A Practical Approach, Second Edition=Ronald D. Ho.pdf

IN VITRO METHODS FOR THE STUDY OF MECHANISMS OF DEVELOPMENTAL TOXICOLOGY 659PostimplantationRabbit WholeEmbryo CultureTime MatingMaternal bloodcollection and removalof the uterusCut alongantimesometrialedge with scissorsUterine WallAmnionVisceral yolk sacEmbryoPull back uterinewalls to exposeconceptusBlood vesselof yolk sacAllantoic placentaCarefully cut aroundconceptus withoutdamaging bloodvessels of the yolk sacTransfer conceptuses individuallyto continuous gassing cultureapparatusCulture 12–48 hr and evaluateFigure 16.4 Schematic diagram of rabbit whole embryo culture. Time-mated pregnant females are anesthetizedon GD 9 prior to opening of the abdomen and removal of maternal blood (to be used for preparation ofculture media). The uterus is removed with the ovaries and cervix attached. Each conceptual swelling isindividually removed and pinned to a silicone filled dish for dissection. A single cut is made along the antimesometrialedge of the uterus, and the overlying uterine tissue is peeled back to expose the embryo. To free theembryo, it is necessary to cut through the placenta, leaving a portion of the placenta attached to maintain theintegrity of the conceptus for culture. Special care must be taken not to damage the visceral yolk sac or anyrelated blood vessels. Embryos are then placed for 24 to 48 h at 37°C in a continuous gassing incubator foroptimal growth at the given developmental stage. Morphological, biochemical, physiological, and molecularendpoints can be evaluated throughout the culture period by removing selected embryos from the culture.Relative size, degree of development, and identification of major structures for the GD 9.5 and GD 10 conceptusesare shown.© 2006 by Taylor & Francis Group, LLC