High-resolution ultrasound biomicroscopy for monitoring ovarian structures in mice

Background: Until recently, the limit of spatial resolution of ultrasound systems has prevented characterization of structures <1 mm. Hence, the study of ovarian follicular development in rodents has been based on one-time histological examination of excised tissues; i.e., longitudinal study of day-to-day ovarian changes has not been possible in mice and rats. The objective was to establish an ultrasonographic approach to study follicular and luteal dynamics in mice and rats. Methods: Experiment 1 was a pilot study to develop methods of immobilization (physical restraint vs. general anesthesia) and determine technical factors affecting ovarian images using ultrasound bio-microscopy in rats vs. mice. The hair coat was removed over the thoraco-lumber area using depilation cream, and a highly viscous acoustic gel was applied while the animals were maintained in sternal recumbency. In Experiment 2, changes in ovarian structures during the estrous cycle were monitored by twice daily ultrasonography in 10 mice for 2 estrous cycles. Results: Ovarian images were not distinct in rats due to attenuation of ultrasound waves. Physical restraint, without general anesthesia, was insufficient for immobilization in mice. By placing the transducer face over the dorsal flank, the kidney was visualized initially as a point of reference. A routine of moving the transducer a few millimetres caudo-laterally from the kidney was established to quickly and consistently localize the ovaries; the total time to scan both ovaries in a mouse was about 10 minutes. By comparing vaginal cytology with non-anesthetized controls, repeated exposure to anesthesia did not affect the estrous cycle. Temporal changes in the number of follicles in 3 different size categories support the hypothesis that follicles >= 20 microns develop in a wavelike fashion. Conclusion: The mouse is a suitable model for the study of ovarian dynamics using transcutaneous ultrasound bio-microscopy. Repeated general anesthesia for examination had no apparent effect on the estrous cycle, and preliminary results revealed a wave-like pattern of ovarian follicle development in mice.