Sham Burn Research Articles

A Shift in Myeloid Cell Phenotype via Down Regulation of Siglec-1 in Island Macrophages of Bone Marrow Is Associated With Decreased Late Erythroblasts Seen in Anemia of Critical Illness

Burn injury has been shown to significantly dampen erythropoiesis in both burn patients and in murine models. Our previous findings elucidated the erythropoietin independent defects in red cell development stages involving erythroid progenitor production and late stage erythroblast enucleation processes. We hypothesized that macrophages (MØ) in erythroblast islands (EBI) could be yet another roadblock impeding erythropoiesis following burn injury. Here we highlight that the methodology to study EBI can be achieved with single cell suspensions using a simple technique such as flow cytometry, as obtaining the central erythroblast island macrophages (EBIMØs) of interest is a delicate process. We elucidated the requisite of EBIMØ from the erythroblast as well as the MØ perspective. In addition to the primary erythropoiesis organ, the bone marrow (BM), spleens were also examined for extra-medullary erythropoiesis. Femurs and spleens were harvested from adult mice (B6D2F1) subjected to 15% total body surface area (TBSA) scald burn (B) or sham burn (S). Total bone marrow cells (TBM) and splenocytes were probed for total erythrons, early and late erythroblasts and EBIMØ by flow cytometry. There was only a marginal increase in the number of EBIMØ after burn, but, between the signatures of EBIMØ, Siglec-1 expression (MFI) was reduced by 40% in B with and a parallel 44% decrease in TBM erythrons in the BM. There were more (2.5-fold) EEBs and less LEBs (2.4-fold) per million TBM cells in B; with a corresponding decrease in Siglec-1 and Ly6G expressions in EBIMØ associated with EEB. Conversely, extra-medullary erythropoiesis was robust in spleens from B. Not only were the numbers of EBIMØs increased in B (p < 0.002), both EEBs and LEBs associated with EBIMØ were higher by 30 and 75%, respectively. Importantly, an increase in Siglec-1 and Vcam1 expressing F480+ splenic macrophages was observed after burn injury. Therefore, stagnant EEBs in the BM after burn injury could be due to low Siglec1 expressing EBIMØ, which perhaps impede their maturation into LEBs and reticulocytes. Repercussion of myeloid cell phenotype specific to BM after burn injury could plausibly account for a defective late stage RBC maturation resulting in anemia of critical illness.Summary Sentence: Characterization of erythroblast island macrophages (EBIMØ) in the bone marrow and spleen at different stages of erythropoiesis after burn injury.

Alteration of oxidative stress and expression of antioxidases in diaphragm of severely burned rats

Objective: To explore the occurrence of oxidative stress and antioxidases expression in diaphragm of severely burned rats, so that the mechanism of respiratory muscle atrophy and dysfunction post-burn injury will be further clarified. Methods: Eighty male Wistar rats (aged 7 to 8 weeks) were divided into sham injury group and burn injury group according to the random number table, with 40 rats in each group. Rats in burn injury group were inflicted with 50% total body surface area full-thickness scald (hereinafter referred to as burn) on the back and abdomen by immersing into 80 ℃ water for 15 s and 8 s respectively. Immediately after injury, 40 mL/kg normal saline was injected through abdomen for resuscitation, and the wounds were treated with iodine. Except for immersing into 37 ℃ warm water and no resuscitation, the other treatments of rats in sham injury group were the same as those of burn injury group. Whole diaphragms of 8 rats per time point per group were collected after anesthesia at post injury hour (PIH) 2 and on post injury day (PID) 1, 3, 7, and 14, and muscle mass was determined. The protein carbonyl content was determined by microplate reader. The protein expressions of catalase, superoxide dismutase 2 (SOD2), and glutathione peroxidase 1 were determined by Western blotting. Data were processed with analysis of variance of factorial design, t test, and Bonferroni correction. Results: (1) There were no statistically significant differences in the diaphragm mass of rats between the 2 groups at PIH 2 and on PID 1 (t=0.453, 0.755, P>0.05). The diaphragm mass of rats in burn injury group started to decrease from PID 3, which was significantly lower than that of sham injury group (t=3.321, P<0.01). The diaphragm mass of rats in burn injury group started to increase from PID 7 to PID 14, which was significantly lower than that of sham injury group (t=4.622, 4.380, P<0.01). (2) Protein carbonyl content in diaphragm of rats in burn injury group at PIH 2, and on PID 1, 3, 7, and 14 [(2.7±0.3), (2.5±0.5), (2.4±0.4), (2.5±0.4), (3.2±0.6) pg/mL] was significantly higher than that of sham injury group respectively [(1.2±0.4), (1.6±0.3), (1.5±0.7), (1.7±0.3), (1.8±0.4) pg/mL, t=5.994, 3.263, 3.666, 3.158, 5.763, P<0.05 or P<0.01]. (3) Protein expressions of catalase in diaphragm of rats in burn injury group on PID 1 and 3 were close to those of sham injury group (t=0.339, 0.324, P>0.05). There were no statistically significant differences in protein expressions of SOD2 in diaphragm of rats between the 2 groups at PIH 2 and on PID 1, 3, 7, and 14 (t=1.446, 1.385, 0.757, 1.561, 0.531, P>0.05). There were no statistically significant differences in protein expressions of glutathione peroxidase 1 in diaphragm of rats in the 2 groups at PIH 2 and on PID 1, 3, and 7 (t=0.200, 0.729, 0.385, 1.559, P>0.05). Conclusions: Continuous oxidative stress and relatively insufficient expression of antioxidases in diaphragm induced by burn injury could be a contributor to diaphragm atrophy.

Role of Endoplasmic Reticulum Stress-Autophagy Axis in Severe Burn-Induced Intestinal Tight Junction Barrier Dysfunction in Mice.

Severe burn injury induces intestinal barrier dysfunction; however, the underlying mechanisms remain elusive. Our previous studies have shown that the intestinal epithelial tight junction (TJ) barrier dysfunction is associated with both endoplasmic reticulum (ER) stress and autophagy in severely burned mice, but the precise role of ER stress and autophagy in the burn-induced intestinal TJ barrier dysfunction needs to be determined. In this study, female C57/BL6 mice were assigned randomly to either sham burn or 30% total body surface area (TBSA) full-thickness burn. The effects of ER stress and autophagy on the intestinal epithelial TJ barrier were validated by inducing or inhibiting both ER stress and autophagy in mice treated with sham burn or burn injury. The intestinal permeability, expression, and localization of TJ proteins, ER stress, and autophagy were assessed by physiological, morphological, and biochemical analyses. The results showed that inducing ER stress with tunicamycin or thapsigargin caused the activation of autophagy, the increase of intestinal permeability, as well as the reduction and reorganization of TJ proteins in the sham-burned mice, and aggravated the burn-induced activation of autophagy, increase of intestinal permeability, as well as the reduction and reorganization of TJ proteins. In contrast, inhibiting ER stress with 4-phenylbutyrate alleviated the burn-induced activation of autophagy, increase of intestinal permeability, as well as the reduction and reorganization of TJ proteins. In addition, inducing autophagy with rapamycin resulted in the increase of intestinal permeability, as well as the reduction and reorganization of TJ proteins in the sham-burned mice, and aggravated the burn-induced increase of intestinal permeability as well as the reduction and reorganization of TJ proteins. However, inhibiting autophagy with 3-methyladenine attenuated the burn-induced increase of intestinal permeability, as well as the reduction and reorganization TJ proteins. It is suggested that the ER stress-autophagy axis contributes to the intestinal epithelial TJ barrier dysfunction after severe burn injury.

Effects of glutamine on skeletal muscle membrane repair in severely burned mice and the functional mechanism

Objective: To observe how glutamine affect the skeletal muscle membrane repair in severely burned mice through promoting the mitsugumin 53 (MG53) dimerization in skeletal muscle and to explore its functional mechanism. Methods: (1) Animal experiments. A total of 179 BALB/c male mice aged 6 to 8 weeks were divided into sham injury group (n=43), burn group (n=73) and burn+ glutamine group (n=63) according to the random number table (the same grouping method below). Mice in sham injury group were sham injured on the back, and mice in burn group and burn+ glutamine group were inflicted with 30% total body surface area full-thickness scald (hereinafter referred to as burn) on the back. Mice in burn+ glutamine group were intragastrically administered with glutamine (1 mg/kg), and the other two groups were given the same amount of amino acid solution once per day for 14 days. On post burn hour 12, 10 mice from burn group were taken for preparation of burn serum, which is used in the following cell experiments. Blood samples were collected from the hearts to prepare serum from 10 mice in sham injury group immediately after burn and from 10 mice in burn group and burn+ glutamine group on post burn day (PBD) 5, 10, and 14, respectively. And then the whole gastrocnemius muscle was harvested after the mice were sacrificed. On PBD 10, the whole flexor brevis digitorum was harvested from 6 mice in the 3 groups respectively after the mice were sacrificed. On PBD 5, 10, and 14, the whole gastrocnemius muscle tissue was harvested from another 9 mice in the 3 groups respectively after the mice were sacrificed. The mass of the whole gastrocnemius muscle of mice was weighed. The total protein content of gastrocnemius muscle of mice was detected by coomassie brilliant blue method. The repair function of myolemma of flexor brevis digitorum of mice was detected by two-photon laser fiber membrane perforating. The serum content of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) of mice was determined with radioimmunoassay. The expressions of MG53 dimer and monomer in gastrocnemius of mice were determined with non-reductive electrophoresis-Western blotting. The protein expressions of endoplasmic reticulum stress sign proteins CCAAT/enhancer binding protein homologous protein (CHOP) and glucose-regulated protein 78 (GRP78) in gastrocnemius of mice were determined with Western blotting. (2) Cell experiments. Mice skeletal muscle precursor cells C2C12 were cultured in vitro, and cells of the second passage were selected for the experiments. The cells were divided into normal control group, burn serum group, and burn serum+ glutamine group, with 3 dishes in each group and 1×10(3) cells in each dish. Cells in normal control group were cultured with 1 mL Dulbecco's modified Eagle medium (DMEM) with fetal bovine serum of volume fraction 10%, cells in burn serum group were cultured with 1 mL DMEM with burn serum of volume fraction 10%, and cells in burn serum+ glutamine group were cultured with 1 mL DMEM with burn serum of volume fraction 10% and 4 μL glutamine with a final molar concentration of 8 mmol/L. After 24 hours of culturing, the repair function of myocyte membrane after differentiation of skeletal muscle precursor cells in mice was detected with the same method before. Another cells were grouped and cultured as before, with 3 wells in each group and 1×10(5) cells in each well. After 24 hours of culturing, the expressions of MG53 dimer and monomer and endoplasmic reticulum stress marker proteins in the cells were detected as before. Data were processed with analysis of variance of factorial design, one-way analysis of variance, least significant difference t test, and Student Newman Keuls test. Results: Animal experiments. (1) Compared with those in sham injury group, the mass and total protein content of gastrocnemius muscle of mice in burn group were significantly decreased on PBD 5, 10, and 14 (P 0.05). Compared with 58.5±1.8 in normal control group, the expression of MG53 dimer of cells in burn serum group was significantly decreased after 24 hours of culturing (14.1±1.4, P<0.05). Compared with that in burn serum group, the expression of MG53 dimer of cells in burn serum+ glutamine group was significantly increased after 24 hours of culturing (30.9±0.6, P<0.05). Compared with those in normal control group, the protein expressions of CHOP and GRP78 of cells were significantly elevated in burn serum group after 24 hours of culturing (P<0.05). Compared with those in burn serum group, the protein expressions of CHOP and GRP78 of cells were significantly reduced in burn serum+ glutamine group after 24 hours of culturing (P<0.05). Conclusions: Glutamine can promote MG53 dimerization by alleviating endoplasmic reticulum stress in severely burned mice. Thus it can accelerate skeletal muscle membrane repair, reduce the local inflammatory reaction of skeletal muscle and consumption of skeletal muscle.

The Role of Beta‐2 Adrenergic Receptors in Cardiac Bioenergetics Following Severe Burns

Severe burn injury is characterized by increased energy expenditure, and altered mitochondrial function is thought to be central to the hypermetabolic response to burns. As catecholamines are an important driver of the hypermetabolic stress response to burn, we set out to determine the role of adrenergic signaling through Beta‐2 adrenergic receptors in cardiac mitochondrial function following burn. We hypothesized that a loss of adrenergic signaling would attenuate cardiac mitochondrial dysfunction in response to burns.We used beta ‐2 adrenergic receptor knockout mice (ADRB2‐KO) and wild type controls (WT). Mice received a 30% of total body surface area scald burn or a sham burn and were sacrificed one week following injury. Hearts were excised and mitochondrial function was measured in saponin‐permeabilized myofiber bundles by high‐resolution respirometry. Data were analyzed using two‐way ANOVA and Bonferroni post‐test to compare all groups.Mitochondrial respiratory capacity was significantly reduced in both WT (P&lt;0.01) and KO (P&lt;0.01) mice following burn, although there was no difference between WT and KO mice. In WT animals, respiration coupled to ATP production was significantly lower in the burn animals compared to shams (p&lt; 0.05). In contrast, respiration coupled to ATP production was not different in sham and burn treated ADRB2‐KO mice. Mitochondrial flux control ratios (leak and couple respiration normalized to maximal uncoupled respiration) were significantly higher in burned WT mice compared to sham (P&lt;0.05). However, mitochondrial flux control was not significantly altered by burn in KO mice.Collectively, these data underscore the role of catecholamines and adrenergic stress in the metabolic response to burns. Specifically, ablation of beta 2 adrenergic signaling in the heart prevents cardiac mitochondrial dysfunction in response to severe burn trauma.Support or Funding InformationThis study was supported by grants from the National Institute of Health (P50‐GM60338, R01‐GM56687, and GM 112936‐01), Shriners Hospitals for Children (80100, 87300, 71008, 70900, 85116, 71000), and SSF grant for Craig Porter. This study was also conducted with the support of the Institute for Translational Sciences at the University of Texas Medical Branch, supported in part by a Clinical and Translational Science Award (UL1TR000071) from the National Center for Advanc‐ing Translational Sciences, National Institutes of Health.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of Exendin-4 on pancreatic islets function in treating hyperglycemia post severe scald injury in rats.

It has been established that glucagon-like peptide 1 (GLP 1) inhibits pancreatic β-cell apoptosis, increases insulin secretion, and improves glucose tolerance in scald injury. However, the effects of Exendin-4, a long-acting incretin similar to GLP 1, remained unclear in severe scald injury. Hence, this study attempted to investigate whether Exendin-4 had similar effects by protecting the histology of pancreas in severely scalded rats. One hundred sixty-two adult Wistar rats were equally randomized to sham burn group, burn group and burn with Exendin-4 treatment group. Rats were subjected to full skin thickness scald injuries (total body surface area: 50%) and were injected subcutaneously with Exendin-4 (4 μg/kg) twice daily. The histological changes of islets, the apoptosis of β cells, the amount of glucagon and insulin, and the concentration of plasma glucagon and insulin were observed; and the intraperitoneal glucose tolerance test was performed as well. The islets and β cells were injured and the number of secretory granules decreased in the scalded rats, but less histopathological changes were seen in the rats treated with Exendin-4. The apoptosis index of treated rats was significantly lower than that of the scalded rats (p < 0.05). There was significant difference in β-cell density postinjury between the two groups (p < 0.05). More insulin and less glucagon in islets and plasma were found in the treated rats (p < 0.05), suggesting improved intraperitoneal glucose tolerance (p < 0.05) and fasting blood glucose (p < 0.05) in this group. Based on our previous finding that GLP-1 could control hyperglycemia by increasing insulin secretion and inhibiting β-cell apoptosis in severe scald injuries, this study further confirmed that Exendin-4 could increase glycemic control following severe scald by preserving the histology of β cells in pancreatic islets and inhibiting their apoptosis.

Beta3-Adrenergic Blockade Restores Erythropoietic Homeostasis in Anemia of Critical Illness

IntroductionDuring anemia of critical illness such as burn injury, erythropoietin (Epo) resistance is accompanied by impaired bone marrow (BM) erythropoiesis. The non-selective β1, β2 -adrenergic receptor (AR) blocker propranolol was effective in mitigating MafB expression in multi potential progenitors and rescuing their erythrocyte commitment in burn patients. However, peripheral HGB levels did not reflect the effect of propranolol perhaps due to defective maturation of late erythroblasts, not regulated by propranolol. Subsequently, we reported that early commitment and late maturation stages are independently orchestrated via discrete β2- and β3-AR mechanisms respectively in the BM of mice with a parallel increase in blood HGB levels. Nonetheless, it is not clear whether commitment of progenitors or maturation of erythroblasts is essential to restore erythropoietic homeostasis after burn injury. In adult mice, the spleen serves as an extra medullary erythropoietic organ under hypoxia and also functions as a primary organ of erythropoietic homeostasis (Bozzini CE et al. Am J Physiol. 1970; 219(3):724). We examined in mice, erythropoietic responses to burn injury in two organs, the spleen and BM to understand the reciprocal relationship, if any, in erythropoiesis between the two adrenergically innervated organs in response to propranolol, and selective β2- and β3- AR blockers. As variation in the size of RBCs is an indication of ineffective maturation, we also evaluated coefficient of variation of RBC size (RDW) in peripheral blood from mice. To validate our animal studies, we also measured RBC parameters in human burn patients.MethodsAnimal study: B6D2F1 mice were randomized into 2 groups, 15% TBSA (total burn surface area) scald burn and sham burn. Burn mice were given β-AR blockers or saline either via Alzet pumps eight hours after injury or by daily injections. All treatments were terminated 24 hours before harvest. Animals were euthanized to obtain spleen, femurs and blood on post burn days (PBD) as specified in results. Single cell suspensions from spleen and BM were characterized for early erythroblasts (CD71+Ter119neg), orthochromatic erythroblasts (Ter119+CD71+Syto16+), reticulocytes (Ter119+CD71+Syto16neg), erythrocytes (Ter119+), and non-erythroid cells (Ter119neg CD71neg) by flow cytometry, and expressed as 103/106 total splenocytes or total BM cells. RDW was measured using HemaTrue analyzer.Human study: Retrospective analysis was done on 19 adult burn patients enrolled in IRB approved study. All patients received standard burn care (SBC) during the study period of seven weeks. As part of another clinical trial, 9/19 patients had received propranolol along with SBC (SBC+PR). Cohorts matched for age, gender and %TBSA.ResultsAnimal study: Splenic erythropoiesis was increased after burn injury on PBD 3, 7, 14 and 21 as measured by erythrocyte and erythroblast production. Propranolol treatment for 14 days via Alzet pump alone or in combination with Epo was not sufficient to reduce splenic erythrocytes and erythroblasts that were elevated after burn injury. Between the selective blockers, only SR59320A (β3-AR) and not butoxamine (β2-AR) significantly reduced splenic erythropoiesis. This was accompanied by a decrease in splenomegaly and a reciprocal increase in BM erythropoiesis only with SR59230A treatment. RDW was significantly increased in blood after burn injury, while MCV remained within normal range. Interestingly, only SR59230A treatment and not propranolol or butoxamine reversed RDW to sham levels.Human study: Hemoglobin levels were higher at PBD 1-3 (due to hemo concentration) and decreased significantly in both groups through PBD 30-48. Mean RDW was normal at PBD 1-3 (Normal range =11.5% -14.5%) in all burn patients. Although RDW seemed to stabilize initially in SBC+PR treated patients at PBD 7-10, it increased to SBC levels by PBD 30-48. Mean corpuscular volume (MCV) remained within normal limits.ConclusionEffective maturation of late erythroblasts in BM is essential for the spleen to sense erythropoietic homeostasis, which is reestablished with β3-AR blockade after burn injury. Results emphasize only β3-AR antagonist restores RDW to sham levels and not β1/ β2-AR blocker. Increased RDW in burn patients corroborates with animal studies. Overall, this model system is suitable to study mechanisms of early and late erythropoiesis after burn injury. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Sodium butyrate inhibits the production of HMGB1 and attenuates severe burn plus delayed resuscitation-induced intestine injury via the p38 signaling pathway

BackgroundInflammatory response triggered by high mobility group box-1 (HMGB1) protein and oxidative stress play critical roles in the intestinal injury after severe burn. Sodium butyrate, a histone deacetylase inhibitor, has potential anti-inflammatory properties, inhibiting the expression of inflammatory mediators such as HMGB1 in diverse diseases. This study was designed to investigate the effects of sodium butyrate on severe burn plus delayed resuscitation-induced intestine injury, intestinal expressions of HMGB1 and intracellular adhesion molecule-1 (ICAM-1), oxidative stress, and signal transduction pathway changes in rats. Materials and methodsFifty-six Sprague-Dawley rats were divided into 3 groups randomly: (1) sham group, animals underwent sham burn; (2) burn group, rats subjected to full-thickness burns of 30% total body surface area (TBSA) and received 2ml/kg/TBSA lactated Ringer solution for resuscitation at 6, 12, and 36h after burn injury; (3) burn plus sodium butyrate (burn+SB) group, animals received burn injury and lactated Ringer solution with sodium butyrate inside for resuscitation in the same manner. Diamine oxidase (DAO) concentration in plasma was measured by enzyme-linked immunosorbent assay. Intestinal fatty acid binding protein (I-FABP) and ICAM-1 expressions in the intestine were analyzed by immunohistochemical method. HMGB1 and p38 mitogen-activated protein kinase (MAPK) expressions in the intestine tissues were examined by Western blot. The intestinal concentration of malondialdehyde (MDA) was also determined. ResultsIntestinal HMGB1 expression was significantly increased in burn group compared with sham group. Sodium butyrate administration significantly inhibited the HMGB1 expression in the intestine, decreased the DAO concentration in plasma, reduced the intestinal I-FABP expression, and improved the intestinal histologic changes induced by burn injury plus delayed resuscitation. Sodium butyrate treatment also markedly reduced the increase of intestinal ICAM-1 expression and MDA content, and inhibited p38 MAPK activity in the intestine of severely burned rats with delayed resuscitation. ConclusionsSodium butyrate inhibits HMGB1 expression which could be attributed to p38 MAPK signal transduction pathway and decreases intestinal inflammatory responses and oxidative stress, thus attenuates burn plus delayed resuscitation-induced intestine injury.

Influences of hydrogen-rich saline on acute kidney injury in severely burned rats and mechanism

Objective: To explore the influences of hydrogen-rich saline on acute kidney injury in severely burned rats and to analyze the related mechanism. Methods: Fifty-six Sprague Dawley rats were divided into sham injury group (n=8), burn group (n=24), and hydrogen-rich saline group (n=24) according to the random number table. Rats in sham injury group were treated by 20 ℃ water bath on the back for 15 s to simulate injury, and rats in burn group and hydrogen-rich saline group were inflicted with 30% total body surface area (TBSA) full-thickness scald (hereinafter referred to as burns) by 100 ℃ water bath on the back for 15 s. Immediately after injury, hydrogen-rich saline at the dose of 10 mL/kg were intraperitoneally injected to the rats in hydrogen-rich saline group at one time, while normal saline with the same dose were intraperitoneally injected to the rats in sham injury group and burn group. At post injury hour (PIH) 6, rats in the 3 groups were intraperitoneally injected with 4 mL·kg(-1)·%TBSA(-1) lactated Ringer's solution for resuscitation. Eight rats from sham injury group at PIH 72 and eight rats from burn group and hydrogen-rich saline group at PIH 6, 24, and 72 were sacrificed respectively after their blood samples from abdominal aorta were collected. Then their kidney tissue was harvested for histopathological observation and renal tubular injury scoring by hematoxylin and eosin staining, serum creatinine and blood urea nitrogen were detected by the clinical blood biochemical analyzer, expression distribution and mRNA expressions of tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and IL-6 in renal tissue were evaluated by immunohistochemical staining and real time fluorescent quantitive reverse transcription polymerase chain reaction respectively, and protein expression of high mobility group protein 1 (HMGB1) was detected by Western blotting. Data were processed with Kruskal-Wallis H test, Dunn test, one-way analysis of variance, Bonferroni test. Results: (1) The renal tubular structure of rats in sham injury group at PIH 72 was complete with no inflammatory cell infiltration and no cellular degeneration or necrosis. Since PIH 6, the changes such as vacuolation and shape change of cells and aggregation of broken protein in renal tubules were observed in rats of burn group, and all these changes deteriorated with time. The renal injury of rats in hydrogen-rich saline group at different post injury time points were relieved compared with those of rats in burn group at the corresponding time points. The renal tubular injury scores of rats in burn group and hydrogen-rich saline group at PIH 6, 24, and 72 were significantly higher than the score in sham injury group at PIH 72 (P 0.05), the levels of serum creatinine of rats in burn group at all the time points and hydrogen-rich saline group at the other time points were significantly higher than the level of serum creatinine of rats in sham injury group at PIH 72 (P 0.05), and the mRNA expression levels of TNF-α, IL-1β, and IL-6 at the other time points in hydrogen-rich saline group were significantly decreased (P 0.05). Compared with those in burn group, the protein expressions of HMGB1 of rats in hydrogen-rich saline group at PIH 6, 24, and 72 were significantly decreased (P<0.05). Conclusions: Hydrogen-rich saline can alleviate the acute kidney injury in severely burned rats through regulating the release of inflammatory cytokines in renal tissue.

Early Hyperbaric Oxygen Treatment Attenuates Burn-Induced Neuroinflammation by Inhibiting the Galectin-3-Dependent Toll-Like Receptor-4 Pathway in a Rat Model.

Hyperbaric oxygen (HBO) treatment has been proven to decrease neuroinflammation in rats. This study aimed to determine the potential mechanism underlying the anti-inflammatory effects of HBO treatment on burn-induced neuroinflammation in rats. Thirty-six adult male Sprague-Dawley (SD) rats were randomly assigned to the following six groups (n = 6 per group): (1) sham burn with sham HBO treatment; (2) sham burn with HBO treatment; (3) burn with one-week sham HBO treatment; (4) burn with two-week sham HBO treatment; (5) burn with one-week HBO treatment; and (6) burn with two-week HBO treatment. SD rats that received third-degree burn injury were used as a full-thickness burn injury model. Subsequently, we analyzed the expression of proteins involved in the galectin-3 (Gal-3)-dependent Toll-like receptor-4 (TLR-4) pathway through enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC) analysis, and Western blotting. A behavior test was also conducted, which revealed that HBO treatment significantly suppressed mechanical hypersensitivity in the burn with HBO treatment group compared to the burn with sham HBO treatment group (p < 0.05). ELISA results showed that tumor necrosis factor α (TNF-α) and interleukin 1 beta (IL-1β) levels in the dorsal horn of the spinal cord and the skin significantly decreased in the burn with HBO treatment group compared with the burn with sham HBO treatment group (p < 0.05). Western blotting results demonstrated that HBO treatment significantly reduced the expression of Gal-3 and TLR-4 in the dorsal horn of the spinal cord in the burn with HBO treatment group compared with the burn with sham HBO treatment group (p < 0.05). IHC analysis showed that the expression of Gal-3, TLR-4, CD68 and CD45 in the dorsal horn of the spinal cord was significantly lower in the burn with HBO treatment group than in the burn with sham HBO treatment group (p < 0.05), and the expression of CD68 and macrophage migration inhibitory factor (MIF) in the right hind paw skin was significantly lower. The expression of vimentin and fibroblast growth factor in the right hind paw skin was significantly higher after HBO treatment (p < 0.05). This study proved that early HBO treatment relieves neuropathic pain, inhibits the Gal-3-dependent TLR-4 pathway, and suppresses microglia and macrophage activation in a rat model.

225 Pharmacological Ameioration of Perturbed Mitophagy Response (Mitophagy Resistance) in Burns

Mitochondrial dysfunctions in skeletal muscles accompanying muscle wasting leads to the poor prognosis of patients with burn injury (BI). The relationship among autophagy, mitochondrial dysfunction, and poor prognosis in BI was not fully determined. Recently, autophagy has been categorized largely into two subsets; non-selective autophagy and selective autophagy including mitophagy. In the latter system, damaged organelles are selectively cleared for their quality control purposes. We have previously discovered that despite the upregulation of basal level of non-selective autophagy, stimulated and selective autophagy (mitophagy) is significantly compromised in BI (state of ‘mitophagy resistance (MR)’). Trehalose, which some stress-resistant organisms including tardigrades utilize to survive under harsh conditions, is known to facilitate autophagy by mTOR-independent pathway. In this study, we investigated whether MR can be pharmacologically ameliorated by trehalose. To study the systemic effect of a whole body BI, a third-degree BI was administered onto the body trunk of wild type mice. Sham-burn (SB) mice served as controls. On post-burn day 3 (PBD3), in vivo microscopy (ivM) was performed to analyze the speed of stimulated mitophagy, mitochondrial membrane potential (MMP) and production of superoxide from mitochondria. For these quantitative ivM analyses, in vivo transfection of a reporter gene construct (mito-Kaede), or staining with MMP-sensitive dye (DiOC6) and/or with MitoSOX, was combined, respectively. For the stimulation of mitophagy, mitochondrial uncoupler, CCCP was intramuscularly injected. The result was compared between mice with BI in the presence or absence of trehalose treatment (2mg/gBW, ip). With mito-Kaede experiment, turnover rate of mitochondria specific to CCCP-induced mitophagy, was successfully monitored. BI mice showed perturbed mitophagy rate than SB (deltaFL/6hr; 18.1% vs. 52.5%), which was significantly ameliorated by trehalose treatment (48.2%, p<0.05). BI also showed decreased basal level of MMP (32% decrease) and increased SO production (243% increase) in response to CCCP as compared to SB, both of which were ameliorated by trehalose treatment (p<0.05). By using novel ivM techniques, we have documented the relationship among MR, MMP loss, and SO production. Successful treatment with trehalose against MR ameliorated MMP loss and SO production, suggesting that MR in BI was likely the cause of muscle dysfunction. Potential efficacy of trehalose against MR was documented for the first time. MR can be a therapeutic target in muscle dysfunctions in BI. It is possible many other organ dysfunctions in critical illnesses may have similar mechanisms.

224 Burn Injury Induced Functional and Morphological Alterations in Skeletal Mitochondria in a Metabolically Characterized Animal Model

Altered mitochondrial biology and function is considered as an underlying mechanism of burn injury induced hypermetabolism and insulin resistance. Our previous work has characterized a burn mouse model with insulin resistance by in vivo clamp method accompanied with muscle insulin signal proteins in skeletal muscle. The present study aimed at exploring the morphology and functional changes of muscle mitochondrial in this characterized burn animal model. C57BL/6 mice (10–12 weeks) received 30% total body surface area (TBSA) full thickness burn injury or sham burn injury. Gastrocnemius and quadriceps muscles were collected on days 1, 3, and 7 after burn injury. Mitochondrial function was assessed by citrate synthase activity, mitochondrial membrane potential and NAD+/NADH ratio. Gene expression related to mitochondrial biogenesis, dynamics and mitochondrial DNA contents were quantified with real time quantitative PCR. The morphological changes were assessed by a quantitative 2-dimensional transmission electron microscopy (TEM). Our studies demonstrated that the major changes occurred on day 3. Mitochondrial dysfunction was revealed with significantly reduced citrate synthase activity (fig A, B, C), mitochondrial membrane potential and NAD+/NADH ratio. This was accompanied with reduced mitochondrial DNA contents and mRNA expression of cytochrome C (CytC), mitochondrial transcription factor A (TFAM), nuclear respiratory factor 1 (NRF1) and fission protein 1(Fis1) (fig D, E). TEM demonstrated that mitochondria became longer and branched with disrupted cristae as compared to sham burn animals (fig F, G). There was a time dependent reduction to number of cristae time-dependently (fig H). Since CytC is the major component of electron transport chain, NRF1 regulates transcription rregulating cellular growth and nuclear genes required for respiration, heme biosynthesis, and mitochondrial DNA transcription and replication, the latter is also regulated by TFAM and Fis1 is one of the mitochondrial fission factors. We conclude that the above molecular and morphological changes mitochondria are part of the mechanisms of metabolic aberrations caused by burn injury.

Early diagnosis using canonical discriminant analysis of innate immune receptor gene expression profiles in a murine infectious or sterile systemic inflammation model.

Infection in patients with systemic inflammation is difficult to diagnose with a single biomarker. We aimed to clarify the time course of change in the gene expression profile of innate immune receptors in infectious or sterile inflammation and to establish an early diagnostic method using canonical discriminant analysis (CDA) of the gene expression profile. To compare infectious and sterile inflammation, we used cecal ligation and puncture (CLP) and 20% full-thickness burn injury (Burn) models. C57BL/6 mice underwent sham treatment (n = 9 × three groups), CLP (n = 12 × three groups), or Burn (n = 12 × three groups) injury. Mice were killed at 6, 12, and 24 hours after injury, and total RNA was extracted from whole blood. We used quantitative real-time polymerase chain reaction to investigate gene expression of innate immune receptors Toll-like receptor 2 (TLR2), TLR4, TLR9, NLRP3 (nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3), and retinoic acid-inducible gene I. To evaluate all gene expression together as patterns, each value was standardized, and CDA was performed at each time point. Gene expression of TLR2 and TLR4 was already significantly increased in both CLP and Burn compared with sham mice at 6 hours after injury (p < 0.05). Gene expression of TLR9 was significantly decreased in CLP compared with sham and Burn mice at 12 hours and 24 hours after injury (p < 0.05) but not at 6 hours. Gene expression of NLRP3 was significantly increased in CLP and Burn compared with sham mice at 6 hours and 24 hours after injury (p < 0.05). In the CDA, each group showed distinctive gene expression patterns at only 6 hours after injury. Each group was clearly classified, and the classification error rates were 0% at all of the time points. Canonical discriminant analysis of the gene expression profile of innate immune receptors could be a novel approach for diagnosing the pathophysiology of complicated systemic inflammation from the early stage of injury.

Polysaccharides from Bupleurum Induce Immune Reversal in Late Sepsis.

Bupleurum chinense, a well-known Traditional Chinese Medicine, has been used for thousands of years in China. In this study, we would suggest that Bupleurum polysaccharides (BPS) could improve the prognosis of sepsis through its impact on redistribution of BMCs, which triggers immune reversal in late sepsis. BALB/c mice were divided into five groups: sham burn group, burn plus P aeruginosa group, burn plus P aeruginosa with BPS (40 mg/kg, 100 mg/kg, and 250 mg/kg) treatment group, and they were sacrificed at post-burn day (PBD) 0, 3, 5, and 7. BMCs, liver cells, and dendritic cells (DCs) were harvested. Flow cytometry was used to determine the change of phenotypes of DCs and isolate these cells. Cytometric beads array was utilized to analyze the level of inflammatory factors. Cell therapy of BMCs, liver cells, and DCs was administrated to explore the protective role of regional organ immunity. BPS could decrease the lethality of burn sepsis in a dose-dependent fashion and increase both the percentage of CD11cCD45RB DCs in bone marrow (BM) and liver and the number of BMCs and liver cells significantly. Cell therapy of BMCs, liver cells, and CD11cCD45RB DCs at PBD7 could protect septic mice from sepsis. BPS has shown its potential in promoting the prognosis of post-burn sepsis through its effect on immune redistribution of BMCs, especially via differentiation of CD11cCD45RB DC cells in BM and nonimmune organs to induce immune reversal in late sepsis.

Effect of parenteral glutamine supplementation combined with enteral nutrition on Hsp90 expression and Peyer's patch apoptosis in severely burned rats.

The aim of this study was to investigate the effects of parenteral glutamine (GLN) supplementation combined with enteral nutrition (EN) on heat shock protein (Hsp) 90 expression and Peyer's patch (PP) apoptosis in severely burned rats. Male Sprague-Dawley (SD) rats were randomly assigned to four groups: Sham burn+EN+GLN-free amino acid (AA; n=10), sham burn+EN+GLN (n=10), burn+EN+AA (n=10), and burn+EN+GLN (n=10). Two hours after a 30% total body surface area (TBSA), full-thickness scald burn injury on the back, burned rats in two of the experimental groups (burn+EN+AA and burn+EN+GLN groups) were fed with a conventional EN solution by oral gavage for 7d. Simultaneously, rats in the burn+EN+GLN group were given 0.35g GLN/kg body weight/d once via a tail vein injection for 7d and rats in the burn+EN+AA group were administered isocaloric/isonitrogenous GLN-free amino acid solution (Tyrosine) for comparison. Rats in two sham burn control groups (sham burn+EN+AA and sham burn+EN+GLN groups) were treated in the same manner except for the burn injury. All rats in the four groups were given 175kcal/kg body wt/d. There was isonitrogenous, isovolumic, and isocaloric intake among the four groups. At the end of the seventh day after completion of the nutritional program, all rats were anesthetized and samples were collected for further analysis. PP apoptosis was measured by terminal deoxyuridine nick-end labeling (TUNEL). The expression of Hsp90 in PPs was analyzed by western blotting. Caspase-3 activity of PPs was also assessed. Levels of proinflammatory cytokines of gut tissues were evaluated by enzyme-linked immunosorbent assay (ELISA). The intestinal immunoglobulin A (IgA) content was also determined by ELISA. The results revealed that intestinal IgA content in rats of the burn+EN+GLN group were significantly increased compared with those in the burn+EN+AA group (P<0.05). The expression of Hsp90 of PPs in rats in the burn+EN+GLN group was significantly upregulated compared with those in the burn+EN+AA group (P<0.05). On the other hand, levels of proinflammatory cytokines of gut tissues, caspase-3 activity, and the number of TUNEL-stained cells of PPs in rats of the burn+EN+GLN group were markedly decreased compared with those of the burn+EN+AA group (P<0.05). The results of this study show that parenteral glutamine supplementation combined with EN may upregulate the expression of Hsp90, reduce caspase-3 activity, lessen the release of proinflammatory cytokines, attenuate PP apoptosis, and improve intestinal IgA response in burned rats. Clinically, therapeutic efforts to improve intestinal immunity may contribute to a favorable outcome in severely burned patients.

Autologous Adipose-Derived Stem Cells Reduce Burn-Induced Neuropathic Pain in a Rat Model.

Background: Burn scar pain is considered as neuropathic pain. The anti-inflammation and anti-neuroinflammation effects of adipose-derived stem cells (ASCs) were observed in several studies. We designed a study using a murine model involving the transplantation of autologous ASCs in rats subjected to burn injuries. The aim was to detect the anti-neuroinflammation effect of ASC transplantation and clarify the relationships between ASCs, scar pain, apoptosis and autophagy. Methods: We randomized 24 rats into 4 groups as followings: Group A and B, received saline injections and autologous transplantation of ASCs 4 weeks after sham burn, respectively; Group C and D, received saline injections and autologous transplantation 4 weeks after burn injuries. A designed behavior test was applied for pain evaluation. Skin tissues and dorsal horn of lumbar spinal cords were removed for biochemical analysis. Results: ASC transplantation significantly restored the mechanical threshold reduced by burn injury. It also attenuated local inflammation and central neuroinflammation and ameliorated apoptosis and autophagy in the spinal cord after the burn injury. Conclusion: In a rat model, autologous ASC subcutaneous transplantation in post-burn scars elicited anti-neuroinflammation effects locally and in the spinal cord that might be related to the relief of post-burn neuropathic pain and attenuated cell apoptosis. Thus, ASC transplantation post-burn scars shows the potential promising clinical benefits.

74 Astaxanthin Pretreatment Attenuates Burn Induced Heart Injury in Rats

Major burn injury results in impairment of left ventricular contractile function. There is convincing evidence to support the acute oxidative stress is involved in the development of burn-related contractile dysfunction. Astaxanthin (ASX) is the strongest antioxidant in nature that shows preventive and therapeutic properties. The aim of present study was to determine whether ASX pretreatment provides protection against burn-induced heart contraction failure. Male Wistar rats were randomly divided into four groups, including sham burn, sham burn+ASX, burn and burn+ASX groups. 30 mg/kg/day of ASX were administered for seven days. The burn and burn+ASX groups were given 40% of total body surface area thermal injury under general anesthesia. The animals were sacrificed to evaluate the left ventricular contractility at 24 hours post-burn. The heart was removed and cannulated to perfuse in a retrograde fashion via the aorta by Langendorff method. The myocardial contractility was measured with a latex balloon inserted into the left ventricle. The contractility index was calculated as the difference between the systolic and end-diastolic pressures as dP/dT. The dP/dT was measured 80 cm of perfusion pressure, and the heart rate was set at 300 beats per minute with an electronic stimulator. The left ventricle max dP/dT was significantly decreased in the burn group compared to those in the sham burn and sham burn+ASX groups (1684±913 mmHg/s vs. 3508±670, 3330±735, respectively, p<0.01). The dP/dT of the burn+ASX group (2727±746 mmHg/s) was significantly higher than that of the burn group (p<0.05). The ASX pretreatment provided better cardiac protection against the burn injury, as evidenced by the improved myocardial contractility.

Changes of content and mRNA expression of gelsolin and proliferation activity of T-lymphocyte in spleen of mice with severe burn injury

Objective: To investigate the changes of content and mRNA expression of gelsolin and proliferation activity of T-lymphocyte in spleen of mice with severe burn injury, so as to determine the optimum intervention time of gelsolin. Methods: Eighty male BALB/c mice were divided into sham injury group and burn group according to the random number table, with 40 mice in each group. Mice in burn group were inflicted with 15% total body surface area full-thickness scald (hereinafter referred to as burn) on the back. Immediately after injury, mice in burn group were hypodermic injected with 1 mL normal saline, with iodophor smeared on back once a day to prevent infection. Mice in sham injury group were sham injured without fluid infusion and smearing iodophor. At post injury hour (PIH) 0 (immediately), 8, 24, 48, and 72, spleen of 8 mice of each group were harvested aseptically, respectively. Proliferation activity of T-lymphocyte was determined with methyl-thiazolyl-tetrazolium colorimetry method; gelsolin content of spleen was determined with enzyme-linked immunosorbent assay; mRNA expression of gelsolin of spleen was determined with real-time fluorescent quantitative reverse transcription polymerase chain reaction. Data were processed with analysis of variance of factorial design, one-way analysis of variance, LSD test and Bonferroni correction. Results: (1) There was no significant difference in proliferation activity of T-lymphocyte in spleen of mice in two groups at PIH 0 (P>0.05). Proliferation activity of T-lymphocyte in spleen of mice in sham injury group was significantly higher than that in burn group at PIH 8, 24, 48, and 72 (with P values below 0.05). There was no significant difference in proliferation activity of T-lymphocyte in spleen of mice in sham injury group at each time point post injury (F=0.756, P>0.05). Proliferation activity of T-lymphocyte in spleen of mice in burn group at PIH 8 was 0.12±0.04, significantly lower than that at PIH 0, 24, 48, and 72 in the same group (0.73±0.07, 0.56±0.07, 0.51±0.09, and 0.59±0.07, respectively, with P values below 0.05). (2) There was no significant difference in gelsolin content of spleen of mice in two groups at PIH 0 (P>0.05). Gelsolin content of spleen of mice in sham injury group was significantly higher than that in burn group at PIH 8, 24, 48, and 72 (with P values below 0.05). There was no significant difference in gelsolin content of spleen of mice in sham injury group at each time point post injury (F=1.083, P>0.05). Gelsolin content of spleen of mice in burn group at PIH 8 was (11.9±2.6) pg/mg, significantly lower than that at PIH 0, 24, 48, and 72 in the same group [(37.7±2.9), (19.9±4.0), (24.1±4.1), and (24.6±4.0) pg/mg, respectively, with P values below 0.05]. (3) There was no significant difference in mRNA expression of gelsolin of spleen of mice in two groups at PIH 0 (P>0.05). The mRNA expressions of gelsolin of spleen of mice in sham injury group were significantly higher than those in burn group at PIH 8, 24, 48, and 72 (with P values below 0.05). There was no significant difference in mRNA expression of gelsolin of spleen of mice in sham injury group at each time point post injury (F=0.413, P>0.05). The mRNA expression of gelsolin of spleen of mice in burn group at PIH 8 was 0.307±0.064, significantly lower than that at PIH 0, 24, 48, and 72 in the same group (0.944±0.023, 0.625±0.091, 0.744±0.104, and 0.821±0.072, respectively, with P values below 0.05). Conclusions: Severe burn injury could induce decrease of proliferation activity of T-lymphocyte and content and mRNA expressions of gelsolin in spleen of mice, and all of them decreased into the lowest at PIH 8. Optimum intervention time of gelsolin for severe burn would be before PIH 8.

Burn-Related Dysregulation of Inflammation and Immunity in Experimental and Clinical Studies.

The purpose of this study was to evaluate burn-related variations of inflammation and immunity. Fifty-five mice were divided randomly into sham burn and burn groups. Eighty-seven hospitalized burn patients were also reviewed. In mice, neutrophils and monocytes were elevated significantly on post burn day (PBD 1). Lymphocytes were reduced on PBDs 1 and 3. Levels of serum tumor necrosis factor-α and interleukin-6 were highest on PBD 1. Interleukin-1β levels were the highest on PBD 3. On PBD 3, CD4CD25T regulatory cells/CD4 cells in spleen were higher. On PBDs 1, 3, 7, and 14, percentage of splenic dendritic cells were significantly lower than the sham burn group. In patients, neutrophils and monocytes were significantly elevated on PBD 1. Levels declined but remained elevated at most days to PBD 7. Lymphocytes in burn groups 1 and 2 were reduced on PBDs 1 and 3, respectively. Our results exhibited that severe burn injury initiated a hyperinflammatory response and immunosuppression. PBDs 1 to 3 were important for changes in inflammation and immunosuppression.

Exercise Altered the Skeletal Muscle MicroRNAs and Gene Expression Profiles in Burn Rats With Hindlimb Unloading.

This study investigated microRNA and target gene profiles under different conditions of burn, bed rest, and exercise training. Male Sprague-Dawley rats (n = 48) were assigned to sham ambulatory, sham hindlimb unloading, burn ambulatory, or burn plus hindlimb unloading groups. Rats received a 40% TBSA scald burn or sham treatments and were ambulatory or hindlimb unloaded. Rats were further assigned to exercise or no exercise. Plantaris tissues were harvested on day 14 and pooled to analyze for microRNA and gene expression profiles. Compared with the sham ambulatory-no exercise group, 73, 79, and 80 microRNAs were altered 2-fold in the burn ambulatory, sham hindlimb unloading, and burn hindlimb unloading groups, all with no exercise, respectively. More than 70% of microRNAs were upregulated in response to burn and hindlimb unloading, whereas 60% microRNA of the profile decreased in hindlimb unloaded burn rats with exercise training. MiR-182 was the most affected in rat muscle. Gene ontology biological process and pathway analysis showed that the oxidative stress pathway was most stimulated in the hindlimb unloaded burn rats; while in response to exercise training, all genes in related pathways such as hypermetabolic, inflammation, and blood coagulation were alleviated. MicroRNAs and transcript gene profiles were altered in burn and hindlimb unloading groups, with additive effects on hindlimb unloaded burn rats. The altered genes' signal pathways were associated with muscle mass loss and function impairment. Muscle improvement with exercise training was observed in gene levels with microRNA alterations as well.