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1.
J Surg Res ; 187(1): 230-236, 2014 03.
Artigo em Inglês | MEDLINE | ID: mdl-24176206

RESUMO

BACKGROUND: Pharmacologic therapy for traumatic brain injury (TBI) has remained relatively unchanged for decades. Ghrelin, an endogenously produced peptide, has been shown to prevent apoptosis and blood-brain barrier dysfunction after TBI. We hypothesize that ghrelin treatment will prevent neuronal degeneration and improve motor coordination after TBI. MATERIALS AND METHODS: A weight drop model created severe TBI in three groups of BALB/c mice: Sham, TBI, and TBI + ghrelin (20 µg intraperitoneal ghrelin). Brain tissue was examined by hematoxylin and eosin and Fluoro-Jade B (FJB) staining to evaluate histologic signs of injury, cortical volume loss, and neuronal degeneration. Additionally, motor coordination was assessed. RESULTS: Ghrelin treatment prevented volume loss after TBI (19.4 ± 9.8 mm(3)versus 71.4 ± 31.4 mm(3); P < 0.05). Similarly, although TBI increased FJB-positive neuronal degeneration, ghrelin treatment decreased FJB staining in TBI resulting in immunohistologic patterns similar to sham. Compared with sham, TBI animals had a significant increase in foot faults at d 1, 3, and 7 (2.75 ± 0.42; 2.67 ± 0.94; 3.33 ± 0.69 versus 0.0 ± 0.0; 0.17 ± 0.19; 0.0 ± 0.0; P < 0.001). TBI + ghrelin animals had significantly decreased foot faults compared with TBI at d 1, 3, and 7 (0.42 ± 0.63; 0.5 ± 0.43; 1.33 ± 0.58; P versus TBI <0.001; P versus sham = NS). CONCLUSIONS: Ghrelin treatment prevented post-TBI cortical volume loss and neurodegeneration. Furthermore, ghrelin improved post-TBI motor deficits. The mechanisms of these effects are unclear; however, a combination of the anti-apoptotic and inflammatory modulatory effects of ghrelin may play a role. Further studies delineating the mechanism of these observed effects are warranted.


Assuntos
Lesões Encefálicas/tratamento farmacológico , Lesões Encefálicas/patologia , Grelina/farmacologia , Transtornos das Habilidades Motoras/tratamento farmacológico , Transtornos das Habilidades Motoras/patologia , Animais , Apoptose/efeitos dos fármacos , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/patologia , Lesões Encefálicas/complicações , Córtex Cerebral/lesões , Córtex Cerebral/patologia , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Transtornos das Habilidades Motoras/etiologia , Degeneração Neural/tratamento farmacológico , Degeneração Neural/patologia , Recuperação de Função Fisiológica/efeitos dos fármacos
2.
J Cell Mol Med ; 17(5): 664-71, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23577721

RESUMO

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.


Assuntos
Queimaduras/patologia , Mitocôndrias Cardíacas/patologia , Miocárdio/patologia , Estimulação do Nervo Vago , Trifosfato de Adenosina/metabolismo , Animais , Apoptose , Fator de Indução de Apoptose/metabolismo , Western Blotting , Queimaduras/prevenção & controle , Citocromos c/metabolismo , Citosol/metabolismo , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias Cardíacas/metabolismo , Dilatação Mitocondrial , Miocárdio/enzimologia , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais , Proteína de Morte Celular Associada a bcl/metabolismo
3.
Am J Pathol ; 181(2): 478-86, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22688057

RESUMO

We have previously shown that vagal nerve stimulation prevents intestinal barrier loss in a model of severe burn injury in which injury was associated with decreased expression and altered localization of intestinal tight junction proteins. α-7 Nicotinic acetylcholine receptor (α-7 nAchR) has been shown to be necessary for the vagus nerve to modulate the systemic inflammatory response, but the role of α-7 nAchR in mediating gut protection remained unknown. We hypothesized that α-7 nAchR would be present in the gastrointestinal tract and that treatment with a pharmacological agonist of α-7 nAchR would protect against burn-induced gut barrier injury. The effects of a pharmacological cholinergic agonist on gut barrier integrity were studied using an intraperitoneal injection of nicotine 30 minutes after injury. Intestinal barrier integrity was examined by measuring permeability to 4-kDa fluorescein isothiocyanate-dextran and by examining changes in expression and localization of the intestinal tight junction proteins occludin and ZO-1. Nicotine injection after injury prevented burn-induced intestinal permeability and limited histological gut injury. Treatment with nicotine prevented decreased expression and altered localization of occludin and ZO-1, as seen in animals undergoing burn alone. Defining the interactions among the vagus nerve, the enteric nervous system, and the intestinal epithelium may lead to development of targeted therapeutics aimed at reducing gut barrier failure and intestinal inflammation after severe injury.


Assuntos
Queimaduras/patologia , Agonistas Colinérgicos/farmacologia , Sistema Nervoso Entérico/metabolismo , Sistema Nervoso Entérico/patologia , Trato Gastrointestinal/inervação , Trato Gastrointestinal/patologia , Receptores Nicotínicos/metabolismo , Animais , Queimaduras/complicações , Queimaduras/metabolismo , Células CACO-2 , Células Cultivadas , Agonistas Colinérgicos/administração & dosagem , Sistema Nervoso Entérico/efeitos dos fármacos , Células Epiteliais/metabolismo , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/metabolismo , Humanos , Inflamação/complicações , Inflamação/metabolismo , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neuroglia , Nicotina/administração & dosagem , Nicotina/farmacologia , Transporte Proteico/efeitos dos fármacos , Proteínas de Junções Íntimas/efeitos dos fármacos , Proteínas de Junções Íntimas/metabolismo , Receptor Nicotínico de Acetilcolina alfa7
4.
J Trauma ; 70(5): 1168-75; discussion 1175-6, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21610431

RESUMO

BACKGROUND: Vagal nerve stimulation (VNS) can have a marked anti-inflammatory effect. We have previously shown that preinjury VNS prevented intestinal barrier breakdown and preserved epithelial tight junction protein expression. However, a pretreatment model has little clinical relevance for the care of the trauma patient. Therefore, we postulated that VNS conducted postinjury would also have a similar protective effect on maintaining gut epithelial barrier integrity. METHODS: Male balb/c mice were subjected to a 30% total body surface area, full-thickness steam burn followed by right cervical VNS at 15, 30, 60, 90, 120, and 150 minutes postinjury. Intestinal barrier dysfunction was quantified by permeability to 4 kDa fluorescein isothiocyanate-Dextran, histologic evaluation, gut tumor necrosis factor-alpha (TNF-α) enzyme-linked immunosorbent assay, and expression of tight junction proteins (myosin light chain kinase, occludin, and ZO-1) using immunoblot and immunoflourescence. RESULTS: Histologic examination documented intestinal villi appearance similar to sham if cervical VNS was performed within 90 minutes of burn insult. VNS done after injury decreased intestinal permeability to fluorescein isothiocyanate-Dextran when VNS was ≤90 minutes after injury. Burn injury caused a marked increase in intestinal TNF-α levels. VNS-treated animals had TNF-α levels similar to sham when VNS was performed within 90 minutes of injury. Tight junction protein expression was maintained at near sham values if VNS was performed within 90 minutes of burn, whereas expression was significantly altered in burn. CONCLUSION: Postinjury VNS prevents gut epithelial breakdown when performed within 90 minutes of thermal injury. This could represent a therapeutic window and clinically relevant strategy to prevent systemic inflammatory response distant organ injury after trauma.


Assuntos
Queimaduras/metabolismo , Mucosa Intestinal/metabolismo , Proteínas de Membrana/metabolismo , Estimulação do Nervo Vago/métodos , Animais , Queimaduras/fisiopatologia , Queimaduras/terapia , Modelos Animais de Doenças , Mucosa Intestinal/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Permeabilidade
5.
Mol Imaging ; 9(1): 30-9, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20128996

RESUMO

Intestinal injury owing to inflammation, severe trauma, and burn is a leading cause of morbidity and mortality. Currently, animal models employed to study the intestinal response to injury and inflammation depend on outdated methods of analysis. Given that these classic intestinal assays are lethal to the experimental animal, there is no ability to study the gut response to injury in the same animal over time. We postulated that by developing an in vivo assay to image intestinal injury using fluorescent dye, it could complement other expensive, time-consuming, and semiquantitative classic means of detecting intestinal injury. We describe a novel in vivo, noninvasive method to image intestinal injury using a charge-coupled device (CCD) camera that allows for serial visual and quantitative analysis of intestinal injury. Our results correlate with traditional, time-consuming, semiquantitative assays of intestinal injury, now allowing the noninvasive, nonlethal assessment of injury over time.


Assuntos
Fluorometria/métodos , Intestinos/lesões , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Animais , Queimaduras/metabolismo , Dextranos/química , Dextranos/metabolismo , Modelos Animais de Doenças , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/química , Fluoresceína-5-Isotiocianato/metabolismo , Histocitoquímica , Mucosa Intestinal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Estatísticas não Paramétricas , Imagem Corporal Total/métodos
6.
Am J Physiol Gastrointest Liver Physiol ; 299(6): G1308-18, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20705905

RESUMO

The enteric nervous system may have an important role in modulating gastrointestinal barrier response to disease through activation of enteric glia cells. In vitro studies have shown that enteric glia activation improves intestinal epithelial barrier function by altering the expression of tight junction proteins. We hypothesized that severe injury would increase expression of glial fibrillary acidic protein (GFAP), a marker of enteric glial activation. We also sought to define the effects of vagal nerve stimulation on enteric glia activation and intestinal barrier function using a model of systemic injury and local gut mucosal involvement. Mice with 30% total body surface area steam burn were used as model of severe injury. Vagal nerve stimulation was performed to assess the role of parasympathetic signaling on enteric glia activation. In vivo intestinal permeability was measured to assess barrier function. Intestine was collected to investigate changes in histology; GFAP expression was assessed by quantitative PCR, by confocal microscopy, and in GFAP-luciferase transgenic mice. Stimulation of the vagus nerve prevented injury-induced intestinal barrier injury. Intestinal GFAP expression increased at early time points following burn and returned to baseline by 24 h after injury. Vagal nerve stimulation prior to injury increased GFAP expression to a greater degree than burn alone. Gastrointestinal bioluminescence was imaged in GFAP-luciferase transgenic animals following either severe burn or vagal stimulation and confirmed the increased expression of intestinal GFAP. Injection of S-nitrosoglutathione, a signaling molecule released by activated enteric glia cells, following burn exerts protective effects similar to vagal nerve stimulation. Intestinal expression of GFAP increases following severe burn injury. Stimulation of the vagus nerve increases enteric glia activation, which is associated with improved intestinal barrier function. The vagus nerve may mediate the signaling that occurs from the central nervous system to the enteric nervous system following gastrointestinal injury.


Assuntos
Queimaduras , Intestinos/citologia , Neuroglia/fisiologia , Pele/lesões , Estimulação do Nervo Vago , Animais , Regulação da Expressão Gênica/fisiologia , Proteína Glial Fibrilar Ácida , Luciferases/genética , Luciferases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Permeabilidade , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
7.
J Trauma ; 68(5): 1059-64, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20453760

RESUMO

BACKGROUND: 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. METHODS: 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. RESULTS: 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). CONCLUSION: 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.


Assuntos
Lesões Encefálicas/complicações , Modelos Animais de Doenças , Doenças do Íleo/prevenção & controle , Estimulação do Nervo Vago/métodos , Análise de Variância , Animais , Permeabilidade Capilar , Sistema Nervoso Central/fisiopatologia , Dextranos , Fluoresceína-5-Isotiocianato/análogos & derivados , Proteína Glial Fibrilar Ácida/análise , Homeostase , Doenças do Íleo/etiologia , Doenças do Íleo/patologia , Doenças do Íleo/fisiopatologia , Mucosa Intestinal/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Necrose , Ratos , Índice de Gravidade de Doença , Método Simples-Cego , Fatores de Tempo , Fator de Necrose Tumoral alfa/análise , Redução de Peso
8.
J Trauma ; 68(6): 1349-54; discussion 1354-6, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20539179

RESUMO

INTRODUCTION: Severe injury can cause intestinal permeability through decreased expression of tight junction proteins, resulting in systemic inflammation. Activation of the parasympathetic nervous system after shock through vagal nerve stimulation is known to have potent anti-inflammatory effects; however, its effects on modulating intestinal barrier function are not fully understood. We postulated that vagal nerve stimulation improves intestinal barrier integrity after severe burn through an efferent signaling pathway, and is associated with improved expression and localization of the intestinal tight junction protein occludin. METHODS: Male balb/c mice underwent right cervical vagal nerve stimulation for 10 minutes immediately before 30% total body surface area, full-thickness steam burn. In a separate arm, animals underwent abdominal vagotomy at the gastroesophageal junction before vagal nerve stimulation and burn. Intestinal barrier injury was assessed by permeability to 4 kDa FITC-dextran, histology, and changes in occludin expression using immunoblotting and confocal microscopy. RESULTS: Cervical vagal nerve stimulation decreased burn-induced intestinal permeability to FITC-dextran, returning intestinal permeability to sham levels. Vagal nerve stimulation before burn also improved gut histology and prevented burn-induced changes in occludin protein expression and localization. Abdominal vagotomy abrogated the protective effects of cervical vagal nerve stimulation before burn, resulting in gut permeability, histology, and occludin protein expression similar to burn alone. CONCLUSION: Vagal nerve stimulation performed before injury improves intestinal barrier integrity after severe burn through an efferent signaling pathway and is associated with improved tight junction protein expression.


Assuntos
Queimaduras/metabolismo , Queimaduras/fisiopatologia , Mucosa Intestinal/metabolismo , Intestinos/fisiopatologia , Proteínas de Membrana/metabolismo , Junções Íntimas/metabolismo , Estimulação do Nervo Vago , Análise de Variância , Animais , Dextranos/farmacocinética , Immunoblotting , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Ocludina , Permeabilidade , Transdução de Sinais
9.
Immunopharmacol Immunotoxicol ; 32(1): 82-91, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19839729

RESUMO

BACKGROUND AND AIM: Pentoxifylline (PTX) has been proven to be an inhibitor of fMLP-induced neutrophil (PMN) oxidative burst and is thought to function by increasing cAMP and Protein kinase A (PKA). We hypothesized that PTX diminishes production of the neutrophil respiratory burst by both PKA-dependent and independent mechanisms. MATERIAL AND METHODS: Human neutrophils were isolated and stimulated with fMLP (1microM) alone or in combination with PTX (2mM). PMN activation was determined by the cytochrome C reduction method in the presence and absence of p38 MAPK (SB203580), ERK (PD98059), and PKA inhibitors (H89). Western blot analysis of Ras, Raf, p38 MAPK, ERK, and Akt was performed in PMNs exposed to fMLP and PTX. Cell membranes were fractionated to measure membrane-associated p47 phox. Treated cells were imaged using confocal microscopy to examine changes in localization of Akt and p47phox. RESULTS: PTX produced a decrease in oxidative burst that was diminished but not abrogated by H89 exposure. The reduction in Ras, Raf, and Akt activation seen with PTX was not effected by the presence of H89. The ability of PTX to attenuate phosphorylation of p38 MAPK and ERK was significantly decreased in the presence of H89, suggesting a PKA-dependent mechanisms. Membrane fractions of neutrophils demonstrate that PTX decreased membrane-associated p47phox, thus diminishing the ability to generate oxidative burst. PTX also decreased membrane localization of Akt and p47phox by confocal microscopy. CONCLUSIONS: PTX attenuates activation of signaling molecules involved in activation of p47phox and suppress the subsequent assembly of the NADPH machinery through both PKA-dependent and PKA-independent mechanisms.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , NADPH Oxidases/metabolismo , Neutrófilos/efeitos dos fármacos , Pentoxifilina/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Explosão Respiratória/efeitos dos fármacos , Regulação para Baixo , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quinases raf/fisiologia , Proteínas ras/fisiologia
10.
Life Sci ; 84(1-2): 18-22, 2009 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-18992758

RESUMO

AIMS: Under normal conditions, the intestinal mucosa acts as a local barrier to prevent the influx of luminal contents. The intestinal epithelial tight junction is comprised of several membrane associated proteins, including zonula occludens-1 (ZO-1) and occludin. Disruption of this barrier can lead to the production of pro-inflammatory mediators and ultimately multiple organ failure. We have previously shown that Pentoxifylline (PTX) decreases histologic gut injury and pro-inflammatory mediator synthesis. We hypothesize that PTX prevents the breakdown of ZO-1 and occludin in an in vitro model of immunostimulated intestinal cell monolayers. MAIN METHODS: Caco-2 human enterocytes were grown as confluent monolayers and incubated under control conditions, or with PTX (2 mM), Cytomix (TNF-alpha, IFN-gamma, IL-1), or Cytomix+PTX for 24 h. Occludin and ZO-1 protein levels were analyzed by Western blot. Confocal microscopy was used to assess the cytoplasmic localization of ZO-1 and occludin. KEY FINDINGS: Cytomix stimulation of Caco-2 cells resulted in a 50% decrease in both occludin and ZO-1 protein. Treatment with Cytomix+PTX restored both occludin and ZO-1 protein to control levels. Confocal microscopy images show that Cytomix caused an irregular, undulating appearance of ZO-1 and occludin at the cell junctions. Treatment with PTX prevented the Cytomix-induced changes in ZO-1 and occludin localization. SIGNIFICANCE: Treatment with PTX decreases the pro-inflammatory cytokine induced changes in the intestinal tight junction proteins occludin and ZO-1. Pentoxifylline may be a useful adjunct in the treatment of sepsis and shock by attenuating intestinal barrier breakdown.


Assuntos
Citocinas/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Proteínas de Membrana/análise , Pentoxifilina/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Fosfoproteínas/análise , Células CACO-2 , Humanos , Mucosa Intestinal/química , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Microscopia Confocal , NF-kappa B/antagonistas & inibidores , Ocludina , Pentoxifilina/uso terapêutico , Sepse/tratamento farmacológico , Proteína da Zônula de Oclusão-1
11.
J Trauma ; 66(1): 17-24; discussion 24-5, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19131801

RESUMO

BACKGROUND: Burn injury can result in loss of intestinal barrier function, leading to systemic inflammatory response syndrome and multiorgan failure. Myosin light chain kinase (MLCK), a tight junction protein involved in the regulation of barrier function, increases intestinal epithelial permeability when activated. Prior studies have shown that tumor necrosis factor (TNF)-alpha activates MLCK, in part through a nuclear factor (NF)-kappa B-dependent pathway. We have previously shown that pentoxifylline (PTX) decreases both TNF-alpha synthesis and NF-kappaB activation in models of shock. Therefore, we postulate that PTX will attenuate activation of the tight junction protein MLCK, which may decrease intestinal tight junction permeability after severe burn. METHODS: Male balb/c mice undergoing a severe burn were randomized to resuscitation with normal saline (NS) or NS + PTX (12.5 mg/kg). Intestinal TNF-alpha levels were evaluated using enzyme linked immunosorbent assay. Gut extracts were obtained to assess MLCK, phosphorylated IKK, IkappaB-alpha, and NF-kappaB p65 levels by immunoblotting. RESULTS: Burn injury increased intestinal MLCK protein levels threefold in animals resuscitated with NS, whereas those receiving PTX had MLCK levels similar to control (p < 0.01). Treatment with PTX attenuated burn-induced intestinal permeability. PTX decreased cytoplasmic IKK, IkappaB-alpha phosphorylation, and nuclear NF-kappaB p65 translocation to sham levels (p < 0.05 vs. NS). CONCLUSION: Treatment with PTX attenuates activation of the tight junction protein MLCK, likely through its ability to decrease local TNF-alpha synthesis and NF-kappaB activation after burn. PTX may have therapeutic utility by decreasing intestinal barrier breakdown after burn.


Assuntos
Queimaduras/metabolismo , Mucosa Intestinal/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Pentoxifilina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Análise de Variância , Animais , Ensaio de Imunoadsorção Enzimática , Immunoblotting , Masculino , Camundongos , Distribuição Aleatória , Fator de Necrose Tumoral alfa/metabolismo
12.
J Trauma ; 67(6): 1162-8, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20009662

RESUMO

BACKGROUND: Intestinal barrier breakdown after severe burn can lead to intestinal inflammation, which may act as the source of the systemic inflammatory response. In vitro intestinal cell studies have shown that mitogen-activated protein kinase (MAPK) signaling is an important modulator of intestinal inflammation. We have previously observed that pentoxifylline (PTX) attenuates burn-induced intestinal permeability and tight junction breakdown. We hypothesized that PTX would limit intestinal barrier breakdown and attenuate inflammatory signaling via the MAPK pathway. METHODS: Male balb/c mice underwent 30% total body surface area full-thickness steam burn. Immediately after burn, animals received an intraperitoneal injection of PTX (12.5 mg/kg) in normal saline or normal saline alone. In vivo intestinal permeability to 4 kDa fluorescein isothiocyanate-dextran was measured. Intestinal extracts were obtained to measure interleukin-6 by enzyme-linked immunosorbent assay, and phosphorylated p38 MAPK, p38 MAPK, phosphorylated extracellular signal-related kinase (1/2) (ERK (1/2)), and ERK (1/2) by immunoblotting. Acute lung injury was assessed by histology at 24 hours after burn. RESULTS: Administration of PTX immediately after injury attenuated burn-induced intestinal permeability. PTX also decreased the burn-induced phosphorylation of p38 MAPK and decreased phosphorylation of ERK (1/2) at 2 hours and 24 hours after injury. Animals given PTX had decreased intestinal interleukin-6 levels. A single dose of PTX also decreased histologic lung injury at 24 hours after burn. CONCLUSION: PTX attenuates burn-induced intestinal permeability and subsequent intestinal inflammation. Use of PTX after burn was also associated with decreased acute lung injury. Because of its compelling anti-inflammatory effects, PTX may be an ideal candidate for use as an immunomodulatory adjunct to resuscitation fluid.


Assuntos
Queimaduras/enzimologia , Absorção Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Pentoxifilina/farmacologia , Ressuscitação/métodos , Animais , Queimaduras/patologia , Queimaduras/fisiopatologia , Inflamação/metabolismo , Intestinos/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Permeabilidade , Fosforilação , Resultado do Tratamento , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
13.
Surgery ; 142(2): 276-83, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17689696

RESUMO

BACKGROUND: The combination of hypertonic saline (HS) and pentoxifylline (PTX) has been shown to synergistically downregulate neutrophil oxidative burst in vitro. We investigated the effects of HS/PTX on human neutrophil mitogen-activated protein kinase (MAPK) signaling and the role of Protein kinase A (PKA) in this process. METHODS: Isolated neutrophils were treated with PTX (2 mmol/L), HS10 (10 mmol/L above isotonicity), and HS40 (40 mmol/L above isotonicity) alone or in combination for determination of intracellular cyclic adenosine monophosphate (cAMP) concentrations. Human neutrophils were stimulated with f-methionyl-leucyl-phenylalanine (fMLP) (1 micromol/L) before the treatments above in both the presence and the absence of PKA inhibition for Western blot analysis of MAPK p38 and extracellular signal-related kinase 1/2 (ERK 1/2) phosphorylation. RESULTS: Concomitant exposure to HS/PTX results in an additive increase in intracellular cAMP. fMLP-induced ERK 1/2 phosphorylation was synergistically attenuated by HS/PTX. Both PTX and HS reduced p38MAPK phosphorylation. No additive effect was observed with combined treatment. Although PKA inhibition abrogated the effects of PTX, HS retained some capacity to attenuate MAPK phosphorylation. CONCLUSION: HS/PTX is more effective in attenuating neutrophil ERK signaling than either component alone, whereas both components alone or in combination produced comparable results with p38MAPK. Although PTX functions primarily through PKA activation, HS may suppress neutrophils through a partially PKA-independent mechanism.


Assuntos
Inibidores Enzimáticos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Neutrófilos/enzimologia , Pentoxifilina/farmacologia , Solução Salina Hipertônica/farmacologia , Tamanho Celular/efeitos dos fármacos , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
14.
J Neurotrauma ; 29(2): 385-93, 2012 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-21939391

RESUMO

Significant effort has been focused on reducing neuronal damage from post-traumatic brain injury (TBI) inflammation and blood-brain barrier (BBB)-mediated edema. The orexigenic hormone ghrelin decreases inflammation in sepsis models, and has recently been shown to be neuroprotective following subarachnoid hemorrhage. We hypothesized that ghrelin modulates cerebral vascular permeability and mediates BBB breakdown following TBI. Using a weight-drop model, TBI was created in three groups of mice: sham, TBI, and TBI/ghrelin. The BBB was investigated by examining its permeability to FITC-dextran and through quantification of perivascualar aquaporin-4 (AQP-4). Finally, we immunoblotted for serum S100B as a marker of brain injury. Compared to sham, TBI caused significant histologic neuronal degeneration, increases in vascular permeability, perivascular expression of AQP-4, and serum levels of S100B. Treatment with ghrelin mitigated these effects; after TBI, ghrelin-treated mice had vascular permeability and perivascular AQP-4 and S100B levels that were similar to sham. Our data suggest that ghrelin prevents BBB disruption after TBI. This is evident by a decrease in vascular permeability that is linked to a decrease in AQP-4. This decrease in vascular permeability may diminish post-TBI brain tissue damage was evident by decreased S100B.


Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/fisiopatologia , Lesões Encefálicas/fisiopatologia , Grelina/fisiologia , Animais , Barreira Hematoencefálica/patologia , Lesões Encefálicas/tratamento farmacológico , Lesões Encefálicas/patologia , Permeabilidade Capilar/efeitos dos fármacos , Permeabilidade Capilar/fisiologia , Modelos Animais de Doenças , Grelina/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos BALB C
15.
PLoS One ; 7(3): e33024, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22403732

RESUMO

Epigenetic marks are fundamental to normal development, but little is known about signals that dictate their placement. Insights have been provided by studies of imprinted loci in mammals, where monoallelic expression is epigenetically controlled. Imprinted expression is regulated by DNA methylation programmed during gametogenesis in a sex-specific manner and maintained after fertilization. At Rasgrf1 in mouse, paternal-specific DNA methylation on a differential methylation domain (DMD) requires downstream tandem repeats. The DMD and repeats constitute a binary switch regulating paternal-specific expression. Here, we define sequences sufficient for imprinted methylation using two transgenic mouse lines: One carries the entire Rasgrf1 cluster (RC); the second carries only the DMD and repeats (DR) from Rasgrf1. The RC transgene recapitulated all aspects of imprinting seen at the endogenous locus. DR underwent proper DNA methylation establishment in sperm and erasure in oocytes, indicating the DMD and repeats are sufficient to program imprinted DNA methylation in germlines. Both transgenes produce a DMD-spanning pit-RNA, previously shown to be necessary for imprinted DNA methylation at the endogenous locus. We show that when pit-RNA expression is controlled by the repeats, it regulates DNA methylation in cis only and not in trans. Interestingly, pedigree history dictated whether established DR methylation patterns were maintained after fertilization. When DR was paternally transmitted followed by maternal transmission, the unmethylated state that was properly established in the female germlines could not be maintained. This provides a model for transgenerational epigenetic inheritance in mice.


Assuntos
Metilação de DNA/genética , Impressão Genômica/genética , Óvulo/metabolismo , Espermatozoides/metabolismo , Animais , Sequência de Bases , Epigênese Genética/genética , Pai , Feminino , Loci Gênicos/genética , Masculino , Camundongos , Camundongos Transgênicos , Família Multigênica/genética , ras-GRF1/genética
16.
Peptides ; 38(1): 94-9, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22960048

RESUMO

The injured intestine is responsible for significant morbidity and mortality after severe trauma and burn; however, targeting the intestine with therapeutics aimed at decreasing injury has proven difficult. We hypothesized that we could use intravenous phage display technology to identify peptide sequences that target the injured intestinal mucosa in a murine model, and then confirm the cross-reactivity of this peptide sequence with ex vivo human gut. Four hours following 30% TBSA burn we performed an in vivo, intravenous systemic administration of phage library containing 10(12) phage in balb/c mice to biopan for gut-targeting peptides. In vivo assessment of the candidate peptide sequences identified after 4 rounds of internalization was performed by injecting 1×10(12) copies of each selected phage clone into sham or burned animals. Internalization into the gut was assessed using quantitative polymerase chain reaction. We then incubated this gut-targeting peptide sequence with human intestine and visualized fluorescence using confocal microscopy. We identified 3 gut-targeting peptide sequences which caused collapse of the phage library (4-1: SGHQLLLNKMP, 4-5: ILANDLTAPGPR, 4-11: SFKPSGLPAQSL). Sequence 4-5 was internalized into the intestinal mucosa of burned animals 9.3-fold higher than sham animals injected with the same sequence (2.9×10(5)vs. 3.1×10(4) particles per mg tissue). Sequences 4-1 and 4-11 were both internalized into the gut, but did not demonstrate specificity for the injured mucosa. Phage sequence 4-11 demonstrated cross-reactivity with human intestine. In the future, this gut-targeting peptide sequence could serve as a platform for the delivery of biotherapeutics.


Assuntos
Queimaduras/complicações , Intestinos/lesões , Biblioteca de Peptídeos , Peptídeos/imunologia , Peptídeos/metabolismo , Sequência de Aminoácidos , Animais , Bacteriófago M13/genética , Reações Cruzadas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Células Epiteliais , Humanos , Mucosa Intestinal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Peptídeos/administração & dosagem , Peptídeos/genética
17.
J Trauma Acute Care Surg ; 72(6): 1562-6, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22695423

RESUMO

BACKGROUND: Traumatic brain injury (TBI) may alter sympathetic tone causing autonomic abnormalities and organ dysfunction. Vagal nerve stimulation (VNS) has been shown to decrease inflammation and distant organ injury after TBI. It is unknown whether VNS may reduce blood-brain barrier (BBB) dysfunction after TBI.We hypothesize that VNS prevents TBI-induced breakdown of the BBB, subsequent brain edema, and neuronal injury. METHODS: A weight-drop model was used to create severe TBI in balb/c mice. Animals were divided into three groups: TBI-TBI only; TBI or VNS--animals that were treated with 10 minutes of VNS immediately before TBI; and sham--animals with opening of the skull but no TBI and VNS treatment. Brain vascular permeability to injected (Mr 70,000) FITC-dextran was measured by radiated fluorescence 6 hours after injury. Injured tissue sections were stained for perivascular aquaporin 4 (AQP-4), an important protein causing BBB--mediated brain edema. Fluorescence was quantified under laser scanning by confocal microscopy. RESULTS: Six hours after TBI, cerebral vascular permeability was increased fourfold compared with sham (mean [SD], 6.6(E+08) [5.5(E+07)] arbitrary fluorescence units [afu] vs. 1.5(E+08) [2.9(E+07)] afu; p G 0.001). VNS prevented the increase in permeability when compared with TBI alone (mean [SD], 3.5 (E+08) [8.3(E+07)] afu vs. 6.6(E+08) [5.5(E+07)] afu; p G 0.05). Perivascular expression of AQP-4 was increased twofold in TBI animals compared with sham (mean [SD], 0.96 [0.12] afu vs. 1.79 [0.37] afu; p G 0.05). Similarly, VNS decreased post-TBI expression of AQP-4 to levels similar to sham (mean [SD], 1.15 [0.12] afu; p G 0.05). CONCLUSION: VNS attenuates cerebral vascular permeability and decreases the up-regulation of AQP-4 after TBI. Future studies are needed to assess the mechanisms by which VNS maintains the BBB.


Assuntos
Aquaporina 4/metabolismo , Barreira Hematoencefálica/fisiopatologia , Lesões Encefálicas/patologia , Lesões Encefálicas/terapia , Dextranos/farmacocinética , Fluoresceína-5-Isotiocianato/análogos & derivados , Estimulação do Nervo Vago/métodos , Análise de Variância , Animais , Aquaporina 4/genética , Barreira Hematoencefálica/metabolismo , Lesões Encefálicas/metabolismo , Modelos Animais de Doenças , Fluoresceína-5-Isotiocianato/farmacocinética , Fluorescência , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Distribuição Aleatória , Valores de Referência , Resultado do Tratamento , Regulação para Cima
18.
Shock ; 36(1): 24-9, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21330948

RESUMO

The role of the Toll-like receptor 4 (TLR4), a component of the innate immune system, in the development of burn-induced acute lung injury (ALI) has not been completely defined. Recent data suggested that an intact TLR4 plays a major role in the development of organ injury in sterile inflammation. We hypothesized that burn-induced ALI is a TLR4-dependent process. Male C57BL/6J (TLR4 wild-type [WT]) and C57BL/10ScN (TLR4 knockout [KO]) mice were subjected to a 30% total body surface area steam burn. Animals were killed at 6 and 24 h after the insult. Lung specimens were harvested for histological examination after hematoxylin-eosin staining. In addition, lung myeloperoxidase (MPO) and intercellular adhesion molecule 1 immunostaining was performed. Lung MPO was measured by an enzymatic assay. Total lung keratinocyte-derived chemoattractant (IL-8) content was measured by enzyme-linked immunosorbent assay. Western blot was performed to quantify phosphorylated IκBα, phosphorylated nuclear factor κB p65 (NF-κBp65), and high mobility group box 1 expression. Acute lung injury, characterized by thickening of the alveolar-capillary membrane, hyaline membrane formation, intraalveolar hemorrhage, and neutrophil infiltration, was seen in WT but not KO animals at 24 h. Myeloperoxidase and intercellular adhesion molecule 1 immunostaining of KO animals was also similar to sham but elevated in WT animals. In addition, a reduction in MPO enzymatic activity was observed in KO mice as well as a reduction in IL-8 levels compared with their WT counterparts. Burn-induced ALI develops within 24 h after the initial thermal insult in our model. Toll-like receptor 4 KO animals were clearly protected and had a much less severe lung injury. Our data suggest that burn-induced ALI is a TLR4-dependent process.


Assuntos
Lesão Pulmonar Aguda/metabolismo , Queimaduras/metabolismo , Queimaduras/fisiopatologia , Receptor 4 Toll-Like/metabolismo , Animais , Western Blotting , Ensaio de Imunoadsorção Enzimática , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas I-kappa B/metabolismo , Molécula 1 de Adesão Intercelular , Interleucina-8/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Inibidor de NF-kappaB alfa , Peroxidase/metabolismo , Fator de Transcrição RelA/metabolismo
19.
Surgery ; 150(3): 379-89, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21783215

RESUMO

BACKGROUND: The purpose of this study was to assess acute lung injury when protection to the gut mucosal barrier offered by vagus nerve stimulation is eliminated by an abdominal vagotomy. METHODS: Male balb/c mice were subjected to 30% total body surface area steam burn with and without electrical stimulation to the right cervical vagus nerve. A cohort of animals were subjected to abdominal vagotomy. Lung histology, myeloperoxidase and ICAM-1 immune staining, myeloperoxidase enzymatic assay, and tissue KC levels were analyzed 24 hours after burn. Additionally, lung IkB-α, NF-kB immunoblots, and NF-kB-DNA binding measured by photon emission analysis using NF-kB-luc transgenic mice were performed. RESULTS: Six hours post burn, phosphorylation of both NF-kB p65 and IkB-α were observed. Increased photon emission signal was seen in the lungs of NF-kB-luc transgenic animals. Vagal nerve stimulation blunted NF-kB activation similar to sham animals whereas abdominal vagotomy eliminated the anti-inflammatory effect. After burn, MPO positive cells and ICAM-1 expression in the lung endothelium was increased, and lung histology demonstrated significant injury at 24 hours. Vagal nerve stimulation markedly decreased neutrophil infiltration as demonstrated by MPO immune staining and enzyme activity. Vagal stimulation also markedly attenuated acute lung injury at 24 hours. The protective effects of vagal nerve stimulation were reversed by performing an abdominal vagotomy. CONCLUSION: Vagal nerve stimulation is an effective strategy to protect against acute lung injury following burn. Moreover, the protective effects of vagal nerve stimulation in the prevention of acute lung injury are eliminated by performing an abdominal vagotomy. These results establish the importance of the gut-lung axis after burn in the genesis of acute lung injury.


Assuntos
Lesão Pulmonar Aguda/patologia , Lesão Pulmonar Aguda/prevenção & controle , Queimaduras/complicações , Trato Gastrointestinal/patologia , Estimulação do Nervo Vago/métodos , Lesão Pulmonar Aguda/etiologia , Animais , Biópsia por Agulha , Queimaduras/diagnóstico , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Immunoblotting , Imuno-Histoquímica , Molécula 1 de Adesão Intercelular/metabolismo , Mucosa Intestinal/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Peroxidase/metabolismo , Distribuição Aleatória , Valores de Referência , Resultado do Tratamento , Vagotomia/métodos
20.
Clinics (Sao Paulo) ; 65(6): 621-8, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20613939

RESUMO

INTRODUCTION: Combining the hemodynamic and immune benefits of hypertonic saline with the anti-inflammatory effects of the phosphodiesterase inhibitor pentoxifylline (HSPTX) as a hemorrhagic shock resuscitation strategy reduces lung injury when compared with the effects of Ringer's lactate (RL). We hypothesized that HSPTX exerts its anti-inflammatory effects by interfering with nuclear factor kappa B/cAMP response element-binding protein (NF-kappaB-CREB) competition for the coactivator CREB-binding protein (CBP) in lung tissue, thus affecting pro-inflammatory mediator production. METHODS: Male Sprague-Dawley rats underwent 60 minutes of hemorrhagic shock to reach a mean arterial blood pressure of 35 mmHg followed by resuscitation with either RL or HSPTX (7.5% HS + 25 mg/kg PTX). After four hours, lung samples were collected. NF-kappaB activation was assessed by measuring the levels of phosphorylated cytoplasmic inhibitor of kappa B (I-kappaB) and nuclear NF-kappaB p65 by western blot. NF-kappaB and CREB DNA-binding activity were measured by electrophoretic mobility shift assay (EMSA). Competition between NF-kappaB and CREB for the coactivator CBP was determined by immunoprecipitation. Interleukin-8 (IL-8) levels in the lung were measured by ELISA. RESULTS: RL resuscitation produced significantly higher levels of lung IL-8 levels, I-kappaB phosphorylation, p65 phosphorylation, and NF-kappaB DNA binding compared with HSPTX. NF-kappaB-CBP-binding activity was similar in both groups, whereas CREB-CBP-binding activity was significantly increased with HSPTX. CREB-DNA binding-activity increased to a greater level with HSPTX compared with RL. DISCUSSION: HSPTX decreases lung inflammation following hemorrhagic shock compared with conventional resuscitation using RL through attenuation of NF-kappaB signaling and increased CREB-DNA binding activity. HSPTX may have therapeutic potential in the attenuation of ischemia-reperfusion injury observed after severe hemorrhagic shock.


Assuntos
Mediadores da Inflamação/metabolismo , Pulmão/metabolismo , Inibidores de Fosfodiesterase/uso terapêutico , Choque Hemorrágico/terapia , Fatores de Transcrição/metabolismo , Animais , Anti-Inflamatórios/uso terapêutico , Modelos Animais de Doenças , Pulmão/patologia , Masculino , NF-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Pentoxifilina/uso terapêutico , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/metabolismo , Ressuscitação/métodos , Solução Salina Hipertônica/uso terapêutico , Choque Hemorrágico/complicações , Choque Hemorrágico/metabolismo
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