The use of ultrasound biomicroscopy for embryo injections

Abstract

Ultrasound biomicroscopy (UBM) is based on the use of > 25 MHz probes enabling high resolution (<100μ) in vivo visualisation of structures up to 1cm deep. It is currently used in ophthalmology and biomedical research (rodents). Ultrasound-guidance is used to improve the accuracy of needle placement for in vivo injections. In developmental biology, the in vitro and in vivo injection of cells, tracer dyes and adenoviral or retroviral vectors into embryos are powerful approaches for studying developmental processes. After implantation, however, the conceptus is encased in the uterus and in vivo injections into the conceptus become a technical challenge when performed in a "blind" manner. In utero microinjection under UBM guidance was shown to be a useful tool for exploring the developmental consequences of altering gene expression and for studying cell lineage or migration during early embryonic and placental development in mice. For example ectoplacental cone region, amniotic cavity and exocoelomic cavity were accurately targeted at E6.5 and E7.5 and a fluorescent bead suspension was injected to study early placental and embryonic development. We have adapted this technique to the rabbit, targeting embryos in vivo at the peri-implantation stage (day 6 to 7.5). As a proof of concept, we performed an experiment on 4 female rabbits. Females were time-mated. On day 6.75, they were anaesthetized and a laparotomy was performed. Uterine horns were exteriorised. Two embryos per doe were injected with sterile saline. Animals were euthanized on Day14. We obtained 2 well-developed live conceptuses after UBM guided embryo injection. In conclusion, UBM makes it possible to puncture rabbit embryos at the peri-implantation stage without compromising normal development. This technique is relevant for any injection or sampling into the embryo with subsequent study of development