Experimental evidence for Parthanatos-like mode of cell death of heat-damaged human skin fibroblasts in a cell culture-based in vitro burn model.

The cellular mechanisms of burn conversion of heat damaged tissue are center of many studies. Even if the molecular mechanisms of heat-induced cell death are controversially discussed in the current literature, it is widely accepted that caspase-mediated apoptosis plays a central role. In the current study we wanted to develop further information on the nature of the mechanism of heat-induced cell death of fibroblasts in vitro. We found that heating of human fibroblast cultures (a 10 s rise from 37 °C to 67 °C followed by a 13 s cool down to 37 °C) resulted in the death of about 50% of the cells. However, the increase in cell death started with a delay, about one hour after exposure to heat, and reached the maximum after about five hours. The lack of clear evidence for an active involvement of effector caspase in the observed cell death mechanism and the lack of observation of the occurrence of hypodiploid nuclei contradict heat-induced cell death by caspase-mediated apoptosis. Moreover, a dominant heat-induced increase in PARP1 protein expression, which correlated with a time-delayed ATP synthesis inhibition, appearance of double-strand breaks and secondary necrosis, indicate a different type of cell death than apoptosis. Indeed, increased translocation of Apoptosis Inducing Factor (AIF) and Macrophage Migration Inhibitory Factor (MIF) into cell nuclei, which correlates with the mentioned enhanced PARP1 protein expression, indicate PARP1-induced, AIF-mediated and MIF-activated cell death. With regard to the molecular actors involved, the cellular processes and temporal sequences, the mode of cell death observed in our model is very similar to the cell death mechanism via Parthanatos described in the literature.

Rescuing the negative effects of aging in burn wounds using tacrolimus applied via microcapillary hydrogel dressing.

INTRODUCTION: Delays in treatment of burn injuries can lead to significant morbidity, loss of function, and poor aesthetic appearance. Preventing conversion from partial- to full-thickness burns may help mitigate these sequelae. The pathophysiology of burn wound conversion remains unknown, but an overactive immune response is thought to be implicated. The purpose of this study was to determine whether downregulating the immune response via tacrolimus can decrease burn wound conversion. METHODS: Assembly of the microfluidic hydrogels was achieved by embedding microfibers within a hydrogel scaffold composed of a gelatin-alginate blend. Tacrolimus stock solution for intraperitoneal injection was made by re-suspending powdered tacrolimus in DMSO at 10 mg/mL. 24 young (2-4 months) and 24 old (>16 months) mice were given partial thickness burns. The treatment cohort received either tacrolimus ointment with a hydrogel dressing (6 young and 6 old) or an intraperitoneal injection of a tacrolimus solution (6 young and 6 old), while the control cohort only received either only the microcapillary hydrogel dressing or an intraperitoneal injection of saline. Mice were euthanized at day 3 after injury and skin samples were taken. Burn depth was evaluated using Vimentin immunostaining. RESULTS: In old mice, intraperitoneal injection of tacrolimus was able to significantly reduce burn wound depth compared to intraperitoneal injection of saline (p = 0.011). Similarly in old mice, topical hydrogel with tacrolimus was able to significantly reduce burn wound depth compared to hydrogel alone (p < 0.001). Topical hydrogel with tacrolimus was able to mitigate the detrimental effects of older age on wound conversion, such that burn wounds of older mice treated with tacrolimus hydrogel dressing had similar burn depths as younger mice (p = 0.240). CONCLUSIONS: Utilizing a combination treatment of tacrolimus and microcapillary hydrogel is able to rescue the negative effects of aging and prevent partial- to full-thickness burn wound conversion. Hopefully these findings will encourage deeper investigation into the possible therapeutic advantages of utilizing immunosuppressive agents to decrease morbidity after burn injuries. Future research will need to specifically investigate IL-2 as an inhibitory target in the acute inflammatory cascade of burn injury.

Clostridium Collagenase Impact on Zone of Stasis Stabilization and Transition to Healthy Tissue in Burns.

Clostridium collagenase has provided superior clinical results in achieving digestion of immediate and accumulating devitalized collagen tissue. Recent studies suggest that debridement via Clostridium collagenase modulates a cellular response to foster an anti-inflammatory microenvironment milieu, allowing for a more coordinated healing response. In an effort to better understand its role in burn wounds, we evaluated Clostridium collagenase's ability to effectively minimize burn progression using the classic burn comb model in pigs. Following burn injury, wounds were treated with Clostridium collagenase or control vehicle daily and biopsied at various time points. Biopsies were evaluated for factors associated with progressing necrosis as well as inflammatory response associated with treatment. Data presented herein showed that Clostridium collagenase treatment prevented destruction of dermal collagen. Additionally, treatment with collagenase reduced necrosis (HMGB1) and apoptosis (CC3a) early in burn injuries, allowing for increased infiltration of cells and protecting tissue from conversion. Furthermore, early epidermal separation and epidermal loss with a clearly defined basement membrane was observed in the treated wounds. We also show that collagenase treatment provided an early and improved inflammatory response followed by faster resolution in neutrophils. In assessing the inflammatory response, collagenase-treated wounds exhibited significantly greater neutrophil influx at day 1, with macrophage recruitment throughout days 2 and 4. In further evaluation, macrophage polarization to MHC II and vascular network maintenance were significantly increased in collagenase-treated wounds, indicative of a pro-resolving macrophage environment. Taken together, these data validate the impact of clostridial collagenases in the pathophysiology of burn wounds and that they complement patient outcomes in the clinical scenario.

Metal chelation attenuates oxidative stress, inflammation, and vertical burn progression in a porcine brass comb burn model.

Oxidative stress and inflammation may mediate cellular damage and tissue destruction as the burn wound continues to progress after the abatement of the initial insult. Since iron and calcium ions play key roles in oxidative stress, this study tested whether topical application of a metal chelator proprietary lotion (Livionex Formulation (LF) lotion), that contains disodium EDTA as a metal chelator and methyl sulfonyl methane (MSM) as a permeability enhancer, would prevent progression or reduce burn wound severity in a porcine model. We have reported earlier that in a rat burn model, LF lotion reduces thermal injury progression. Here, we used the porcine brass comb burn model that closely mimics the human condition for contact burns and applied LF lotion every 8 h starting 15 min after the injury. We found that LF lotion reduces the depth of cell death as assessed by TUNEL staining and blood vessel blockage in the treated burn sites and interspaces. The protein expression of pro-inflammatory markers IL-6, TNF-a, and TNFα Converting Enzyme (TACE), and lipid aldehyde production (protein-HNE) was reduced with LF treatment. LF lotion reversed the burn-induced decrease in the aldehyde dehydrogenase (ALDH-1) expression in the burn sites and interspaces. These data show that a topically applied EDTA-containing lotion protects both vertical and horizontal burn progression when applied after thermal injury. Curbing burn wound conversion and halting the progression of second partial burn to third-degree full-thickness burn remains challenging when it comes to burn treatment strategies during the acute phase. Burn wound conversion can be reduced with targeted treatments to attenuate the oxidative and inflammatory response in the immediate aftermath of the injury. Our studies suggest that LF lotion could be such a targeted treatment.

Comprehensive perception of burn conversion: a literature review.

Burn conversion is the conversion of stasis zone into both greater burn area and burn depth. It may hamper the patient's condition since morbidity and mortality are expected to be higher with the increase of burn size. To gain a comprehensive perception of burn conversion, this study aims to collect the latest updates regarding therapy, diagnosis, etiology and prognosis. A literature search was carried out on online databases, namely PubMed, SCOPUS and PROQUEST. The keywords "Burns AND (Conversion OR Progression OR Expansion)" were formulated. The inclusion and exclusion criteria were applied. Twenty-six articles were found, which were divided into diagnosis of burn conversion (11%), etiology of burn conversion (8%), prognosis of burn conversion (0%), and therapy of burn conversion (81%). All of the research was performed on animals. One of the best tools to diagnose burn conversion was the forwardlooking infrared (FLIR) imaging, having sensitivity up to 96% and specificity up to 100% to predict scar depth ≥3 mm. The main etiology was ischemia, reactive oxygen species and inflammation. Most of the research regarding therapy showed benefit in preventing burn conversion. However, no side effects were investigated and not all of the research was statistically significant. More research on burn conversion prognosis and treatment side effects should be performed. Further research involving trials in humans should be conducted, since animal and human trials may differ.

L'approfondissement d'une brûlure est la conversion de la zone d'ischémie en zone nécrosée, résultant en l'extension en profondeur et /ou en surface d'icelle, obérant ainsi les pronostics vital et fonctionnel. Cette étude a pour but de faire le point sur les données actuelles de la science pour ce qui en concerne l'étiologie, le diagnostic, le traitement et le pronostic. Nous avons exploré la littérature dans Pubmed, Scopus et Proquest, avec les mots clés « burns ¼ AND (conversion OR progression OR expansion), ce qui a ramené 26 articles. Le diagnostic était le sujet de 11% d'entre eux, l'étiologie de 8%, le traitement de 81%. Aucun ne traitait du pronostic. Le meilleur outil diagnostic est l'imagerie Forward Looking InfraRed (FLIR), qui a les meilleures sensibilité (jusqu'à 96%) et spécificité (jusqu'à 100%) pour prédire une atteinte sur plus de 3 mm de profondeur. Les causes principales sont l'ischémie, les radicaux oxygénés réactifs et l'inflammation. Le meilleur traitement est préventif, toutes les recherches ne donnant pas des résultats significatifs et aucune ne s'intéressant aux effets secondaires des options étudiées. Ainsi, la recherche, en particulier chez l'humain, doit s'intensifier dans ce champ.

Safety profile of atorvastatin in the role of burn wound injury conversion.

BACKGROUND: Atorvastatin could be beneficial in the treatment of burn patients to prevent burn wound progression from partial to full thickness. Our primary aim is to evaluate the safety of atorvastatin in burn patients. METHODS: Single center retrospective chart review of burn patients receiving atorvastatin during admission May 2016-May 2019 with historic controls was performed. Demographics, burn total body surface area, atorvastatin doses, creatinine phosphokinase, aspartate aminotransferase levels and adverse events were analyzed. RESULTS: 48 burn patients received atorvastatin during admission. Nine patients experienced elevated CK or AST levels during admission, but did not correlate with timing of atorvastatin administration and were comparable to levels in control patients. No adverse events associated with atorvastatin were identified. CONCLUSIONS: Atorvastatin administered to patients with burn injuries was not associated with any adverse events or attributable lab abnormalities. We believe that atorvastatin is safe to use in patients with burns and can be safely studied to determine the drug's effect on the prevention of burn wound conversion.

Eosinophilic recruitment in thermally injured older animals is associated with worse outcomes and higher conversion to full thickness burn.

BACKGROUND: Partial burn injury in older patients is associated with higher rates of morbidity, mortality, and conversion to full thickness burn (Finnerty et al., 2009; Pham et al., 2009). Both human and mouse models demonstrate an altered systemic immune response in older subjects, however less is known about the localized response (Jeschke et al., 2016; Farinas et al., 2018; Mohs et al., 2017). We hypothesized that a mouse model could demonstrate differences in the localized inflammatory response of the old. METHODS: Six old (66 weeks) and young (8 weeks) mice received partial thickness thermal burns. Localized and systemic expression of nine chemokines (TNFalpha, MCP-1, MIP-2, S100A9, EGF, IL-10, RANTES, G-CSF, and EOTAXIN) were evaluated at day 3 after burn using Luminex analysis. Vimentin immunostaining was used to evaluate injury depth. RESULTS: Vimentin staining demonstrated increased burn depth in old mice (449±38µm) as compared to young (166±18µm) (p<0.05). Both groups exhibited increased localized expression of EOTAXIN after burn (p<0.05), however expression in old mice (83.6±6.1pg/ml) was lower than that of young (126.8±18.7pg/ml) (p<0.05). Systemically, however, old mice had increased baseline EOTAXIN expression (1332.40±110.78pg/ml) compared to young (666.12±45.8pg/ml) (p<0.005). CONCLUSIONS: EOTAXIN is one of the primary chemoattractants for selective eosinophilic recruitment and activation. While eosinophils are important for wound healing, a hyperactive eosinophilic response can result in tissue damage. We hypothesize that the increased baseline serum EOTAXIN in the old may prime their hyperactive response, and may contribute to their worse clinical outcomes. Long-term eosinophil activation requires further study, however our findings indicate a role for EOTAXIN and eosinophils in burn response.

Indocyanine green dye angiography accurately predicts survival in the zone of ischemia in a burn comb model.

INTRODUCTION: Surgical evaluation of burn depth is performed via clinical observation, with only moderate reliability. While perfusion analysis has been proposed to enhance accuracy, no perfusion study has attempted to predict burn extension into the area of ischemia surrounding the original insult. We examined whether laser Doppler imaging (LDI) and indocyanine green (ICG) angiography predicted survival in the zone of ischemia in a porcine hot comb burn model. METHODOLOGY: Six full-thickness wounds were created on 5 female Yorkshire swine using a validated porcine hot comb burn model. 4 full-thickness burns were created separated by 3 unburned interspaces that represent the zone of ischemia. The interspaces between each comb burn were monitored using LDI and ICG Angiography at 1, 4, 24, and 48 h after burn. Interspace survival was assessed via gross observation and blinded histological readings 7 days after injury. RESULTS: ICG Angiographic assessments of burn perfusion were significantly different in viable vs. non-viable interspace perfusion at 1 h, 4 h, and 48 h. Temporal plotting of a trend-line derived from quantitative perfusion measurements rendered two distinct graphs, allowing for the derivation of a predictive algorithm to separate viable and non-viable interspaces. LDI revealed no such prognostic trend. CONCLUSION: Results from a validated porcine burn comb model suggest that ICG angiography has significant potential in the prediction of burn progression early after burn. However, the full potential of this technology cannot be determined until completion of clinical trials.