Proceedings of The Physiological Society

King's College London (2011) Proc Physiol Soc 22, C15

Oral Communications

Endogenous stem cells in the postnatal mouse cortex following Traumatic Brain Injury

A. I. Ahmed1,2, A. Shtaya1,2, M. Zaben1,2, E. Owens1, W. P. Gray1,2

1. Clinical Neurosciences, University of Southampton, Southampton, United Kingdom. 2. Wessex Neurological Centre, Southampton University Hospitals NHS Trust, Southampton, United Kingdom.

Interest to promote regeneration of the injured nervous system has recently turned towards the use of endogenous stem cells. Following a robust stab injury, mature astrocytes in the adult mouse cortex proliferate and contribute to gliogenesis in the mouse cortex in vivo, but also neurogenesis in sphere cultures in vitro (Buffo 2008). This raises two questions critical for efforts to harness endogenous stem cells for repair. Firstly, what cues are involved in driving these precursor cells out of quiescence following injury? Secondly, what are the environmental signals that promote gliogenesis rather than neurogenesis in the injured cortex? We investigate whether a source of potential endogenous stem cells that resides in the cortex is activated following Traumatic Brain Injury (TBI), and correlate signaling pathways with this activity. Using a validated organotypic stretch injury model (Morrison 2006), cultured postnatal mouse cortico-hippocampal slices were stretched to induce an injury equivalent to a severe TBI. In uninjured cortex, proliferating cells cultured in vitro (number of neurospheres per well) are virtually absent in older mice (equivalent postnatal day 15 compared to postnatal day 8). However, following a severe stretch injury, this neurosphere forming capacity is increased in the hippocampus, and significantly, is also restored in the cortex (3.0 +/- 0.3 spheres per well in control and 6.0 +/- 0.5 in the injured hippocampus after 11 days in vitro and 0.5 +/- 0.1 in control and 7.1 +/- 1.3 in injured cortex; p<0.01 2-way Anova with Bonferroni post-hoc). Moreover, these proliferating cells derived from the injured cortex are multipotent. To determine the endogenous source of these cells following TBI, we examined cells from the injured cortex that express the protein Glial Fibrillary Acidic Protein (GFAP). Using cortical cells that express GFP in GFAP cells (Nolte 2001), flow cytometry was used to separate injured cells into GFP positive and negative populations. Positive cells accounted for the majority of proliferating neurospheres formed (3.4 +/- 1.0 in the positive population compared to 0.7 +/- 0.2; p<0.001 student t-test). The Sonic Hedgehog (Shh) signaling pathway is activated following a cortical freeze injury (Amankulor 2009) and contributes towards cellular proliferation. In our TBI model, there is a transient upregulation of the Shh receptor, Patched 1, 6 hours post-injury, which may contribute to the proliferative effect observed after TBI. Our results indicate that a source of endogenous stem cells residing in the postnatal mouse cortex proliferate in vitro only following Traumatic Brain Injury (TBI). Moreover, these proliferating cells are multipotent and are derived mostly from GFAP expressing cells. This raises the possibility of utilising an endogenous source of stem cells for repair following TBI.

Where applicable, experiments conform with Society ethical requirements