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For patients with lower limb amputations, prostheses are immensely helpful for mobility and the ability to perform job-related or recreational activities. However, the skin covering the amputation stump is typically transposed from adjacent areas of the leg and lacks the weight-bearing capacity that is only found in the specialized skin covering the palms and soles (a.k.a. volar skin). As a result, the skin tissue in direct contact with the prosthesis frequently breaks down, leading to the development of painful sores and other complications that limit, and often preclude, the use of prostheses. Transplanting volar skin onto amputation stumps could be a solution to these problems, but traditional skin transplantation techniques cause substantial morbidity at the donor site, such as pain and scarring, which are especially problematic for volar skin given the critical functional importance of the volar skin areas. We previously developed the technology to collect and engraft full-thickness skin tissue while avoiding long-term donor site morbidity, by harvesting the skin in the form of small (~0.5 mm diameter) cores that we termed "micro skin tissue columns" (MSTCs), so that each donor wound is small enough to heal quickly and without clinically appreciable scarring or other long-term abnormalities. The goal of this study was to establish whether a similar approach could be used to confer the structural and molecular characteristics of volar skin ectopically to other skin areas. In a human-to-mouse xenograft model, we show the long-term persistence of various human plantar MSTC-derived cell types in the murine recipient. Then in an autologous porcine model, we harvested MSTCs from the bottom of the foot and transplanted them onto excision wounds on the animals' trunks. The healing processes at both the donor and graft sites were monitored over 8 weeks, and tissue samples were taken to verify volar-specific characteristics by histology and immunohistochemistry. The volar donor sites were well-tolerated, healed rapidly, and showed no signs of scarring or any other long-term defects. The graft sites were able to maintain volar-specific histologic features and expression of characteristics protein markers, up to the 8-week duration of this study. These results suggest that MSTC grafting could be a practical approach to obtain autologous donor volar skin tissue, confer volar skin characteristics ectopically to nonvolar skin areas, improve the load-bearing capacity of amputation stump skin, and ultimately enhance mobility and quality-of-life for lower limb amputees.
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Transplante de Pele , Pele , Suporte de Carga , Animais , Transplante de Pele/métodos , Camundongos , Pele/metabolismo , Humanos , Feminino , Masculino , SuínosRESUMO
It has long been known that sensitivity to cold-induced damage can vary greatly between different cell types, with lethal temperatures reportedly ranging from -2°C for canine osteocytes, to -70°C for mammary adenocarcinomas. This suggests that, for certain applications, "therapeutic windows" may exist wherein the cryosurgery temperature could be controlled to specifically target more cold-sensitive cell types, while sparing less sensitive cells. However, this potential selectivity has not been developed into practical clinical treatments, in part because of a lack of available investigative tools that can provide consistent, reproducible cooling within the desired temperature range. Here we describe an experimental cryosurgery tool that allows user control over the three key cryosurgery parameters - temperature, pressure, and duration. The tool is composed of inexpensive components that are generally accessible in most laboratory settings, and could be a practical investigative tool for developing and optimizing novel topical cryosurgery approaches.
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OBJECTIVE: In this study, we evaluated the effectiveness of antimicrobial blue light (aBL; 410 nm wavelength) against ß-lactamase-carrying bacteria and the effect of aBL on the activity of ß-lactamases. METHODS: Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae strains carrying ß-lactamases as well as a purified ß-lactamase enzymes were studied. ß-lactamase activity was assessed using a chromogenic cephalosporin hydrolysis assay. Additionally, we evaluated the role of porphyrins in the photoreaction, as well as protein degradation by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Finally, we investigated the bactericidal effect of combined aBL-ceftazidime exposure against a metallo-ß-lactamase expressing P. aeruginosa strain. RESULTS: Our study demonstrated that aBL effectively killed ß-lactamase-producing bacteria and reduced ß-lactamase activity. After an aBL exposure of 1.52 J/cm2, a 50% reduction in enzymatic activity was observed in P. aeruginosa. Additionally, we found a 40% decrease in the photoreaction activity of porphyrins following an aBL exposure of 64.8 J/cm2. We also revealed that aBL reduced ß-lactamase activity via protein degradation (after 136.4 J/cm2). Additionally, aBL markedly improved the bactericidal effect of ceftazidime (by >4-log10) in the metallo-ß-lactamase P. aeruginosa strain. CONCLUSION: Our results provide evidence that aBL compromises bacterial ß-lactamase activity, offering a potential approach to overcome ß-lactam resistance in bacteria.
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Precision-cut liver slices (PCLS) are increasingly used as a model to investigate anti-fibrotic therapies. However, many studies use PCLS from healthy animals treated with pro-fibrotic stimuli in culture, which reflects only the early stages of fibrosis. The effects of different culture conditions on PCLS from cirrhotic animals has not been well characterized and there is no consensus on optimal methods. In this study, we report a method for the collection and culture of cirrhotic PCLS and compare the effect of common culture conditions on viability, function, and gene expression. Additionally, we compared three methods of RNA isolation and identified a protocol with high yield and purity. We observed significantly increased albumin production when cultured with insulin-transferrin-selenium and dexamethasone, and when incubated on a rocking platform. Culturing with insulin-transferrin-selenium and dexamethasone maintained gene expression closer to the levels in fresh slices. However, despite stable viability and function up to 4 days, we found significant changes in expression of key genes by day 2. Interestingly, we also observed that cirrhotic PCLS maintain viability in culture longer than slices from healthy animals. Due to the influence of matrix stiffness on fibrosis and hepatocellular function, it is important to evaluate prospective anti-fibrotic therapies in a platform that preserves tissue biomechanics. PCLS from cirrhotic animals represent a promising tool for the development of treatments for chronic liver disease.
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Dexametasona , Cirrose Hepática , Fígado , Animais , Ratos , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Cirrose Hepática/genética , Dexametasona/farmacologia , Masculino , RNA/isolamento & purificação , RNA/genética , RNA/metabolismo , Insulina/metabolismo , Insulina/farmacologia , Ratos Sprague-Dawley , Selênio/farmacologia , Técnicas de Cultura de Tecidos/métodosRESUMO
Fractional laser ablation is a technique developed in dermatology to induce remodeling of skin scars by creating a dense pattern of microinjuries. Despite remarkable clinical results, this technique has yet to be tested for scars in other tissues. As a first step toward determining the suitability of this technique, we aimed to (1) characterize the response to microinjuries in the healthy and cirrhotic liver, and (2) determine the underlying cause for any differences in response. Healthy and cirrhotic rats were treated with a fractional laser then euthanized from 0 h up to 14 days after treatment. Differential expression was assessed using RNAseq with a difference-in-differences model. Spatial maps of tissue oxygenation were acquired with hyperspectral imaging and disruptions in blood supply were assessed with tomato lectin perfusion. Healthy rats showed little damage beyond the initial microinjury and healed completely by 7 days without scarring. In cirrhotic rats, hepatocytes surrounding microinjury sites died 4-6 h after ablation, resulting in enlarged and heterogeneous zones of cell death. Hepatocytes near blood vessels were spared, particularly near the highly vascularized septa. Gene sets related to ischemia and angiogenesis were enriched at 4 h. Laser-treated regions had reduced oxygen saturation and broadly disrupted perfusion of nodule microvasculature, which matched the zones of cell death. Our results demonstrate that the cirrhotic liver has an exacerbated response to microinjuries and increased susceptibility to ischemia from microvascular damage, likely related to the vascular derangements that occur during cirrhosis development. Modifications to the fractional laser tool, such as using a femtosecond laser or reducing the spot size, may be able to prevent large disruptions of perfusion and enable further development of a laser-induced microinjury treatment for cirrhosis.
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Isquemia , Cirrose Hepática , Animais , Ratos , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Masculino , Isquemia/metabolismo , Isquemia/patologia , Fígado/metabolismo , Fígado/patologia , Terapia a Laser/métodos , Ratos Sprague-Dawley , Hepatócitos/metabolismoRESUMO
BACKGROUND: Autofluorescence photography can detect specific light-tissue interactions and record important pathophysiological changes associated with nonmelanoma skin cancer (NMSC), which has been ascribed to the fluorescence of an aromatic amino acid, tryptophan. OBJECTIVE: To assess the impact of a novel, autofluorescence imaging (AFI) device on margin control for NMSCs before Mohs micrographic surgery (MMS) in an effort to decrease overall operating time. METHODS: Before the initial stage of MMS, NMSCs were measured with a 2-mm margin as standard of care (normal margin). The tumor was then imaged with the AFI device. A 2-mm margin was drawn around the fluorescent area captured by the AFI device and was referred to as the camera margin. The tumor was excised based on the normal margin and evaluated on frozen histological section. RESULTS: Imaging based on the AFI device resulted in appropriate recommendations for margin control in 8 of 11 tumors. Four of these tumors did not fluoresce and demonstrated a lack of tumor residuum on stage I specimen, as anticipated. There were no side effects from the AFI device. CONCLUSION: This is an initial pilot study that supports the use of a novel, noninvasive imaging device to help with margin assessment before MMS. On optimization, this device has potential to extend applicability to surgical excisions for tumors that do not fulfill criteria for MMS.
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Margens de Excisão , Cirurgia de Mohs , Imagem Óptica , Neoplasias Cutâneas , Humanos , Neoplasias Cutâneas/cirurgia , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/diagnóstico por imagem , Projetos Piloto , Imagem Óptica/métodos , Imagem Óptica/instrumentação , Masculino , Feminino , Idoso , Carcinoma Basocelular/cirurgia , Carcinoma Basocelular/patologia , Carcinoma Basocelular/diagnóstico por imagem , Carcinoma de Células Escamosas/cirurgia , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/diagnóstico por imagem , Pessoa de Meia-Idade , Idoso de 80 Anos ou maisRESUMO
Post-inflammatory hyperpigmentation (PIH) is a hypermelanosis that often occurs secondary to skin irritation or injury, especially in darker skin tones, for which there is currently a lack of effective treatment options. Few preclinical models are available to study PIH. Here, we show that the Yucatan miniature pig consistently develops PIH after skin injuries. Skin wounds were produced on Yucatan pigs by needle punches, full-thickness excisions, or burns. Wound sites were monitored and photographed regularly. Tissue samples were collected after 24 weeks and processed for histology/immunohistochemistry. Skin pigmentation and histologic changes were quantified by computer-assisted image analyses. All injury methods resulted in hyperpigmentation. Melanin content at the histologic level was quantified in the larger (burn and excision) wounds, showing a significant increase compared to uninjured skin. Increased melanin was found for both epidermal and dermal regions. Dermal melanin deposits were primarily clustered around the papillary vasculature, and were associated not with melanocytes but with leukocytes. The Yucatan miniature pig model recapitulates key clinical and histologic features of PIH in humans, including skin hyperpigmentation at both gross and histologic levels, and persistence of dermal melanin subsequent to injury. This model could be used to further our understanding of the etiology of PIH, and for new therapy development.
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Modelos Animais de Doenças , Hiperpigmentação , Melaninas , Porco Miniatura , Animais , Suínos , Hiperpigmentação/patologia , Hiperpigmentação/metabolismo , Melaninas/metabolismo , Pele/patologia , Pele/metabolismo , Inflamação/patologia , Pigmentação da Pele , Feminino , HumanosRESUMO
Wound research has typically been performed without regard for where the wounds are located on the body, despite well-known heterogeneities in physical and biological properties between different skin areas. The skin covering the palms and soles is highly specialized, and plantar ulcers are one of the most challenging and costly wound types to manage. Using primarily the porcine model, we show that plantar skin is molecularly and functionally more distinct from nonplantar skin than previously recognized, with unique gene and protein expression profiles, broad alterations in cellular functions, constitutive activation of many wound-associated phenotypes, and inherently delayed healing. This unusual physiology is likely to play a significant but underappreciated role in the pathogenesis of plantar ulcers as well as the last 25+ years of futility in therapy development efforts. By revealing this critical yet unrecognized pitfall, we hope to contribute to the development of more effective therapies for these devastating nonhealing wounds.
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Fenótipo , Pele , Cicatrização , Animais , Cicatrização/fisiologia , Suínos , Pele/patologia , Pele/lesões , Pele/metabolismo , Modelos Animais de Doenças , Úlcera do Pé/fisiopatologia , Úlcera do Pé/patologia , Humanos , Feminino , Fenômenos Fisiológicos da Pele , PéRESUMO
BACKGROUND: People with Neurofibromatosis Type 1 (NF1) suffer disfigurement and pain when hundreds to thousands of cutaneous neurofibromas (cNFs) appear and grow throughout life. Surgical removal of cNFs under anesthesia is the only standard therapy, leaving surgical scars. OBJECTIVE: Effective, minimally-invasive, safe, rapid, tolerable treatment(s) of small cNFs that may prevent tumor progression. METHODS: Safety, tolerability, and efficacy of 4 different treatments were compared in 309, 2-4 mm cNFs across 19 adults with Fitzpatrick skin types (FST) I-IV: radiofrequency (RF) needle coagulation, 755 nm alexandrite laser with suction, 980 nm diode laser, and intratumoral injection of 10 mg/mL deoxycholate. Regional pain, clinical responses, tumor height and volume (by 3D photography) were assessed before, 3 and 6 months post-treatment. Biopsies were obtained electively at 3 months. RESULTS: There was no scarring or adverse events > grade 2. Each modality significantly (P < .05) reduced or cleared cNFs, with large variation between tumors and participants. Alexandrite laser and deoxycholate were fast and least painful; 980 nm laser was most painful. Growth of cNFs was not stimulated by treatment(s) based on height and volume values at 3 and 6 months compared to baseline. LIMITATIONS: Intervention was a single treatment session; dosimetry has not been optimized. CONCLUSIONS: Small cNFs can be rapidly and safely treated without surgery.
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Neurofibroma , Neurofibromatose 1 , Neuroma , Neoplasias Cutâneas , Adulto , Humanos , Estudos Prospectivos , Neurofibroma/cirurgia , Resultado do Tratamento , Neoplasias Cutâneas/cirurgia , Neurofibromatose 1/complicações , Neurofibromatose 1/terapia , Cicatriz , Dor , Ácido DesoxicólicoRESUMO
In the age of antimicrobial resistance, the urgency by which novel therapeutic approaches need to be introduced into the clinical pipeline has reached critical levels. Antimicrobial blue light (aBL), as an alternative approach, has demonstrated promise as a stand-alone therapeutic method, albeit with a limited window of antimicrobial activity. Work by others indicates that treatment with antibiotics increases the production of reactive oxygen species (ROS) which may, in part, contribute to the bactericidal effects of antibiotics. These findings suggest that there may be potential for synergistic interactions with aBL, that similarly generates ROS. Therefore, in this study, the mechanism of aBL is investigated, and the potential for aBL to synergistically promote antibiotic activity is similarly evaluated. Furthermore, the translatability of using aBL and chloramphenicol in combination within a mouse model of Acinetobacter baumanii burn infection is assessed. It is concluded that porphyrins and hydroxyl radicals driven by "free iron" are paramount to the effectiveness of aBL; and aBL is effective at promoting multiple antibiotics in different multidrug-resistant bacteria. Moreover, rROS up-regulation, and promoted antibiotic uptake are observed during aBL+antibiotic exposure. Lastly, aBL combined with chloramphenicol appears to be both effective and safe for the treatment of A. baumannii burn infection. In conclusion, aBL may be a useful adjunct therapy to antibiotics to potentiate their action.
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Anti-Infecciosos , Queimaduras , Animais , Camundongos , Antibacterianos/farmacologia , Radical Hidroxila , Luz Azul , Espécies Reativas de Oxigênio , Queimaduras/microbiologia , Cloranfenicol/farmacologia , BactériasRESUMO
Cutaneous bacterial wound infections typically involve gram-positive cocci such as Staphylococcus aureus (SA) and usually become biofilm infections. Bacteria in biofilms may be 100-1000-fold more resistant to an antibiotic than the clinical laboratory minimal inhibitory concentration (MIC) for that antibiotic, contributing to antimicrobial resistance (AMR). AMR is a growing global threat to humanity. One pathogen-antibiotic resistant combination, methicillin-resistant SA (MRSA) caused more deaths globally than any other such combination in a recent worldwide statistical review. Many wound infections are accessible to light. Antimicrobial phototherapy, and particularly antimicrobial blue light therapy (aBL) is an innovative non-antibiotic approach often overlooked as a possible alternative or adjunctive therapy to reduce antibiotic use. We therefore focused on aBL treatment of biofilm infections, especially MRSA, focusing on in vitro and ex vivo porcine skin models of bacterial biofilm infections. Since aBL is microbicidal through the generation of reactive oxygen species (ROS), we hypothesized that menadione (Vitamin K3), a multifunctional ROS generator, might enhance aBL. Our studies suggest that menadione can synergize with aBL to increase both ROS and microbicidal effects, acting as a photosensitizer as well as an ROS recycler in the treatment of biofilm infections. Vitamin K3/menadione has been given orally and intravenously worldwide to thousands of patients. We conclude that menadione/Vitamin K3 can be used as an adjunct to antimicrobial blue light therapy, increasing the effectiveness of this modality in the treatment of biofilm infections, thereby presenting a potential alternative to antibiotic therapy, to which biofilm infections are so resistant.
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Anti-Infecciosos , Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Infecção dos Ferimentos , Humanos , Vitamina K 3/farmacologia , Vitamina K 3/uso terapêutico , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/uso terapêutico , Espécies Reativas de Oxigênio/farmacologia , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Anti-Infecciosos/farmacologia , Biofilmes , Infecções Estafilocócicas/tratamento farmacológico , Testes de Sensibilidade MicrobianaRESUMO
BACKGROUND: Extracorporeal membrane oxygenators (ECMO) are currently utilized to mechanically ventilate blood when lung or lung and heart function are impaired, like in cases of acute respiratory distress syndrome (ARDS). ARDS can be caused by severe cases of carbon monoxide (CO) inhalation, which is the leading cause of poison-related deaths in the United States. ECMOs can be further optimized for severe CO inhalation using visible light to photo-dissociate CO from hemoglobin (Hb). In previous studies, we combined phototherapy with an ECMO to design a photo-ECMO device, which significantly increased CO elimination and improved survival in CO-poisoned animal models using light at 460, 523, and 620 nm wavelengths. Light at 620 nm was the most effective in removing CO. OBJECTIVE: The aim of this study is to analyze the light propagation at 460, 523, and 620 nm wavelengths and the 3D blood flow and heating distribution within the photo-ECMO device that increased CO elimination in CO-poisoned animal models. METHODS: Light propagation, blood flow dynamics, and heat diffusion were modeled using the Monte Carlo method and the laminar Navier-Stokes and heat diffusion equations, respectively. RESULTS: Light at 620 nm propagated through the device blood compartment (4 mm), while light at 460 and 523 nm only penetrated 48% to 50% (~2 mm). The blood flow velocity in the blood compartment varied with regions of high (5 mm/s) and low (1 mm/s) velocity, including stagnant flow. The blood temperatures at the device outlet for 460, 523, and 620 nm wavelengths were approximately 26.7°C, 27.4°C, and 20°C, respectively. However, the maximum temperatures within the blood treatment compartment rose to approximately 71°C, 77°C, and 21°C, respectively. CONCLUSIONS: As the extent of light propagation correlates with efficiency in photodissociation, the light at 620 nm is the optimal wavelength for removing CO from Hb while maintaining blood temperatures below thermal damage. Measuring the inlet and outlet blood temperatures is not enough to avoid unintentional thermal damage by light irradiation. Computational models can help eliminate risks of excessive heating and improve device development by analyzing design modifications that improve blood flow, like suppressing stagnant flow, further increasing the rate of CO elimination.
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Intoxicação por Monóxido de Carbono , Oxigenação por Membrana Extracorpórea , Síndrome do Desconforto Respiratório , Animais , Intoxicação por Monóxido de Carbono/terapia , Oxigenadores de Membrana , Oxigenação por Membrana Extracorpórea/métodos , Fototerapia/métodos , Síndrome do Desconforto Respiratório/terapiaRESUMO
Foot ulceration annually affects millions of patients and accounts for billions of dollars in medical expenses in the US alone. Many previous studies have investigated co-morbidities associated with impaired healing, such as microbial infection, compromised circulation, and diabetes. By comparison, little is known about how wound healing proceeds in plantar skin, despite its many unique specializations related to its load-bearing function. One of the main challenges in modeling plantar wounds is the difficulty in maintaining wound dressings, as animals generally have a low tolerance to wearing bandages on their feet. With assistance from the MGH Center for Comparative Medicine, we developed a positive reinforcement-based behavioral training regimen that successfully induced tolerance for plantar dressings in swine, which is a critical first step towards enabling in vivo study of the wound healing process in this highly specialized skin area. This training program will be described in detail in this manuscript.
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Bandagens , Cicatrização , Suínos , AnimaisRESUMO
Cutaneous pain is a common symptom of skin disease, and available therapies are inadequate. We developed a neural selective and injectable method of cryoneurolysis with ice slurry, which leads to a long-lasting decrease in mechanical pain. The aim of this study is to determine whether slurry injection reduces cutaneous pain without inducing the side effects associated with conventional cryoneurolysis. Using the rat sciatic nerve, we examined the effects of slurry on nerve structure and function in comparison with the effects of a Food and Drug Administrationâapproved cryoneurolysis device (Iovera). Coherent anti-Stokes Raman scattering microscopy and immunofluorescence staining were used to investigate histological effects on the sciatic nerve and on downstream cutaneous nerve fibers. Complete Freund's Adjuvant model of cutaneous pain was used to study the effect of the slurry on reducing pain. Structural changes in myelin induced by slurry were comparable with those induced by Iovera, which uses much colder temperatures. Compared with that of Iovera, the decrease in mechanical pain due to slurry was less profound but lasted longer without signs of dysesthesia. Slurry did not cause a reduction of epidermal nerve fibers or a change in thermal pain sensitivity. Slurry-treated rats showed reduced cutaneous mechanical pain in response to Complete Freund's Adjuvant. Slurry injection can be used to successfully reduce cutaneous pain without causing dysesthesia.
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Gelo , Dermatopatias , Ratos , Animais , Adjuvante de Freund/farmacologia , Ratos Sprague-Dawley , Parestesia , Dor/etiologiaRESUMO
Cryoneurolysis is an opioid-sparing therapy for long-lasting and reversible reduction of pain. We developed a nerve-selective method for cryoneurolysis by local injection of ice-slurry (- 5 to - 6 °C) that induced decrease in nocifensive response starting from about a week after treatment and lasting up to 8 weeks. In this study, we test the hypothesis that injection of colder slurry leads to faster onset of analgesia. Colder slurry (- 9ºC) was injected around the rat sciatic nerve to induce cryoneurolysis. Hematoxylin and Eosin (H&E) staining was used to examine histologic effects on surrounding tissues. Coherent anti-Stokes Raman scattering (CARS) microscopy was used to study effects on myelin sheaths. Functional tests were used to assess changes in sensory and motor function in the treated hind paw. No inflammation or scarring was detected in surrounding skin and muscle tissues at day 7 post slurry injection. Functional tests showed rapid onset reduction in mechanical pain sensitivity starting from day 1 and lasting up to day 98. CARS imaging demonstrated disintegration of myelin sheaths post treatment followed by complete recovery of nerve structure by day 140. In this study we showed that colder slurry (- 9 °C) produces more rapid onset and longer duration of analgesia, while remaining nerve-selective.
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Analgesia , Manejo da Dor , Ratos , Animais , Nervo Isquiático , Bainha de Mielina , DorRESUMO
Objectives: There is growing evidence that excess adipose tissue within the head and neck contributes to obstructive sleep apnea (OSA), particularly in obese patients. This subset of the population is often difficult to treat with surgical therapies. We theorized that a novel, transcervical method of injectable cryoablation using ice-slurry can achieve low temperatures without causing neurovascular damage or airway distress in a swine model. Methods: Four Yorkshire pigs were injected with ice-slurry comprised of normal saline and 10% glycerol cooled to -6°C via a transcervical, ultrasound guided approach. Direct laryngoscopy was used to confirm accurate placement of the slurry. Thermocouple placement at the needle-tip was used to measure temperatures at injection site. Swine were monitored for clinical signs of tongue necrosis and airway edema for 2 months, and then euthanized. Twelve biopsy samples from the base of the tongue were collected for histology. These were assessed for presence of tissue damage, inflammation and collagen formation by a blinded board-certified pathologist. Results: Tongue tissue temperature below 10°C was achieved for 13.5 ± 1.1 min. Minimum tissue temperature was -4 ± 0.6°C. There was no clinical or pathological evidence of tongue damage to include damage to the lingual nerve or artery. There was some histologic evidence of new collagen formation in areas of the tongue. Conclusions: Transcervical ultrasound-guided ice-slurry injection is feasible, well-tolerated at temperatures previously shown to be capable of selectively targeting adipose tissue in the base of the tongue in a preclinical swine model, without causing neurovascular damage or airway distress when properly injected.
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Background: Cryoneurolysis uses tissue cooling as an opioid-sparing, long-lasting treatment for peripheral nerve pain. A nerve-selective method for cryoneurolysis by local injection of ice-slurry was developed to allow cryoneurolysis to be performed with a standard needle and syringe, similar to peripheral nerve blocks. Since the treatment of patients with chronic pain may require repeated injections, we investigated the safety and tolerance of repeated treatments in a rat model. Methods: Three repeated ice-slurry treatments, given 6 weeks apart were performed around the rat sciatic nerve. Nerve and surrounding tissues were collected up to 4 months after the third treatment for analysis. Coherent anti-Stokes Raman scattering (CARS) microscopy was used to study effects on myelin sheaths and axon structure. Immunofluorescence (IF) staining was used to study effects on axon density. Hematoxylin and Eosin (H&E) staining was used to examine histologic effects on sciatic nerve and surrounding tissue. Results: Histologic and CARS image analysis of nerve tissue collected months after three injections demonstrated recovery of nerve structure, myelin organization and axon density to baseline levels, without any residual inflammation, scarring or neuroma formation. No inflammation or scarring was detected in surrounding skin and muscle tissues. Conclusion: Repeated ice-slurry injections cause temporary, nerve-selective and reversible changes in the peripheral nerve. There was no histologic damage to surrounding skin and muscle tissues. Repeated treatments with injectable ice-slurry for cryoneurolysis appear to be safe and well tolerated. Clinical studies for patients with chronic pain are warranted.
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BACKGROUND: Autologous fractional full-thickness skin grafting is a method of harvesting full-thickness skin with reduced donor site morbidity compared with conventional skin grafting. OBJECTIVE: To demonstrate that full-thickness skin microbiopsies can be harvested with minimal scarring or complications. MATERIALS AND METHODS: In a nonrandomized, self-controlled, pilot trial, subjects ( n = 8) underwent tissue harvesting of full-thickness skin columns of 200, 400, 500, 600, 800 µm, 1, and 2 mm diameters. The extent of scarring was measured by using the Patient and Observer Scar Assessment Scale and blinded evaluation of photographs at 6 weeks postprocedure. Pain visual analog scale (VAS) and side effects were recorded. RESULTS: When present, scars were first observed after 2 to 4 weeks, much more often for wounds >400 µm ( p < .001). Blinded dermatologists increasingly identified clinical scarring on photographs with larger harvested microcolumn diameters ( p < .001). Median VAS pain score was 0 (range 0-4). All subjects rated the procedure safe and tolerable. CONCLUSION: Harvesting full-thickness skin microcolumns is well-tolerated over a wide range of column diameters. At diameters of less than 500 µm, side effects including scarring are minimal.
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Queimaduras , Cicatriz , Queimaduras/complicações , Cicatriz/etiologia , Cicatriz/patologia , Humanos , Dor/etiologia , Pele/patologia , Transplante de Pele/efeitos adversos , Transplante de Pele/métodos , CicatrizaçãoRESUMO
Vibrio vulnificus is an invasive marine bacterium that causes a variety of serious infectious diseases. With the increasing multidrug-resistant variants, treatment of V. vulnificus infections is becoming more difficult. In this study, we explored antimicrobial blue light (aBL; 405 nm wavelength) for the treatment of V. vulnificus infections. We first assessed the efficacy of aBL against five strains of V. vulnificus in vitro. Next, we identified and quantified intracellular porphyrins in V. vulnificus to provide mechanistic insights. Additionally, we measured intracellular reactive oxygen species (ROS) production and bacterial membrane permeabilization following aBL exposures. Lastly, we conducted a preclinical study to investigate the efficacy and safety of aBL for the prevention and treatment of burn infections caused by V. vulnificus in mice. We found that aBL effectively killed V. vulnificus in vitro in both planktonic and biofilm states, with up to a 5.17- and 4.57-log10 CFU reduction being achieved, respectively, following an aBL exposure of 216 J/cm2. Protoporphyrin IX and coproporphyrins were predominant in all the strains. Additionally, intracellular ROS was significantly increased following aBL exposures (P < 0.01), and there was evidence of aBL-induced permeabilization of the bacterial membrane (P < 0.0001). In the preclinical studies, we found that female mice treated with aBL 30 min after bacterial inoculation showed a survival rate of 81% following 7 days of observation, while only 28% survival was observed in untreated female mice (P < 0.001). At 6 h post-inoculation, an 86% survival was achieved in aBL-treated female mice (P = 0.0002). For male mice, 86 and 63% survival rates were achieved when aBL treatment was given 30 min and 6 h after bacterial inoculation, respectively, compared to 32% survival in the untreated mice (P = 0.0004 and P = 0.04). aBL did not reduce cellular proliferation or induce apoptosis. We found five cytokines were significantly upregulated in the males after aBL treatment, including MCSF (P < 0.001), MCP-5 (P < 0.01), TNF RII (P < 0.01), CXCL1 (P < 0.01), and TIMP-1 (P < 0.05), and one in the females (TIMP-1; P < 0.05), suggesting that aBL may induce certain inflammatory processes. In conclusion, aBL may potentially be applied to prevent and treat V. vulnificus infections.