Comparison of a modified mid-coronal sectioning technique and Wilson's technique when conducting eye and brain examinations in rabbit teratology studies.

BACKGROUND: There are two methods used when examining fetal rabbit eyes and brain in teratology studies. One method employs prior fixation before serial sectioning (Wilson's technique) and the other uses fresh tissue (mid-coronal sectioning). METHODS: We modified the mid-coronal sectioning technique to include removal of eyes and brain for closer examination and to increase the number of structures that can be evaluated and compared it to the Wilson's technique. We found that external examination of the head, in conjunction with either sectioning method, is equally sensitive in identifying developmental defects. We evaluated 40,401 New Zealand White (NZW) and Dutch-Belted (DB) rabbit fetuses for external head alterations, of which 28,538 fetuses were further examined for eye and brain alterations using the modified mid-coronal sectioning method (16,675 fetuses) or Wilson's technique (11,863 fetuses). The fetuses were from vehicle control or drug-treated pregnant rabbits in embryo-fetal development studies conducted to meet international regulatory requirements for the development of new drugs. RESULTS: Both methods detected the more common alterations (microphthalmia and dilated lateral cerebral ventricles) and other less common findings (changes in size and/or shape of eye and brain structures). CONCLUSIONS: While both methods are equally sensitive at detecting common and rare developmental defects, the modified mid-coronal sectioning technique eliminates the use of chemicals and concomitant fixation artifacts that occur with the Wilson's technique and allows for examination of 100% intact fetuses thereby increasing potential for detecting eye and brain alterations as these findings occur infrequently in rabbits.

Concordance between alizarin red stained skeleton and micro-CT skeleton evaluation methods: A case study in New Zealand White rabbits.

OBJECTIVES: The objective of this study was to examine the fetal skeletons using both alizarin red stain and micro-computed tomography (CT) images; investigate differences, and to determine if the conclusions of the study were the same regardless of the examination method. METHODS: A candidate drug was given orally by gavage to pregnant New Zealand White rabbits on gestation day (GD) 7 to GD 19 (mating = GD 0) at doses of 0 (control), 0.02, 0.5, 5, and 15 mg/kg/day. Maternal toxicity was evident at ≥0.02 mg/kg/day. The 199 fetal skeletons (totaling 50,546 skeletal elements) obtained at cesarean delivery on GD29 were first stained with Alizarin Red S, then imaged by a Siemens Inveon micro-CT scanner. All fetal skeletons were examined by both methods, without knowledge of dose group, and the results were compared. RESULTS: In total, 33 types of skeletal abnormalities were identified. There was 99.8% concordance of results comparing stain to micro-CT. Ossification of the middle phalanx of the forepaw digit 5 showed the greatest difference between the two methods. CONCLUSION: Overall, micro-CT imaging is a realistic, and robust alternative to skeletal staining to examine fetal rabbit skeletons in developmental toxicity studies.

No developmental toxicity observed with dolutegravir in rat whole embryo culture.

BACKGROUND: An in vitro rat whole embryo culture study investigated whether direct exposure to dolutegravir (TivicayTM ) during the critical period for neural tube development would result in abnormal development. METHODS: Dolutegravir (DTG), and HIV integrase inhibitor, was administered at 0 (vehicle), 5.3 µg/mL and 9.3 µg/mL on Gestation Day (GD) 9 through 11 (approximate 40 hour exposure period) along with positive (Valproic Acid) and negative (Penicillin G) controls. The DTG concentrations tested were selected based on clinical exposure at the maximum human recommended dose and maximum feasible concentration that could be formulated under the experimental conditions. RESULTS: Approximately 6% of DTG present in the culture media was absorbed into the embryos, demonstrating embryonic exposure at a similar level to that observed in a rat DTG placental transfer study. There was no effect in either the DTG or Penicillin G groups on visceral yolk sac size/morphology, embryo size, somite number and embryo morphology at any concentration tested. Valproic Acid, by contrast, produced statistically significant decreases in visceral yolk sac size, embryo size and somite number along with defects in visceral yolk sac and embryonic morphology, including neural tube defects (NTDs), in all embryos. CONCLUSION: DTG at the maximum human recommended dose administered to rats in a whole embryo culture assay did not produce any abnormal effects, while the positive control Valproic Acid produced abnormal effects, including neural tube defects.

The use of micro-CT imaging to examine and illustrate fetal skeletal abnormalities in Dutch Belted rabbits and to prove concordance with Alizarin Red stained skeletal examination.

OBJECTIVES: In our laboratory we evaluated the use of micro-computed tomography (micro-CT) using a high resolution acquisition protocol and fetuses obtained on Gestation Day (GD) 29 (mating = GD 0). METHODS: To show concordance between traditional Alizarin Red S stain and micro-CT skeletal examination methods, 103 fetuses from 19 untreated Dutch belted rabbits were obtained by cesarean section and stored frozen. The fetuses were thawed, imaged and examined digitally by micro-CT, then stained and re-examined using traditional methods. RESULTS: A total of 12 individual malformations and 35 unique variations were detected by both methods. Differences in the extent of ossification were found in only 51 of 26,196 bones while 99.8% of the observations were identical. Of the 51 differences, 31 were an unossified fifth medial phalanx of the forepaw indicating that very low-density skeletal bones may be visible by Alizarin Red stain but not by micro-CT scan. To establish this methodology under pharmaceutical testing conditions, we obtained and imaged by micro-CT Alizarin Red S stained abnormal fetal rabbit skeletons previously exposed to a drug candidate associated with craniofacial malformations in humans. All of the types of skeletal abnormalities first identified by staining were also detected by micro-CT examination. Representative images of these 66 different fetal skeletal abnormalities were characterized, and compiled to illustrate visual concordance between micro-CT scanned and traditional Alizarin Red S stained skeletons. CONCLUSION: Micro-CT imaging is an accurate, reliable and robust method that can be used as an alternative to stain when examining fetal rabbit skeletons in developmental toxicity studies.