Introduction and aims: Investigating blood-brain barrier function in the developing animal can be challenging given the small size of animals and sometimes an in utero setting of experimentation. It necessitates for careful considerations of experimental methodologies in order not to endanger the physiological well-being of the foetus or the newborn animal. These experiments can also be technically difficult requiring different routes of administration into the developing animal. Our aim is to provide methodologies that can be used to successfully complete such experiments.

Methods: To help we have tested and validated a range of molecular weight tracers that can easily be visualised in tissues (down to electron microscopic level) and used to assess BBB integrity. Together with quantifiable markers, experiments can detect both global (across whole brain or brain region) and local changes in BBB function at high resolution. In addition, it might be necessary to administer markers into the developing animal in different ways, depending on species and age of animals, such as injections into the embryo (in utero), foetal membrane vessels or the retroorbital plexus.

Results and Conclusions: Different techniques are often optimal in different settings of experimentation in the developing animal. Optimising the experimental methods will allow for better validity of results and improved animal ethics. By using these techniques, we have over the years revealed changes in BBB function, both spatially and temporally, under various pathophysiological states in the developing animal such as hypoxia-ischemia, neonatal infection/inflammation or following intraventricular haemorrhage.