Abdikarim Abdullahi

Animal models in burn research

Burn injury is a severe form of trauma affecting more than 2 million people in North America each year. Burn trauma is not a single pathophysiological event but a devastating injury that causes structural and functional deficits in numerous organ systems. Due to its complexity and the involvement of multiple organs, in vitro experiments cannot capture this complexity nor address the pathophysiology. In the past two decades, a number of burn animal models have been developed to replicate the various aspects of burn injury, to elucidate the pathophysiology, and to explore potential treatment interventions. Understanding the advantages and limitations of these animal models is essential for the design and development of treatments that are clinically relevant to humans. This review aims to highlight the common animal models of burn injury in order to provide investigators with a better understanding of the benefits and limitations of these models for translational applications. While many animal models of burn exist, we limit our discussion to the skin healing of mouse, rat, and pig. Additionally, we briefly explain hypermetabolic characteristics of burn injury and the animal model utilized to study this phenomena. Finally, we discuss the economic costs associated with each of these models in order to guide decisions of choosing the appropriate animal model for burn research.

Burn Induces Browning of the Subcutaneous White Adipose Tissue in Mice and Humans

SUMMARY Burn is accompanied by long-lasting immunometabolic alterations referred to as hypermetabolism that are characterized by a considerable increase in resting energy expenditure and substantial whole-body catabolism. In burned patients, the length and magnitude of the hypermetabolic state is the highest of all patients and associated with profoundly increased morbidity and mortality. Unfortunately, the mechanisms involved in hypermetabolism are essentially unknown. We hypothesized that the adipose tissue plays a central role for the induction and persistence of hypermetabolism post-burn injury. Here, we show that burn induces a switch in the phenotype of the subcutaneous fat from white to beige, with associated characteristics such as increased mitochondrial mass and UCP1 expression. Our results further demonstrate the significant role of catecholamines and interleukin-6 in this process. We conclude that subcutaneous fat remodeling and browning represent an underlying mechanism that explains the elevated energy expenditure in burn-induced hypermetabolism.

Pathophysiologic response to burns in the elderly

Over the last decades advancements have improved survival and outcomes of severely burned patients except one population, elderly. The Lethal Dose 50 (LD50) burn size in elderly has remained the same over the past three decades, and so has morbidity and mortality, despite the increased demand for elderly burn care. The objective of this study is to gain insights on why elderly burn patients have had such a poor outcome when compared to adult burn patients. The significance of this project is that to this date, burn care providers recognize the extreme poor outcome of elderly, but the reason remains unclear. In this prospective translational trial, we have determined clinical, metabolic, inflammatory, immune, and skin healing aspects. We found that elderly have a profound increased mortality, more premorbid conditions, and stay at the hospital for longer, p < 0.05. Interestingly, we could not find a higher incidence of infection or sepsis in elderly, p > 0.05, but a significant increased incidence of multi organ failure, p < 0.05. These clinical outcomes were associated with a delayed hypermetabolic response, increased hyperglycemic and hyperlipidemic responses, inversed inflammatory response, immune-compromisation and substantial delay in wound healing predominantly due to alteration in characteristics of progenitor cells, p < 0.05. In summary, elderly have substantially different responses to burns when compared to adults associated with increased morbidity and mortality. This study indicates that these responses are complex and not linear, requiring a multi-modal approach to improve the outcome of severely burned elderly.

Burn plus lipopolysaccharide augments endoplasmic reticulum stress and NLRP3 inflammasome activation and reduces PGC-1α in liver.

ABSTRACT Extensively burned patients often suffer from sepsis (especially caused by Pseudomonas aeruginosa), which may prolong metabolic derangement, contribute to multiple organ failure, and increase mortality. The molecular and cellular mechanisms of such infection-related metabolic derangement and organ dysfunction are unclear. We have previously shown that severely burned patients have significant and persisting hepatic endoplasmic reticulum (ER) stress. We hypothesized that ER stress and the unfolded protein response correlate with NOD-like receptor, pyrin domain containing 3 (NLRP3) inflammasome activation in burn. These may trigger profound metabolic changes in the liver, which form the pathological basis of liver damage and liver dysfunction after burn injury. A two-hit rat model was established by a 60% total body surface area scald burn and intraperitoneal injection of P. aeruginosa–derived lipopolysaccharide (LPS) 3 days after burn. One day later, animals were killed, and liver tissue samples were collected for gene expression and protein analysis of NLRP3 inflammasome activation, ER stress, and glucose and lipid metabolism. Liver damage was assessed by plasma markers (alanine aminotransferase and aspartate aminotransferase) and liver immunohistochemical analysis. Our results showed that burn injury and LPS injection induced inflammasome activation in liver and augmented hepatic ER stress and liver damage. Although there was an increased metabolic demand after burn, hepatic NLRP3 inflammasome activation corresponded to inhibition of PGC-1&agr; (peroxisome proliferator-activated receptor &ggr;-coactivator 1&agr;) and its upstream regulators protein kinase A catalyst unit, AMP-activated protein kinase &agr;, and sirtuin-1 may provide a mechanism for the enhanced metabolic derangement after major burn injury plus sepsis. In conclusion, burn + LPS augments inflammasome activation and ER stress in liver, which in turn contribute to postburn metabolic derangement.

Glucose Control in Severely Burned Patients Using Metformin: An Interim Safety and Efficacy Analysis of a Phase II Randomized Controlled Trial.

Objective: To determine whether metformin can achieve glucose control no worse than insulin (noninferiority) without the danger of hypoglycemia (superiority). In addition, to assess whether metformin has any additional effects on lipolysis and inflammation that will enhance burn recovery (superiority). Summary Background Data: Hyperglycemia and insulin resistance after burn injury are associated with increased morbidity and mortality. Insulin administration improves postburn infections, severity of sepsis, and morbidity, but also causes a 4–5-fold increase in hypoglycemia, which is associated with a 9-fold increase in mortality. Methods: Severely burned adult patients with burns over 20% total body surface area (TBSA) burn were prospectively randomized in this Phase II clinical trial to either metformin or insulin (standard of care) treatment. Primary outcomes were glucose levels and incidence of hypoglycemia. Secondary outcomes included glucose and fat metabolism, and clinical outcomes. Results: Forty-four patients were enrolled in this Phase II clinical trial, 18 metformin and 26 insulin patients. Demographics, burn size, concomitant injuries, and mortality were comparable between both groups. Metformin controlled blood glucose as equally as insulin with no difference between the 2 treatment groups, P > 0.05. While there was a 15% incidence of hypoglycemia in the insulin group, there was only 1 mild hypoglycemic episode (6%) in the metformin group, P < 0.05. Oral glucose tolerance tests at discharge revealed that metformin significantly improved insulin sensitivity, P < 0.05. Furthermore, metformin had a strong antilipolytic effect after burn injury when compared with insulin and was associated with significantly reduced inflammation, P < 0.05. Conclusions: Metformin decreases glucose equally as effective as insulin without causing hypoglycemia, with additional benefits including improved insulin resistance and decreased endogenous insulin synthesis when compared with insulin controls. These results indicate that metformin is safe in burn patients and further supports the use of metformin in severely burned patients for postburn control of hyperglycemia and insulin resistance.

Il-6 Signal From the Bone Marrow is Required for the Browning of White Adipose Tissue Post Burn Injury

ABSTRACT The hypermetabolic stress response after burn contributes to multi-organ failure, sepsis, morbidity, and mortality. The cytokine interleukin 6 (IL-6) has been hypothesized to mediate not only white adipose tissue (WAT) browning in burns, but also other hypermetabolic conditions. In addition to its inflammatory effects, IL-6 also acts as a metabolic mediator that affects metabolic tissues. Therefore, we sought to uncover the origin of circulating IL-6 post burn injury that regulates WAT browning. WAT and sera samples were collected from both adult burn patients admitted to the Ross Tilley Burn Centre at Sunnybrook Hospital and mice subjected to a burn injury. Collected tissues were analyzed for browning markers and metabolic state via histology, gene expression, and resting energy expenditure. Increased WAT browning was observed in burn patients as well as mice subjected to burn injury. Circulating IL-6 levels were significantly elevated post burn injury in mice (<0.05) and in burn patients (<0.05), the latter of which was positively correlated with elevated REE. Genetic loss of whole body IL-6 in mice prevented burn-induced WAT browning. Transplanting IL-6 knockout (KO) mice with bone marrow (BM) from wild-type (WT) mice, recovered the browning phenotype in these mice, as evaluated by increased uncoupling protein 1 (UCP1) expression (<0.05). Conversely, transplanting irradiated WT mice with BM from IL-6 KO mice impaired burn induced browning with no significant expression of UCP1. Together, our findings implicate BM derived IL-6 as the source controlling browning of WAT post burn injury. Thus, targeting IL-6 is a promising target for hypermetabolism in burns.

Taming the Flames: Targeting White Adipose Tissue Browning in Hypermetabolic Conditions

In this era of increased obesity and diabetes prevalence, the browning of white adipose tissue (WAT) has emerged as a promising therapeutic target to induce weight loss and improve insulin sensitivity in this population. The browning process entails a shift in the WAT from primarily storing excess energy to the dissipation of energy as heat. However, this idealistic view of WAT browning being the savior of the metabolic syndrome has been criticized by studies in burn and cancer patients that have shown browning to be detrimental rather than beneficial. In fact, in the context of hypermetabolic states, the browning of WAT has presented with substantial clinical adverse outcomes related to cachexia, hepatic steatosis, and muscle catabolism. Therefore, the previous thought construct of understanding browning as an all-beneficial physiologic event has now been met with skepticism. In this review, we focus on current knowledge of browning of WAT and its adverse metabolic alterations during hypermetabolic states. We also discuss the regulators and signaling pathways involved in the browning process and their potential for being targeted by new or existing drugs to inhibit or alleviate browning, potentially leading to decreased hypermetabolism and improved clinical outcomes. Lastly, the imminent clinical applications of pharmacological agents are explored in the perspective of attenuating WAT browning and its associated adverse side effects reported in burn patients.

Lipidomic analysis enables prediction of clinical outcomes in burn patients

Recent discoveries have highlighted the novel metabolic functions of adipose tissue in enhancing hypermetabolism after trauma. As the exact function and expression profiles of serum lipids and free fatty acids (FFA) are essentially unknown, we determined the lipidomic expression profile after burn in correlation to clinical outcomes to identify important lipid mediators affecting post-burn outcomes. We conducted a prospective cohort study with 46 adult burn patients and 5 healthy controls at the Ross Tilley Burn Center in Toronto, Canada. Patients were stratified based on major demographic and clinical variables, including age, burn severity, mortality, and sepsis. Serum FFAs and inflammatory markers were measured during acute hospital stay. We found that FFAs were acutely elevated post-burn and returned to baseline over time. Greater burn severity and age were associated with an impaired acute response in unsaturated FFAs and pro-inflammatory cytokines. Elevations in saturated and mono-unsaturated FFAs correlated significantly to increased mortality. In summary, persistent elevation of unsaturated lipids was associated with a functionally altered inflammatory-immunological milieu and worse clinical outcomes. The present lipidomic analysis indicates profound alterations in the lipid profile after burn by characterizing key lipids as potential diagnostic and outcome indicators in critically injured patients.

Modeling Acute ER Stress in vivo and in vitro.

ABSTRACT The endoplasmic reticulum (ER) is a critical organelle that synthesizes secretory proteins and serves as the main calcium storage site of the cell. The accumulation of unfolded proteins at the ER results in ER stress. Although the association between ER stress and the pathogenesis of many metabolic conditions have been well characterized using both in vivo and in vitro models, no standardized model concerning ER stress exists. Here, we report a standardized model of ER stress using two well-characterized ER stress-inducing agents, thapsigargin and tunicamycin. Our aim in this current study was 2-fold: to characterize and establish which agent is optimal for in vitro use to model acute ER stress and to evaluate which agent is optimal for in vivo use. To study the first aim we used two well-established metabolic cell lines; human hepatocellular carcinoma (HepG2s) and differentiated mouse adipocytes (3T3-L1). In the second aim we utilized C57BL/6J mice that were randomized into three treatment groups of sham, thapsigargin, and tunicamycin. Our in vitro results showed that tunicamycin worked as a rapid and efficacious inducer of ER stress in adipocytes consistently, whereas thapsigargin and tunicamycin were equally effective in inducing ER stress in hepatocytes. In regards to our in vivo results, we saw that tunicamycin was superior in not only inducing ER stress but also recapturing the metabolic alterations associated with ER stress. Thus, our findings will help guide and inform researchers as to which ER stress agent is appropriate with regards to their model.

Metformin adapts its cellular effects to bioenergetic status in a model of metabolic dysfunction

Thermal injury induces a complex immunometabolic response, characterized by hyperglycemia, extensive inflammation and persistent hypermetabolism. It has been suggested that attenuation of the hypermetabolic response is beneficial for patient wellbeing. To that effect, metformin represents an attractive therapeutic agent, as its effects on glycemia, inflammation and bioenergetics can improve outcomes in burn patients. Therefore, we studied metformin and its effects on mitochondrial bioenergetics in a murine model of thermal injury. We set out to determine the impact of this agent on mitochondrial hypermetabolism (adult mice) and mitochondrial dysfunction (aged mice). Seahorse respirometry complimented by in-gel activity assays were used to elucidate metformin’s cellular mechanism. We found that metformin exerts distinctly different effects, attenuating the hypermetabolic mitochondria of adult mice while significantly improving mitochondrial bioenergetics in the aged mice. Furthermore, we observed that these changes occur both with and without adenosine monophosphate kinase (AMPK) activation, respectively, and analyzed damage markers to provide further context for metformin’s beneficial actions. We suggest that metformin has a dual role following trauma, acting via both AMPK-dependent and independent pathways depending on bioenergetic status. These findings help further our understanding of metformin’s biomolecular effects and support the continued use of this drug in patients.