|
We applied the Case cryo-imaging system to detect signals of developmental toxicity in transgenic mouse fetuses
resulting from maternal exposure to a developmental environmental toxicant (2,3,7,8-tetrachlorodibenzo-p-dioxin,
TCDD). We utilized a fluorescent transgenic mouse model that expresses Green Fluorescent Protein (GFP) exclusively
in smooth muscles under the control of the smooth muscle gamma actin (SMGA) promoter (SMGA/EGFP mice kindly
provided by J. Lessard, U. Cincinnati). Analysis of cryo-image data volumes, comprising of very high-resolution
anatomical brightfield and molecular fluorescence block face images, revealed qualitative and quantitative
morphological differences in control versus exposed fetuses. Fetuses randomly chosen from pregnant females euthanized
on gestation day (GD) 18 were either manually examined or cryo-imaged. For cryo-imaging, fetuses were embedded,
frozen and cryo-sectioned at 20 μm thickness and brightfield color and fluorescent block-face images were acquired with
an in-plane resolution of ≈15 μm. Automated 3D volume visualization schemes segmented out the black embedding
medium and blended fluorescence and brightfield data to produce 3D reconstructions of all fetuses. Comparison of
Treatment groups TCDD GD13, TCDD GD14 and control through automated analysis tools highlighted differences not
observable by prosectors performing traditional fresh dissection. For example, severe hydronephrosis, suggestive of
irreversible kidney damage, was detected by cryoimaging in fetuses exposed to TCDD. Automated quantification of total
fluorescence in smooth muscles revealed suppressed fluorescence in TCDD-exposed fetuses. This application
demonstrated that cryo-imaging can be utilized as a routine high-throughput screening tool to assess the effects of
potential toxins on the developmental biology of small animals.
|
