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ABSTRACT: Mitochondria play a central role in the integration and execution of a wide variety of apoptotic signals. In the present study, we examined the deleterious effects of burn injury on heart tissue. We explored the effects of vagal nerve stimulation (VNS) on cardiac injury in a murine burn injury model, with a focus on the protective effect of VNS on mitochondrial dysfunction in heart tissue. Mice were subjected to a 30% total body surface area, full-thickness steam burn followed by right cervical VNS for 10 min. and compared to burn alone. A separate group of mice were treated with the M3 -muscarinic acetylcholine receptor (M3 -AchR) antagonist 4-DAMP or phosphatidylinositol 3 Kinase (PI3K) inhibitor LY294002 prior to burn and VNS. Heart tissue samples were collected at 6 and 24 hrs after injury to measure changes in apoptotic signalling pathways. Burn injury caused significant cardiac pathological changes, cardiomyocyte apoptosis, mitochondrial swelling and decrease in myocardial ATP content at 6 and 24 hrs after injury. These changes were significantly attenuated by VNS. VNS inhibited release of pro-apoptotic protein cytochrome C and apoptosis-inducing factor from mitochondria to cytosol by increasing the expression of Bcl-2, and the phosphorylation level of Bad (pBad(136) ) and Akt (pAkt(308) ). These protective changes were blocked by 4-DAMP or LY294002. We demonstrated that VNS protected against burn injury-induced cardiac injury by attenuating mitochondria dysfunction, likely through the M3 -AchR and the PI3K/Akt signalling pathways.
Journal of Cellular and Molecular Medicine 04/2013; · 4.13 Impact Factor
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ABSTRACT: Intestinal barrier breakdown following traumatic brain injury (TBI) is characterized by increased intestinal permeability, leading to bacterial translocation, and inflammation. The hormone ghrelin may prevent intestinal injury and have anti-inflammatory properties. We hypothesized that exogenous ghrelin prevents intestinal injury following TBI. A weight-drop model created severe TBI in three groups of anesthetized Balb/c mice. Group TBI: animals underwent TBI only; Group TBI/ghrelin: animals were given 10 μg of ghrelin intraperitoneally prior and 1 h following TBI; Group sham: no TBI or ghrelin injection. Intestinal permeability was measured 6 h following TBI by detecting serum levels of FITC-Dextran after injection into the intact ileum. The terminal ileum was harvested for histology, expression of the tight junction protein MLCK and inflammatory cytokine TNF-α. Permeability increased in the TBI group compared to the sham group (109.7 ± 21.8 μg/mL vs. 32.2 ± 10.1 μg/mL; p < 0.002). Ghrelin prevented TBI-induced permeability (28.3 ± 4.2 μg/mL vs. 109.7 ± 21.8 μg/mL; p < 0.001). The intestines of the TBI group showed blunting and necrosis of villi compared to the sham group, while ghrelin injection preserved intestinal architecture. Intestinal MLCK increased 73% compared to the sham group (p < 0.03). Ghrelin prevented TBI-induced MLCK expression to sham levels. Intestinal TNF-α increased following TBI compared to the sham group (46.2 ± 7.1 pg/mL vs. 24.4 ± 2.2 pg/mL p < 0.001). Ghrelin reduced TNF-α to sham levels (29.2 ± 5.0 pg/mL; p = NS). We therefore conclude that ghrelin prevents TBI-induced injury, as determined by intestinal permeability, histology, and intestinal levels of TNF-α. The mechanism for ghrelin mediating intestinal protection is likely multifactorial, and further studies are needed to delineate these possibilities.
Journal of neurotrauma 12/2010; 27(12):2255-60. · 4.25 Impact Factor
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ABSTRACT: Hypertonic saline (HS) restores prostaglandin E(2) (PGE(2))-induced T-cell suppression in the presence of 1100 microM arginine. However, under arginine-free culture conditions, HS dose not restore T-cell proliferation. Therefore, we wanted to determine if HS can restore PGE(2)-induced T-cell suppression in the presence of 80 microM of arginine, the physiologically relevant arginine concentration. We also wanted to determine the concentration of arginine that induces HS restoration of PGE(2)-suppressed T-cell proliferation and whether HS restoration of T-cell dysfunction is dependent on the injection time of HS.
Jurkat cells were cultured in media containing 0, 40, 80, 400, 800, or 1100 microM arginine. In both the PGE(2)-stimulated and HS-treated group, we measured cell proliferation using MTT assay and arginase activity. We also measured cell proliferation relative to HS injection time.
In 80 microM arginine, HS did not restore Jurkat cell proliferation that had been suppressed by PGE(2). Increased concentrations of arginine in the media increased MTT cell proliferation. In 800 microM arginine media, HS restored PGE(2)-suppressed Jurkat cell proliferation to normal. HS restored PGE(2)-suppressed Jurkat cell proliferation when it was added at 2 h, similar to at same time and 1 h after PGE(2) stimulation.
In order to restore PGE(2)-suppressed Jurkat cell proliferation, HS requires at least 800 microM arginine. HS restored PGE(2)-suppressed Jurkat cell proliferation even though HS was added at 2 h after PGE(2) stimulation.
Journal of Surgical Research 09/2010; 163(1):e17-22. · 2.25 Impact Factor
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ABSTRACT: It has been well established that laparoscopic surgery presents several clinical benefits, including reduced pain and a shorter hospital stay. These effects have been associated with a decrease in the inflammatory response. Previous studies have demonstrated that reduced inflammation after laparoscopic surgery is the product of carbon dioxide insufflation, which decreases peritoneal pH. The objective of this study was to investigate the cellular and molecular mechanisms responsible for the reduced response after exposure to acidic environments.
A murine macrophage line (J744) was incubated in culture medium at pH 6.0 or pH 7.4 for 3 h at 37°C. Then, cells were stimulated with lipopolysaccharide (LPS) at pH 7.4, the expression of TNF-α (qRT-PCR or enzyme-linked immunosorbent assay (ELISA) and intracellular pH were measured. In addition, CD14 and Toll-like receptor 4 expression and NF-κB nuclear translocation were analyzed.
A significant decrease in LPS-induced TNF-α expression levels was observed in cells pre-incubated at pH 6.0 in comparison with cells at neutral pH conditions. This decrease in TNF-α levels was not associated with a reduction in cell surface expression of CD14 and Toll-like receptor 4. Exposure to an extracellular acidic environment resulted in a reduction of IκB phosphorylation and NF-κB nuclear translocation, secondary to a significant drop in cytosolic pH.
These observations provide a potential mechanism for the reduced expression of TNF-α after exposure to low extracellular pH, which may be related to acidification after CO(2) insufflation during laparoscopic surgery. In addition, extracellular acidic pH environments could emerge as an important regulator of macrophage function.
Journal of Surgical Research 09/2010; 173(1):127-34. · 2.25 Impact Factor
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ABSTRACT: Traumatic brain injury (TBI) causes gastrointestinal dysfunction and increased intestinal permeability. Regulation of the gut barrier may involve the central nervous system. We hypothesize that vagal nerve stimulation prevents an increase in intestinal permeability after TBI.
Balb/c mice underwent a weight drop TBI. Selected mice had electrical stimulation of the cervical vagus nerve before TBI. Intestinal permeability to 4.4 kDa FITC-Dextran was measured 6 hours after injury. Ileum was harvested and intestinal tumor necrosis factor-alpha and glial fibrillary acidic protein (GFAP), a marker of glial activity, were measured.
TBI increased intestinal permeability compared with sham, 6 hours after injury (98.5 microg/mL +/- 12.5 vs. 29.5 microg/mL +/- 5.9 microg/mL; p < 0.01). Vagal stimulation prevented TBI-induced intestinal permeability (55.8 +/- 4.8 microg/mL vs. 98.49 microg/mL +/- 12.5; p < 0.02). TBI animals had an increase in intestinal tumor necrosis factor-alpha 6 hours after injury compared with vagal stimulation + TBI (45.6 +/- 8.6 pg/mL vs. 24.1 +/- 1.4 pg/mL; p < 0.001). TBI increased intestinal GFAP 6.2-fold higher than sham at 2 hours and 11.5-fold higher at 4 hours after injury (p < 0.05). Intestinal GFAP in vagal stimulation + TBI animals was also 6.7-fold higher than sham at 2 hours, however, intestinal GFAP was 18.0-fold higher at 4 hours compared with sham and 1.6-fold higher than TBI alone (p < 0.05).
In a mouse model of TBI, vagal stimulation prevented TBI-induced intestinal permeability. Furthermore, vagal stimulation increased enteric glial activity and may represent the pathway for central nervous system regulation of intestinal permeability.
The Journal of trauma 05/2010; 68(5):1059-64. · 2.48 Impact Factor
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ABSTRACT: Objective. Previous work suggests that supplementation of Ringer's lactate (RL) resuscitation with pentoxifylline (PTX) abrogates distant organ injury after hemorrhage. The aim of this work was to determine whether PTX can effectively attenuate resuscitation-induced intestinal injury and pro-inflammatory mediator production. Material and methods. Male Sprague–Dawley rats underwent 60 min of hypotension followed by resuscitation with RL or RL + PTX (25 mg/kg). The ileum was examined for injury. Nitric acid and pro-inflammatory cytokines were measured using an enzyme-linked immunosorbent assay. Inducible nitric oxide synthase (iNOS) and phosphorylation of nuclear factor (NF)-κB and signal transducer and activator of transcription (STAT)-3 were evaluated using Western blotting. Results. RL resuscitation led to a marked increase in all parameters over the control. The addition of PTX significantly reduced histologic injury and levels of iNOS, NO, tumor necrosis factor-α, interleukin-6, and cytokine-induced neutrophil chemoattractant (p<0.05). RL-induced phosphorylation of cytoplasmic IκB-α, nuclear NF-κB, and STAT3 was abrogated with PTX supplementation. Conclusion. Intestinal injury and activation of iNOS-mediated pathways associated with RL resuscitation were significantly reduced when PTX was used as an adjunct to standard RL treatment.
07/2009; 5(1):51-58.
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ABSTRACT: The use of engineered tissue for the treatment of a variety of acute to chronic wounds has become a clinical standard, and a better understanding of the cellular mechanisms of re-vascularization and barrier integrity could enhance clinical outcomes. Here, we focus on the characterization of the re-vascularization of acellular grafts such as Integra in an animal model to better understand the physiological properties of blood vessels growing in the collagen-glycosaminoglycan matrix vs. wound margins. While Integra has been extensively studied in pre-clinical models, the re-modeling mechanisms of the capillary bed under these matrices are not well understood. Therefore, our first objective was to quantify the kinetics of re-vascularization. The second objective was to assess changes in vascular permeability (VP) of the wound bed compared to normal adjacent skin. The third objective was to establish a non-invasive and quantitative assay for the measurement of VP to facilitate the rapid and reproducible characterization of vascular integrity. Using an excisional wound model in mice, we characterize the appearance, growth, and maturation of blood vessels in an Integra graft over 28 days after surgery. Initial appearance of blood vessels in the graft was observed at 7 days, with angiogenesis peaking between 7 and 14 days. The onset of VP coincided with the increase in re-vascularization of the wound bed and there was a sustained elevation of VP that declined to baseline by 28 days. We propose a non-invasive strategy to assess VP of the wound capillary bed will facilitate a better understanding of the cell and molecular basis of angiogenesis in wound healing.
Burns: journal of the International Society for Burn Injuries 06/2009; 35(6):811-7. · 1.95 Impact Factor
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ABSTRACT: T-cell dysfunction after trauma is characterized by decreased T-cell proliferation. Hypertonic saline (HS) restores T-cell proliferation by an unknown mechanism. Arginine and regulation of arginine metabolism plays an important role in normal T-cell function. We hypothesize that HS restoration of T-cell dysfunction is dependent on an arginine mediated mechanism.
Jurkat cells were cultured in both 0 mM and 1.14 mM arginine media. Cell proliferation was suppressed using prostaglandin E2 (PGE(2)) and treated with HS at 20 and 40 mM above isotonicity. Arginase activity was blocked by norNOHA. Cell proliferation, arginase activity, and nitrite accumulation were measured.
PGE(2) caused a 15.0% inhibition of Jurkat cell proliferation compared with control (P<0.05). HS reversed PGE(2) suppressed Jurkat cell proliferation to normal. PGE(2) suppression decreased mean arginase activity (66.5+/-15 nmol/min/mg) compared with controls (98.4+/-14 nmol/min/mg) (P<0.05). Cells treated with HS had higher arginase activity (123.8+/-38 nmol/min/mg) then PGE(2) suppressed cells and controls (P <.05). Conversely, nitrite was decreased by 14.5% +/- 3.1% in HS treated cells compared with PGE(2) suppression (P<0.05). HS did not restore PGE(2) cell suppression when arginase I was blocked by norNOHA, nor when cells were cultured in arginine-free media.
Arginine is essential in restoring Jurkat cell proliferation by HS. HS may restore T-cell dysfunction by increasing arginine transport and arginine metabolism by arginase I. HS treatment will not restore suppressed T-cell proliferation without adequate extracellular concentrations of arginine.
Journal of Surgical Research 06/2009; 156(1):83-9. · 2.25 Impact Factor
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ABSTRACT: Traumatic brain injury (TBI) can lead to several physiologic complications including gastrointestinal dysfunction. Specifically, TBI can induce an increase in intestinal permeability, which may lead to bacterial translocation, sepsis, and eventually multi-system organ failure. However, the exact mechanism of increased intestinal permeability following TBI is unknown. We hypothesized that expression of tight junction protein ZO-1 and occludin, responsible for intestinal architectural and functional integrity, will decrease following TBI and increase intestinal permeability. BALB/c mice underwent a weight drop TBI model following anesthesia. Brain injury was confirmed by a neurologic assessment and gross brain pathology. Six hours following injury, FITC-dextran (25 mg 4.4 kDa FITC-dextran) was injected into the intact lumen of the isolated ileum. Intestinal permeability was measured in plasma 30 min following injection, by using spectrophotometry to determine plasma FITC-dextran concentrations. Whole ileum extracts were used to measure expression of tight junction proteins ZO-1 and occludin by Western blot. TBI caused a significant increase in intestinal permeability (110.0 microg/mL +/-22.2) compared to sham animals (29.4 microg/mL +/- 9.7) 6 h after injury (p = 0.016). Expression of ZO-1 was decreased by 49% relative to sham animals (p < 0.02), whereas expression of occludin was decreased by 73% relative to sham animals (p < 0.001). An increase in intestinal permeability corresponds with decreased expression of tight junction proteins ZO-1 and occludin following TBI. Expression of intestinal tight junction proteins may be an important factor in gastrointestinal dysfunction following brain injury.
Journal of neurotrauma 04/2009; 26(8):1353-9. · 4.25 Impact Factor
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ABSTRACT: Introduction:
Under the trimodal distribution, most trauma deaths occur within the first hour. Determination of cause of death without autopsy
review is inaccurate. The goal of this study is to determine cause of death, in hourly intervals, in trauma patients who died
in the first 24 h, as determined by autopsy.
Materials and Methods:
Trauma deaths that occurred within 24 h at a Level I trauma center were reviewed over a six-year period ending December 2005.
Timing of death was separated into 0–1, 1–3, 3–6, 6–12 and 12–24 h intervals. Cause of death was determined by clinical course
and AIS scores, and was confirmed by autopsy results.
Results:
Overall, 9,388 trauma patients were admitted, of which 185 deaths occurred within 24 h, with 167 available autopsies. Blunt
and penetrating were the injury mechanisms in 122 (73%) and 45 (27%) patients, respectively. Of 167 deaths, 73 (43.7%) occurred
within the first hour. Brain injury, when compared to other body areas, was the most likely cause of death in all hourly intervals,
but hemorrhage was as or more important than brain injury as the cause of death during the first 3 h and up to 6 h. No deaths
were attributable to hemorrhage after 12 h.
Conclusions:
The temporal distribution of the cause of death varies in the first 24 h after admission. Hemorrhage should not be overlooked
as the cause of death, even after survival beyond 1 h. Understanding the temporal relationship of causes of early death can
aid in the targeting of management and surgical training to optimize patient outcome.
European Journal of Trauma and Emergency Surgery 01/2009; 35(1):26-30. · 0.33 Impact Factor
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Todd W Costantini,
William H Loomis,
James G Putnam,
Dana Drusinsky,
Jessica Deree,
Sunghyuk Choi,
Paul Wolf,
Andrew Baird,
Brian Eliceiri,
Vishal Bansal,
Raul Coimbra
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ABSTRACT: Loss of intestinal barrier function after burn injury allows movement of intraluminal contents across the mucosa, which can lead to the development of distant organ injury and multiple organ failure. Tight junction function is highly regulated by membrane-associated proteins including occludin and zonula occludens protein 1 (ZO-1), which can be modulated by systemic inflammation. We hypothesized that (1) burn injury leads to gut barrier injury, and (2) phosphodiesterase inhibition will attenuate these burn-induced changes. Male balb/c mice undergoing a 30% steam burn were randomized to resuscitation with normal saline or normal saline + pentoxifylline (PTX; 12.5 mg/kg). Intestinal injury was assessed by histological diagnosis and TNF-alpha levels using enzyme-linked immunosorbent assay. Intestinal permeability was assessed by measuring the plasma concentration of fluorescein isothiocyanate-dextran after intraluminal injection in the distal ileum. Occludin and ZO-1 levels were analyzed by immunoblotting and immunohistochemistry. Thirty percent total body surface area (TBSA) burn results in a significant increase in intestinal permeability. Treatment with PTX after burn attenuates intestinal permeability to sham levels. Burn injury resulted in a marked decrease in the levels of tight junction proteins occludin and ZO-1 at 6 and 24 h. The use of PTX after burn significantly decreases the breakdown of occludin and ZO-1. Pentoxifylline also attenuates the burn-induced increase in plasma and intestinal TNF-alpha. Confocal microscopy demonstrates that PTX attenuates the burn-induced reorganization of occludin and ZO-1 away from the tight junction. Pentoxifylline attenuates burn-induced intestinal permeability and decreases the breakdown and reorganization of intestinal occludin and ZO-1. Therefore, phosphodiesterase inhibition may be a useful adjunct strategy in the attenuation of burn-induced gut barrier injury.
Shock (Augusta, Ga.) 10/2008; 31(4):416-22. · 2.87 Impact Factor
