Implementación de un protocolo para evaluar la reactividad contráctil ex-vivo del íleon de rata en baño de tejido aislado
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Date
2021
Authors
Asunción Alvarez, Holbert Daniel
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Publisher
Universidad Nacional de Trujillo
Abstract
La farmacología ha utilizado el sistema de baño de tejido aislado durante muchos años para la caracterización de receptores y su transducción de señales. Particularmente, el íleon aislado, es uno de los modelos más empleados en este campo de investigación. El objetivo de este estudio fue la implementación de un protocolo experimental para evaluar la reactividad contráctil ex-vivo del íleon de rata en baño de tejido aislado. El protocolo inicia detallando los pasos para montar el baño de tejido aislado, así como su calibración, ya que esta es una etapa crucial para poder obtener datos fiables de investigación. Los segmentos ileales se obtuvieron de Rattus norvegicus Holtzman machos (n = 9) y para registrar la reactividad contráctil se empleó un sistema PowerLab 26T. El protocolo inició evaluando el efecto de una sustancia desconocida sobre el tono basal. En segundo lugar, se demostró que usando segmentos pre-contraídos con acetilcolina (ACh 10-5 M), serotonina (5-HT 10-5 M) y BaCl2 80 mM se podría estudiar el efecto espasmolítico (efecto terapéutico) de una sustancia.
Así mismo, para entender el efecto antiespasmódico (efecto preventivo) se construyeron curvas dosis-respuesta frente a ACh (10-10 M-10-4 M) con la finalidad de conocer si la sensibilidad (pEC50) del tejido es modifica o no mediante la adición de una sustancia exógena. Luego, para evaluar el rol del influjo de Ca2+ extracelular en la respuesta contráctil a ACh, se elaboró un protocolo usando una solución Tyrode libre de Ca2+. Finalmente, para estudiar si los efectos antiespasmódicos de las sustancias ensayadas estaban asociadas con su actividad antioxidante, los segmentos ileales se pre-incubaron durante 20 min con la muestra, antes de la contracción con peróxido de hidrógeno (H2O2) como sustancia pro-oxidante
ABSTRACT The isolated tissue bath system has been used by pharmacology for many years for the characterization of receptors and their signal transduction. In particular, the isolated ileum is one of the most widely used models in this field of research. The objective of this study was the implementation of an experimental protocol to evaluate the ex-vivo contractile reactivity of rat ileum in an isolated tissue bath. The protocol begins by detailing the steps to assemble the isolated tissue bath, as well as its calibration, since this is a crucial stage in order to obtain reliable research data. The ileal segments were obtained from male Rattus norvegicus Holtzman (n = 9) and a PowerLab 26T system was used to record contractile reactivity. The protocol began by evaluating the effect of an unknown substance on the basal tone. Second, it was demonstrated that using pre-contracted segments with acetylcholine (10-5 M ACh), serotonin (10-5 M 5-HT) and 80 mM BaCl2, the spasmolytic effect (therapeutic effect) of a substance could be studied. Likewise, to understand the antispasmodic effect (preventive effect), dose-response curves were constructed against ACh (10-10 M-10-4 M) in order to know if the sensitivity (pEC50) of the tissue is modified or not by means of the addition of an exogenous substance. Then, to evaluate the role of the influx of extracellular Ca2+ in the contractile response to ACh, a protocol was developed using a Ca2+ -free Tyrode solution. Finally, to study whether the antispasmodic effects of the tested substances were associated with their antioxidant activity, the ileal segments were pre-incubated for 20 min with the sample, before contraction with hydrogen peroxide (H2O2) as pro-oxidant substance.
ABSTRACT The isolated tissue bath system has been used by pharmacology for many years for the characterization of receptors and their signal transduction. In particular, the isolated ileum is one of the most widely used models in this field of research. The objective of this study was the implementation of an experimental protocol to evaluate the ex-vivo contractile reactivity of rat ileum in an isolated tissue bath. The protocol begins by detailing the steps to assemble the isolated tissue bath, as well as its calibration, since this is a crucial stage in order to obtain reliable research data. The ileal segments were obtained from male Rattus norvegicus Holtzman (n = 9) and a PowerLab 26T system was used to record contractile reactivity. The protocol began by evaluating the effect of an unknown substance on the basal tone. Second, it was demonstrated that using pre-contracted segments with acetylcholine (10-5 M ACh), serotonin (10-5 M 5-HT) and 80 mM BaCl2, the spasmolytic effect (therapeutic effect) of a substance could be studied. Likewise, to understand the antispasmodic effect (preventive effect), dose-response curves were constructed against ACh (10-10 M-10-4 M) in order to know if the sensitivity (pEC50) of the tissue is modified or not by means of the addition of an exogenous substance. Then, to evaluate the role of the influx of extracellular Ca2+ in the contractile response to ACh, a protocol was developed using a Ca2+ -free Tyrode solution. Finally, to study whether the antispasmodic effects of the tested substances were associated with their antioxidant activity, the ileal segments were pre-incubated for 20 min with the sample, before contraction with hydrogen peroxide (H2O2) as pro-oxidant substance.
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Keywords
Baño de tejido aislado. Antiespasmódico. Espasmolítico. Ileon. Farmacología experimental