Mechanisms of Microvascular Response to Laser Pulses

“Selective photothermolysis” is widely used for treating vascular lesions. In order to understand mechanisms of response, we investigated fast events during pulsed laser treatment of microvessels. A high-speed (2000 fps) CCD camera and microscope were used to image hamster cheek pouch microvessels during and after 532 nm and 1064 nm laser pulse exposures. Pulse duration and fluence were varied systematically (1–50 ms, 0–600 J per cm2). Threshold fluences for fast events were determined. On a millisecond time-scale, a specific series of fast events occur, which are wavelength, fluence, irradiance, and pulse duration dependent. In order of increasing fluence we observed: blood coagulation, vasoconstriction, thread-like appearance of the treated vascular segment, vessel disappearance, intravascular cavitation, bubble formation, vessel wall rupture and hemorrhage, and shrinkage of perivascular tissue. With increasing pulse duration, the threshold fluences for coagulation, vessel disappearance, and cavitation increase, and cavitation becomes less violent, conforming to the vessel lumen. Intravascular cavitation did not always rupture the vessel wall, and is not the mechanism for immediate vessel disappearance, a desired endpoint for treating vascular lesions. The apparent mechanism for immediate vessel disappearance is contraction of intravascular blood and perivascular collagen after thermal denaturation. This study suggests that detecting fast events in humans, in real time, may provide useful feedback signals for “smarter” laser devices

Thermal Injury Causes DNA Damage and Lethality in Unheated Surrounding Cells: Active Thermal Bystander Effect

Direct heat exposure to cells causes protein degradation and DNA damage, which can lead to genetic alteration and cell death, but little is known about heat-induced effects on the surrounding tissue. After burns or laser surgery, loss of viability in the surrounding tissue has been explained by a temperature gradient due to heat diffusion. This study shows that, in the absence of any direct heating, heat diffusion, or cell-to-cell contact, “bystander” cells that share the medium with heat-exposed cells exhibit DNA damage, apoptosis, and loss of viability. We coin this phenomenon “active thermal bystander effect” (ATBE). Significant ATBE was induced by fibroblasts exposed for 10minutes to a temperature range of 44–50°C (all P<0.011). The ATBE was not induced by cells heated to lethality above 54°C and immediate medium exchange did not suppress the effect. Therefore, the thermal bystander effect appears to be an active process in which viable, heat-injured cells induce a signal cascade and/or mediator that damages or kills surrounding bystander cells. The ATBE may have clinical relevance for acute burn trauma, hyperthermic treatments, and distant tissue damage after localized heat stress

Endogenous Skin Fluorescence Includes Bands that may Serve as Quantitative Markers of Aging and Photoaging

Aging and photoaging cause distinct changes in skin cells and extracellular matrix. Changes in hairless mouse skin as a function of age and chronic UVB exposure were investigated by fluorescence excitation spectroscopy. Fluorescence excitation spectra were measured in vivo, on heat-separated epidermis and dermis, and on extracts of mouse skin to characterize the absorption spectra of the emitting chromophores. Fluorescence excitation spectra obtained in vivo on 6 wk old mouse skin had maxima at 295, 340, and 360 nm; the 295 nm band was the dominant band. Using heat separated tissue, the 295 nm band predominantly originated in the epidermis and the bands at 340 and 360 nm originated in the dermis. The 295 nm band was assigned to tryptophan fluorescence, the 340 nm band to pepsin digestable collagen cross-links fluorescence and the 360 nm band to collagenase digestable collagen cross-links fluorescence. Fluorescence excitation maxima remained unchanged in chronologically aged mice (34–38 wk old), whereas the 295 nm band decreased in intensity with age and the 340 nm band increased in intensity with age. In contrast, fluorescence excitation spectra of chronically UVB exposed mice showed a large increase in the 295 nm band compared with age-matched controls and the bands at 340 and 350 nm were no longer distinct. Two new bands appeared in the chronically exposed mice at 270 nm and at 305 nm. These reproducible changes in skin autofluorescence suggest that aging causes predictable alterations in both epidermal and dermal fluorescence, whereas chronic UV exposure induces the appearance of new fluorphores

Selective Photothermolysis of Cutaneous Pigmentation by Q-switched Nd: YAG Laser Pulses at 1064, 532. and 355nm

Exposure of skin to nanosecond-domain laser pulses affects the pigmentary system by a process called selective photothermolysis, in which melanosomes and pigmented cells are preferentially altered. Due to the broad absorption spectrum of melanin, this effect may occur with wavelengths that penetrate to vastly different depths within tissue, potentially producing different biologic responses. The effects of single near-ultraviolet (355nm), visible (532nm), and near infrared (1064nm) pulses of 10–12nsec duration were determined in guinea pig skin using gross, histologic, and electron microscopic observations. Threshold response in pigmented skin was a transient immediate ash-white discoloration, requiring 0.11, 0.20 and 1.0J/cm2, at 355, 532, and 1064 nm, respectively. At each wavelength, melanosomes were reputed within keratinocytes and melanocytes, with cytoplasmic and nuclear alterations. Delayed epidermal depigmentation occurred, followed by gradual repigmentation. Deep follicular cells were altered only at 532 and 1064 nm, which produced permanent leukotrichia. The action spectrum for threshold response was consistent with mechanisms implied by selective photothermolysis. These data may be useful for consideration of treatment for cutaneous pigmentation abnormalities or unwanted follicular pigmentation, or both

A micro-sterile inflammation array as an adjuvant for influenza vaccines

There is an urgent need of adjuvants for cutaneous vaccination. Here we report that micro-sterile inflammation induced at inoculation sites can augment immune responses to influenza vaccines in animal models. The inoculation site is briefly illuminated with a handheld, non-ablative fractional laser before the vaccine is intradermally administered, which creates an array of self-healing microthermal zones (MTZs) in the skin. The dying cells in the MTZs send “danger” signals that attract a large number of antigen-presenting cells, in particular, plasmacytoid dendritic cells (pDCs) around each MTZ forming a micro-sterile inflammation array. A pivotal role for pDCs in the adjuvanticity is ascertained by significant abrogation of the immunity after systemic depletion of pDCs, local application of a TNF-α inhibitor, or null mutation of IFN regulatory factor7 (IRF7). In contrast to conventional adjuvants that cause persistent inflammation and skin lesions, micro-sterile inflammation enhances efficacy of influenza vaccines, yet with diminished adverse effects

Longitudinal, 3D in vivo imaging of sebaceous glands by coherent anti-Stokes Raman scattering microscopy –normal function and response to cryotherapy

Sebaceous glands perform complex functions, and are centrally involved in the pathogenesis of acne vulgaris. Current techniques for studying sebaceous glands are mostly static in nature, whereas the gland’s main function – excretion of sebum via the holocrine mechanism – can only be evaluated over time. We present a longitudinal, real-time alternative – the in vivo, label-free imaging of sebaceous glands using Coherent Anti-Stokes Raman Scattering (CARS) microscopy, which is used to selectively visualize lipids. In mouse ears, CARS microscopy revealed dynamic changes in sebaceous glands during the holocrine secretion process, as well as in response to damage to the glands caused by cooling. Detailed gland structure, plus the active migration of individual sebocytes and cohorts of sebocytes were measured. Cooling produced characteristic changes in sebocyte structure and migration. This study demonstrates that CARS microscopy is a promising tool for studying the sebaceous gland and its associated disorders in three-dimensions in vivo

An elastic second skin

We report the synthesis and application of an elastic, wearable crosslinked polymer layer (XPL) that mimics the properties of normal, youthful skin. XPL is made of a tunable polysiloxane-based material that can be engineered with specific elasticity, contractility, adhesion, tensile strength and occlusivity. XPL can be topically applied, rapidly curing at the skin interface without the need for heat- or light-mediated activation. In a pilot human study, we examined the performance of a prototype XPL that has a tensile modulus matching normal skin responses at low strain (<40%), and that withstands elongations exceeding 250%, elastically recoiling with minimal strain-energy loss on repeated deformation. The application of XPL to the herniated lower eyelid fat pads of 12 subjects resulted in an average 2-grade decrease in herniation appearance in a 5-point severity scale. The XPL platform may offer advanced solutions to compromised skin barrier function, pharmaceutical delivery and wound dressings

Fractional Skin Harvesting: Autologous Skin Grafting without Donor-site Morbidity

Background: Conventional autologous skin grafts are associated with significant donor-site morbidity. This study was conducted to determine feasibility, safety, and efficacy of a new strategy for skin grafting based on harvesting small columns of full-thickness skin with minimal donor-site morbidity. Methods: The swine model was used for this study. Hundreds of full-thickness columns of skin tissue (~700 µm diameter) were harvested using a custom-made harvesting device, and then applied directly to excisional skin wounds. Healing in donor and graft sites was evaluated over 3 months by digital photographic measurement of wound size and blinded, computer-aided evaluation of histological features and compared with control wounds that healed by secondary intention or with conventional split-thickness skin grafts (STSG). Results: After harvesting hundreds of skin columns, the donor sites healed rapidly without scarring. These sites reepithelialized within days and were grossly and histologically indistinguishable from normal skin within 7 weeks. By contrast, STSG donor sites required 2 weeks for reepithelialization and retained scar-like characteristics in epidermal and dermal architecture throughout the experiment. Wounds grafted with skin columns resulted in accelerated reepithelialization compared with ungrafted wounds while avoiding the “fish-net” patterning caused by STSG. Conclusion: Full-thickness columns of skin can be harvested in large quantities with negligible long-term donor-site morbidity, and these columns can be applied directly to skin wounds to enhance wound healing

Reconstitution of full‐thickness skin by microcolumn grafting

Abstract In addition to providing a physical barrier, skin also serves a diverse range of physiological functions through different specialized resident cell types/structures, including melanocytes (pigmentation and protection against ultraviolet radiation), Langerhans cells (adaptive immunity), fibroblasts (maintaining extracellular matrix, paracrine regulation of keratinocytes), sweat glands (thermoregulation) and hair follicles (hair growth, sensation and a stem cell reservoir). Restoration of these functional elements has been a long‐standing challenge in efforts to engineer skin tissue, while autologous skin grafting is limited by the scarcity of donor site skin and morbidity caused by skin harvesting. We demonstrate an alternative approach of harvesting and then implanting μm‐scale, full‐thickness columns of human skin tissue, which can be removed from a donor site with minimal morbidity and no scarring. Fresh human skin microcolumns were used to reconstitute skin in wounds on immunodeficient mice. The restored skin recapitulated many key features of normal human skin tissue, including epidermal architecture, diverse skin cell populations, adnexal structures and sweat production in response to cholinergic stimulation. These promising preclinical results suggest that harvesting and grafting of microcolumns may be useful for reconstituting fully functional skin in human wounds, without donor site morbidity. © 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd