Cutaneous burn injury represents a major risk factor for the development of traumatic ectopic bone formation following blast-related extremity injury.

Blast-related traumatic heterotopic ossification (tHO) impacts clinical outcomes in combat-injured patients, leading to delayed wound healing, inflammatory complications, and reduced quality of life. Blast injured patients often have significant burns. This study investigated whether a partial thickness thermal burn injury exacerbates blast-related tHO in a clinically relevant polytrauma animal model. Adult male Sprague Dawley rats were subjected to an established model involving a whole-body blast overpressure exposure (BOP), complex extremity trauma followed by hind limb amputation (CET) followed by the addition of a 10 % total body surface area (TBSA) second degree thermal burn (BU). Micro-CT scans on post-operative day 56 showed a significant increase in HO volume in the CET + BU as compared to the CET alone injury group (p < .0001; 22.83 ± 3.41 mm3 vs 4.84 ± 5.77 mm3). Additionally, CET + BU concomitant with BOP significantly increased HO (p < .0001; 34.95 ± 7.71 mm3) as compared to CET + BU alone, confirming BOP has a further synergistic effect. No HO was detectable in rats in the absence of CET. Serum analysis revealed similar significant elevated (p < .0001) levels of pro-inflammatory markers (Cxcl1 and Il6) at 6 h post-injury (hpi) in the CET + BU and BOP + CET + BU injury groups as compared to naïve baseline values. Real-time qPCR demonstrated similar levels of chondrogenic and osteogenic gene expression in muscle tissue at the site of injury at 168 hpi in both the CET + BU and BOP+CET + BU injury groups. These results support the hypothesis that a 10 % TBSA thermal burn markedly enhances tHO following acute musculoskeletal extremity injury in the presence and absence of blast overpressure. Furthermore, the influence of BOP on tHO cannot be accounted for either in regards to systemic inflammation induced from remote injury or inflammatory-osteo-chondrogenic expression changes local to the musculoskeletal trauma, suggesting that another mechanism beyond BOP and BU synergistic effects are at play. Therefore, these findings warrant future investigations to explore other mechanisms by which blast and burn influence tHO, and testing prophylactic measures to mitigate the local and systemic inflammatory effects of these injuries on development of HO.

Inhibition of focal adhesion kinase 2 results in a macrophage polarization shift to M2 which attenuates local and systemic inflammation and reduces heterotopic ossification after polysystem extremity trauma.

Introduction: Heterotopic ossification (HO) is a complex pathology often observed in combat injured casualties who have sustained severe, high energy polytraumatic extremity injuries. Once HO has developed, prophylactic therapies are limited outside of surgical excision. Tourniquet-induced ischemia injury (IR) exacerbates trauma-mediated musculoskeletal tissue injury, inflammation, osteogenic progenitor cell development and HO formation. Others have shown that focal adhesion kinase-2 (FAK2) plays a key role in regulating early inflammatory signaling events. Therefore, we hypothesized that targeting FAK2 prophylactically would mitigate extremity trauma induced IR inflammation and HO formation. Methods: We tested whether the continuous infusion of a FAK2 inhibitor (Defactinib, PF-573228; 6.94 µg/kg/min for 14 days) can mitigate ectopic bone formation (HO) using an established blast-related extremity injury model involving femoral fracture, quadriceps crush injury, three hours of tourniquet-induced limb ischemia, and hindlimb amputation through the fracture site. Tissue inflammation, infiltrating cells, osteogenic progenitor cell content were assessed at POD-7. Micro-computed tomography imaging was used to quantify mature HO at POD-56. Results: In comparison to vehicle control-treated rats, FAK2 administration resulted in no marked wound healing complications or weight loss. FAK2 treatment decreased HO by 43%. At POD-7, marked reductions in tissue proinflammatory gene expression and assayable osteogenic progenitor cells were measured, albeit no significant changes in expression patterns of angiogenic, chondrogenic and osteogenic genes. At the same timepoint, injured tissue from FAK-treated rats had fewer infiltrating cells. Additionally, gene expression analyses of tissue infiltrating cells resulted in a more measurable shift from an M1 inflammatory to an M2 anti-inflammatory macrophage phenotype in the FAK2 inhibitor-treated group. Discussion: Our findings suggest that FAK2 inhibition may be a novel strategy to dampen trauma-induced inflammation and attenuate HO in patients at high risk as a consequence of severe musculoskeletal polytrauma.

Systemic inflammation induced from remote extremity trauma is a critical driver of secondary brain injury.

Blast exposure, commonly experienced by military personnel, can cause devastating life-threatening polysystem trauma. Despite considerable research efforts, the impact of the systemic inflammatory response after major trauma on secondary brain injury-inflammation is largely unknown. The aim of this study was to identify markers underlying the susceptibility and early onset of neuroinflammation in three rat trauma models: (1) blast overpressure exposure (BOP), (2) complex extremity trauma (CET) involving femur fracture, crush injury, tourniquet-induced ischemia, and transfemoral amputation through the fracture site, and (3) BOP+CET. Six hours post-injury, intact brains were harvested and dissected to obtain biopsies from the prefrontal cortex, striatum, neocortex, hippocampus, amygdala, thalamus, hypothalamus, and cerebellum. Custom low-density microarray datasets were used to identify, interpret and visualize genes significant (p < 0.05 for differential expression [DEGs]; 86 neuroinflammation-associated) using a custom python-based computer program, principal component analysis, heatmaps and volcano plots. Gene set and pathway enrichment analyses of the DEGs was performed using R and STRING for protein-protein interaction (PPI) to identify and explore key genes and signaling networks. Transcript profiles were similar across all regions in naïve brains with similar expression levels involving neurotransmission and transcription functions and undetectable to low-levels of inflammation-related mediators. Trauma-induced neuroinflammation across all anatomical brain regions correlated with injury severity (BOP+CET > CET > BOP). The most pronounced differences in neuroinflammatory-neurodegenerative gene regulation were between blast-associated trauma (BOP, BOP+CET) and CET. Following BOP, there were few DEGs detected amongst all 8 brain regions, most were related to cytokines/chemokines and chemokine receptors, where PPI analysis revealed Il1b as a potential central hub gene. In contrast, CET led to a more excessive and diverse pro-neuroinflammatory reaction in which Il6 was identified as the central hub gene. Analysis of the of the BOP+CET dataset, revealed a more global heightened response (Cxcr2, Il1b, and Il6) as well as the expression of additional functional regulatory networks/hub genes (Ccl2, Ccl3, and Ccl4) which are known to play a critical role in the rapid recruitment and activation of immune cells via chemokine/cytokine signaling. These findings provide a foundation for discerning pathophysiological consequences of acute extremity injury and systemic inflammation following various forms of trauma in the brain.

Tourniquet-induced ischemia creates increased risk of organ dysfunction and mortality following delayed limb amputation.

Tourniquets are critical for the control of traumatic extremity hemorrhage. In this study, we sought to determine, in a rodent blast-related extremity amputation model, the impact of prolonged tourniquet application and delayed limb amputation on survival, systemic inflammation, and remote end organ injury. Adult male Sprague Dawley rats were subjected to blast overpressure (120±7 kPa) and orthopedic extremity injury consisting femur fracture, one-minute soft tissue crush injury (20 psi), ± 180 min of tourniquet-induced hindlimb ischemia followed by delayed (60 min of reperfusion) hindlimb amputation (dHLA). All animals in the non-tourniquet group survived whereas 7/21 (33%) of the animals in the tourniquet group died within the first 72 h with no deaths observed between 72 and 168 h post-injury. Tourniquet induced ischemia-reperfusion injury (tIRI) likewise resulted in a more robust systemic inflammation (cytokines and chemokines) and concomitant remote pulmonary, renal, and hepatic dysfunction (BUN, CR, ALT. AST, IRI/inflammation-mediated genes). These results indicate prolonged tourniquet application and dHLA increases risk of complications from tIRI, leading to greater risk of local and systemic complications including organ dysfunction or death. We thus need enhanced strategies to mitigate the systemic effects of tIRI, particularly in the military prolonged field care (PFC) setting. Furthermore, future work is needed to extend the window within which tourniquet deflation to assess limb viability remains feasible, as well as new, limb-specific or systemic point of care tests to better assess the risks of tourniquet deflation with limb preservation in order to optimize patient care and save both limb and life.

Systematic Identification of the Optimal Housekeeping Genes for Accurate Transcriptomic and Proteomic Profiling of Tissues following Complex Traumatic Injury.

Trauma triggers critical molecular and cellular signaling cascades that drive biological outcomes and recovery. Variations in the gene expression of common endogenous reference housekeeping genes (HKGs) used in data normalization differ between tissue types and pathological states. Systematically, we investigated the gene stability of nine HKGs (Actb, B2m, Gapdh, Hprt1, Pgk1, Rplp0, Rplp2, Tbp, and Tfrc) from tissues prone to remote organ dysfunction (lung, liver, kidney, and muscle) following extremity trauma. Computational algorithms (geNorm, Normfinder, ΔCt, BestKeeper, RefFinder) were applied to estimate the expression stability of each HKG or combinations of them, within and between tissues, under both steady-state and systemic inflammatory conditions. Rplp2 was ranked as the most suitable in the healthy and injured lung, kidney, and skeletal muscle, whereas Rplp2 and either Hprt1 or Pgk1 were the most suitable in the healthy and injured liver, respectively. However, the geometric mean of the three most stable genes was deemed the most stable internal reference control. Actb and Tbp were the least stable in normal tissues, whereas Gapdh and Tbp were the least stable across all tissues post-trauma. Ct values correlated poorly with the translation from mRNA to protein. Our results provide a valuable resource for the accurate normalization of gene expression in trauma-related experiments.

The Role of Estrogen and Thyroid Hormones in Zebrafish Visual System Function.

Visual system development is a highly complex process involving coordination of environmental cues, cell pathways, and integration of functional circuits. Consequently, a change to any step, due to a mutation or chemical exposure, can lead to deleterious consequences. One class of chemicals known to have both overt and subtle effects on the visual system is endocrine disrupting compounds (EDCs). EDCs are environmental contaminants which alter hormonal signaling by either preventing compound synthesis or binding to postsynaptic receptors. Interestingly, recent work has identified neuronal and sensory systems, particularly vision, as targets for EDCs. In particular, estrogenic and thyroidogenic signaling have been identified as critical modulators of proper visual system development and function. Here, we summarize and review this work, from our lab and others, focusing on behavioral, physiological, and molecular data collected in zebrafish. We also discuss different exposure regimes used, including long-lasting effects of developmental exposure. Overall, zebrafish are a model of choice to examine the impact of EDCs and other compounds targeting estrogen and thyroid signaling and the consequences of exposure in visual system development and function.

Time dependent effects of prolonged hyperglycemia in zebrafish brain and retina.

Prolonged hyperglycemia causes long-term vision complications and an increased risk of cognitive deficits. High blood sugar also confers an osmotic load/stress to cells. We assessed behavioral and neurochemical changes in zebrafish brain and retina following prolonged hyperglycemia for 4-weeks or 8-weeks. At each time point, behavior was assessed using 3-chamber choice task and optomotor response; tissue was then collected and levels of inflammatory markers, tight junction proteins, and neurotransmitters determined using Western Blots. After 4-weeks, brain levels of v-rel reticuloendotheliosis viral oncogene homolog A (avian) (RelA; NF-kB subunit), IkB kinase (IKK), and glial fibrillary acidic protein (GFAP) were significantly elevated; differences in zonula occludens-1 (ZO-1), claudin-5, glutamic acid decarboxylase (GAD), and tyrosine hydroxylase (TH) were not significant. In retina, significant differences were observed only for TH (decreased), Rel A (increased), and GFAP (increased) levels. Glucose-specific differences in initial choice latency and discrimination ratios were also observed. After 8-weeks, RelA, GAD, and TH were significantly elevated in both tissues; IKK and GFAP levels were also elevated, though not significantly. ZO-1 and claudin-5 levels osmotically decreased in retina but displayed an increasing trend in glucose-treated brains. Differences in discrimination ratio were driven by osmotic load. OMRs increased in glucose-treated fish at both ages. In vivo analysis of retinal vasculature suggested thicker vessels after 4-weeks, but thinner vessels at 8-weeks. In vitro, glucose treatment reduced formation of nodes and meshes in 3B-11 endothelial cells, suggesting a reduced ability to form a vascular network. Overall, hyperglycemia triggered a strong inflammatory response causing initial trending changes in tight junction and neuronal markers. Most differences after 4-weeks of exposure were observed in glucose-treated fish suggesting effects on glucose metabolism independent of osmotic load. After 8-weeks, the inflammatory response remained and glucose-specific effects on neurotransmitter markers were observed. Osmotic differences impacted cognitive behavior and retinal protein levels; protein levels in brain displayed glucose-driven changes. Thus, we not only observed differential sensitivities of retina and brain to glucose-insult, but also different cellular responses, suggesting hyperglycemia causes complex effects at the cellular level and/or that zebrafish are able to compensate for the continued high blood glucose levels.

Alternate Immersion in Glucose to Produce Prolonged Hyperglycemia in Zebrafish.

Zebrafish (Danio rerio) are an excellent model to investigate the effects of chronic hyperglycemia, a hallmark of Type II Diabetes Mellitus (T2DM). This alternate immersion protocol is a noninvasive, step-wise method of inducing hyperglycemia for up to eight weeks. Adult zebrafish are alternately exposed to sugar (glucose) and water for 24 hours each. The zebrafish begin treatment in a 1% glucose solution for 2 weeks, then a 2% solution for 2 weeks, and finally a 3% solution for the remaining 4 weeks. Compared to water-treated (stress) and mannitol-treated (osmotic) controls, glucose-treated zebrafish have significantly higher blood sugar levels. The glucose-treated zebrafish show blood sugar levels of 3-times that of controls, suggesting that after both four and eight weeks hyperglycemia can be achieved. Sustained hyperglycemia was associated with increased Glial Fibrillary Acidic Protein (GFAP) and increased nuclear factor Kappa B (NF-kB) levels in retina and decreased physiological responses, as well as cognitive deficits suggesting this protocol can be used to model disease complications.

The Three-Chamber Choice Behavioral Task using Zebrafish as a Model System.

Neurodegenerative diseases are age-dependent, debilitating, and incurable. Recent reports have also correlated hyperglycemia with changes in memory and/or cognitive impairment. We have modified and developed a three-chamber choice cognitive task similar to that used with rodents for use with hyperglycemic zebrafish. The testing chamber consists of a centrally located starting chamber and two choice compartments on either side, with a shoal of conspecifics used as the reward. We provide data showing that once acquired, zebrafish remember the task at least 8 weeks later. Our data indicate that zebrafish respond robustly to this reward, and we have identified cognitive deficits in hyperglycemic fish after 4 weeks of treatment. This behavioral assay may also be applicable to other studies related to cognition and memory.

Alarming Cargo: The Role of Exosomes in Trauma-Induced Inflammation.

Severe polytraumatic injury initiates a robust immune response. Broad immune dysfunction in patients with such injuries has been well-documented; however, early biomarkers of immune dysfunction post-injury, which are critical for comprehensive intervention and can predict the clinical course of patients, have not been reported. Current circulating markers such as IL-6 and IL-10 are broad, non-specific, and lag behind the clinical course of patients. General blockade of the inflammatory response is detrimental to patients, as a certain degree of regulated inflammation is critical and necessary following trauma. Exosomes, small membrane-bound extracellular vesicles, found in a variety of biofluids, carry within them a complex functional cargo, comprised of coding and non-coding RNAs, proteins, and metabolites. Composition of circulating exosomal cargo is modulated by changes in the intra- and extracellular microenvironment, thereby serving as a homeostasis sensor. With its extensively documented involvement in immune regulation in multiple pathologies, study of exosomal cargo in polytrauma patients can provide critical insights on trauma-specific, temporal immune dysregulation, with tremendous potential to serve as unique biomarkers and therapeutic targets for timely and precise intervention.

Using a variant of the optomotor response as a visual defect detection assay in zebrafish.

We describe a visual stimulus that can be used with both larval and adult zebrafish (Danio rerio). This protocol is a modification of a standard visual behavior analysis, the optomotor response (OMR). The OMR is often used to determine the spatial response or to detect directional visuomotor deficiencies. An OMR can be generated using a high contrast grated pattern, typically vertical bars. The spatial sensitivity is measured by detection and response to a change in grating bar width and is reported in cycles per degree (CPD). This test has been used extensively with zebrafish larvae and adults to identify visual- and/or motor-based mutations. Historically, when tested in adults, the grated pattern was presented from a vertical perspective, using a rotating cylinder around a holding tank, allowing the grating to be seen solely from the sides and front of the organism. In contrast, OMRs in zebrafish larvae are elicited using a stimulus projected below the fish. This difference in methodology means that two different experimental set-ups are required: one for adults and one for larvae. Our visual stimulus modifies the stimulation format so that a single OMR stimulus, suitable for use with both adults and larvae, is being presented underneath the fish. Analysis of visuomotor responses using this method does not require costly behavioral tracking software and, using a single behavioral paradigm, allows the observer to rapidly determine visual spatial response in both zebrafish larvae and adults.

The role of neutrophil extracellular traps and TLR signaling in skeletal muscle ischemia reperfusion injury.

Ischemia reperfusion (IR) injury results in devastating skeletal muscle fibrosis. Here, we recapitulate this injury with a mouse model of hindlimb IR injury which leads to skeletal muscle fibrosis. Injury resulted in extensive immune infiltration with robust neutrophil extracellular trap (NET) formation in the skeletal muscle, however, direct targeting of NETs via the peptidylarginine deiminase 4 (PAD4) mechanism was insufficient to reduce muscle fibrosis. Circulating levels of IL-10 and TNFα were significantly elevated post injury, indicating toll-like receptor (TLR) signaling may be involved in muscle injury. Administration of hydroxychloroquine (HCQ), a small molecule inhibitor of TLR7/8/9, following injury reduced NET formation, IL-10, and TNFα levels and ultimately mitigated muscle fibrosis and improved myofiber regeneration following IR injury. HCQ treatment decreased fibroadipogenic progenitor cell proliferation and partially inhibited ERK1/2 phosphorylation in the injured tissue, suggesting it may act through a combination of TLR7/8/9 and ERK signaling mechanisms. We demonstrate that treatment with FDA-approved HCQ leads to decreased muscle fibrosis and increased myofiber regeneration following IR injury, suggesting short-term HCQ treatment may be a viable treatment to prevent muscle fibrosis in ischemia reperfusion and traumatic extremity injury.