments with a Fraxel re:store laser system on the forearm with pulse energies ranging from 10 to 70 mJ. The treatment sites are imaged at 1-h increments up to 40 h using confocal microscope z-stacks using 10-mum-depth spacing. At least five individual microscopic treatment zones are imaged per subject, time point, and treatment energy. Images are analyzed for tissue structure and morphology to classify each lesion as healed or not healed, depending on epidermal re-epithelialization at each time point and treatment energy. Probit analysis is used to statistically determine the ED(50) and ED(84) probabilities for a positive dose response (healed lesion) as a function of treatment energy. Confocal observations reveal epidermal keratinocyte migration patterns confirmed with histological analysis using hematoxylin and eosin (HE) and lactate dehydrogenase (LDH) staining at 10 mJ at 0, 7, 16, and 24-h post-treatment. Results indicate that more time is required to conclude re-epithelialization with larger lesion sizes (all less than 500 mum) corresponding to higher treatment energies. For the entire pulse energy range tested, epidermal re-epithelialization concludes between 10 to 22-h post-treatment for ED(50) and 13 to 28 h for ED(84).

In vivo histological evaluation of a novel ablative fractional resurfacing device.

BACKGROUND AND OBJECTIVES: A novel carbon dioxide (CO(2)) laser device employing ablative fractional resurfacing was tested on human skin in vivo for the first time. STUDY DESIGN/MATERIALS AND METHODS: An investigational 30 W, 10.6 microm CO(2) laser system was focused to a 1/e(2) spot size of 120 microm to generate an array of microscopic treatment zones (MTZ) in human forearm skin. A range of pulse energies between 5 and 40 mJ was tested and lesion dimensions were assessed histologically using hematoxylin and eosin. Wound healing of the MTZ's was assessed immediately-, 2-day, 7-day, 1-month, and 3-month post treatment. The role of heat shock proteins was examined by immunohistochemistry. RESULTS: The investigational CO(2) laser system created a microscopic pattern of ablative and thermal injury in human skin. The epidermis and part of the dermis demonstrated columns of thermal coagulation that surrounded tapering ablative zones lined by a thin eschar layer. Changing the pulse energy from 5 to 30 mJ resulted in a greater than threefold increase in lesion depth and twofold increase in width. Expression of heat shock protein (hsp)72 was detected as early as 2 days post-treatment and diminished significantly by 3 months. In contrast, increased expression of hsp47 was first detected at 7 days and persisted at 3 months post-treatment. CONCLUSION: The thermal effects of a novel investigational ablative CO(2) laser system utilizing fractional resurfacing were characterized in human forearm skin. We confirmed our previous ex vivo findings and show for the first time in-vivo, that a controlled array of microscopic treatment zones of ablation and coagulation could be deposited in human skin by varying treatment pulse energy. Immunohistochemical studies of heat shock proteins revealed a persistent collagen remodeling response lasting at least 3 months. We successfully demonstrated the first in-vivo use of ablative fractional resurfacing (AFR) treatment on human skin.

The effects of pulse energy variations on the dimensions of microscopic thermal treatment zones in nonablative fractional resurfacing.

BACKGROUND AND OBJECTIVES: We examined the effects of pulse energy variations on the dimensions of microscopic thermal injury zones (MTZs) created on human skin ex vivo and in vivo using nonablative fractional resurfacing. MATERIALS AND METHODS: A Fraxel SR laser system emitting at 1,550 nm provided an array of microscopic spots at variable densities. Pulse energies ranging from 4.5 to 40 mJ were tested on human abdominal skin ex vivo and in vivo. Tissue sections were stained with hematoxylin and eosin (H&E) or nitro blue tetrazolium chloride (NBTC) and MTZ dimensions were determined. Ex vivo and in vivo results were compared. Dosimetry analyses were made for the surface treatment coverage calculation as a function of pulse energy and collagen coagulation based on H&E stain or cell necrotic zone based on NBTC stain. RESULTS: Each MTZ was identified by histological detection of a distinct region of loss of tissue birefringence and hyalinization, representing collagen denaturation and cell necrosis within the irradiated field immediately, 1, 3, and 7 days after treatment. At high pulse energies, the MTZ depth could exceed 1 mm and width approached 200 microm as assessed by H&E. NBTC staining revealed viable interlesional tissue. In general, no statistically significant difference was found between in vivo and ex vivo depth and width measurements. CONCLUSIONS: The Fraxel SR laser system delivers pulses across a wide range of density and energy levels. We determined that increases in pulse energy led to increases in MTZ depth and width without compromising the structure or viability of interlesional tissue.

Ex vivo histological characterization of a novel ablative fractional resurfacing device.

BACKGROUND AND OBJECTIVES: We introduce a novel CO(2) laser device that utilizes ablative fractional resurfacing for deep dermal tissue removal and characterize the resultant thermal effects in skin. STUDY DESIGN/MATERIALS AND METHODS: A prototype 30 W, 10.6 microm CO(2) laser was focused to a 1/e(2) spot size of 120 microm and pulse duration up to 0.7 milliseconds to achieve a microarray pattern in ex vivo human skin. Lesion depth and width were assessed histologically using either hematoxylin & eosin (H&E) or lactate dehydrogenase (LDH) stain. Pulse energies were varied to determine their effect on lesion dimensions. RESULTS: Microarrays of ablative and thermal injury were created in fresh ex vivo human skin irradiated with the prototype CO(2) laser device. Zones of tissue ablation were surrounded by areas of tissue coagulation spanning the epidermis and part of the dermis. A thin condensed lining on the interior wall of the lesion cavity was observed consistent with eschar formation. At 23.3 mJ, the lesion width was approximately 350 microm and depth 1 mm. In this configuration, the cavities were spaced approximately 500 microm apart and interlesional epidermis and dermis demonstrated viable tissue by LDH staining. CONCLUSION: A novel prototype ablative CO(2) laser device operating in a fractional mode was developed and its resultant thermal effects in human abdominal tissue were characterized. We discovered that controlled microarray patterns could be deposited in skin with variable depths of dermal tissue ablation depending on the treatment pulse energy. This is the first report to characterize the successful use of ablative fractional resurfacing as a potential approach to dermatological treatment.

Laser-induced transepidermal elimination of dermal content by fractional photothermolysis.

The wound healing process in skin is studied in human subjects treated with fractional photothermolysis. In-vivo histological evaluation of vacuoles formed over microthermal zones (MTZs) and their content is undertaken. A 30-W, 1550-nm single-mode fiber laser system delivers an array of 60 microm or 140 microm 1e2 incidence microbeam spot size at variable pulse energy and density. Treatments span from 6 to 20 mJ with skin excisions performed 1-day post-treatment. Staining with hematoxylin and eosin demonstrates an intact stratum corneum with vacuolar formation within the epidermis. The re-epithelialization process with repopulation of melanocytes and keratinocytes at the basal layer is apparent by 1-day post-treatment. The dermal-epidermal (DE) junction is weakened and separated just above zones of dermal coagulation. Complete loss of dermal cell viability is noted within the confines of the MTZs 1-day post-treatment, as assessed by lactate dehydrogenase. All cells falling outside the irradiation field remain viable. Content within the epidermal vacuoles stain positively with Gomori trichrome, suggesting a dermal origin. However, the positive staining could be due to loss of specificity after thermal alteration. Nevertheless, this dermal extrusion hypothesis is supported by very specific positive staining with an antihuman elastin antibody. Fractional photothermolysis creates microthermal lesions that allow transport and extrusion of dermal content through a compromised DE junction. Some dermal material is incorporated into the microepidermal necrotic debris and shuttled up the epidermis to eventually be exfoliated through the stratum corneum. This is the first report of a nonablative laser-induced transport mechanism by which dermal content can be predictably extruded biologically through the epidermis. Thus, treatment with the 1550-nm fiber laser may provide the first therapeutic option for clinical indications, including pigmentary disorders such as medically recalcitrant melasma, solar elastosis, as well as depositional diseases such as mucinosis and amyloidosis.