The cGAS-STING pathway connects mitochondrial damage to inflammation in burn-induced acute lung injury in rat.

OBJECTIVE: Damage associated molecular patterns (DAMPs) are pathological mediators linking local tissue damage to systemic inflammation in various diseases. Some DAMPs, such as mitochondrial DNA (mtDNA), can be recognized by the cytoplasmic cGAS protein to trigger the activation of the stimulator of interferon genes (STING)-dependent innate immune pathway responsible for infection or sterile inflammation. The objective of our study was to evaluate the association between circulating mtDNA and cGAS-STING pathway activation in mediating inflammation following burn injury. METHODS: 48 adult Sprague-Dawley male rats were divided into eight groups (Sham, 2, 4, 8, 12, 24, 48, 72 h after burn injury). The animals underwent 40% total body surface area scald injury to produce a full-thickness burn. Plasma samples were collected via cardiac puncture under deep anesthesia. Tissues were harvested and placed in formalin, followed by paraffin embedment. Total plasma DNA was isolated followed by measurement of mtDNA using quantitative polymerase chain reaction. Haemotoxylin-Eosin stain and Western blot was used for lung histology and protein assays, respectively. Statistical analyses were performed using ANOVA and student's t-test and represented as mean ± s.d. RESULTS: Plasma mtDNA trended upward at early time-points following burn injury with peak levels at 8 h after burn when compared to the control group (345 ± 83.4 copies/µl vs. 239 ± 43.1 copies/µl, p = 0.07) and followed a bell-shaped distribution. Lung slices from burned rats showed acute injury marked by increased inflammatory infiltrate, with the maximum changes seen at 24 h, accompanied with significant upregulation of neutrophil elastase (p = 0.04). Compared with sham animals, cGAS and STING protein levels in lung tissue were up-regulated at 4 and 8 h after burn (p = 0.03 and p < 0.001, respectively). CONCLUSION: Activation of the cGAS-STING pathway by increased plasma mtDNA is an important pathway driving neutrophil infiltration in burn-induced acute lung injury in rats. A further understanding of the STING-mediated immunopathology in lung and other susceptible organs may be important for the development of novel therapies for burn injury.

Tau Is Elevated in Pediatric Patients on Extracorporeal Membrane Oxygenation.

Neurologic injury is a known and feared complication of extracorporeal membrane oxygenation (ECMO). Neurologic biomarkers may have a role in assisting in early identification of such. Axonal biomarker tau has not been investigated in the pediatric ECMO population. The objective of this study is to evaluate plasma levels of tau in pediatric patients supported with ECMO. Eighteen patients requiring ECMO support in a quaternary pediatric intensive care unit at a university-affiliated children's hospital from October 2015 to February 2017 were enrolled. Patients undergoing extracorporeal cardiopulmonary resuscitation or recent history of bypass were excluded. Plasma tau was measured using enzyme-linked immunosorbent assay. Neuroimaging was reviewed for acute neurologic injury, and tau levels were analyzed to assess for correlation. Tau was significantly higher in ECMO patients than in control subjects. Sixty-one percent of subjects had evidence of acute brain injury on neuroimaging, but tau level did not correlate with injury. Subjects with multifocal injury all experienced infarction and had significantly higher tau levels on ECMO day 3 than patients with isolated injury. In addition, peak tau levels of neuro-injured subjects were compared with controls and noninjured ECMO subjects using receiver operating curve analysis. This study demonstrates preliminary evidence of axonal injury in pediatric ECMO patients.

Optimization of low-abundance protein extraction and abundant protein removal from defatted soybean meal.

The aim of this study was to optimize the conditions for the extraction of low-abundance proteins (LAPs) and the removal of abundant proteins (APs; ß-conglycinin and glycinin) from soybean meal. Single factor and orthogonal experiments were designed to determine the effects of four factors (isopropanol concentration, total extraction time, ultrasonic power, and ultrasonic time) on protein concentration in isopropanol extracts. Proteins in the isopropanol supernatant and the cold acetone precipitate of isopropanol were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS). The results showed that the optimal conditions were 50% isopropanol, ultrasonic pretreatment for 15 min at 350 W, and a total extraction time of 1 h. Under these conditions, the protein concentration in the isopropanol extracts reached 0.8081 g/L. Many LAPs were detected, including ß-amylase, soybean agglutinin, soybean trypsin inhibitor, fumarylacetoacetase-like, phospholipase D alpha 1-like, oleosin, and even some unknown soybean proteins. The soybean APs (ß-conglycinin and glycinin) were not found. The method may be useful for discovering new soybean proteins and extracting enough LAPs of soybean to allow further studies of their physiological effects on animals without the influence of APs.

Establishment of primary bovine intestinal epithelial cell culture and clone method.

The aim of this study was to establish bovine intestinal epithelial cell (BIEC) line and provide a novel clone cell method. Although various strategies of bovine cell culture and clone techniques have been reported, these methods remain not established. Here, we culture successfully primary BIECs and establish a novel clone cell method. Our result showed that BIECs could be successfully cultured and passaged about generation 5. These cellular aggregates and clusters were adherent loosely at day 2 of culture. Cell aggregates and clusters start to proliferate after approximately 4 d. The BIECs showed positive reaction against cytokeratin 18, E-cadherin, and characteristics of epithelial-like morphology. In addition, the fatty acid-binding proteins (FABPs), villin, and intestinal peptidase (IP) band were positive in BIECs. Our results suggest that the establishment of culturing and clone BIEC methods will apply to isolate and clone other primary cells. These BIECs could therefore contribute to the study of bovine intestinal nutrient absorption and regulation, immune regulation, and the pathogenesis of the bovine intestinal disease, which will provide intestinal cell model in vitro.

Adult obese mice suffer from chronic secondary brain injury after mild TBI.

BACKGROUND: A traumatic brain injury (TBI) event is a devastating injury to the brain that may result in heightened inflammation, neurodegeneration, and subsequent cognitive and mood deficits. TBI victims with co-morbidities such as heart disease, diabetes, or obesity may be more vulnerable to the secondary brain injury that follows the initial insult. Compared to lean individuals, obese subjects tend to have worse clinical outcomes and higher mortality rates after trauma. METHODS: To elucidate whether obesity predisposes individuals to worse outcomes after TBI, we subjected adult lean and obese male/female mice to a mild TBI. The injury was administered using a controlled skull impact (CSI) device. Lean or obese 6-month-old C57 BL/6 mice were subjected once to a mild TBI. Additionally, at day 30 after injury, both the lean and obese mice were tested for increased anxiety using the open field test. RESULTS: At day 30 after TBI, compared to the lean mice, we found heightened microglial (MG) activation in the cerebral cortex, corpus callosum, and hypothalamus. Another compelling finding was that, compared to the non-injured obese male control mice, the obese TBI mice had a decrease in the rate of weight gain and serum corticosterone levels at day 30 after injury. Additionally, the injured obese mice displayed higher levels of anxiety as determined by a significant decrease in time spent in the non-peripheral zones in the open field test. In contrast to the obese males, the obese female mice did not exhibit increases in the number of active MG in the brain, changes in weight gain/corticosterone levels, or increased anxiety at day 30 after TBI. CONCLUSIONS: The data presented here suggests that obese mice have worse outcomes compared to lean mice after mild TBI. Also, the obese males have worse outcomes than the injured female mice. This data may explain the sequela of chronic secondary brain injury in obese adults after a single mild TBI. Also, this report may help shape how the overweight/obese populations are monitored over the days and months following a TBI.

Severe burn increased skeletal muscle loss in mdx mutant mice.

BACKGROUND: Severe burn causes muscle mass loss and atrophy. The balance between muscle cell death and growth maintains tissue homeostasis. We hypothesize that preexisting cellular structural defects will exacerbate skeletal muscle mass loss after burn. Using a Duchenne muscular dystrophy (mdx) mutant mouse, we investigated whether severe burn caused more damage in skeletal muscle with preexisting muscle disease. METHODS: The mdx mice and wild-type (WT) mice received 25% total body surface area scald burn. Gastrocnemius (GM), tibialis anterior, and gluteus muscles were obtained at days 1 and 3 after burn. GM muscle function was measured on day 3. Animals without burn served as controls. RESULTS: Wet tissue weight significantly decreased in tibialis anterior and gluteus in both mdx and WT mice after burn (P < 0.05). The ratio of muscle to body weight decreased in mdx mutant mice (P < 0.05) but not WT. Isometric force was significantly lower in mdx GM, and this difference persisted after burn (P < 0.05). Caspase-3 activity increased significantly after burn in both the groups, whereas HMGB1 expression was higher in burn mdx mice (P < 0.05). Proliferating cell nuclear antigen decreased significantly in mdx mice (P < 0.05). Myogenic markers pax7, myoD, and myogenin increased after burn in both the groups and were higher in mdx mice (P < 0.05). CONCLUSIONS: More muscle loss occurred in response to severe burn in mdx mutant mice. Cell turnover in mdx mice after burn is differed from WT. Although markers of myogenic activation are elevated in mdx mutant mice, the underlying muscle pathophysiology is less tolerant of traumatic injury.

Effects of exercise on soleus in severe burn and muscle disuse atrophy.

BACKGROUND: Muscle loss is a sequela of severe burn and critical illness with bed rest contributing significantly to atrophy. We hypothesize that exercise will mitigate muscle loss after burn with bed rest. MATERIALS AND METHODS: Male rats were assigned to sham ambulatory (S/A), burn ambulatory (B/A), sham hindlimb unloading (S/H), or burn hindlimb unloading (B/H). Rats received a 40% scald burn or sham and were ambulatory or placed in hindlimb unloading, a model of bed rest. Half from each group performed twice daily resistance climbing. Hindlimb isometric forces were measured on day 14. RESULTS: Soleus mass and muscle function were not affected by burn alone. Mass was significantly lower in hindlimb unloading (79 versus 139 mg, P < 0.001) and no exercise (103 versus 115 mg, P < 0.01). Exercise significantly increased soleus mass in B/H (86 versus 77 mg, P < 0.01). Hindlimb unloading significantly decreased muscle force in the twitch (12 versus 31 g, P < 0.001), tetanic (55 versus 148 g, P < 0.001), and specific tetanic measurements (12 versus 22 N/cm(2), P < 0.001). Effects of exercise on force depended on other factors. In B/H, exercise significantly increased twitch (14 versus 8 g, P < 0.05) and specific tetanic force (14 versus 7 N/cm(2), P < 0.01). Fatigue index was lower in ambulatory (55%) and exercise (52%) versus hindlimb (69%, P = 0.03) and no exercise (73%, P = 0.002). CONCLUSIONS: Hindlimb unloading is a significant factor in muscle atrophy. Exercise increased the soleus muscle mass, twitch, and specific force in this model. However, the fatigue index decreased with exercise in all groups. This suggests exercise contributes to functional muscle change in this model of disuse and critical illness.

Serum Levels of Neurofilament-H are Elevated in Patients Suffering From Severe Burns.

In previous studies, after injury, burn patients experienced an increase in neuro-inflammation, edema, and neuronal cell death. As demonstrated in other brain injury models, fluid-based biomarkers such as phosphorylated neurofilament-H (pNFL-H) have been shown to correlate with injury severity. In this study the authors hypothesized that burn-injured patients have an increase in pNFL-H in the blood during the acute and chronic time-points after injury. In this prospective clinical study, blood (8 cc) was collected from burn patients (n = 36; TBSA 10-60%) at Parkland hospital, Dallas, Texas, on days 1, 7, and 14 after injury. The serum levels of pNFL-H were measured using the enzyme-linked immunoassay. Compared to noninjured controls, the burn patients exhibited a significant increase in the serum levels of pNFL-H on days 7 (P < .0001) and 14 (P < .0001) after burn injury. No significant increase was observed on day 1 (P < .07) after injury. A positive correlation between TBSA and pNFL-H levels was observed for day 14 (r = .55; P < .03). Additionally, using the receiver operating characteristic analysis, the authors determined the area under the curve was 98% for both day 7 and 14. In conclusion, this study describes the serum profile of pNFL-H in patients suffering from severe burns during the acute (day 1) and chronic (days 7 and 14) time-points. These results suggest that detection of pNFL-H may be useful in determining which individuals suffer from nerve cell degeneration after burn.

Hypertonic HBOC-201 decreases neutrophil activation after hemorrhagic shock.

OBJECTIVE: To evaluate neutrophil activation after exposure to standard HBC-201 (suspended in lactate Ringer's solution) versus HBOC-201 suspended in hypertonic 7.5% saline solution. METHODS: We use plasma and tissue obtained from pigs subjected to controlled hemorrhagic shock and an ex vivo model of stimulated human whole blood. The pigs were resuscitated with the following (n = 8 per group) standard HBOC-201, or hypertonic HBOC-201. We used HTS 7.5%, Ringer's lactate as control resuscitation. Human blood was stimulated with same fluids. We measured the following neutrophil markers; IL-8, H2O2 in pig plasma, MPO in pig tissue, and H2O2, IL-8, and CD11b/CD18 in human whole blood. RESULTS: H2O2 and IL-8 as well as tissue MPO were significantly decreased in pigs resuscitated with HT-HBOC-201 and HT 7.5%. Ex vivo experiments blood diluted with HTS and HT-HBOC-201 revealed lower expression of CD11b/CD18, H2O2, and IL-8. Blood diluted with HBOC-201 had a higher CD11b/CD18 expression than blood diluted with LR solution. CONCLUSION: Our in vivo and ex vivo experiments indicate that HBOC-201 suspended in hypertonic 7.5% saline solution is associated with significantly less neutrophil activation when compared to standard HBOC-201 suspended in lactate Ringer's solution.

A TREM-1 Polymorphism A/T within the Exon 2 Is Associated with Pneumonia in Burn-Injured Patients.

Background. The triggering receptor expressed in myeloid cells (TREM-1) is a key mediator in the activation of the local inflammatory response during lung infections. We aimed to evaluate the effect of a functionally relevant TREM-1 single nucleotide polymorphism within the exon 2 (A→T) on the development of pneumonia in burn patients. Objective. To determine whether a single nucleotide polymorphism (SNP) within the exon 2 (A→T) in the TREM-1 gene is associated with ventilator-associated pneumonia (VAP) in burn-injured patients. Methods. 540 patients with ≥10% total body surface area (TBSA) burn injuries or inhalation injury were prospectively enrolled. The influence of a polymorphism (A→T) in exon 2 of the TREM-1 gene was evaluated for association with increased risk of pneumonia by logistic regression analysis. Measurements and Main Results. 209 patients met criteria for VAP. Multivariate regression analysis showed that, after adjustment for potential confounders, we found that carriage of the TREM-1 T allele is associated with more than a 3-fold increased risk of VAP (OR 6.3, 95% CI 4-9). Conclusions. A TREM-1 single nucleotide polymorphism within the exon 2 (A→T) is associated with the development of pneumonia in burn patients.

Examination with next-generation sequencing technology of the bacterial microbiota in bronchoalveolar lavage samples after traumatic injury.

BACKGROUND: We examined the microbiota of bronchoalveolar lavage (BAL) samples with next-generation sequencing (NGS) technology to determine whether its results correlate with those of standard culture methods or affect patient outcome or both. METHODS: We collected BAL samples in the surgical intensive care unit (SICU) as part of the standard of care for intubated individuals who had a Clinical Pulmonary Infection Score (CPIS)≥6 points. A portion of the BAL fluid was sequenced for the 16S region of ribosomal deoxyribonucleic acid (rDNA) with the Roche 454 FLX Titanium sequencer. Sequences were analyzed through a data-analysis pipeline to identify the appropriate taxonomic designation (∼species) of each 16s sequence. The bacterial microbiota of each BAL sample was compared with the bacteria identified in the sample through standard culture methods. Correlations between the taxonomic diversity of the microbiota and clinical outcome were examined through linear regression and Pearson correlation. RESULTS: Bronchoalveolar lavage samples from 12 individuals in the SICU who had a CPIS≥6 points were examined through 454 pyrosequencing. The number of phylotypes (∼species) in the samples ranged from 15 to 129. The number of phyla in the BAL samples ranged from 3 to 14. There was little correlation between the bacteria identified by NGS and those identified with standard culture methods. The same predominant bacterial strain was identified by both culture and sequencing in only a single sample. The correlation between patient days on a ventilator and the number of species in BAL samples was significant (r=0.7435, p=0.0056; r2=0.5528). CONCLUSIONS: Increasing diversity of the bacterial microbiota in BAL samples correlates with the duration of mechanical ventilation. Bacteria identified through standard culture methods were not well correlated with the findings of NGS.

Resveratrol decreases inflammation in the brain of mice with mild traumatic brain injury.

BACKGROUND: Following a mild traumatic brain injury (TBI) event, the secondary brain injury that persists after the initial blow to the head consists of excitotoxicity, decreased cerebral glucose levels, oxidant injury, mitochondrial dysfunction, inflammation, and neuronal cell death. To date, there are no effective interventions used at decreasing secondary brain injury after mild TBI. METHODS: In this study, male mice were treated with either placebo or resveratrol (100 mg/kg) at 5 minutes and 12 hours after mild TBI. The mice were injured using the controlled cortical impact device. In this closed-head model, a midline incision was made to access the skull and the impactor tip was aligned on the sagittal suture midway between the bregma and lambda sutures. The mice were injured at a depth of 2.0 mm, velocity of 4 m/s, and a delay time of 100 milliseconds. At 72 hours following injury, the animals were intracardially perfused with 0.9% saline followed by 10% phosphate-buffered formalin. The whole brain was removed, sliced, and stained for microglial activation (Iba1). In addition, using the enzyme-linked immunosorbent assay, tissue levels of interleukin 6 (IL-6) and IL-12 were measured in the cerebral cortex and hippocampus. RESULTS: In this study, we found that in the placebo treatment group, there was a significant increase in Iba1 staining in the brain. The levels of microglial activation was reduced by resveratrol in the cerebral cortex (p < 0.001), corpus callosum (p < 0.001), and dentate gyrus (p < 0.005) brain regions after mild TBI. In addition to Iba1, resveratrol decreased the brain levels of IL-6 (p < 0.0001) and IL-12 (p < 0.004), which were observed in the hippocampus of the placebo group. In our model, no increase of IL-6 or IL-12 was observed in the cerebral cortex following TBI. CONCLUSION: Resveratrol given acutely after TBI results in a decrease in neuroinflammation. These results suggest that resveratrol may be beneficial in reducing secondary brain injury after experiencing a mild TBI.

[Recession of both horizontal rectus muscles in Duane Retraction Syndrome with significant globe retraction].

OBJECTIVE: To evaluate the effect of Recession of both horizontal rectus muscles in Duane Retraction Syndrome with significant globe retraction. METHODS: Sixteen cases with DRS were summarized retrospectively. All patients had undergone surgery with recession of both horizontal rectus muscles. All clinical records, including sex, age, types of DRS, clinical features, surgical methods and clinical outcomes were analyzed. All patients were followed up for 3 months to 1 year. RESULTS: Fifteen cases had only monocular involvement while one had both eyes. The number of type I DRS was 3 cases, 1 case was esotropia while others were orthotropic in primary position. Type III DRS was observed 13 cases. Esotropia was seen in 6 cases (7 eyes), exotropia of 1 cases and orthotropic in primary position of 6 cases. 10 cases exhibited marked face turn. An upshoot or downshoot and variable severity of retraction of globe were found in all patients on attempt adduction of the affected eye. All patients had undergone surgery with recession of both horizontal rectus muscles. The medical rectus muscles were recessed from 5 mm to 7 mm and lateral rectus muscles 3 mm to 9 mm simultaneously, which was based on the amount of primary position deviation. Among these 2 cases were combined with Y-splitting of lateral rectus muscle. After surgery, all patients were orthotropic in primary position. Their symptom of unacceptable abnormal head position, significant globe retraction, noticeable narrowing of the palpebral fissure and significant upshoot or downshoot were ameliorated or disappeared. Especially the recession of lateral rectus muscle in addition to Y-splitting combining with the simultaneous medial rectus recession resulted in further amelioration of globe retraction in addition to upshoot and downshoot. CONCLUSION: Recession of both horizontal rectus muscles is effective in the treatment of significant globe retraction in Duane syndrome. Type III DRS with significant globe retraction but has no marked deviation and face turn can adopt this method to ameliorate their aspect. The method of lateral rectus muscle in addition to Y-splitting plays an important role in the treatment of upshoot and downshoot.

Estrone is neuroprotective in rats after traumatic brain injury.

In various animal and human studies, early administration of 17ß-estradiol, a strong antioxidant, anti-inflammatory, and anti-apoptotic agent, significantly decreases the severity of injury in the brain associated with cell death. Estrone, the predominant estrogen in postmenopausal women, has been shown to be a promising neuroprotective agent. The overall goal of this project was to determine if estrone mitigates secondary injury following traumatic brain injury (TBI) in rats. Male rats were given either placebo (corn oil) or estrone (0.5 mg/kg) at 30 min after severe TBI. Using a controlled cortical impact device in rats that underwent a craniotomy, the right parietal cortex was injured using the impactor tip. Non-injured control and sham animals were also included. At 72 h following injury, the animals were perfused intracardially with 0.9% saline followed by 10% phosphate-buffered formalin. The whole brain was removed, sliced, and stained for TUNEL-positive cells. Estrone decreased cortical lesion volume (p<0.01) and neuronal injury (p<0.001), and it reduced cerebral cortical levels of TUNEL-positive staining (p<0.0001), and decreased numbers of TUNEL-positive cells in the corpus callosum (p<0.03). We assessed the levels of ß-amyloid in the injured animals and found that estrone significantly decreased the cortical levels of ß-amyloid after brain injury. Cortical levels of phospho-ERK1/2 were significantly (p<0.01) increased by estrone. This increase was associated with an increase in phospho-CREB levels (p<0.021), and brain-derived neurotrophic factor (BDNF) expression (p<0.0006). In conclusion, estrone given acutely after injury increases the signaling of protective pathways such as the ERK1/2 and BDNF pathways, decreases ischemic secondary injury, and decreases apoptotic-mediated cell death. These results suggest that estrone may afford protection to those suffering from TBI.

IL-10 polymorphism associated with decreased risk for mortality after burn injury.

OBJECTIVE: Evaluation of single nucleotide polymorphisms (SNPs) in the interleukin-10 promoter (-592 and -819) on risk for death after burn injury. METHODS: Association between the IL-10 SNPs and outcome after burn injury was evaluated in a cohort of 265 patients from Parkland Hospital, Dallas, TX with ≥ 15% TBSA burns without non-burn trauma (ISS ≤ 16), traumatic or anoxic brain injury or spinal cord injury, who survived >48 h under an IRB-approved protocol. Clinical data were collected prospectively and genotyping was conducted by TaqMan assay. Whole blood from 31 healthy volunteers was stimulated with LPS (100 ng/mL) to determine the level of IL-10 expression for each allele by enzyme-linked immunosorbent assay (ELISA). RESULTS: After adjustment for percent total body surface area (TBSA) burned, inhalation injury, age, gender, and race/ethnicity, carriage of ­592A and/or ­819T was significantly associated (P = 0.014) with a decreased risk for death (adjusted odds ratio: 0.404; 95% CI: 0.197-0.829). As the candidate SNPs were in complete linkage disequilibrium, it was not possible to distinguish which allele was associated with decreased mortality risk. Age, inhalation injury, and full-thickness burn size were significantly associated with increased risk for death. In the LPS stimulated blood of healthy controls, carriage of the -592A and/or -819T allele demonstrated a trend for decreased levels of IL-10 (P = 0.079). CONCLUSION: Carriage of the ­592A and/or ­819T allele in the IL-10 promoter appears to reduce the risk for death after burn injury.