The immune cell profile of the developing rat brain.

Little is known about the peripheral immune cell (PIC) profile of the developing brain despite growing appreciation for these cells in the mature nervous system. To address this gap, the PIC profile, defined as which cells are present, where they are located, and for how long, was examined in the developing rat using spectral flow cytometry. Select regions of the rat brain (cerebellum, hippocampus, and hypothalamus) were examined at embryonic day 20, and postnatal days 0, 7 and 16. At their peak (E20), PICs were most abundant in the cerebellum, then the hippocampus and hypothalamus. Within the PIC pool, monocytes were most prevalent in all regions and time points, and shifted from being majority classical at E20 to non-classical by PN7. T cells increased over time, and shifted from majority cytotoxic to T-helper cells by PN7. This suggests the PIC profile transitions from reactive to adaptive and surveilling in the second postnatal week. NK cells and mast cells increased temporarily, and mast cells were restricted to the hippocampus and hypothalamus, suggesting they may play a specific role in the development of those regions. Mimicking a viral infection by administration of Poly I:C increased the influx of PICs into the neonatal brain, particularly of NK cells and in the case of males only, non-classical monocytes. This work provides a map for researchers as they study immune cell contributions to healthy and pathological brain development.

Generation of an Iba1-EGFP Transgenic Rat for the Study of Microglia in an Outbred Rodent Strain.

Neuroscience has been transformed by the ability to genetically modify inbred mice, including the ability to express fluorescent markers specific to cell types or activation states. This approach has been put to particularly good effect in the study of the innate immune cells of the brain, microglia. These specialized macrophages are exceedingly small and complex, but also highly motile and mobile. To date, there have been no tools similar to those in mice available for studying these fundamental cells in the rat brain, and we seek to fill that gap with the generation of the genetically modified Sprague Dawley rat line: SD-Tg(Iba1-EGFP)Mmmc Using CRISPR-Cas/9 technology, we knocked in EGFP to the promoter of the gene Iba1 With four male and three female founders confirmed by quantitative PCR analysis to have appropriate and specific insertion, we established a breeding colony with at least three generations of backcrosses to obtain stable and reliable Iba1-EGFP expression. The specificity of EGFP expression to microglia was established by flow cytometry for CD45low/CD11b+ cells and by immunohistochemistry. Microglial EGFP expression was detected in neonates and persisted into adulthood. Blood macrophages and monocytes were found to express low levels of EGFP, as expected. Last, we show that EGFP expression is suitable for live imaging of microglia processes in acute brain slices and via intravital two-photon microscopy.