RESUMEN

Polymorphonuclear leukocyte infiltration into tissues in host defense and inflammatory disease causes increased vascular permeability and edema formation through unknown mechanisms. Here, we report the involvement of a paracrine mechanism in neutrophil-evoked alteration in endothelial barrier function. We show that upon neutrophil adhesion to the endothelial lining, leukocytic beta2 integrin signaling triggers the release of neutrophil-borne heparin-binding protein (HBP), also known as CAP37/azurocidin, a member of the serprocidin family of neutrophil cationic proteins. HBP induced Ca++-dependent cytoskeletal rearrangement and intercellular gap formation in endothelial-cell monolayers in vitro, and increased macromolecular efflux in microvessels in vivo. Moreover, selective inactivation of HBP prevented the neutrophils from inducing endothelial hyperpermeability. Our data suggest a fundamental role of neutrophil-derived HBP in the vascular response to neutrophil trafficking in inflammation. Targeting this molecule in inflammatory disease conditions offers a new strategy for prevention of endothelial barrier dysfunction caused by misdirected leukocyte activation.

Asunto(s)

Proteínas Sanguíneas/metabolismo , Permeabilidad Capilar/fisiología , Proteínas Portadoras/metabolismo , Neutrófilos/metabolismo , Animales , Péptidos Catiónicos Antimicrobianos , Proteínas Sanguíneas/farmacología , Calcio/metabolismo , Proteínas Portadoras/farmacología , Bovinos , Permeabilidad de la Membrana Celular , Células Cultivadas , Citoesqueleto/fisiología , Endotelio Vascular/citología , Humanos

RESUMEN

Hypertonic saline prevents vascular adherence of neutrophils and ameliorates ischemic tissue injury. We hypothesized that hypertonic saline attenuates N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated expression of adhesion molecules on human polymorphonuclear leukocytes (PMNLs). fMLP-stimulated up-regulation of beta2-integrins was diminished by hypertonic saline but not by hypertonic choline chloride-, mannitol-, or sucrose-modified Hanks' buffered salt solution. Shedding of L-selectin was decreased by hypertonic saline and choline chloride but not by hypertonic mannitol or sucrose. When the effects of hypertonic sodium chloride- and choline chloride-modified media were compared, neither solution affected fMLP-receptor binding but both equally inhibited fMLP-stimulated increase in intracellular calcium, ionophore A23187, and phorbol myristate acetate (PMA)-stimulated numerical up-regulation of beta2-integrins. Analysis of mitogen-activated protein (MAP) kinases p38 and p44/42 for phosphorylation revealed that hypertonic solutions did not differ in preventing fMLP-stimulated increases in phospho-p38 and phospho-p44/42. Resting PMNLs shrunk by hypertonic saline increased their volume during incubation and further during chemotactic stimulation. Addition of amiloride further enhanced inhibition of up-regulation of beta2-integrins. No fMLP-stimulated volume changes occurred in PMNLs exposed to hypertonic choline chloride, resulting in significant cell shrinkage. Results suggest a sodium-specific inhibitory effect on up-regulation of beta2-integrins of fMLP-stimulated PMNLs, which is unlikely to be caused by alterations of fMLP receptor binding, decrease in cytosolic calcium, attenuation of calcium or protein kinase C-dependent pathways, suppression of p38- or p44/42 MAP kinase-dependent pathways, or cellular ability to increase or decrease volumes.

Asunto(s)

Moléculas de Adhesión Celular/biosíntesis , Neutrófilos/química , Solución Salina Hipertónica/farmacología , Antígenos CD18/efectos de los fármacos , Antígenos CD18/metabolismo , Calcio/metabolismo , Moléculas de Adhesión Celular/efectos de los fármacos , Humanos , Selectina L/efectos de los fármacos , Selectina L/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , N-Formilmetionina Leucil-Fenilalanina/farmacología , Fosforilación/efectos de los fármacos , Cloruro de Sodio/farmacología , Regulación hacia Arriba/efectos de los fármacos

RESUMEN

The immunomodulator Linomide (roquinimex) ameliorates the development of numerous inflammatory and immunological diseases, including sepsis, arthritis, and encephalomyelitis. However, the mechanism underlying this protective effect of Linomide remains unclear. In this study, we wanted to evaluate the effect of Linomide treatment on the different steps in the extravasation process of leukocytes stimulated by tumor necrosis factor alpha (TNF-alpha) in vivo. For this purpose, we used intravital microscopy in the mouse cremaster muscle microcirculation. We found that pretreatment with Linomide dose-dependently (3-300 mg/kg) reduced TNF-alpha-induced leukocyte adhesion and tissue recruitment. Notably, at 300 mg/kg response to TNF-alpha was nearly abolished, i.e. leukocyte adhesion was decreased by 83% and recruitment by 86%. In fact, the anti-inflammatory effect of this dose of Linomide corresponded in magnitude to the potency of 10 mg/kg of dexamethasone. Moreover, administration of Linomide did not alter the systemic leukocyte counts. On the other hand, 1-10 mg/kg of dexamethasone decreased the circulating number of mononuclear leukocytes by 77%. Taken together, our novel findings demonstrate that Linomide is a potent inhibitor of leukocyte adhesion and recruitment in cytokine-activated tissues. These data may help explain the documented protection provided by Linomide in inflammatory diseases characterized by cytokine and leukocyte accumulation.

Asunto(s)

Adyuvantes Inmunológicos/farmacología , Movimiento Celular/efectos de los fármacos , Hidroxiquinolinas/farmacología , Leucocitos/efectos de los fármacos , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Animales , Adhesión Celular/efectos de los fármacos , Dexametasona/farmacología , Relación Dosis-Respuesta a Droga , Glucocorticoides/farmacología , Recuento de Leucocitos , Leucocitos/citología , Leucocitos/inmunología , Masculino , Ratones , Microcirculación/efectos de los fármacos , Microscopía por Video , Músculo Esquelético/irrigación sanguínea , Músculo Esquelético/efectos de los fármacos , Selectina-P/biosíntesis , Selectina-P/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factor de Necrosis Tumoral alfa/farmacología

RESUMEN

Gram-negative sepsis and subsequent endotoxic shock after surgery remain problematic in the United States and throughout the world. While morphine is widely prescribed for postoperative trauma pain management, there are reports that morphine may compromise the immune system and contribute to postoperative sepsis. The current study tested the hypothesis that morphine attenuates leukocyte rolling and sticking in both arterioles and venules via nitric oxide production. Nude mice implanted with slow-release morphine pellets were used in this study. The dorsal skinfold chamber model for intravital fluorescence microscopy on awake mice was used. Leukocyte/endothelial interactions were evaluated after bolus injection of oxidized low density lipoprotein. Morphine was found to significantly attenuate leukocyte rolling and sticking in both the arterial and venular side of the microcirculation. This attenuation was reversed by simultaneous implantation of naloxone pellets. The mechanisms of this attenuation were further investigated by administration of the nitric oxide synthase inhibitors NG-nitro-l-arginine (NOLA) and aminoguanidine (AG) in drinking water. NOLA was found to significantly reverse this morphine-induced attenuation of leukocyte rolling and sticking in both arterioles and venules. However, AG did not have the same effect. The results indicate that morphine interferes with leukocyte/endothelial cell interactions via stimulation of nitric oxide production.

Asunto(s)

Analgésicos Opioides/farmacología , Comunicación Celular/efectos de los fármacos , Comunicación Celular/fisiología , Endotelio Vascular/fisiología , Leucocitos/fisiología , Morfina/farmacología , Animales , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Endotelio Vascular/citología , Inhibidores Enzimáticos/farmacología , Guanidinas/farmacología , Leucocitos/citología , Ratones , Ratones Pelados , Nitroarginina/farmacología

RESUMEN

Ductal structures of the adult pancreas contain stem cells that differentiate into islets of Langerhans. Here, we grew pancreatic ductal epithelial cells isolated from prediabetic adult non-obese diabetic mice in long-term cultures, where they were induced to produce functioning islets containing alpha, beta and delta cells. These in vitro-generated islets showed temporal changes in mRNA transcripts for islet cell-associated differentiation markers, responded in vitro to glucose challenge, and reversed insulin-dependent diabetes after being implanted into diabetic non-obese diabetic mice. The ability to control growth and differentiation of islet stem cells provides an abundant islet source for beta-cell reconstitution in type I diabetes.

Asunto(s)

Diabetes Mellitus Tipo 1/terapia , Trasplante de Células Madre Hematopoyéticas , Trasplante de Islotes Pancreáticos , Islotes Pancreáticos/citología , Conductos Pancreáticos/citología , Células Madre/citología , Animales , Supervivencia Celular , Células Epiteliales/citología , Células Epiteliales/patología , Islotes Pancreáticos/irrigación sanguínea , Ratones , Ratones Endogámicos NOD , Neovascularización Fisiológica , Conductos Pancreáticos/patología , Estado Prediabético/patología

RESUMEN

Neutrophil-borne heparin-binding protein (HBP) is a multifunctional protein involved in the progression of inflammation. HBP is stored in neutrophil granules and released upon stimulation of the cells in proximity to endothelial cells. HBP affects endothelial cells in multiple ways; however, the molecular and cellular mechanisms underlying the interaction of HBP with these cells are unknown. Affinity isolation and enzymatic degradation demonstrated that HBP released from human neutrophils binds to endothelial cell-surface proteoglycans, such as syndecans and glypican. Flow cytometry indicated that a significant fraction of proteoglycan-bound HBP is taken up by the endothelial cells, and we used radiolabeled HBP to determine the internalization rate of surface-bound HBP. Confocal and electron microscopy revealed that internalized HBP is targeted to perinuclear compartments of endothelial cells, where it colocalizes with mitochondria. Western blotting of isolated mitochondria from HBP-treated endothelial cells showed that HBP is present in 2 forms - 28 and 22 kDa. Internalized HBP markedly reduced growth factor deprivation-induced caspase-3 activation and protected endothelial cells from apoptosis, suggesting that uptake and intracellular routing of exogenous HBP to mitochondria contributes to the sustained viability of endothelial cells in the context of locally activated neutrophils.

Asunto(s)

Apoptosis/fisiología , Proteínas Sanguíneas/metabolismo , Proteínas Portadoras/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/fisiología , Mitocondrias/metabolismo , Neutrófilos/fisiología , Proteoglicanos/metabolismo , Péptidos Catiónicos Antimicrobianos , Apoptosis/efectos de los fármacos , Transporte Biológico , Células Cultivadas , Cromatografía de Afinidad , Heparina/metabolismo , Humanos , Cinética , Leucotrieno B4/farmacología , N-Formilmetionina Leucil-Fenilalanina/farmacología , Proteoglicanos/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Venas Umbilicales

RESUMEN

Reactive oxygen species have been identified as key mediators of leukocyte/endothelial cell interaction under various pathological conditions and diseases such as ischemia/reperfusion injury, inflammation, and after exposure to cigarette smoke. Consequently, antioxidants have been shown to successfully prevent the sequelae of these conditions, ranging from tissue infarction to atherogenesis. In this study we investigated whether, via its established superoxide dismutase-like activity, a novel polynitroxyl hemoglobin-based oxygen carrier (PNH), could affect the stimulation of leukocyte rolling and adhesion to endothelial cells in response to cigarette smoke. Using the dorsal skin fold chamber model for intravital microscopic observation of leukocyte/endothelium and -/platelet interactions in hamsters, we could demonstrate that cigarette smoke exposure elicited in control animals the rolling and adhesion of leukocytes along the endothelium of postcapillary venules and also of arterioles, as well as the formation of leukocyte/platelet aggregates. In contrast to the hemoglobin based oxygen carrier (HBOC) alone, that showed no therapeutic benefit, PNH significantly inhibited these proadhesive processes secondary to cigarette smoke. Also, PNH significantly reduced the formation of leukocyte/platelet aggregates in the blood stream of the cigarette smoke-exposed animals. These effects are not due to changes in microhemodynamic conditions, because wall shear rates remained unchanged in all three groups of animals.

Asunto(s)

Hemoglobinas/metabolismo , Leucocitos/fisiología , Oxígeno/metabolismo , Fumar/efectos adversos , Animales , Adhesión Celular , Cricetinae , Método Doble Ciego , Endotelio Vascular , Femenino , Radicales Libres , Leucocitos/citología , Mesocricetus , Microcirculación

RESUMEN

Vitamin E has been supplemented to the diets of farm animals to improve fertility, health, growth rates and quality of animal products. Because of the positive experience obtained in farm animals, vitamin E has been added in increasing amounts to the diets of laboratory animals. Today, vitamin E levels in standard rodent maintenance diets range from 30 mg/kg (France, United States), 90-120 mg/kg (Netherlands, United Kingdom) to as much as 200 mg/kg (Germany). While increasing fertility and health of laboratory animals, these vitamin E supplements affect diverse pathophysiological conditions and thus the outcome of animal models of disease. Because of the large variability of vitamin E levels between laboratories within and between different countries, results obtained in established animal models may no longer be comparable and/or reproducible. Researchers should be aware of these vitamin E supplements and carefully control for potential effects in their respective animal models that involve--or may involve--the generation of reactive oxygen species.

Asunto(s)

Suplementos Dietéticos , Vitamina E/farmacología , Animales , Animales de Laboratorio , Modelos Animales de Enfermedad , Reproducibilidad de los Resultados

RESUMEN

STUDY DESIGN: The effect of intravenous administration of hypertonic saline on leukocyte adhesion after compression injury of the spinal cord was evaluated. OBJECTIVES: To investigate changes in leukocyte adhesion after spinal cord injury and to evaluate the effect of hypertonic saline on this process. SUMMARY OF BACKGROUND DATA: Leukocytes have been thought to exacerbate tissue injury after ischemia-reperfusion. Downregulating and reducing the number of circulating leukocytes has attenuated tissue damage in various models of cerebral ischemia. Recently, investigators have reported that leukocytes exacerbate injury in the spinal cord after trauma. Other recent findings have indicated that hypertonic saline may play a role in decreasing leukocyte adhesion and activation. METHODS: Sprague-Dawley rats were anesthetized, and a C3-C5 laminectomy was performed. Injury was caused by 35 g of compression applied to the cord for 10 minutes. Animals were divided into three groups: sham treated, untreated, and treated. The treated animals received 7.5% hypertonic saline (5 mL/kg, intravenously) 5 minutes after the injury. Sticking leukocytes and shear rate were measured using fluorescence microscopy. RESULTS: Administration of 7.5% hypertonic saline after injury significantly decreased the number of sticking leukocytes in the venules and arterioles. Shear rate was unchanged between the groups. CONCLUSIONS: The results show that an increase in leukocyte adhesion after a compressive injury is attenuated by the administration of 7.5% hypertonic saline. The decrease in adhesion cannot be attributed to changes in the shearing forces, because no significant change was observed in the shear rate. Hypertonic saline may interfere with leukocytes directly by interfering with their ability to swell and thus may prevent activation.

Asunto(s)

Leucocitos/fisiología , Solución Salina Hipertónica/uso terapéutico , Compresión de la Médula Espinal/terapia , Animales , Adhesión Celular , Leucocitos/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Compresión de la Médula Espinal/sangre , Compresión de la Médula Espinal/fisiopatología , Factores de Tiempo

RESUMEN

After ischemia-reperfusion, polymorphonuclear leukocytes (PMNLs) become activated by inflammatory mediators, adhere to the vascular endothelium via the interaction of specific adhesion molecules, and cause tissue injury by the release of cytotoxic oxygen radicals and enzymes. Results obtained in animal experiments suggest a key role for PMNLs in ischemia-reperfusion injury of transplanted livers; therefore, we studied the expression of adhesion molecules on circulating PMNLs (beta2-integrins [CD18] and L-selectin [CD62L]) in 20 patients undergoing orthotopic liver transplantation (study group). To determine the effects of surgical trauma to the liver in the absence of ischemia and reperfusion, the expression of PMNL adhesion molecules was measured in 10 patients scheduled for elective partial liver resection without hepatic vascular exclusion (control group). Patients were classified as responders or nonresponders based on changes in the expression of adhesion molecules elicited by reperfusion. In the control group, all patients remained nonresponders, showing that surgical trauma of the liver alone does not cause activation of circulating PMNLs. In contrast, 8 of 20 patients in the study group were classified as responders. In responders, postoperative serum liver enzyme activities were significantly higher than in nonresponders, indicating that activation of PMNL was associated with damage to hepatocellular integrity. Because expression of adhesion molecules was already changed during surgery, monitoring of the expression of beta2-integrins and L-selectin on circulating PMNLs during orthotopic liver transplantation might be useful in prediction of early graft dysfunction.

Asunto(s)

Células Sanguíneas/metabolismo , Antígenos CD18/metabolismo , Selectina L/metabolismo , Trasplante de Hígado , Neutrófilos/metabolismo , Adolescente , Adulto , Anciano , Fenómenos Fisiológicos Sanguíneos , Frío , Femenino , Hepatectomía/métodos , Humanos , Interleucinas/análisis , Recuento de Leucocitos , Hígado/fisiopatología , Masculino , Persona de Mediana Edad , Concentración Osmolar , Proteínas Recombinantes , Factor de Necrosis Tumoral alfa/análisis , Factor de Necrosis Tumoral alfa/farmacología

RESUMEN

In a defect wound model in rats local application of hyaluronan improves proliferation of granulation tissue and epithelialization under diabetic conditions, thus, supporting its use in therapy of problem wounds in diabetes.

Asunto(s)

Diabetes Mellitus Experimental/fisiopatología , Ácido Hialurónico/administración & dosificación , Cicatrización de Heridas/efectos de los fármacos , Heridas y Lesiones/fisiopatología , Administración Tópica , Animales , Diabetes Mellitus Experimental/patología , Tejido de Granulación/patología , Tejido de Granulación/fisiopatología , Microscopía Fluorescente , Ratas , Heridas y Lesiones/patología

RESUMEN

Increasing clinical and experimental evidence suggests that traumatic brain injury (TBI) elicits an acute inflammatory response. In the present study we investigated whether white blood cells (WBC) are activated in the cerebral microcirculation early after TBI and whether WBC accumulation affects the posttraumatic cerebrovascular response. Twenty-four anesthetized rabbits had chronic cranial windows implanted 3 weeks before experimentation. Animals were divided into four experimental groups and were studied for 7 hours (groups I, IIa, and III) or 2 hours (group IIb). Intravital fluorescence videomicroscopy was used to visualize WBC (rhodamine 6G, intravenously), pial vessel diameters, and blood-brain barrier (BBB) integrity (Na+-fluorescein) at 6 hours (groups I, IIa, and III) or 1 hour (group IIb) after TBI. Group I (n = 5) consisted of sham-operated animals. Groups IIa (n = 7) and IIb (n = 5) received fluid-percussion injury at 1 hour. Group III (n = 7) received fluid-percussion injury and 1 mg/kg anti-adhesion monoclonal antibody (MoAb) "IB4" 5 minutes before injury. Venular WBC sticking, intracranial pressure (ICP), and arterial vessel diameters increased significantly for 6 hours after trauma. IB4 reduced WBC margination and prevented vasodilation. Intracranial pressure was not reduced by treatment with IB4. Blood-brain barrier damage occurred at 1 hour but not at 6 hours after TBI and was independent of WBC activation. This first report using intravital videomicroscopy to study the inflammatory response after TBI reveals upregulated interaction between WBC and cerebral endothelium that can be manipulated pharmacologically. White blood cell activation is associated with pial arteriolar vasodilation. White blood cells do not induce BBB breakdown less than 6 hours after TBI and do not contribute to posttraumatic ICP elevation. The role of WBC more than 6 hours after TBI should be investigated further.

Asunto(s)

Lesiones Encefálicas/sangre , Lesiones Encefálicas/fisiopatología , Circulación Cerebrovascular/fisiología , Leucocitos/fisiología , Animales , Arteriolas/fisiopatología , Presión Sanguínea/fisiología , Barrera Hematoencefálica/fisiología , Lesiones Encefálicas/patología , Dióxido de Carbono , Adhesión Celular/fisiología , Presión Intracraneal/fisiología , Recuento de Leucocitos , Microcirculación/fisiología , Piamadre/irrigación sanguínea , Conejos , Volumen de Ventilación Pulmonar

RESUMEN

UNLABELLED: We explored the hypothesis that brain damage after cardiac arrest caused by ventricular fibrillation (VF) needs different therapies than that after asphyxiation, which has been studied less thoroughly. In 67 healthy mongrel dogs of both sexes cardiac arrest (at normothermia) by ventricular fibrillation (no blood flow lasting 10 min) or asphyxiation (no blood flow lasting 7 min) was reversed by normothermic external cardiopulmonary resuscitation, followed by intermittent positive-pressure ventilation for 20 h, and intensive care to 96 h. To ameliorate ischemic brain damage, the calcium entry blocker lidoflazine or a solution of free radical scavengers (mannitol and L-methionine in dextran 40) plus magnesium sulphate, was given intravenously immediately upon restoration of spontaneous circulation. Outcome was evaluated as functional deficit, brain creatine kinase (CK) leakage into the cerebrospinal fluid (CSF) and brain morphologic changes. Lidoflazine seemed to improve cerebral outcome after VF but not after asphyxiation. Free radical scavengers plus magnesium sulphate seemed to improve cerebral outcome after asphyxiation, but not after VF. After VF, scattered ischemic neuronal changes in multiple brain regions dominated, and total brain histopathologic damage scores correlated with final neurologic deficit scores at 96 h (r = 0.66) and with peak CK levels in CSF (r = 0.81). After asphyxiation, in addition to the same ischemic neuronal changes, microinfarcts occurred, and there was no correlation between total brain histopathologic damage scores and neurologic deficit scores or CK levels in CSF. CONCLUSIONS: Different mechanisms of cardiac arrest, which cause different morphologic patterns of brain damage, may need different cerebral resuscitation treatments.

Asunto(s)

Asfixia/complicaciones , Isquemia Encefálica/prevención & control , Reanimación Cardiopulmonar/métodos , Paro Cardíaco/etiología , Fibrilación Ventricular/complicaciones , Animales , Isquemia Encefálica/etiología , Bloqueadores de los Canales de Calcio/uso terapéutico , Perros , Femenino , Depuradores de Radicales Libres/uso terapéutico , Paro Cardíaco/terapia , Lidoflazina/uso terapéutico , Sulfato de Magnesio/uso terapéutico , Masculino , Respiración con Presión Positiva

RESUMEN

Cigarette smoking within minutes induces leukocyte adhesion to the vascular wall and formation of intravascular leukocyte-platelet aggregates. We find this is inhibited by platelet-activating factor (PAF) receptor antagonists, and correlates with the accumulation of PAF-like mediators in the blood of cigarette smoke-exposed hamsters. These mediators were PAF-like lipids, formed by nonenzymatic oxidative modification of existing phospholipids, that were distinct from biosynthetic PAF. These PAF-like lipids induced isolated human monocytes and platelets to aggregate, which greatly increased their secretion of IL-8 and macrophage inflammatory protein-1alpha. Both events were blocked by a PAF receptor antagonist. Similarly, blocking the PAF receptor in vivo blocked smoke-induced leukocyte aggregation and pavementing along the vascular wall. Dietary supplementation with the antioxidant vitamin C prevented the accumulation of PAF-like lipids, and it prevented cigarette smoke-induced leukocyte adhesion to the vascular wall and formation of leukocyte-platelet aggregates. This is the first in vivo demonstration of inflammatory phospholipid oxidation products and it suggests a molecular mechanism coupling cigarette smoke with rapid inflammatory changes. Inhibition of PAF-like lipid formation and their intravascular sequela by vitamin C suggests a simple dietary means to reduce smoking-related cardiovascular disease.

Asunto(s)

Ácido Ascórbico/farmacología , Plaquetas/fisiología , Monocitos/fisiología , Neutrófilos/fisiología , Factor de Activación Plaquetaria/análogos & derivados , Factor de Activación Plaquetaria/fisiología , Receptores de Superficie Celular , Receptores Acoplados a Proteínas G , Fumar/sangre , Animales , Antioxidantes/farmacología , Azepinas/farmacología , Plaquetas/efectos de los fármacos , Adhesión Celular , Agregación Celular , Quimiocina CCL4 , Cricetinae , Humanos , Interleucina-8/sangre , Proteínas Inflamatorias de Macrófagos/sangre , Monocitos/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Factor de Activación Plaquetaria/antagonistas & inhibidores , Factor de Activación Plaquetaria/farmacología , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Glicoproteínas de Membrana Plaquetaria/antagonistas & inhibidores , Glicoproteínas de Membrana Plaquetaria/fisiología , Valores de Referencia , Factores de Tiempo , Triazoles/farmacología

RESUMEN

BACKGROUND: Traumatic brain injury (TBI) induces an acute inflammatory response characterized by early recruitment of inflammatory cells (white blood cells). Rapid resuscitation of TBI with hypertonic saline/dextran (HS/DEX) yields promising results in clinical and experimental studies. The purpose of this paper was to test the hypothesis that HS/DEX exerts its effects in part through a modulation of the acute inflammatory response to TBI. METHODS: Rabbits equipped with chronic cranial windows underwent fluid-percussion injury and were followed up for 6 hours. Intravital fluorescence videomicroscopy technique was used to visualize white blood cell trafficking and to measure pia vessel diameters and venular shear rates. Three groups were studied: sham (group I, n = 5), trauma (group II, n = 7), and trauma and 4 mL/kg 7.2% NaCl/10% dextran 60 IV over 5 minutes at 10 minutes after TBI (group III, n = 7). RESULTS: TBI in groups II and III led to significant increases of intracranial pressure. Arteriolar diameters after trauma increased by 17 +/- 8% at 6 hours in group II. Infusion of HS/DEX completely prevented this secondary diameters increase. At 6 hours, the increase of "sticking" white blood cells in group III was reduced by approximately 90% compared with group II. CONCLUSIONS: Whether the anti-inflammatory effect of HS/DEX plays a role in reducing delayed brain damage (> 6 hours after TBI) or other systemic complications of TBI arises as an important question and should be investigated further.

Asunto(s)

Lesiones Encefálicas/fisiopatología , Lesiones Encefálicas/terapia , Circulación Cerebrovascular/efectos de los fármacos , Dextranos/uso terapéutico , Microcirculación/efectos de los fármacos , Sustitutos del Plasma/uso terapéutico , Solución Salina Hipertónica/uso terapéutico , Enfermedad Aguda , Animales , Lesiones Encefálicas/inmunología , Preescolar , Modelos Animales de Enfermedad , Combinación de Medicamentos , Evaluación Preclínica de Medicamentos , Humanos , Inflamación , Leucocitos/inmunología , Conejos , Factores de Tiempo

RESUMEN

BACKGROUND: This manuscript describes the results of three studies designed to test the hypothesis that the intravenous administration of hypertonic saline could help to preserve spinal cord function after injury. METHODS: A static compression model was used to injure rat spinal cords. Somatosensory evoked potentials and spinal cord blood flow changes were monitored in the acute studies. The first study compared the effects of administration of hypertonic saline with isotonic saline solutions. The second study evaluated the effect of hypertonic saline administration at 5, 15, and 60 minutes after injury. A chronic injury model was evaluated in the third study. Spontaneous voiding, neurologic function, and evidence of histologic changes were evaluated. RESULTS AND CONCLUSIONS: The administration of hypertonic saline after spinal cord injury increased blood flow and helped preserve spinal cord function in the acute models. The rate of recovery with the chronic model was significantly faster in hypertonic saline treated animals.

Asunto(s)

Solución Salina Hipertónica/uso terapéutico , Traumatismos de la Médula Espinal/terapia , Enfermedad Aguda , Animales , Enfermedad Crónica , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Potenciales Evocados Somatosensoriales/efectos de los fármacos , Femenino , Hemodinámica/efectos de los fármacos , Humanos , Flujometría por Láser-Doppler , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Traumatismos de la Médula Espinal/fisiopatología , Factores de Tiempo

RESUMEN

Alterations in leukocyte/endothelium interaction due to phototoxic effects of the fluorescent dyes acridine orange (AO) and rhodamine 6G (Rh6G) were studied by intravital microscopy using the dorsal skinfold model in awake Syrian golden hamsters. AO (0.5 mg/kg/min; constant IV infusion) and Rh6G (0.1 micromol/kg; bolus IV) were administered via an indwelling venous catheter. Five to seven arterioles (35-55 microm) and postcapillary venules (30-65 microm) were investigated in each animal. Vessels were exposed four times for 30 sec to continuous light of the appropriate excitation wavelength with a 10-15-min time interval between exposures. Animals were randomly assigned to five experimental groups (five distinct light energy levels). AO and Rh6G induced leukocyte rolling/sticking in postcapillary venules and arterioles when exposed to high light energy levels. AO, but not Rh6G, induced arteriolar vasospasm when exposed to high light energies. The potential phototoxic effect of AO and Rh6G is demonstrated, as assessed by the stimulation of leukocyte-endothelium interaction and arteriolar vasospasm in vivo. This study underscores the necessity to optimize microscopic set-ups for intravital microscopy, to reduce the excitation light energy level significantly, and to perform stringent control experiments, ruling out an artificial phototoxicity-induced stimulation of leukocyte adhesion.

Asunto(s)

Adhesión Celular/efectos de la radiación , Leucocitos/efectos de la radiación , Luz , Naranja de Acridina/farmacología , Animales , Arteriolas/citología , Arteriolas/efectos de los fármacos , Cricetinae , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Femenino , Colorantes Fluorescentes , Leucocitos/citología , Mesocricetus , Microscopía Fluorescente , Rodaminas/farmacología , Vasoconstricción/efectos de los fármacos

RESUMEN

In pancreatic islet transplantation, the adhesion of activated leukocytes to endothelial cells and the loss of microvascular integrity represent the critical microcirculatory events, which promote loss of graft function due to rejection. With the view that oxygen radicals may contribute to graft rejection, we studied the effect of the antioxidant vitamin E on microvascular rejection of islet grafts. Islets were transplanted syngeneically and xenogeneically (rat) into dorsal skin-fold chambers of hamsters, which received a non-vitamin-E-supplemented laboratory chow. Treated animals with xenografts were fed with a diet supplemented with vitamin E in a low (150 mg/kg) and high (8000 mg/kg) concentration. Intravital fluorescence microscopy demonstrated complete vascularization of syngeneic grafts at day 10 after transplantation, intact islet microcirculation at day 20 with a functional capillary density of 653 +/- 6 cm-1, and only few leukocytes adherent to the endothelial lining of the islets' microvasculature (88 +/- 23 mm-2). Xenogeneic islets showed initial signs of rejection at day 6, including adhesion of leukocytes to the microvascular endothelium (610 +/- 110 mm-2) and loss of endothelial integrity. After 20 days, functional capillary density was significantly lower (173 +/- 68 cm-1) when compared with syngeneic grafts, indicating failure of graft acceptance. Supplementation of the diet with low and high concentrations of vitamin E resulted in a significant (P < 0.05) reduction of xenograft leukocyte-endothelium interaction (146 +/- 29 mm-2 and 109 +/- 42 mm-2) at day 6 after transplantation and and adequate development of functional capillary density at day 20 (478 +/- 36 cm-1 and 539 +/- 86 cm-1; P < 0.05), indicating prevention of microvascular rejection. We conclude that dietary supplementation of the lipophilic antioxidant vitamin E attenuates leukocyte-endothelial cell interactions, preserves microvascular integrity, and thus inhibits microvascular rejection in a dose-dependent fashion. Our study underscores the pivotal mediator role of reactive oxygen species in islet xenograft rejection and, furthermore, suggests that dietary vitamin E may act as an adjunct anti-rejection treatment in clinical islet transplantation.

Asunto(s)

Rechazo de Injerto/prevención & control , Trasplante de Islotes Pancreáticos/patología , Vitamina E/uso terapéutico , Animales , Cricetinae , Dieta , Rechazo de Injerto/patología , Rechazo de Injerto/fisiopatología , Humanos , Mesocricetus , Neovascularización Fisiológica/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Trasplante Heterólogo

RESUMEN

Adhesion molecules on polymorphonuclear leukocytes (PMNL) play an important role in nonspecific defense mechanisms directed at invading microorganisms. When local infection, however, cannot be controlled, a systemic inflammatory response syndrome (SIRS) ensues which may progress to septic shock and multiple organ failure, these being major determinants of the patient's outcome. In the present study, the expression of beta 2-integrins and L-selectin on blood PMNL was measured on subsequent days in patients with sepsis (n = 17) and in healthy volunteers (n = 15). beta 2-Integrins and L-selectin molecules were detected by flow cytometry, using the monoclonal antibodies IB4 (anti-CD18) and Dreg200 (anti-CD62L), respectively. Adhesion molecules were determined at baseline immediately after blood collection and also 45 min after incubation of cells in vitro at body temperature to allow for spontaneous regulation. In addition, PMNL were activated by receptor-dependent and receptor-independent stimuli to characterize stimulus-specific adhesion molecule expression. In parallel with the measurement of adhesion molecules, severity of sepsis was assessed by the Elebute score. The results demonstrate significant differences in the basal, spontaneous and stimulus-induced expression of adhesion molecules between healthy volunteers, survivors (n = 11) and nonsurvivors (n = 6). Moreover, when survivors and nonsurvivors with severe sepsis (Elebute score > 12) were compared, basal expressions of both beta 2-integrins and L-selectin were significantly lower in patients who did not survive. Thus, measurement of adhesion molecules on circulating PMNL may be useful to identify septic patients at high risk for lethal outcome.

Asunto(s)

Antígenos CD18/metabolismo , Selectina L/metabolismo , Neutrófilos/metabolismo , Sepsis/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Calcimicina/farmacología , Femenino , Humanos , Masculino , Persona de Mediana Edad , N-Formilmetionina Leucil-Fenilalanina/farmacología , Índice de Severidad de la Enfermedad , Sobrevivientes , Acetato de Tetradecanoilforbol/farmacología , Factor de Necrosis Tumoral alfa/farmacología

RESUMEN

The time course of blood-brain barrier (BBB) breakdown after traumatic brain injury (TBI) has important implications for therapy. This study was conducted in order to test post-traumatic BBB dysfunction in a model of fluid-percussion induced TBI in rabbits at 1 and 6 hours after TBI and relate it to white blood cell (WBC) activation. Ten anesthetized rabbits had chronic cranial windows implanted three weeks prior to experimentation. Fluid-percussion injury (3.5 atm.) was induced and animals were followed for 1 or 6 h. Intravital fluorescence videomicroscopy was used to assess BBB permeability and WBC adhesion to pial venules. Na(+)-fluorescein was infused continuously over 30 min at either 30 min (Group I, n = 5) or 5.5 h (Group II, n = 5) after TBI. Microvascular permeability in individual postcapillary venules was assessed qualitatively at 1 and 30 min after start of infusion. TBI led to a transient mean arterial blood pressure (MAP) surge after trauma and a progressive increase in the number of sticking WBCs per mm2 vessel wall. Na(+)-fluorescein extravasation was observed in 4 out of 5 Group I animals and in none of Group II. BBB breakdown was not associated with WBC sticking. We conclude that after fluid-percussion injury the BBB is damaged at 1 h post-trauma and that its function is restored 6 h later. Increased WBC sticking at 6 h is not associated with BBB breakdown. Whether WBCs may cause vascular permeability changes at a later point needs further investigation.

Asunto(s)

Barrera Hematoencefálica/fisiología , Lesiones Encefálicas/patología , Leucocitos/fisiología , Animales , Adhesión Celular/fisiología , Microscopía por Video , Conejos