RESUMEN
The pathophysiology of pressure ulcers is not yet fully understood. Widely used experimental models lead to pressure ulcers in the skin and the subcutaneous tissue only. The aim of the present study was to develop a clinically relevant deep pressure ulcer model in nude mice involving all relevant tissue layers. Balb/c nude mice were anesthetized and a steel disk was implanted under the great gluteus muscle. Full recovery was allowed for 10 days. Pressure was then periodically applied (2 hours compression, 1 hour recovery) using a Neodymium magnet in conjunction with the disk in the unanesthetized mouse. Cycles were repeated for 1, 4, 6, 8, or 10 times. Controls underwent the same procedure without placement of the magnet. The pressure ulcer grade was found to be cycle dependent,with the highest degree after 10 cycles. Histological evaluations revealed signs of necrosis in the skin and subcutaneous fat after four, and in the muscle after eight and 10 cycles of compression. Polymorphnuclear granulocyte infiltration in certain layers was found to be dependent on the number of cycles. We conclude that this clinically relevant nude mouse model can be used to investigate the mechanism of pressure ulcer development and to study new therapeutic approaches.
Asunto(s)
Modelos Animales de Enfermedad , Úlcera por Presión/patología , Úlcera por Presión/fisiopatología , Animales , Femenino , Masculino , Ratones , Ratones DesnudosRESUMEN
Reactive oxygen species (ROS) are dangerous intermediates of cellular oxygen metabolisms, and are involved in pathogenesis of a wide range of diseases. Superoxide Dismutases (SODs) are an important part of antioxidant defense systems in mammalian cells capable of reducing the harmful effect of ROS on human tissues. Unfortunately, intravenously administered SOD shows a biological half-life of a few minutes, and enteral administration fails due to biodegradation of the enzyme in the gastrointestinal system. The aim of our study was to improve biological half-life of recombinant human Cu/Zn SOD (rhSOD) within systemic circulation by liposomal encapsulation and aerosolization into the lungs. We studied the feasibility of a "needle-free" route of drug administration via the lungs combined with the sustained release effect of liposomes in an experimental pig model. We studied 14 anesthetized pigs separated into three groups. The first group (n = 5) received 15 mg aerosolized liposomal rhSOD. The second group (n = 4) received 15 mg intravenously injected liposomal rhSOD. The third group (n = 5) served as an untreated control. We determined rhSOD concentration as well as activity within the lungs by the use of bronchoalveolar lavages (BALs). RhSOD plasma concentrations were determined by blood sampling. In animals treated with aerosolized liposomal rhSOD plasma concentration of the enzyme increased and formed a plateau ranging from 19 to 21 ng/mL over the whole observational period (5 h). At the end of the experiment 5 h after completion of aerosol administration 95.2% of peak plasma concentration was found in this group. Three and 5 h after completion of aerosolization leucocytes (p = 0.54, 0.40) in BALs as well as PaO2 (p = 0.44, 0.35), PaCO(2) (p = 0.83, 0.75), and pH (p = 0.07, 0.07) in arterial blood remained unchanged compared to baseline. In animals treated with intravenously injected liposomal rhSOD, plasma concentration of the enzyme substantially increased to 3987 ng/mL but rapidly decreased over the observational period (5 h). At the end of the experiment 14.1% of peak plasma concentration was found in this group. Aerosolization of liposomal rhSOD leads to long-term and uniform uptake into systemic circulation without acute deleterious effects on respiratory tract. Compared to intravenously administered liposomal rhSOD, biologic half-life within systemic circulation was substantially prolonged in aerosol-treated animals. It could be a feasible strategy for administration of radical scavenging enzymes for treatment of systemic diseases.
Asunto(s)
Depuradores de Radicales Libres/administración & dosificación , Superóxido Dismutasa/administración & dosificación , Superóxido Dismutasa/farmacocinética , Administración por Inhalación , Aerosoles , Animales , Animales Recién Nacidos , Líquido del Lavado Bronquioalveolar/química , Preparaciones de Acción Retardada , Femenino , Depuradores de Radicales Libres/efectos adversos , Depuradores de Radicales Libres/sangre , Depuradores de Radicales Libres/farmacocinética , Semivida , Inyecciones Intravenosas , Liposomas , Masculino , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/efectos adversos , Proteínas Recombinantes/sangre , Proteínas Recombinantes/farmacocinética , Superóxido Dismutasa/efectos adversos , Superóxido Dismutasa/sangre , PorcinosRESUMEN
BACKGROUND: Treatment of traumatic liver and spleen rupture is a major challenge for the surgeon. Because of their excellent blood supply and tissue structure, rupture of the liver and spleen is often associated with massive abdominal hemorrhage. Frequently the surgeon's only feasible option is partial or total resection of the organ. The purpose of this study was to test the hemostatic efficacy of gelatin thrombin granules (FloSeal) in a standardized severe traumatic liver and spleen rupture model in swine (representing a grade IV-V rupture) during severe hemorrhagic shock and coagulation disorder. METHODS: Standardized combined penetrating liver and spleen rupture was inflicted in 10 anesthetized swine. Hemorrhagic shock was induced after heparinization. Gelatin thrombin granules were used to treat both the ruptured liver and the ruptured spleen. Blood loss, hemostasis, and 48 hours survival rate were quantified. Cardiorespiratory parameters, activated clotting time, and plasma fibrinogen level were monitored. After 1 hour and 48 hours a second look evaluation was performed to detect any postoperative hemorrhage. Ruptures were then examined macroscopically and histologically. RESULTS: Hemostasis was achieved with FloSeal in all swine. The mean amount of FloSeal used was 14 mL +/- 2.5 mL. Macroscopic and histologic findings after 48 hours showed excellent clot integration into the surrounding tissue without any adverse effects. CONCLUSION: Gelatin thrombin granules (FloSeal) are effective in treating severe penetrating rupture of the liver and spleen even during hemorrhagic shock, retransfusion conditions, and coagulation disorder.
Asunto(s)
Esponja de Gelatina Absorbible/uso terapéutico , Hígado/lesiones , Choque Hemorrágico/terapia , Rotura del Bazo/terapia , Animales , Trastornos de la Coagulación Sanguínea/etiología , Trastornos de la Coagulación Sanguínea/terapia , Modelos Animales de Enfermedad , Femenino , Hemoglobinas/metabolismo , Hemostáticos , Hepatopatías/complicaciones , Hepatopatías/terapia , Masculino , Rotura/complicaciones , Rotura/terapia , Choque Hemorrágico/etiología , Rotura del Bazo/complicaciones , Sus scrofa , Trombina/uso terapéuticoRESUMEN
Human adipose-derived stem cells (ASC) can be expanded in an undifferentiated state or differentiated along the osteogenic, chondrogenic, adipogenic, myogenic, endothelial and neurogenic lineage. To test their in vivo and in situ regenerative potential, their fate needs to be traced after application in suitable defect models. Non-invasive imaging systems allow for real time tracking of labelled cells in the living animal. We have evaluated a bioluminescence cell tracking approach to visualise ASC labelled with luciferase in the living animal. Two procedures have been tested to efficiently label human stem cells with a reporter gene (luciferase, green fluorescent protein), namely lipofection with Lipofectamine 2000 and electroporation with a Nucleofector device. With both lipofection and nucleofection protocols, we have reached transfection efficiencies up to 60%. Reporter gene expression was detectable for 3 weeks in vitro and did not interfere with the phenotype and the stem cell properties of the cells. By means of a highly sensitive CCD camera, we were able to achieve real time imaging of cell fate for at least 20 days after application (intravenous, intramuscular, intraperitoneal, subcutaneous) in nude mice. Moreover, we were able to influence cell mobility by choosing different modes of application such as enclosure in fibrin matrix. The optical imaging system with transient transfection is an elegant cell-tracking concept to follow survival and fate of human stem cells in small animals.
Asunto(s)
Tejido Adiposo/citología , Técnicas Citológicas/métodos , Células Madre/citología , Animales , Diferenciación Celular , Proliferación Celular , Genes Reporteros , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Hibridación Fluorescente in Situ , Luciferasas/metabolismo , Ratones , Ratones Desnudos , Osteogénesis , Transfección , Imagen de Cuerpo EnteroRESUMEN
Although reamed intramedullary nailing has been one of the greatest advances in modern fracture care, the concomitant increase in medullary cavity pressure leads to intravasation of bone marrow content into the blood stream, an effect that can evoke serious systemic reactions. A newly developed rinsing-suction-reamer (RSR) was able to substantially reduce the pressure and bone marrow intravasation content during experimental femoral nailing. We investigated the pathophysiological effects using the RSR, testing the hypothesis that by reducing marrow fat embolization, RSR would also reduce the activation of coagulation compared with the universal AO-Reamer (AOR) and comparable to external fixation. Twenty-two pigs were treated with either simulated external fixation or reamed femoral nailing using AOR or RSR. During surgery, the intramedullary pressure was measured and intravasation of medullary material was quantified. After surgery, the pigs were kept anaesthetised and monitored for 6 h. At defined intervals, serological, hematological, and hemodynamic parameters were evaluated. The RSR was significantly superior when compared to AOR with regard to the generation of intramedullary pressure and fat embolization; however, with external fixation the values were even lower. The evaluation of other parameters revealed no clear differences between the two reamers and the external fixator. The pig model showed that RSR led to a significant reduction of the intramedullary increase in pressure and fat intravasation compared to AOR. Although the reduction of fat embolism by RSR is not associated with pathophysiological changes, RSR can have advantages for the treatment of femoral fractures.
Asunto(s)
Clavos Ortopédicos , Fracturas del Fémur , Fijación Intramedular de Fracturas/instrumentación , Fijación Intramedular de Fracturas/métodos , Animales , Médula Ósea , Modelos Animales de Enfermedad , Embolia Grasa/etiología , Embolia Grasa/patología , Embolia Grasa/fisiopatología , Fijadores Externos/efectos adversos , Fracturas del Fémur/fisiopatología , Fracturas del Fémur/cirugía , Fijación Intramedular de Fracturas/efectos adversos , Hemodinámica , Pulmón/patología , Presión , PorcinosRESUMEN
Administration of a single bolus of endotoxin is a model of sepsis response in experimental animal studies. Large animal species, such as pigs and sheep, are more sensitive to endotoxin administration due to an initial excessive pulmonary hypertensive response frequently resulting in acute right heart dysfunction. We investigated whether infusion of high-dose endotoxin in pigs but administered in an increasing dose results in inflammatory response without excessive pulmonary hypertension and right heart dysfunction. Piglets of both sexes weighing 25 to 30 kg were anesthetized and mechanically ventilated. After instrumentation and baseline measurements, animals received an infusion of total 500 microg kg(-1) i.v. endotoxin (Escherichia coli LPS) over 2 h in an increasing dose of 0.5 to 12 microg kg(-1) min(-1). Hemodynamic, respiratory, and oxygenation parameters were measured every hour. At 1 and 5 h following endotoxin, plasma levels of inflammatory and organ damage parameters were measured. Endotoxin infusion induced progressive arterial hypoxemia, an increase in peak inspiratory pressure, sustained pulmonary hypertension, and systemic hypotension that persisted throughout the experiment. Endotoxin plasma levels peaked at 1 h following infusion and declined toward baseline values at 5 h thereafter. In contrast, plasma levels of nitrite/nitrate, IL-1ra (as marker of cytokine response), remained markedly increased at 5 h after endotoxin infusion as compared with baseline values. Plasma markers of organ damage were significantly increased. Our data show that the dosing of endotoxin in an increasing manner in pigs produces a reliable model of an experimental sepsis response and organ dysfunction without immediate overwhelming pulmonary hypertension resulting in cardiovascular failure.
Asunto(s)
Endotoxinas/fisiología , Hipertensión Pulmonar/prevención & control , Síndrome de Dificultad Respiratoria/inducido químicamente , Animales , Relación Dosis-Respuesta a Droga , Endotoxinas/administración & dosificación , Infusiones Intravenosas , PorcinosRESUMEN
PURPOSE: To investigate whether analysis of a washout curve of contrast material obtained with serial computed tomography (CT) enables differentiation between hydrostatic pulmonary edema and pulmonary edema caused by increased capillary permeability. MATERIALS AND METHODS: The institutional committee on animal experiments approved this study, which was performed in accordance with designated guidelines. Chest CT was performed in 12 piglets after induction of anesthesia and start of mechanical ventilation. Dynamic CT was performed before and after induction of hydrostatic edema (n = 5) or oleic acid-induced increased vascular permeability edema (n = 7). Scans were obtained over 240 seconds during inspiratory breath holding at a single representative subcarinal level in the lungs. This anatomic level was kept constant and included areas of normal ventilation before and after induction of pulmonary edema and areas of ground-glass opacity and consolidation after induction of pulmonary edema. Measured lung attenuation in the regions of interest was normalized to that before contrast material injection and plotted as a function of time. Statistical analysis was performed by using two-way analysis of variance with repeated measures. RESULTS: In general, before induction of pulmonary edema, attenuation of normally aerated lung areas did not increase after the initial peak of enhancement during the first pass of contrast material. In animals with hydrostatic edema, no attenuation changes in areas of ground-glass opacity were observed after the initial peak. Conversely, lung attenuation increased continuously in animals with oleic acid-induced high-permeability pulmonary edema (P = .002). After induction of lung edema, pulmonary enhancement measured in lung regions with normal ventilation or consolidation did not change in either group. Pulmonary fluid accumulation 90 minutes after induction of edema did not significantly differ between groups. CONCLUSION: Dynamic contrast-material enhanced CT can help differentiate between permeability and hydrostatic lung edema in an animal model.
Asunto(s)
Síndrome de Dificultad Respiratoria/diagnóstico por imagen , Tomografía Computarizada por Rayos X , Animales , Femenino , Masculino , Porcinos , Tomografía Computarizada por Rayos X/métodosRESUMEN
Human fibrin sealant (FS) has been proven effective for skin grafting after severe burn, however no systematic evaluation of application conditions has been performed so far. In order to find the optimal FS amount for fixation of skin grafts to deep defects, we created four full thickness wounds (8 cmx4 cm) on the dorsum of six male pigs. Wounds were covered with unmeshed split thickness skin grafts and fixed either with a thin layer (0.05 ml/cm2) or a thick layer (0.15 ml/cm2) of fibrin sealant (FS) without additional sutures. Sutures served as controls. FS was used as a slow clotting spray (4-5 IUthrombin/ml). Outcome measurements revealed that hematoma formation (day of surgery) was more extensive and occurred more frequently in the suture group as compared to FS 0.05 ml/cm2 (p<0.05). Areas of graft dislocation tended to be larger in the suture group versus the FS 0.05 ml/cm2. The FS 0.05 ml/cm2 graft take on day 5 appeared to be enhanced in comparison to the suture group. Excellent outcome was notable on the final observation day (day 21) in the FS 0.05 ml/cm2 group with a take of 99.7% (IQR 96.1-100%). Corresponding values in the FS 0.15 ml/cm2 group were 96.9% (IQR 92.2-99%) and 95.9% (IQR 93.2-98%) in the suture group. The results indicate, that the usage of a sprayed thin FS layer (0.05 ml/cm2) in a slow clotting rate (4-5 IUthrombin/ml) is an appropriate fixation method in split thickness skin transplantation.
Asunto(s)
Quemaduras/cirugía , Adhesivo de Tejido de Fibrina/uso terapéutico , Trasplante de Piel/métodos , Animales , Supervivencia de Injerto , Masculino , Distribución Aleatoria , Porcinos , Resultado del Tratamiento , Cicatrización de Heridas/efectos de los fármacosRESUMEN
We investigated whether mechanical ventilation after aspiration is deleterious when started before surfactant therapy. Gas exchange and lung mechanics were measured in rabbits after aspiration either mechanically ventilated before or after lavage with diluted surfactant or Ringer's solution. Lung injury was induced by intratracheal instillation of 2 mL/kg of a betain/HCl pepsin mixture. After 30 min of spontaneous breathing, ventilation was started in 12 rabbits, which were then treated by lavage with diluted surfactant (15 mL/kg body weight; 5.3 mg/mL, group MVpre S) or with Ringer's solution (1 mL/kg; group MVpre R). Another 12 rabbits were treated by lavage while spontaneously breathing and were then connected to the ventilator (MVpost S and MVpost R). Sham control rabbits were mechanically ventilated for 4 h. At the end of experiment, PaO2/FiO2 ratio in MVpost S was five times higher than in MVpre S (P=0.0043). Lung mechanics measurements showed significant difference between MVpre S and MVpost S (P=0.0072). There was histopathologic evidence of decreased lung injury in MVpost S. Immediate initiation of ventilation is harmful when lung injury is induced by aspiration. Further investigations are needed to clarify whether the timing of lavage with diluted surfactant has an impact on the treatment of patients with aspiration or comparable types of direct lung injury.