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

DEVELOPMENTAL TOXICITY TESTING — METHODOLOGY 241for 1 week, rinsed twice with tap water, and stored in 70% isopropyl alcohol. Another decalcifyingfixative that does not contain picric acid is Harrison’s, with similar benefits to Davidson’s. Mostlaboratories utilize the Staples’s technique 68 or modifications of the Staples’s technique 77,81 forvisceral examination of rodent fetuses. Wilson’s technique cannot be used for rabbit fetuses sincecurrent guidelines require both visceral and skeletal examination of all fetuses of that species.f. Craniofacial ExaminationPrior to or after visceral examination (for each fetus scheduled for craniofacial examination), thehead is removed and placed in Bouin’s solution or another decalcifying fixative (see above) forfixation and decalcification. Rodent heads may be put individually in a scintillation vial approximatelythree-quarters full of fixative. Because rabbits have identification numbers on top of theirheads, the heads from an entire litter may be put in a large container for fixation. (Some laboratoriesperform only a single midcoronal section on rabbit heads at sacrifice; this provides information oneye structure and on the status of the lateral ventricles of the cerebrum [e.g., is there hydrocephaly?]but does not provide information on other areas of the fore-, mid-, and hindbrain.) Rodent headsshould remain in the fixative for approximately 72 h prior to examination, and rabbit heads shouldremain for about a week. The following recipe may be used to prepare 6 L of Bouin’s fixative:Saturated picric acid (57.13 g in 4200 ml distilled water)37% Formaldehyde (1428 ml)Glacial acetic acid (286 ml)The equipment necessary for craniofacial evaluation includes a dissecting microscope, scalpelsor razor blades, and forceps. The head is removed from the bottle and blotted dry. Up to seven cutsmay be made using a sharp, clean blade. They should always be made in the sequence listed, butadditional cuts may be added if a structure appears abnormal, is missed, or must be verified asmissing. The cuts should be smooth and perpendicular to the cutting surface. The following descriptionsand the head cuts are modified from Wilson 17,69 and van Julsingha and Bennett 82 (Figure 7.7).Before the heads are cut, they are examined for any grossly apparent abnormalities that shouldbe more carefully explored as the cutting proceeds. The first cut is made with the head turned noseupward (use a pair of blunt forceps to grip the head) and exposes the tongue, palate, upper lips,and lower jaw. It is a ventrodorsal section (i.e., horizontal section) beginning at the mouth andcoursing immediately inferior to the ears. The tongue should be lifted from the palate after the cut ismade, and the palate and upper lips examined for incomplete closure (clefting). The pattern of therugae should be examined for correct closure of the palate (the rugae should not be misaligned wherethey meet in the midline). The nasopharyngeal opening, the cochlea of the ears, and the base of thebrainstem (medulla oblongata) may also be visualized. The flat surface produced by this cut simplifiesthe remaining slices by stabilizing the head, which should now be turned over (anterodorsal side up).The second cut is made about half way between the tip of the nose and the foremost corner ofthe eye slits. The nasal passages, nasal conchae, nasal septum, palate, and insertions for the vibrissaeshould be visible on either side of the cut.The third cut is made through the eyes. Tooth primordia and the Harderian glands may bevisible, in addition to the nasal septum, nasal passages, nasal conchae, and palate. Both eyes,including cornea, lens, and retina, are visible in cross-section. The cerebral hemispheres and theanterior-most portions of the two lateral ventricles are readily visualized, as are the mandibularrami on either side. Occasionally, the interventricular foramen may be bisected, and a small portionof the thalamus and third ventricle will be visible. Other structures that should be located in thissection include the optic nerves and/or the optic chiasma, the nasopharynx, the soft palate, andcranial nerves V and VII.© 2006 by Taylor & Francis Group, LLC