Brain capillarization in diving mammals: a selection of staining and quantification methods

Abstract

Diving species can cope with acute and repeated hypoxia through adaptations that are absent in non-diving animals. One of the greatest challenges to deal with during diving is the lowering of the arterial oxygen pressure (PaO2), which causes a decrease in the driving force for the oxygen diffusion from the capillaries to the cells. My hypothesis is that the marine mammalian brain shows improved brain blood flow through a denser capillarization which shortens the distance between the capillaries and the cells, allowing the lower PO2 gradient to achieve a sufficient rate of O2 supply to the neural tissue. To study this, a reliable method for determining the capillary density needs to be found. The principal aim of this project is to validate a method to stain and visualize the capillaries and to use this as an initial test to verify the hypothesis that the seal brain shows higher capillarization, when compared to terrestrial mammals as a general adaptation to hypoxia. Several studies have shown that there is a relation between the metabolic activity and the degree of capillarization of a certain tissue. This hypothesis, that brain regions with different metabolic demands (i.e. grey and white matter) can show dissimilar levels of capillarization, is here tested as well. The brains of 2 harp seals (Pagophilus groenlandicus), 1 hooded seal (Cystophora cristata) and 2 reindeer (Rangifer tarandus) were collected. Samples were taken from the frontal and visual cortex, the hippocampus, the cerebellum and the medulla, since these regions were previously reported to have different capillary densities. After a detailed analysis of different staining techniques, capillaries were identified by immunostaining the collagen IV of their basement membrane and visualized at the confocal microscope. The images obtained were then subjected to two different quantification methods and results were compared. The method that is indicated as “automatized method” turned out to be more reliable and also easier to apply to the images. Since the anti-collagen IV technique gave a good quality stain in both the species studied here, it is concluded that its use and the following application of the “automatized method” as a quantification method is a reliable combination for assessing the degree of capillarization of cerebral tissue. In general, both the hypothesis that diving species have an enhanced brain capillarization and that regional differences occur between cerebral regions are confirmed but further investigations with a higher number of samples are needed to better assess this hypothesis.