RESUMEN
The presence and progression of a neuromuscular pathology can impact on the contractile force production of a muscle. Hence, measurements of force production can be an important tool for the evaluation of disease progression. In this chapter, we describe how to perform in situ function testing on the tibialis anterior muscle using a murine model. Performing neuromuscular in situ function testing allows force measurements to be recorded in a physiologically relevant environment.
Asunto(s)
Músculo Esquelético , Fenómenos Fisiológicos del Sistema Nervioso , Ratones , Animales , Músculo Esquelético/fisiología , Contracción Muscular/fisiologíaRESUMEN
Vascularization is a major hurdle for growing three-dimensional tissue engineered constructs. This study investigated the mechanisms involved in hypoxic preconditioning of primary rat myoblasts in vitro and their influence on local angiogenesis postimplantation. Primary rat myoblast cultures were exposed to 90 min hypoxia at <1% oxygen followed by normoxia for 24 h. Real time (RT) polymerase chain reaction evaluation indicated that 90 min hypoxia resulted in significant downregulation of miR-1 and miR-206 (p < 0.05) and angiopoietin-1 (p < 0.05) with upregulation of vascular endothelial growth factor-A (VEGF-A; p < 0.05). The miR-1 and angiopoietin-1 responses remained significantly downregulated after a 24 h rest phase. In addition, direct inhibition of miR-206 in L6 myoblasts caused a significant increase in VEGF-A expression (p < 0.05), further establishing that changes in VEGF-A expression are influenced by miR-206. Of the myogenic genes examined, MyoD was significantly upregulated, only after 24 h rest (p < 0.05). Preconditioned or control myoblasts were implanted with Matrigel™ into isolated bilateral tissue engineering chambers incorporating a flow-through epigastric vascular pedicle in severe combined immunodeficiency mice and the chamber tissue harvested 14 days later. Chambers implanted with preconditioned myoblasts had a significantly increased percentage volume of blood vessels (p = 0.0325) compared with chambers implanted with control myoblasts. Hypoxic preconditioned myoblasts promote vascularization of constructs via VEGF upregulation and downregulation of angiopoietin-1, miR-1 and miR-206. The relatively simple strategy of hypoxic preconditioning of implanted cells - including non-stem cell types - has broad, future applications in tissue engineering of skeletal muscle and other tissues, as a technique to significantly increase implant site angiogenesis.
Asunto(s)
Regulación hacia Abajo , Implantes Experimentales , MicroARNs/genética , Mioblastos/patología , Neovascularización Fisiológica , Ingeniería de Tejidos/instrumentación , Regulación hacia Arriba , Factor A de Crecimiento Endotelial Vascular/genética , Animales , Biomarcadores/metabolismo , Hipoxia de la Célula/genética , Células Cultivadas , Desmina/metabolismo , Regulación hacia Abajo/genética , Masculino , Ratones SCID , MicroARNs/metabolismo , Desarrollo de Músculos/genética , Mioblastos/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Sprague-Dawley , Andamios del Tejido/química , Regulación hacia Arriba/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Insoluble, pure protein particles could be advantageous as single-entity vaccines or as carriers for small peptide epitopes. Dense gas anti-solvent precipitation was employed to produce pure protein particles which were found to be insoluble in water. As particulate and multimerized antigens are more immunogenic and hence more advantageous for vaccination, particles were produced via this method using ovalbumin as a model antigen. The particles produced had a mean diameter of approximately 300nm, and remained as discrete particles at low pH. At neutral pH or in the presence of electrolyte, the particles exhibited predictable flocculation behaviour to produce aggregates 1-5microm in diameter. Immunisation of mice with these flocculates elicited specific ovalbumin antibody production, T-cell proliferation and a cytotoxic T-cell response, all in the absence of adjuvant. Thus, dense gas processing could be used as a generic method to produce pure protein particulate vaccines.
Asunto(s)
Adyuvantes Inmunológicos , Formación de Anticuerpos/inmunología , Antígenos/inmunología , Inmunidad Celular/inmunología , Material Particulado/inmunología , Vacunas/inmunología , Animales , Antígenos/química , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Proliferación Celular , Química Farmacéutica , Pollos , Inmunización , Inyecciones Intradérmicas , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Electrónica de Rastreo , Muramidasa/inmunología , Ovalbúmina/inmunología , Tamaño de la Partícula , Linfocitos T/inmunología , Vacunas/químicaRESUMEN
The soldierfish (Gymnapistes marmoratus), which is related to the stonefish (Synanceia spp.), inhabits the western, southern and lower eastern coastlines of Australia. We have previously found that G. marmoratus venom possesses pharmacological activity similar to Synanceia trachynis venom (Hopkins, B.J., Hodgson, W.C., 1998. Cardiovascular studies on venom from the soldierfish (Gymnapistes marmoratus). Toxicon 36, 973-872; Church, J.E., Hodgson, W.C., 2000. Dose-dependent cardiovascular and neuromuscular effects of stonefish (Synanceja trachynis) venom. Toxicon 38, 391-407), namely an action at muscarinic receptors and adrenoceptors. The aim of this study was to determine the effectiveness of Synanceia antivenom in neutralising the in vitro and in vivo cardiovascular activity of G. marmoratus venom. Venom extract (0.4-12 microg protein/ml) caused dose- and endothelium-dependent relaxation in porcine U46619-precontracted coronary arteries. This relaxation was abolished by 10 min prior exposure of the tissue to Synanceia antivenom (3 units/ml). In rat paced (5 ms, 2 Hz, 7-12 V) left atria, G. marmoratus venom extract (40 microg protein/ml) produced a transient negative, followed by a sustained positive inotropic response. In spontaneously beating right atria, venom extract (40 microg protein/ml) produced similar changes in rate. Prior incubation of venom extract with Synanceia antivenom (1 unit/4 microg venom extract protein, 10 min) significantly attenuated both components of the inotropic response, and abolished the positive chronotropic response. In the anaesthetised rat, venom extract (400 microg protein/kg, i.v.) produced a transient depressor response, followed by a more sustained pressor response. Prior incubation of venom extract with Synanceia antivenom (1 unit/4 microg venom extract protein, 10 min) significantly attenuated both components of the in vivo response. As Synanceia antivenom neutralised the cardiovascular activity of G. marmoratus venom both in vitro and in vivo, we suggest that the venoms of the two species may share a similar component(s).
Asunto(s)
Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/antagonistas & inhibidores , Antivenenos/uso terapéutico , Venenos de los Peces/antagonistas & inhibidores , Peces Venenosos , Corazón/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Análisis de Varianza , Animales , Presión Sanguínea/efectos de los fármacos , Vasos Coronarios/efectos de los fármacos , Ratas , Porcinos , Vasoconstrictores/antagonistas & inhibidoresRESUMEN
There has been recent debate regarding the labile nature of stonefish venoms and the pharmacology of their breakdown products. The present study examined the cardiovascular and neuromuscular effects of lyophilised venom, and conducted a preliminary investigation of freshly milked venom. Lyophilised venom (20 microg/ml) caused endothelium-dependent relaxation in rat aortae that was abolished by atropine (0.1 microM). In contrast, an endothelium-independent contractile response occurred in porcine coronary arteries. However, in the presence of atropine (10 nM), this became a relaxation response which was attenuated by the B2 antagonist FR-173657 (0.1 microM) or by a combination of idazoxan (1 microM) and propranolol (1 microM). In rat isolated atria, lyophilised venom (4 microg/ml) caused a biphasic inotropic response consisting of an initial decrease, and then increase, in force which were attenuated by atropine (0.5 microM) and propranolol (5 microM), respectively. The increase in force produced by venom was unaffected by reserpine pre-treatment suggesting a direct action at adrenoceptors. In the anaesthetised rat, lyophilised venom (1-300 microg/kg, i.v.), caused a dose-dependent depressor response, with a subsequent pressor response at higher concentrations (30-300 microg/kg, i.v.). In the presence of atropine (1 mg/kg, i.v.), the depressor response to venom was abolished, a transient pressor response unmasked and the secondary pressor response augmented. In the additional presence of prazosin (50 microg/kg, i.v.), the transient pressor response was abolished and the secondary pressor response attenuated. Lyophilised venom had no significant effect on nerve-evoked (10 microg/ml) or directly-evoked (100 microg/ml) twitches of the chick biventer cervicis muscle preparation. Milked venom (1 microl/ml) caused a biphasic response (i.e., an initial relaxation followed by contraction) in rat aortae, a contraction in porcine coronary arteries, complete cessation of rat isolated atrial activity and markedly inhibited both nerve-evoked and directly-evoked twitches of the chick biventer cervicis muscle preparation. In the anaesthetised rat, milked venom (15 microl/kg, i.v.) caused immediate cardiovascular collapse. It appears that the cardiovascular effects of stonefish venom are mediated by a dose-dependent action at muscarinic receptors and adrenoceptors.