Frozen-sectioning yields similar results as traditional methods for fetal cephalic examination in the rat
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Updated: Aug 14, 2026
Preparation of Newborn Rat Brain Tissue for Ultrastructural Morphometric Analysis of Synaptic Vesicle Distribution at Nerve Terminals
Published on: June 7, 2019
A Barry Astroff1, Sylvia E Ray, Leanna M Rowe
1Eli Lilly and Company, Lilly Research Laboratories, Greenfield, Indiana 46140, USA. astroff_barry@Lilly.com
Background:
An integral component of developmental toxicity studies is the evaluation of fetal anatomy, which consists of external, visceral, and skeletal examinations. The visceral examination includes an assessment of the fetal head which, in the rat, is typically completed after chemical fixation. Because chemical fixation requires approximately 7 days, a comprehensive visceral examination, including the head, of the fetal rodent cannot be completed at the time of cesarean section. An alternative method to chemical fixation was desired, whereby one could complete an overall visceral examination at the time of cesarean section. In addition, the method would also have to present fetal cranial structures in a manner consistent with that derived after chemical fixation.
Methods:
Pregnant Sprague-Dawley rats were administered either 6-aminonicotinamide (6AN), 6 or 12 mg/kg on gestation day 13, or all-trans retinoic acid (RA) 5 or 25 mg/kg on gestation days 9 and 10, untreated dams served as a control group. On gestation day 20, fetuses were sacrificed and removed via cesarean section and one-half of the fetuses from each litter were placed in Bouin's solution (chemical fixation) and the remaining fetuses maintained under refrigeration until just before frozen tissue preparation (freeze fixation). Sectioning of the fetal head was similarly conducted after either chemical or frozen-fixation. Fetal cranial findings observed after chemical fixation were compared to those observed after frozen-fixation in the untreated control and 6AN and RA-treated groups.
Results:
The incidence and severity of the cranial findings, including effects on the eye, brain, and palate, were similarly observed, regardless of fixation method.
Conclusions:
A frozen sectioning method for evaluation of the fetal head, yielding results comparable to those derived after chemical fixation, is described. This procedure provides a viable alternative to chemical fixation, and allows the teratologist to complete a comprehensive fetal visceral examination at the time of cesarean section.
