Burn-induced organ dysfunction: Vagus nerve stimulation attenuates organ and serum cytokine levels

Introduction

The interaction of the CNS and the immune system is well known. A parasympathetic anti-inflammatory pathway has recently been described. Both electrical and pharmacological parasympathetic stimulation attenuate proinflammatory mediator generation. Burn induces abacterial cytokine generation and we sought to evaluate whether parasympathetic stimulation after experimental burn decreases cardiodepressive mediator generation.

Material and methods

A 30% TBSA full-thickness rat burn model was used. After microsurgical preparation of the cervical portion of the vagus nerve, we performed electric vagus nerve stimulation. Serum was harvested and organ samples of heart and liver were homogenized. Samples were subjected to sandwich-ELISA specific for TNF-α, IL-1β and IL-6. Heart rate measurements were done using left ventricular microcatheterization. Statistical analysis was done using Student's t-tests and analysis of variance (ANOVA).

Results

Burn induced a significant rise of TNF-α, IL-1β and IL-6 in organ homogenates and serum. After cervical vagal electrostimulation, serum and organ homogenate levels of proinflammatory cytokines were markedly reduced compared to burn controls. Left ventricular microcatheter assessment demonstrated no cardiodepressive effect of the vagal stimulation itself.

Conclusion

Our results encourage further research regarding the neuroimmunologic background of burn, possibly leading to the development of a novel therapeutic approach to burn-induced organ dysfunction and immunodysregulation.     

Isotetrandrine protects against lipopolysaccharide-induced acute lung injury by suppression of mitogen-activated protein kinase and nuclear factor-kappa B

Background

Mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) signaling pathways are pleiotropic regulator of many genes involved in lipopolysaccharide (LPS)-induced acute lung injury (ALI). The present study aimed to reveal the protective effect of isotetrandrine (ITD), a small molecule inhibitor, on various aspects of LPS-induced inflammation in vitro and in vivo.

Methods

In vitro, RAW 264.7 cells were pretreated with different dose of ITD 1 h before treatment with 1 mg/L of LPS. In vivo, to induce ALI, male BALB/c mice were injected intranasally with LPS and treated with ITD (20 and 40 mg/kg) 1 h before LPS.

Results

In vitro, the cytokine levels of tumor necrosis factor-α, interleukin (IL)-1β, and IL-6 in supernatant were reduced by ITD. Meanwhile, in vivo, pulmonary inflammatory cell infiltration, myeloperoxidase activity, total cells, neutrophils, macrophages, along with the levels of tumor necrosis factor-α, IL-1β, and IL-6 in bronchoalveolar lavage fluid were dose-dependently attenuated by ITD. Furthermore, our data showed that ITD significantly inhibited the activation of MAPK and NF-κB, which are induced by LPS in ALI model.

Conclusions

These results suggested that ITD dose-dependently suppressed the severity of LPS-induced ALI by inactivation of MAPK and NF-κB, which may involve the inhibition of tissue oxidative injury and pulmonary inflammatory process.     

Small Interfering RNA Targeting Nuclear Factor Kappa B to Prevent Vein Graft Stenosis in Rat Models

Background

Intimal hyperplasia plays an important role in vein graft stenosis. Inflammatory injury, especially nuclear factor kappaB (NF-κB) gene activation, is highly involved in stenosis progression. We examined whether neointimal hyperplasia and vein graft stenosis could be inhibited by silencing the NF-κB gene with small interference RNA (siRNA).

Methods

Sixty adult male Sprague-Dawley rats were randomly divided into a normal vein group, a vein graft group, a scrambled siRNA group, and an NF-κB siRNA group. We performed reverse interpositional grafting of the autologous external jugular vein to the abdominal aorta. Vein grafts were treated with liposome and gel complexes containing NF-κB siRNA or scrambled siRNA. The levels of monocyte chemoattractant protein -1, tumor necrosis factor-α, and NF-κB p65 in vessel tissues were evaluated after surgery for content of proliferating cell nuclear antigen (PCNA) and vascular wall thickness.

Results

NF-κB siRNA treated vein graft showed less neointimal formation and fewer positive PCNA cells (P < .05). In addition there were lower levels of, NF-κB p65 protein and of inflammatory mediators (P < .05) compared with the vein graft group.

Conclusion

Our study suggested that siRNA transfection suppressed NF-κB expression, reduced inflammatory factors, lessened neointimal proliferation, and suppressed PCNA.     

Inhibition of Na/H exchanger 1 by cariporide alleviates burn-induced multiple organ injury

Background

Severe burns initiate an inflammatory response characterized by the upregulation of proinflammatory cytokine, which contributes to multiple organ injury. Na⁺/H⁺ exchanger 1 (NHE1) plays a significant role in several inflammatory processes. This study was designed to investigate the role of NHE1 in burn-induced inflammation and multiple organ injury.

Materials and methods

Rats were subjected to a 30% total body surface area full-thickness burn. Cariporide was used to assess the function of NHE1 in burn-induced multiple organ injury by biochemical parameters, histologic changes, and inflammatory cytokine production.

Results

We found that NHE1 expression was significantly increased after burn injury. Inhibition of NHE1 by cariporide attenuated burn-induced edema and tissue injury in heart, lung, kidney, and small intestine. Cariporide also inhibited plasma levels of tumor necrosis factor α, interleukin 6, and myeloperoxidase activity.

Conclusions

These results indicate that NHE1 inhibition prevents burn-induced multiple organ injury. The salutary effects afforded by NHE1 inhibition, at least in part, are mediated by attenuating systemic inflammatory response.     

The vagus nerve alters the pulmonary dendritic cell response to injury

Background

We have shown previously that vagal nerve stimulation (VNS) protects against burn-induced acute lung injury (ALI). Although the mobilization and activation of immune cells is central to tissue injury caused by the systemic inflammatory response, the specific inflammatory cell populations that are modulated by VNS have yet to be fully defined. The purpose of this study was to assess whether VNS alters inflammatory cell recruitment to the lung after severe burn injury.

Materials and methods

Male C57BL/6 mice were subjected to 30% total body surface area steam burn with and without electrical stimulation of the right cervical vagus nerve. The relative levels of pulmonary dendritic cells (DC) and macrophages were compared at 4 h versus 24 h after burn injury. Lung tissue injury was characterized by histology to assess changes in lung architecture, and measure the protein levels of interleukin 6 and transforming growth factor-β1.

Results

Severe burn caused an increase in pulmonary DC recruitment at 4 h after injury that persisted at 24 h after severe burn, whereas there was no change in the number of pulmonary macrophages. In contrast, VNS limited the burn-induced recruitment of pulmonary DC. VNS prevented histologic lung injury and attenuated the release of interleukin 6 and transforming growth factor-β1 in the lung after burn injury.

Conclusions

VNS is an effective method to limit pulmonary DC recruitment to the lung and prevent ALI after burn injury. Identifying strategies to limit inflammatory cell recruitment to the lung may have clinical utility in preventing ALI in severely burned patients.     

Correlation between circulating proteasome activity,total protein and c-reactive protein levels following burn in children

Aim of the study

To characterize burn-induced changes following burn in children by analyzing circulating proteasome (c-proteasome) activity in the plasma in correlation with total protein and c-reactive protein levels in the plasma, and the severity of the burn.

Methods

Fifty consecutive children scalded by hot water who were managed at the Department of Pediatric Surgery after primarily presenting with burns in 4–20% TBSA were included into the study. The children were aged 9 months up to 14 years (mean age 2.5 ± 1 years). Patients were divided into groups according to the pediatric injury severity score used by American Burns Association. Plasma proteasome activity was assessed using Suc-Leu-Leu-Val-Tyr-AMC peptide substrate, 2–6 h, 12–16 h, 3 days, 5 days, and 7 days after injury. 20 healthy children consecutively admitted for planned inguinal hernia repair served as controls.

Results

Statistically significant elevation of plasma c-proteasome activity was noted in all groups of burned children 12–16 h after the injury. We found a strong negative correlation of c-proteasome activity with total protein levels, and positive correlation with CRP levels 12–16 h after burn. We also found stronger correlation between c-proteasome activity and severity of burn, than CRP level and severity of burn 12–16 h, and 3 days after the burn. Correlations were statistically significant.

Conclusions

This study characterized circulating 20S proteasome activity levels after burn. C-proteasome activity elevate after burn and correlate negatively with plasma total protein level, thus plasma 20S proteasome activity could be additional biomarker of tissue damage in burn in pediatric population.     

Burn induces a Th-17 inflammatory response at the injury site

Introduction

The high incidence of morbidity and mortality following major burn can be in part attributed to immune dysfunction and wound healing complications. Inflammation plays a major role in the complex process of wound repair. Recently, a novel class of T-helper cells, termed Th-17 cells, has been found to secrete the pro-inflammatory cytokines IL-17 and IL-22. The Th-17 response also involves other cytokines, such as IL-6 and TGF-β, which have been shown to be associated with burn-induced inflammation. Nonetheless, the relationships between the Th-17 response and post-burn inflammation are unknown.

Methods

C57BL/6 male mice (n = 5-6/group) were subjected to a major burn (25% TBSA) or sham procedure. Three hours thereafter, skin samples were collected (uninjured skin and burn skin) and processed for the determination of Th-17 cytokine (IL-6, IL-17, IL-22, IL-23, IL-27, and TGF-β) levels by ELISA.

Results

At 3 h after burn a significant (∼3-fold) increase in tissue levels of IL-17 and IL-22 was observed at the burn site as compared to sham skin. The burn-induced Th-17 response was independent of statistically significant changes in other Th-17 cytokines (i.e., IL-6, IL-23, IL-27 and TGF-β).

Conclusions

These findings indicate the development of a robust Th-17 response at the burn site that may play an important role in subsequent immune and wound healing derangements.     

Effects of sleep deprivation on pain-related factors in the temporomandibular joint

Background

The objective of this study was to investigate the effects of experimental sleep deprivation (SD) on the temporomandibular joint (TMJ) in rats by examining pain-related factors and to determine the possible involvement of estrogen and NF (nuclear factor) κB signaling in the TMJ synovial membrane.

Methods

The influence of SD, conducted in rats using the modified multiple platform method, was estimated by observing behavioral manifestations and examining changes in serum hormone levels. The morphologic changes of synovial tissue were observed with light microscopy and the serum levels of estrogen were measured by radioimmunoassay. Activation of NF-κB in the synovial membrane was examined using an immunofluorescence technique, and the expression levels of interleukin (IL) 1β, IL-6, tumor necrosis factor α, cyclooxygenase 2, and inducible nitric oxide synthase were measured with real-time polymerase chain reaction.

Results

The SD group showed evidence of elevated anxiety and stress, and increased plasma levels of estradiol compared with the control group. The activity of NF-κB was significantly enhanced and translocation of NF-κB p65 was evident in the synovial membrane after SD. The expression of pain-related factors IL-1β, IL-6, cyclooxygenase-2, tumor necrosis factor α, and inducible nitric oxide synthase in the synovial membrane significantly increased after SD.

Conclusions

These results indicate that SD increases serum levels of estrogen and induces alterations in pain-related factors in the TMJ. The NF-κB pathway has been associated with the regulation of these inflammatory cytokines and plays an important role in temporomandibular disorders.     

Sclerosis therapy of bronchial artery attenuates acute lung injury induced by burn and smoke inhalation injury in ovine model

Introduction

In burned sheep, we showed more than a 10-fold increase in bronchial blood flow following smoke inhalation. It was previously reported that sclerosis of the bronchial artery prior to smoke exposure reduces the pathophysiology of the inhalation insult. We hypothesized that sclerosis of the bronchial artery after insult attenuates smoke/burn-induced acute lung injury.

Methods

Through an incision at the 4th intercostal space, a catheter was placed via the esophageal artery into the bronchial artery such that the bronchial blood flow remained intact. Acute lung injury was induced by a 40% total body surface area, 3rd degree cutaneous burn and smoke inhalation. Adult female sheep (n = 18, 35.6 ± 1.0 kg) were divided into three groups following the injury: (1) sclerosis group: 1 h after injury, 4 mL of 70% ethanol was injected into bronchial artery via bronchial catheter, n = 6; (2) control group: 1 h after injury, an equal dose of saline was injected into bronchial artery via the bronchial catheter, n = 6; (3) sham group: no injury and no treatment, n = 6. The experiment was conducted in awake animals for 24 h.

Results

Bronchial blood flow, measured by microspheres, was significantly reduced after ethanol injection in the sclerosis group. Pulmonary function, evaluated by measurement of blood gas analysis, pulmonary mechanics, and pulmonary transvascular fluid flux, was severely impaired in the control group. However, pulmonary function was significantly improved by bronchial artery sclerosis.

Conclusion

The results of our study clearly demonstrate a crucial role of enhanced bronchial circulation in thermal injury-related morbidity. Decreasing bronchial circulation using pharmacological agents may be an effective strategy in management of burn patients with concomitant smoke inhalation injury.     

Skeletal muscle satellite cell activation following cutaneous burn in rats

Background

Cutaneous burn distant from skeletal muscles induces atrophy; however, its effect on muscle stem cells resident in skeletal muscle (satellite cells) distal to burn is not known.

Methods

Satellite cell activation was measured in predominantly fast-twitch [tibialis anterior, extensor digitorum longus (EDL), plantaris, and gastrocnemius] and slow-twitch (soleus) muscles of rats that received either 40% total body surface area full-thickness scald burn or sham burn to the trunk area by determining bromodeoxyuridine incorporation, MyoD, and Pax7 immunohistochemistry in vivo ≤48 h after burn. To determine the effects of circulating factors on satellite cell activation, satellite cell cultures were treated with serum from sham or burn rats.

Results

In vivo activation of satellite cells was increased in fast muscles isolated from burn as compared to sham animals, whereas a significant response was not seen in slow muscles. Serum taken from animals in the burn group increased the activation of satellite cells isolated from both sham and burn animals in vitro, suggesting that circulating factors have the potential to increase satellite cell activation following burn.

Conclusions

Increases in satellite cell activation in muscles distal to burn are fiber-type-dependent, and circulating factors may play a role in the activation of satellite cells following burn. A better understanding of the impact of burn on satellite cell functionality will allow us to identify the cellular mechanisms of long-term muscle atrophy.     

Suppressive effects of levobupivacaine on endotoxin-induced microglial activation

Background

We sought to elucidate the effects of levobupivacaine on modulating endotoxin-induced upregulation of inflammatory mediators and activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs) signaling pathways in activated microglia.

Materials and methods

Confluent murine microglia (BV-2) were treated with endotoxin (lipopolysaccharide, 50 ng/mL) or endotoxin plus levobupivacaine (5, 25, or 50 μM) and denoted as the LPS, LPS + L(5), LPS + L(25), and LPS + L(50) groups, respectively. Levobupivacaine was administered immediately after endotoxin. Control groups were run simultaneously.

Results

The concentrations of inflammatory mediators, including macrophage inflammatory protein-2 (P = 0.023 and 0.016), tumor necrosis factor-α (P = 0.025 and 0.020), interleukin (IL)-1β (P = 0.018 and 0.014), IL-6 (P = 0.029 and 0.023), nitric oxide (P = 0.025 and 0.026), and prostaglandin E₂ (P = 0.028 and 0.016) of the LPS + L(25) and LPS + L(50) groups were significantly lower than those of the LPS group. The concentrations of macrophage inflammatory protein-2 (P = 0.035), IL-1β (P = 0.024), nitric oxide (P = 0.031), and prostaglandin E₂ (P = 0.036) but not tumor necrosis factor-α and interleukin-6 of the LPS + L(5) group were also significantly lower than those of the LPS group. These data revealed that effects of endotoxin on upregulating inflammatory mediators were inhibited by levobupivacaine. Moreover, effects of endotoxin on activating NF-κB, including inhibitor-κB degradation, NF-κB nuclear translocation, and NF-κB–DNA binding, were also inhibited by levobupivacaine. Similarly, effects of endotoxin on activating MAPKs, including extracellular signal–regulated kinase, c-jun N-terminal kinase, and p38 MAPK, were also significantly inhibited by levobupivacaine.

Conclusions

Levobupivacaine significantly inhibited endotoxin-induced upregulation of inflammatory mediators and activation of NF-κB and MAPKs signaling pathways in activated microglia.     

Cinanserin reduces plasma extravasation after burn plasma transfer in rats

Background

Thermal injuries greater than 20% body surface area (BSA) lead to systemic edema and hypovolemic shock. Capillary leakage is induced by different immunomodulative cytokines. Serotonin (5-HT) plays an important role in inflammation, vasodilatation and vasoconstriction and many other pathways such as systemic inflammation in endotoxemia and burns. Cinanserin, a specific 5-HT₂ receptor blocking agent was administered to observe whether burn induced systemic edema can be reduced.

Methods

Donor animals underwent thermal injury (100 °C water, 30% BSA, 12 s) for positive controls and negative controls underwent a shamburn procedure (37 °C water, 30% BSA, 12 s). Donor rat-plasma was transferred to healthy individuals after bolus injection of Cinanserin (5 mg/kg body weight) was performed in recipient animals. Intravital microscopy was performed in mesenteric venules (0/60/120 min) to asses systemic edema by FITC-albumin extravasation. Additionally, leukocyte activation (cells/mm²) was observed.

Results

Burnplasma-transfer results in systemic capillary leakage that is not observed in sham burn controls. Intraveneous application of Cinanserin significantly reduces systemic burn edema to shamburn levels. Leukocyte-endothelial interactions are significantly reduced by administration of Cinanserin.

Conclusion

Specific 5-HT₂ antagonism reduces systemic burn edema and leukocyte activation after plasma transfer. Reduction of capillary leakage may be partially mediated by leukocyte dependent as well as independent mechanisms. Future studies need to evaluate specific 5-HT₂ receptor subtypes to distinguish between local and systemic effects of serotonin antagonists.     

Inhibitory effects of astragalin on lipopolysaccharide-induced inflammatory response in mouse mammary epithelial cells

Background

Tea brewed from the leaves of persimmon or Rosa agrestis have several medical functions including treating allergy, antiatopic dermatitis, and anti-inflammatory effects. The objective of this study was to investigate the molecular mechanisms of astragalin, a main flavonoid component isolated from these herbs, in modifying lipopolysaccharide (LPS)-induced signaling pathways in primary cultured mouse mammary epithelial cells (mMECs).

Materials and methods

The mMECs were treated with LPS in the absence or presence of different concentrations of astragalin. The expression of proinflammatory cytokines tumor necrosis factor α, and interleukin 6, as well as nitric oxide production were determined by enzyme-linked immunosorbent assay and Griess reaction, respectively. Cyclooxygenase-2, inducible nitric oxide synthase, toll-like receptor 4 (TLR4), nuclear factor-κB (NF-κB), inhibitor protein of NF-κB (IκBα), P38, extracellular signal-regulated kinase, and c-Jun N-terminal kinase were measured by Western blot.

Results

The results showed that astragalin suppressed the expression of tumor necrosis factor α, interleukin 6, and nitric oxide in a dose-dependent manner in mMECs. Western blot results showed that the expression of inducible nitric oxide synthase and cyclooxygenase-2 was inhibited by astragalin. Besides, astragalin efficiently decreased LPS-induced TLR4 expression, NF-κB activation, IκBα degradation, and the phosphorylation of p38, extracellular signal-regulated kinase in BMECs.

Conclusions

Our results indicated that astragalin exerts anti-inflammatory properties possibly via the inactivation of TLR4-mediated NF-κB and mitogen-activated protein kinases signaling pathways in LPS-stimulated mMECs. Thus, astragalin may be a potential therapeutic agent for bovine mastitis.     

nNOS expression in the brain of rats after burn and the effect of the ACE inhibitor captopril

Objective

To investigate the role of endogenous neuronal nitric oxide synthase (nNOS) on brain injury after burn and the effects of the captopril.

Methods

Wistar albino rats (200–250 g) were exposed on the dorsal surface to 90 °C (burn) or 25 °C (sham) water for 10 s. The ACE group was treated with intraperitoneal 10 mg/kg captopril immediately after burn and this treatment was repeated twice daily. At the end of the 24 h brain samples were taken. nNOS was studied in brain areas by immunohistochemistry.

Results

There was no difference between the cerebellar and hypothalamic areas the nNOS expression of all groups. nNOS expression increased in the frontal cortex, striatum and midbrain in the burn group compared to the control group. In the frontal cortex, nNOS expression significantly decreased after ACE inhibitor treatment (p < 0.05). The striatal nNOS of the ACE group significantly increased when compared to the control group (p = 0.001). In the midbrain of the animals, nNOS decreased in the ACE group. Hippocampal nNOS expression did not change after burn and significantly increased after ACE inhibitor therapy (p < 0.05).

Conclusions

Our data showed that the pathophysiological events following burn appear to be related to an acute inflammatory reaction which is associated with nNOS in the frontal cortex, striatum and midbrain, and captopril treatment abrogates the nNOS response in the frontal cortex and midbrain.