Proceedings of The Physiological Society

Future Physiology (Leeds, UK) (2017) Proc Physiol Soc 39, PC14

Poster Communications

Characterisation of Nestin-GFP expression in the spinal cord of transgenic mice

C. Colquhoun1, J. Deuchars1, S. A. Deuchars1

1. University of Leeds, Leeds, United Kingdom.

Ependymal cells (ECs) have recently become of interest due to their activation and multipotency following injury to the central nervous system. Following spinal cord injury (SCI) ECs proliferate, differentiate, and migrate towards the site of injury (Mothe & Tator 2005). These progeny may form part of the glial scar in SCI (Meletis et al. 2008), however the driving force behind their behaviour is yet to be determined. Transgenic mice in which the neural stem cells of the embryonic and adult central nervous system express green fluorescent protein (GFP) have proved to be a useful tool to study neurogenesis (3). Nestin is an intermediate filament protein known to mark neural stem and progenitor cells (Lendahl et al. 1990). In these animals the regulatory elements of the nestin gene were used to create a reporter line, thereby allowing the identification and isolation of such cells. While these cells have been characterised in the brain (Mignone et al. 2004), the identity of GFP-expressing cells has not been confirmed elsewhere within the CNS. In this study immunohistochemistry using antibodies against EC markers, and Fluorogold retrograde labelling were used to characterise the Nestin-GFP expression in transgenic mice. Adult transgenic mice (N = 3, 4-6 weeks) were injected intraperitoneally (I.P.) with 0.1 mL Fluorogold (50 mg/kg) and terminally anaesthetised with sodium pentabarbitone (60 mg/kg) I.P. and perfused transcardially with 4% paraformaldehyde. Antibodies used to label ECs in transverse slices (40 µm) include: CD24, CXCR4, Galectin-3, NKA-α1, Sox2, and vimentin. Nestin-GFP expression is seen throughout the spinal cord in the white and grey matter, intensifying at the central canal. FG labelling colocalized with Nestin-GFP in the white and grey matter of the spinal cord and brain, identifying these cells as pericytes. A sub-population of GFP-positive cells at the central canal of the spinal cord were not FG-positive, and these cells colocalized with markers of ECs, confirming their identity. 100% of GFP-labelled cells (n = 6 slices) colocalized with markers of ECs, however cell counts are ongoing. Additionally, Nestin-GFP-positive ECs were found to colocalize with antibodies against CXCR4 and galectin-3, indicating potential targets for the manipulation of endogenous stem cells following SCI. Nestin-GFP expression within the brain supported the identification of these cells as ECs. GFP-positive cells largely devoid of FG labelling were located at the subgranular zone within the dentate gyrus, supporting previous studies (Encinas et al. 2011). Our results indicate this transgenic line will be a useful tool for studying neurogenesis in the intact and damaged spinal cord, as well as the manipulation of ECs following injury. Further insight into Nestin-GFP expression in an injured spinal cord could help untangle EC behaviour and any mechanisms hindering repair.

Where applicable, experiments conform with Society ethical requirements