Trans-Uterine Tracer Injections in Mammalian Foetuses

Intra-uterine manipulation of mammalian foetuses for experimental purposes was first described at the beginning of this century (Wolff, 1919). Though numerous publications have appeared since which bear witness to the feasibility of intra-uterine manipulation, its application has remained rather restricted. In this paper we describe a technique for the trans-uterine injection of neuronal tracers into rat foetuses. Uterine wall and foetal membranes are pierced only with a micro pipette, and are thus left virtually intact, preventing loss of amniotic fluid. Surgical mortality is 24% overall, but the experimental success rate is much lower (23%). Even so these results are comparable to more complicated procedures, because the technique is simple (i.e. requires no micro surgical skill), and because up to 6 foetuses can be injected per dam. Technical problems, such as foetal anaesthesia and the detection of false-negative results due to imponderable factors are discussed.     

Intra-Uterine Silastic Embedding of Mammalian Foetuses for Surgical Immobilization

Since the beginning of this century intra uterine operations were performed upon foetal mammals in order to study regeneration and development. Although methods became more refined, manipulation of the animals remained difficult and stereotaxic methods were never developed. In this study we describe a method to improve intra-uterine manipulation and stereotaxis by replacing amniotic fluid with silicone rubber, which immobilises the foetus. Survival times up to 24 hours were achieved, sufficient for neuronal tracing or for electrophysiological recording. All experiments were performed at embryonic day 18 (E18).     

Age Determination of Bovine Foetuses

The need for methods for age determination of foetuses is defined. X-ray examination of the foetal head is recommended as the surest basis for age determination. Calculations are made concerning the correlation between cranial length and foetal age, and the methods used for measurements are described in detail. Measurements of the metacarpus and metatarsus may also be used, but they give a poorer correlation. A formula is worked out for age determination based on cranial measurements. On x-ray photographs, tooth development is seen to occur in an obvious sequence. This is described. However, the interpretation of these observations is subjective and requires control material for comparison. The same is true for the sequence of ossification of the skeleton, which in foetuses < 120 days gives the surest basis for evaluation. Here, too, a wide range of control material for comparison is necessary. The sequence in the appearance of body hair — development and eruption — is noted, as far as the head is concerned. This alone gives an insecure basis for evaluation. By using the 4 systems of parameters mentioned, the age of a foetus can be determined within an error of ± 4—5 days.     

Intravenous Injections in Neonatal Mice

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are technically feasible and routine. However, some mouse models can have early onset of disease with a rapid progression that makes administration of potential therapies difficult. The temporal (or facial) vein is just anterior to the ear bud in mice and is clearly visible for the first two days after birth on either side of the head using a dissecting microscope. During this window, the temporal vein can be injected with volumes up to 50 μl. The injection is safe and well tolerated by both the pups and the dams. A typical injection procedure is completed within 1-2 min, after which the pup is returned to the home cage. By the third postnatal day the vein is difficult to visualize and the injection procedure becomes technically unreliable. This technique has been used for delivery of adeno-associated virus (AAV) vectors, which in turn can provide almost body-wide, stable transgene expression for the life of the animal depending on the viral serotype chosen.