Optical properties of biomimetic probes engineered from erythrocytes.

Light-activated theranostic materials offer a potential platform for optical imaging and phototherapeutic applications. We have engineered constructs derived from erythrocytes, which can be doped with the FDA-approved near infrared (NIR) chromophore, indocyanine green (ICG). We refer to these constructs as NIR erythrocyte-mimicking transducers (NETs). Herein, we investigated the effects of changing the NETs mean diameter from micron- (≈4 µm) to nano- (≈90 nm) scale, and the ICG concentration utilized in the fabrication of NETs from 5 to 20 µM on the resulting absorption and scattering characteristics of the NETs. Our approach consisted of integrating sphere-based measurements of light transmittance and reflectance, and subsequent utilization of these measurements in an inverse adding-doubling algorithm to estimate the absorption (µ a) and reduced scattering (µ s') coefficients of these NETs. For a given NETs diameter, values of µ a increased over the approximate spectral band of 630-860 nm with increasing ICG concentration. Micron-sized NETs produced the highest peak value of µ a when using ICG concentrations of 10 and 20 µM, and showed increased values of µ s' as compared to nano-sized NETs. Spectral profiles of µ s' for these NETs showed a trend consistent with Mie scattering behavior for spherical objects. For all NETs investigated, changing the ICG concentration minimally affected the scattering characteristics. A Monte Carlo-based model of light distribution showed that the presence of these NETs enhanced the fluence levels within simulated blood vessels. These results provide important data towards determining the appropriate light dosimetry parameters for an intended light-based biomedical application of NETs.

The Role of Laser Speckle Imaging in Port-Wine Stain Research: Recent Advances and Opportunities.

Here, we review our current knowledge on the etiology and treatment of port-wine stain (PWS) birthmarks. Current treatment options have significant limitations in terms of efficacy. With the combination of 1) a suitable preclinical microvascular model, 2) laser speckle imaging (LSI) to evaluate blood-flow dynamics, and 3) a longitudinal experimental design, rapid preclinical assessment of new phototherapies can be translated from the lab to the clinic. The combination of photodynamic therapy (PDT) and pulsed-dye laser (PDL) irradiation achieves a synergistic effect that reduces the required radiant exposures of the individual phototherapies to achieve persistent vascular shutdown. PDL combined with anti-angiogenic agents is a promising strategy to achieve persistent vascular shutdown by preventing reformation and reperfusion of photocoagulated blood vessels. Integration of LSI into the clinical workflow may lead to surgical image guidance that maximizes acute photocoagulation, is expected to improve PWS therapeutic outcome. Continued integration of noninvasive optical imaging technologies and biochemical analysis collectively are expected to lead to more robust treatment strategies.

Label-free optical-resolution photoacoustic microscopy of superficial microvasculature using a compact visible laser diode excitation.

We have developed laser-diode-based optical-resolution photoacoustic microscopy (LD-OR-PAM) of superficial microvasculature which has the desirable properties of being compact, low-cost, and label-free. A 300-mW visible pulsed laser diode was operated at a 405 ± 5 nm wavelength with a pulse energy as low as 52 nJ. By using a 3.6 MHz ultrasound transducer, the system was tested on carbon fibers with a lateral resolution of 0.95 µm and an SNR of 38 dB. The subcutaneous microvasculature on a mouse back was imaged without an exogenous contrast agent which demonstrates the potential of the proposed prototype for skin chromophores. Our eventual goal is to offer a practical and affordable multi-wavelength functional LD-OR-PAM instrument suitable for clinical applications.

Sustained activation of c-Jun N-terminal and extracellular signal-regulated kinases in port-wine stain blood vessels.

BACKGROUND: Port-wine stain (PWS) is a congenital, progressive vascular malformation but the pathogenesis remains incompletely understood. OBJECTIVE: We sought to investigate the activation status of various kinases, including extracellular signal-regulated kinase, c-Jun N-terminal kinase, AKT, phosphatidylinositol 3-kinase, P70 ribosomal S6 kinase, and phosphoinositide phospholipase C γ subunit, in PWS biopsy tissues. METHODS: Immunohistochemistry was performed on 19 skin biopsy samples from 11 patients with PWS. RESULTS: c-Jun N-terminal kinase, extracellular signal-regulated kinase, and P70 ribosomal S6 kinase in pediatric and adult PWS blood vessels were consecutively activated. Activation of AKT and phosphatidylinositol 3-kinase was found in many adult hypertrophic PWS blood vessels but not in infants. Phosphoinositide phospholipase C γ subunit showed strong activation in nodular PWS blood vessels. LIMITATION: Infantile PWS sample size was small. CONCLUSION: Our data suggest a subsequent activation profile of various kinases during different stages of PWS: (1) c-Jun N-terminal and extracellular signal-regulated kinases are firstly and consecutively activated in all PWS tissues, which may contribute to both the pathogenesis and progressive development of PWS; (2) AKT and phosphatidylinositol 3-kinase are subsequently activated, and are involved in the hypertrophic development of PWS blood vessels; and (3) phosphoinositide phospholipase C γ subunit is activated in the most advanced stage of PWS and may participate in nodular formation.

In vivo laser cartilage reshaping with carbon dioxide spray cooling in a rabbit ear model: a pilot study.

BACKGROUND/OBJECTIVES: Similar to conventional cryogen spray cooling, carbon dioxide (CO2) spray may be used in combination with laser cartilage reshaping (LCR) to produce cartilage shape change while minimizing cutaneous thermal injury. Recent ex vivo evaluation of LCR with CO2 cooling in a rabbit model has identified a promising initial parameter space for in vivo safety and efficacy evaluation. This pilot study aimed to evaluate shape change and cutaneous injury following LCR with CO2 cooling in 5 live rabbits. STUDY DESIGN/MATERIALS AND METHODS: The midportion of live rabbit ears were irradiated with a 1.45 µm wavelength diode laser (12 J/cm(2)) with simultaneous CO2 spray cooling (85 millisecond duration, 4 alternating heating/cooling cycles per site, 5 to 6 irradiation sites per row for 3 rows per ear). Experimental and control ears (no LCR) were splinted in the flexed position for 30 days following exposure. A total of 5 ears each were allocated to the experimental and control groups. RESULTS: Shape change was observed in all irradiated ears (mean 70 ± 3°), which was statistically different from control (mean 37 ± 11°, P = 0.009). No significant thermal cutaneous injury was observed, with preservation of the full thickness of skin, microvasculature, and adnexal structures. Confocal microscopy and histology demonstrated an intact and viable chondrocyte population surrounding irradiated sites. CONCLUSIONS: LCR with CO2 spray cooling can produce clinically significant shape change in the rabbit auricle while minimizing thermal cutaneous and cartilaginous injury and frostbite. This pilot study lends support for the potential use of CO2 spray as an adjunct to existing thermal-based cartilage reshaping modalities. An in vivo systematic evaluation of optimal laser dosimetry and cooling parameters is required.

Topical rapamycin systematically suppresses the early stages of pulsed dye laser-induced angiogenesis pathways.

BACKGROUND: Administration of topical rapamycin (RPM) suppresses the regeneration and revascularization of photocoagulated blood vessels induced by pulsed dye laser (PDL). OBJECTIVE: To systematically elucidate the molecular pathophysiology of the inhibition of PDL-induced angiogenesis by topical RPM in a rodent model. METHODS: The mRNA expression profiles of 86 angiogenic genes and phosphorylation levels of ribosomal protein S6 kinase (P70S6K) in rodent skin were examined with or without topical RPM administration post-PDL exposure. RESULTS: The PDL-induced systematic increases in transcriptional levels of angiogenic genes showed a peak expression at days 3-7 post-PDL in rodent skin. Topical application of 1% RPM significantly and systematically suppressed the PDL-induced increase in mRNA levels of the examined angiogenic genes during the first five days post-PDL. The phosphorylation levels of P70S6K increased after PDL exposure but those increases were suppressed by the topical RPM. After topical application, RPM penetrated to an approximate depth of 768.4 µm into rodent skin. CONCLUSION: Topical application of 1% RPM can significantly and systematically suppress the PDL-induced early stage of angiogenesis via inhibition of the AKT/mTOR/P70S6K pathway in a rodent model.

Temperature Profile Measurement From Radiofrequency Nasal Airway Reshaping Device.

OBJECTIVE: Nasal airway obstruction (NAO) is caused by various disorders including nasal valve collapse (NVC). A bipolar radiofrequency (RF) device (VivAer®, Aerin Medical, Sunnyvale, CA) has been used to treat NAO through RF heat generation to the upper lateral cartilage (ULC). The purpose of this study is to measure temperature elevations in nasal tissue, using infrared (IR) radiometry to map the spatial and temporal evolution of temperature. STUDY DESIGN: Experimental and computational. METHODS: Composite porcine nasal septum was harvested and sectioned (1 mm and 2 mm). The device was used to heat the cartilage in composite porcine septum. An IR camera (FLIR® ExaminIR, Teledyne, Wilsonville, OR) was used to image temperature on the back surface of the specimen. These data were incorporated into a heat transfer finite element model that also calculated tissue damage using Arrhenius rate process. RESULTS: IR temperature imaging showed peak back surface temperatures of 49.57°C and 42.21°C in 1 and 2 mm thick septums respectively. Temperature maps were generated demonstrating the temporal and spatial evolution of temperature. A finite element model generated temperature profiles with respect to time and depth. Rate process models using Arrhenius coefficients showed 30% chondrocyte death at 1 mm depth after 18 s of RF treatment. CONCLUSION: The use of this device creates a thermal profile that may result in thermal injury to cartilage. Computational modeling suggests chondrocyte death extending as deep as 1.4 mm below the treatment surface. Further studies should be performed to improve dosimetry and optimize the heating process to reduce potential injury. Laryngoscope, 134:1063-1070, 2024.

In vivo, high-resolution, three-dimensional imaging of port wine stain microvasculature in human skin.

BACKGROUND AND OBJECTIVES: Port-wine stain (PWS) is a congenital, progressive vascular malformation of the dermis. The use of optical coherence tomography (OCT) for the characterization of blood vessels in PWS skin has been demonstrated by several groups. In the past few years, advances in OCT technology have greatly increased imaging speed. Sophisticated numerical algorithms have improved the sensitivity of Doppler OCT dramatically. These improvements have enabled the noninvasive, high-resolution, three-dimensional functional imaging of PWS skin. Here, we demonstrate high-resolution, three-dimensional, microvasculature imaging of PWS and normal skin using Doppler OCT technique. STUDY DESIGN/MATERIALS AND METHODS: The OCT system uses a swept source laser which has a central wavelength of 1,310 nm, an A-line rate of 50 kHz and a total average power of 16 mW. The system uses a handheld imaging probe and has an axial resolution of 9.3 µm in air and a lateral resolution of approximately 15 µm. Images were acquired from PWS subjects at the Beckman Laser Institute and Medical Clinic. Microvasculature of the PWS skin and normal skin were obtained from the PWS subject. RESULTS: High-resolution, three-dimensional microvasculature of PWS and normal skin were obtained. Many enlarged PWS vessels are detected in the dermis down to 1.0 mm below the PWS skin surface. In one subject, the blood vessel diameters range from 40 to 90 µm at the epidermal-dermal junction and increase up to 300-500 µm at deeper regions 700-1,000 µm below skin surface. The blood vessels close to the epidermal-dermal junction are more uniform, in terms of diameter. The more tortuous and dilated PWS blood vessels are located at deeper regions 600-1,000 µm below the skin surface. In another subject example, the PWS skin blood vessels are dilated at very superficial layers at a depth less than 500 µm below the skin surface. The PWS skin vessel diameters range from 60 to 650 µm, with most vessels having a diameter of around 200 µm. CONCLUSIONS: OCT can be used to quantitatively image in vivo skin micro-vasculature. Analysis of the PWS and normal skin blood vessels were performed and the results can provide quantitative information to optimize laser treatment on an individual patient basis.

Ex vivo investigations of laser auricular cartilage reshaping with carbon dioxide spray cooling in a rabbit model.

Laser cartilage reshaping (LCR) with cryogen spray cooling is a promising modality for producing cartilage shape change while reducing cutaneous thermal injury. However, LCR in thicker tissues, such as auricular cartilage, requires higher laser power, thus increasing cooling requirements. To eliminate the risks of freeze injury characteristic of high cryogen spray pulse rates, a carbon dioxide (CO2) spray, which evaporates rapidly from the skin, has been proposed as the cooling medium. This study aims to identify parameter sets which produce clinically significant reshaping while producing minimal skin thermal injury in LCR with CO2 spray cooling in ex vivo rabbit auricular cartilage. Excised whole rabbit ears were mechanically deformed around a cylindrical jig and irradiated with a 1.45-µm wavelength diode laser (fluence 12-14 J/cm(2) per pulse, four to six pulse cycles per irradiation site, five to six irradiation sites per row for four rows on each sample) with concomitant application of CO2 spray (pulse duration 33-85 ms) to the skin surface. Bend angle measurements were performed before and after irradiation, and the change quantified. Surface temperature distributions were measured during irradiation/cooling. Maximum skin surface temperature ranged between 49.0 to 97.6 °C following four heating/cooling cycles. Significant reshaping was achieved with all laser dosimetry values with a 50-70 °C difference noted between controls (no cooling) and irradiated ears. Increasing cooling pulse duration yielded progressively improved gross skin protection during irradiation. CO2 spray cooling may potentially serve as an alternative to traditional cryogen spray cooling in LCR and may be the preferred cooling medium for thicker tissues. Future studies evaluating preclinical efficacy in an in vivo rabbit model are in progress.

Current and Emerging Imaging Techniques for Neurofibromatosis Type 1-Associated Cutaneous Neurofibromas.

A consistent set of measurement techniques must be applied to reliably and reproducibly evaluate the efficacy of treatments for cutaneous neurofibromas (cNFs) in people with neurofibromatosis type 1 (NF1). cNFs are neurocutaneous tumors that are the most common tumor in people with NF1 and represent an area of unmet clinical need. This review presents the available data regarding approaches in use or development to identify, measure, and track cNFs, including calipers, digital imaging, and high-frequency ultrasound sonography. We also describe emerging technologies such as spatial frequency domain imaging and the application of imaging modalities such as optical coherence tomography that may enable the detection of early cNFs and prevention of tumor-associated morbidity.

High-resolution imaging of microvasculature in human skin in-vivo with optical coherence tomography.

In this paper, the features of the intensity-based Doppler variance (IBDV) method were analyzed systemically with a flow phantom. The effects of beam scanning density, flow rate and the time interval between neighboring A-lines on the performance of this method were investigated. The IBDV method can be used to quantify the flow rate and its sensitivity can be improved by increasing the time interval between the neighboring A-lines. A higher sensitivity IBDV method that applies the algorithm along the slower scan direction was proposed. In comparison to laser speckle imaging maps of blood flow, we demonstrated the ability of the method to identify vessels with altered blood flow. In clinical measurements, we demonstrated the ability of the method to image vascular networks with exquisite spatial resolution and at depths up to 1.2 mm in human skin. These results collectively demonstrated the potential of the method to monitor the microvasculature during disease progression and in response to therapeutic intervention.

Topical rapamycin suppresses the angiogenesis pathways induced by pulsed dye laser: molecular mechanisms of inhibition of regeneration and revascularization of photocoagulated cutaneous blood vessels.

BACKGROUND AND OBJECTIVES: Pulsed dye laser (PDL) is the most effective treatment for port wine stain (PWS) birthmarks. However, regeneration and revascularization of photocoagulated blood vessels may result in poor therapeutic outcome. We have recently shown that rapamycin (RPM), an angiogenesis inhibitor, can reduce the regeneration and revascularization of photocoagulated blood vessels. Herein, we attempt to further elucidate the molecular pathophysiology on the inhibition of the regeneration and revascularization of photocoagulated blood vessels by topical RPM in an animal model. MATERIALS AND METHODS: Two separate skin areas on each hamster were irradiated by PDL. After PDL exposure, topical RPM was applied daily to one of the randomly selected test sites. PDL, PDL + RPM and normal skin test sites were biopsied on day 3 after PDL exposure. The total ribonucleic acid (RNA) and protein were extracted from biopsied skin samples and quantified. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and immunoblot were subsequently performed to quantify the mRNA and protein levels of hypoxia-inducible factor-1alpha (HIF-1α), vascular endothelial growth factor (VEGF) and ribosomal protein S6 kinase (S6). The phosphorylation levels of S6 and AKT were also evaluated by immunoblot. RESULTS: The mRNA and protein levels of HIF-1α, VEGF, and S6 significantly increased after PDL exposure as compared to the normal hamster skin. Topical application of 1% RPM suppressed the PDL-induced increase in mRNA and protein levels of those genes on day 3 post-PDL exposure. The phosphorylation levels of S6 and AKT increased after PDL exposure but the increases were suppressed by the topical application of RPM. CONCLUSION: The increase in expression of HIF-1α, VEGF, and S6 after PDL-exposure suggests that angiogenesis pathways play very active roles in the process of skin blood vessel regeneration and revascularization. Topical application of 1% RPM can suppress the angiogenesis pathways and, therefore, reduce the regeneration and revascularization of photocoagulated blood vessels.

Photocoagulation of dermal blood vessels with multiple laser pulses in an in vivo microvascular model.

BACKGROUND/OBJECTIVES: Current laser therapy of port wine stain (PWS) birthmarks with a single laser pulse (SLP) does not produce complete lesion removal in the majority of patients. To improve PWS therapeutic efficacy, we evaluated the performance of an approach based on multiple laser pulses (MLP) to enhance blood vessel photocoagulation. STUDY DESIGN: The hamster dorsal window chamber model was used. Radiant exposure (RE), pulse repetition rate (f(r)), total number of pulses (n(p)), and length of vessel irradiated were varied. Blood vessels in the window were irradiated with either SLP with RE of 4-7 J/cm(2) or MLP with RE per pulse of 1.4-5.0 J/cm(2), f(r) of 0.5-26.0 Hz, and n(p) of 2-5. The laser wavelength was 532 nm and pulse duration was 1 ms. Either a 2 mm vessel segment or entire vessel branch was irradiated. Digital photographs and laser speckle images of the window were recorded before and at specific time points after laser irradiation to monitor laser-induced blood vessel structural and functional changes, respectively. RESULTS: We found that: (1) for a SLP approach, the RE required to induce blood vessel photocoagulation was 7 J/cm(2) as compared to only 2 J/cm(2) per pulse for the MLP approach; (2) for MLP, two pulses at a repetition rate of 5 Hz and a RE of 3 J/cm(2) can induce photocoagulation of more than 80% of irradiated blood vessel; and (3) irradiation of a longer segment of blood vessel resulted in lower reperfusion rate. CONCLUSIONS: The MLP approach can induce blood vessel photocoagulation at much lower RE per pulse as compared to SLP. The 5 Hz f(r) and the need for two pulses are achievable with modern laser technology, which makes the MLP approach practical in the clinical management of PWS birthmarks.

Membrane Cholesterol Enrichment of Red Blood Cell-Derived Microparticles Results in Prolonged Circulation.

Particles fabricated from red blood cells (RBCs) can serve as vehicles for delivery of various biomedical cargos. Flipping of phosphatidylserine (PS) from the inner to the outer membrane leaflet normally occurs during the fabrication of such particles. PS externalization is a signal for phagocytic removal of the particles from circulation. Herein, we demonstrate that membrane cholesterol enrichment can mitigate the outward display of PS on microparticles engineered from RBCs. Our in-vitro results show that the phagocytic uptake of cholesterol-enriched particles by murine macrophages takes place at a lowered rate, resulting in reduced uptake as compared to RBC-derived particles without cholesterol enrichment. When administered via tail-vein injection into healthy mice, the percent of injected dose (ID) per gram of extracted blood for cholesterol-enriched particles was ∼1.5 and 1.8 times higher than the particles without cholesterol enrichment at 4 and 24 h, respectively. At 24 h, ∼43% ID/g of the particles without cholesterol enrichment was eliminated or metabolized while ∼94% ID/g of the cholesterol-enriched particles were still retained in the body. These results indicate that membrane cholesterol enrichment is an effective method to reduce PS externalization on the surface of RBC-derived particles and increase their longevity in circulation.

High speed photo-mediated ultrasound therapy integrated with OCTA.

Photo-mediated Ultrasound Therapy (PUT), as a new anti-vascular technique, can promote cavitation activity to selectively destruct blood vessels with a significantly lower amount of energy when compared to energy level required by other laser and ultrasound treatment therapies individually. Here, we report the development of a high speed PUT system based on a 50-kHz pulsed laser to achieve faster treatment, decreasing the treatment time by a factor of 20. Furthermore, we integrated it with optical coherence tomography angiography (OCTA) for real time monitoring. The feasibility of the proposed OCTA-guided PUT was validated through in vivo rabbit experiments. The addition of OCTA to PUT allows for quantitative prescreening and real time monitoring of treatment response, thereby enabling implementation of individualized treatment strategies.

Intensity-based modified Doppler variance algorithm: application to phase instable and phase stable optical coherence tomography systems.

The traditional phase-resolved Doppler method demonstrates great success for in-vivo imaging of blood flow and blood vessels. However, the phase-resolved method always requires high phase stability of the system. In phase instable situations, the performance of the phase-resolved methods will be degraded. We propose a modified Doppler variance algorithm that is based on the intensity or amplitude value. Performances of the proposed algorithm are compared with traditional phase-resolved Doppler variance and color Doppler methods for both phase stable and phase instable systems. For the phase instable situation, the proposed algorithm demonstrates images without phase instability induced artifacts. In-vivo imaging of window-chamber hamster skin is demonstrated for phase instable situation with a spectrometer-based Fourier domain OCT system. A microelectromechanical systems (MEMS) based swept source OCT (SSOCT) system is also used to demonstrate the performance of the proposed method in a phase instable situation. The phase stability of the SSOCT system is analyzed. In-vivo imaging of the blood vessel of human skin is demonstrated with the proposed method and the SSOCT system. For the phase stable situation, the proposed algorithm also demonstrates comparable performance with traditional phase-resolved methods. In-vivo imaging of the human choroidal blood vessel network is demonstrated with the proposed method under the phase stable situation. Depth-resolved fine choroidal blood vessel networks are shown.

Wide-field functional imaging of blood flow and hemoglobin oxygen saturation in the rodent dorsal window chamber.

The rodent dorsal window chamber is a widely used in vivo model of the microvasculature. The model consists of a 1cm region of exposed microvasculature in the rodent dorsal skin that is immobilized by surgically implanted titanium frames, allowing the skin microvasculature to be visualized. We describe a detailed protocol for surgical implantation of the dorsal window chamber which enables researchers to perform the window chamber implantation surgery. We further describe subsequent wide-field functional imaging of the chamber to obtain hemodynamic information in the form of blood oxygenation and blood flow on a cm size region of interest. Optical imaging techniques, such as intravital microscopy, have been applied extensively to the dorsal window chamber to study microvascular-related disease and conditions. Due to the limited field of view of intravital microscopy, detailed hemodynamic information typically is acquired from small regions of interest, typically on the order of hundreds of µm. The wide-field imaging techniques described herein complement intravital microscopy, allowing researchers to obtain hemodynamic information at both microscopic and macroscopic spatial scales. Compared with intravital microscopy, wide-field functional imaging requires simple instrumentation, is inexpensive, and can give detailed metabolic information over a wide field of view.

Numerical optimization of sequential cryogen spray cooling and laser irradiation for improved therapy of port wine stain.

BACKGROUND AND OBJECTIVE: Despite application of cryogen spray (CS) precooling, customary treatment of port wine stain (PWS) birthmarks with a single laser pulse does not result in complete lesion blanching for a majority of patients. One obvious reason is nonselective absorption by epidermal melanin, which limits the maximal safe radiant exposure. Another possible reason for treatment failure is screening of laser light within large PWS vessels, which prevents uniform heating of the entire vessel lumen. Our aim is to identify the parameters of sequential CS cooling and laser irradiation that will allow optimal photocoagulation of various PWS blood vessels with minimal risk of epidermal thermal damage. STUDY DESIGN AND METHODS: Light and heat transport in laser treatment of PWS are simulated using a custom 3D Monte Carlo model and 2D finite element method, respectively. Protein denaturation in blood and skin are calculated using the Arrhenius kinetic model with tissue-specific coefficients. Simulated PWS vessels with diameters of 30-150 µm are located at depths of 200-600 µm, and shading by nearby vessels is accounted for according to PWS histology data from the literature. For moderately pigmented and dark skin phototypes, PWS blood vessel coagulation and epidermal thermal damage are assessed for various parameters of sequential CS cooling and 532-nm laser irradiation, i.e. the number of pulses in a sequence (1-5), repetition rate (7-30 Hz), and radiant exposure. RESULTS: Simulations of PWS treatment in darker skin phototypes indicate specific cooling/irradiation sequences that provide significantly higher efficacy and safety as compared to the customary single-pulse approach across a wide range of PWS blood vessel diameters and depths. The optimal sequences involve three to five laser pulses at repetition rates of 10-15 Hz. CONCLUSIONS: Application of the identified cooling/irradiation sequences may offer improved therapeutic outcome for patients with resistant PWS, especially in darker skin phototypes.

Longitudinal, multimodal functional imaging of microvascular response to photothermal therapy.

Although studies have shown that photothermal therapy can coagulate selectively abnormal vasculature, the ability of this method to achieve consistent, complete removal of the vasculature is questionable. We present the use of multimodal, wide-field functional imaging to study, in greater detail, the biological response to selective laser injury. Specifically, a single-platform instrument capable of coregistered fluorescence imaging and laser speckle imaging was utilized to monitor vascular endothelial growth factor gene expression and blood flow, respectively, in a transgenic rodent model. Collectively, the longitudinal, in vivo data collected with our instrument suggest that the biological response to selective laser injury involves early-stage redistribution of blood flow, followed by increased vascular endothelial growth factor promoter activity to stimulate pro-angiogenic events.

Long-term blood vessel removal with combined laser and topical rapamycin antiangiogenic therapy: implications for effective port wine stain treatment.

BACKGROUND AND OBJECTIVES: Complete blanching of port wine stain (PWS) birthmarks after laser therapy is rarely achieved for most patients. We postulate that the low therapeutic efficacy or treatment failure is caused by regeneration and revascularization of photocoagulated blood vessels due to angiogenesis associated with the skin's normal wound healing response. Rapamycin (RPM), an antiangiogenic agent, has been demonstrated to inhibit growth of pathological blood vessels. Our objectives were to (1) investigate whether topical RPM can inhibit reperfusion of photocoagulated blood vessels in an animal model and (2) determine the effective RPM concentration required to achieve this objective. STUDY DESIGN/MATERIALS AND METHODS: For both laser-only and combined laser and RPM treated animals, blood vessels in the dorsal window chambers implanted on golden Syrian hamsters were photocoagulated with laser pulses. Structural and flow dynamics of blood vessels were documented with color digital photography and laser speckle imaging to evaluate photocoagulation and reperfusion. For the combined treatment group, topical RPM was applied to the epidermal side of the window daily for 14 days after laser exposure. RESULTS: In the laser-only group, 23 out of 24 photocoagulated blood vessels reperfused within 5-14 days. In the combined treatment group with different RPM formulae and concentrations, the overall reperfusion rate of 36% was much lower as compared to the laser-only group. We also found that the reperfusion rate was not linearly proportional to the RPM concentration. CONCLUSIONS: With topical RPM application, the frequency of vessel reperfusion was considerably reduced, which implies that combined light and topical antiangiogenic therapy might be a promising approach to improve the treatment efficacy of PWS birthmarks.