Porcine models are frequently used for burn healing studies; however, factors including anatomic location and lack of standardised wound methods can impact the interpretation of wound data. The objectives of this study are to examine the influence of anatomical locations on the uniformity of burn creation and healing in porcine burn models. To optimise burn parameters on dorsal and ventral surfaces, ex vivo and in situ euthanized animals were first used to examine the location-dependence of the burn depth and contact time relationship. The location-dependent healing in vivo was then examined using burn and excisional wounds at dorsal, ventral, caudal and cranial locations. Lactate dehydrogenase (LDH) and H&E were used to assess burn depth and wound re-epithelialization. We found that burn depth on the ventral skin was significantly deeper than that of the dorsal skin at identical thermal conditions. Compared with burns created ex vivo, burns created in situ immediately post-mortem were significantly deeper in the ventral location. In live animals, 2 out of 12 burn wounds were fully re-epithelialized after 14 days in contrast to complete re-epithelialization of all excisional wounds. Among the burn wounds, those at the cranial-dorsal site exhibited faster healing than at the caudal-dorsal site. This study showed that anatomical location is an important consideration for the consistency of burn depth creation and healing. These data support symmetric localization of treatment and control for comparative assessment of burn healing in porcine models to prevent misinterpretation of results and increase the translatability of findings to humans.

BACKGROUND:Delayed indocyanine green fluorescence imaging is under investigation in various clinical disease processes. Understanding the mechanisms of indocyanine green accumulation and retention is essential to correctly interpreting and analyzing imaging data. The purpose of this scoping review was to synthesize what is known about the mechanism of indocyanine green retention at the cellular level to better understand the clinical nuances of delayed indocyanine green imaging and identify critical gaps in our knowledge to guide future studies. METHODS:We performed a scoping review of 7,087 citations after performing database searches of PubMed, Scopus, the Cochrane Library, and the Web of Science Core Collection electronic databases. Studies were eligible for inclusion if they were peer-reviewed original research discussing the mechanism of indocyanine green retention in the results section in disease processes involving inflammation and/or necrosis, including cancer, and were available in English. Data were extracted using Covidence software. RESULTS:Eighty-nine studies were included in the final analysis. Several features of indocyanine green retention were identified. CONCLUSION:We identified several mechanistic features involved in indocyanine green accumulation in diseased tissue that overall had distinct mechanisms of indocyanine green retention in tumors, nontumor inflammation, and necrosis. Our study also reveals new insights on how inflammatory infiltrate influences indocyanine green fluorescence imaging. These findings are noteworthy because they add to our understanding of how fluorescence-guided surgery may be optimized based on the pathology of interest via specific indocyanine green dosing and timing of image acquisition.

Abstract Introduction No objective technique exists to distinguish necrotic from viable tissue, risking over-excision in burns and loss of wound healing potential. Using delayed fluorescence imaging of indocyanine green (ICG), a method called second window ICG (SWIG), we have shown that high dose ICG persists in burn wounds in both animals and patients 24 hours after infusion. The necrotic avidity of ICG is proposed as the underlying mechanism, although inflammation also likely has a role. The objective of this study is to examine the hypothesis at both the tissue and cellular levels. Methods To examine the localization of ICG in burn tissue, 4-mm biopsies taken from burn patients who underwent SWIG were embedded and sequentially sectioned for H&E (tissue architecture) and LDH (tissue viability) and ICG fluorescence imaging. To examine whether ICG preferentially binds to necrotic cells, primary skin cells (fibroblasts or keratinocytes) were subjected to thermal injury at 65ºC for 0 (untreated), 2.5 or 5 minutes. The cells were subsequently incubated with ICG at concentrations ranging from 0.5 to 5 μg/mL at 37ºC for 30 minutes. A necrosis and apoptosis detection kit with Yo-Pro™-1 and propidium iodide (PI) was used to differentiate live, apoptotic and necrotic cell populations in flow cytometry. The ICG florescence intensity of each cell population was measured with flow cytometry. Results The fluorescence microscopic imaging revealed that ICG intensity was most intense in the superficial severely thermally-injured tissue and gradually decreased with depth. This zone of high ICG intensity was co-localized with high cellularity (shown in H&E) consisting of a mixture of nonviable skin cells (shown in LDH) and inflammatory infiltrates. Using flow cytometry, the optimal concentration of ICG with the highest ICG florescence median intensity (FMI) and the best separation of live, apoptotic and necrotic cell populations was 2.5 μg/mL, which was used for subsequent flow cytometry experiments. For both fibroblasts and keratinocytes, the ICG FMI was significantly higher in the necrotic cell population than that of either the live or apoptotic cell population, suggesting higher affinity of ICG to necrotic cells. The ICG FMI of necrotic cells with longer heat exposure (5 minutes) was significantly higher than that of necrotic cells with shorter heat exposure (2.5 minutes), suggesting that the binding sites for ICG increase with the degree of thermal injury. Conclusions This study demonstrates the necrotic avidity of ICG in burned skin tissue and two primary skin cell types. Further studies are needed to study the ICG binding characteristics with inflammatory cell populations and single cell suspensions dissociated from human burn tissue. Applicability of Research to Practice This, along with our pilot study of SWIG in patients, supports the feasibility of SWIG in guiding surgical decision-making in burn excision by defining thresholds of fluorescence intensity values.

Abstract Introduction Objective determination of burn wound healing potential remains elusive and significantly impacts decision making for surgery, the extent of tissue excised intraoperatively and the use of donor site-sparing alternative tissue therapies. Indocyanine green angiography (ICGA) has promise as an adjunct to evaluate healing potential, but feasibility has limited adoption in clinical practice. Delayed fluorescence imaging of indocyanine green (ICG), in a method called second-window ICG (SWIG), is a new technique used intraoperatively to guide tumor resection via increased peritumoral endothelial permeability. The objective of this study is to examine ICGA and SWIG fluorescence in burns requiring excision and grafting, and to correlate SWIG fluorescence to microscopic localization of inflamed and necrotic tissue. Methods Deep partial thickness, indeterminate depth or full thickness burns were identified in adult patients scheduled for excision and grafting. 24 hours prior to surgery, baseline bright light and fluorescence images were obtained before the administration of up to 5 mg/kg ICG intravenously. ICGA was performed within 5 minutes of infusion initiation. On the day of surgery, bright light and SWIG fluorescence images were obtained before and after burn excision. The excised tissue was imaged ex-vivo to determine the presence of fluorescence in the tissue compared to that remaining within the wound bed. Excised tissue was processed for histologic analysis of cellular architecture, viability, inflammation and necrosis. Macroscopic ICGA and SWIG fluorescence images were compared to the associated microscopic tissue sections to determine the presence of inflammatory infiltrate, localization of non-viable tissue, and co-localization of ICG fluorescence. Results ICGA imaging performed preoperatively demonstrated variable fluorescence throughout the burns without a clear cutoff value to delineate deep partial versus full thickness burns. SWIG imaging revealed a speckled fluorescence pattern prior to burn excision that became diffuse after excision suggesting a potential utility of SWIG to intraoperatively identify excision completion. ICGA and SWIG fluorescence demonstrated an inverse relationship, and SWIG fluorescence was associated with non-viable tissue. Conclusions ICGA imaging alone was unreliable to delineate the need for surgical intervention. SWIG imaging of burn injuries may represent a valuable tool to guide the extent of excision intraoperatively and reduce unnecessary excision of viable tissue. Further studies are needed to understand SWIG fluorescence at the inflammation-necrosis border and how ICGA fluorescence along with SWIG can synergistically improve detection of healing potential in burn patients.

Wound cleansing agents are routine in wound care and preoperative preparation. Antiseptic activity intends to prevent contaminating microbes from establishing an infection while also raising concerns of cytotoxicity and delayed wound healing. We evaluated the cytotoxicity of five clinically used wound cleaning agents (saline, povidone iodine, Dove® and Dial® soaps, and chlorhexidine gluconate [CHG]) using both an ex vivo and in vivo human skin xenograft mouse model, in contrast to classical in vitro models that lack the structural and compositional heterogeneity of human skin. We further established an ex vivo wound contamination model inoculated with ~100 cells of Pseudomonas aeruginosa or Staphylococcus aureus to evaluate antimicrobial efficacy. Scanning electron microscopy and confocal microscopy were used to evaluate phenotypic and spatial characteristics of bacterial cells in wound tissue. CHG significantly reduced metabolic activity of the skin explants, while all treatments except saline affected local cellular viability. CHG cytotoxicity persisted and progressed over 14 days, impairing wound healing in vivo. Within the contamination model, CHG treatment resulted in a significant reduction of P. aeruginosa wound surface counts at 24 h post-treatment. However, this effect was transient and serial application of CHG had no effect on both P. aeruginosa or S. aureus microbial growth. Microscopy revealed that viable cells of P. aeruginosa reside deep within wound tissue post-CHG application, likely serving as a reservoir to re-populate the tissue to a high bioburden. We reveal concerning cytotoxicity and limited antimicrobial activity of CHG in human skin using clinically relevant models, with the ability to resolve spatial localization and temporal dynamics of tissue viability and microbial growth.

Abstract Introduction Chlorhexidine (CHG) is ubiquitous in surgical perioperative care. In vivo studies of CHG cytotoxicity on human skin are lacking. Given the use of CHG for daily wound cares and as a presurgical scrub, including donor site preparation, we sought to identify if CHG cytotoxicity would persist in a clinically relevant in vivo human skin xenograft model. Methods Human skin tissues were obtained from elective surgeries. Partial thickness wounds were created ex vivo in human skin using a 4 mm punch biopsy. 2% CHG (treatment) or PBS (control) was applied to the wounds for 30 minutes followed by rinsing the tissue +/- mechanical disruptive irrigation. Tissues were cultured at the air-liquid interface for 24 hours in culture media after treatment and tissue viability was performed using an MTT assay. For in vivo studies, athymic mice (n=4) were grafted on bilateral flanks with human skin. Eight weeks after engraftment and normalization of skin architecture, 4 mm partial thickness wounds were created on each xenograft (2 per mouse – treatment and control). 2% CHG was applied daily for 2 minutes followed by irrigation with PBS in the treatment wound. The control wound received PBS application and irrigation. The xenografts received treatment daily for 14 days to mimic daily wound cares, and digital images were obtained to document presence of infection and gross wound healing. On day 14, the xenografts were harvested and stained for lactate dehydrogenase and H&E to assess cell viability and wound re-epithelization, respectively. Results An MTT assay on ex vivo human skin wounds showed that CHG treated groups (irrigation or non-irrigation) had lower cell viability compared to the PBS treated group, however irrigation mitigates the cytotoxicity of CHG on human skin. In the in vivo xenograft study, no signs of infection were identified in either PBS or CHG treated wounds throughout the study. The wound size appeared larger on gross inspection in the CHG treated group compared to the PBS group as early as day 2.Microscopically, the PBS treated wounds were fully re-epithelialized (n=2) or had significantly more re-epithelialization (n=2) than the CHG treated wounds (n=4) after 14 days of treatment. The PBS-treated wounds were viable throughout the tissue, indicating the irrigation procedure was not harmful to the cells. In the CHG-treated wounds, nonviable cells were observed in the dermis beneath the wound that was directly in contact with CHG suggesting penetration of CHG contributes to cytotoxicity in acute wounds. Conclusions Daily CHG use is cytotoxic to human skin and impedes wound healing.

BACKGROUND: No objective technique exists to distinguish necrotic from viable tissue, risking over-excision in burns and loss of wound healing potential. Second window indocyanine green (SWIG) is a novel fluorescence-imaging modality being studied to identify residual solid tumors during oncological surgery. SWIG has also been shown to have avidity for necrosis in animal models, but translation of these findings to humans is lacking. The objective of this study was to evaluate SWIG in the identification of burn wound necrosis and compare it with previously published indocyanine green angiography (ICGA) techniques. STUDY DESIGN: This study used mouse, human skin xenograft and human patient burn models. Brightfield and SWIG near-infrared imaging were performed on macroscopic tissue samples, which were then cryopreserved, sectioned, and analyzed for microscopic fluorescence. SWIG fluorescence findings were correlated to visual assessment of the burn wound as well as histological markers of necrosis using hematoxylin and eosin and lactate dehydrogenase stains. RESULTS: We found that SWIG identified burn necrosis in a manner dependent on the dose and timing of indocyanine green (ICG) administration and had an inverse fluorescence signal compared with ICGA. Furthermore, SWIG fluorescence identified the interface of viable and nonviable tissue. CONCLUSION: Our study confirmed that ICGA is an inconsistent and nonstandardized modality to evaluate burn injuries. In contrast, SWIG imaging is a potential imaging modality to objectively prognosticate burn wound healing potential and guide intraoperative burn excision. Further studies are needed to define ratios of fluorescence intensity values to guide surgical decision-making in burn excision and to better define how ICG is retained in necrotic tissue to enhance utility of SWIG in other disease processes.