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Neonatal Intraventricular Haemorrhage Downregulates Notch and Activates Proliferation in the Subventricular Zone

Presented at the Neonatal Society 2015 Summer Meeting (programme).

Dawes WJDawes WJ, Marino S

Blizard Institute, Barts and the London School of Medicine, Queen Mary University of London, 4 Newark Street, London E1 2AT

Background: The clinical outcome and pathological impact of Intraventricular Haemorrhage (IVH) in premature neonates is unclear. It is hypothesised that IVH has the potential to impact on cortical development but evidence in support of this is lacking. Our aim was to design a mouse model that could specifically explore the impact of GMH on the Neural Stem Progenitor Cells (NSPC) within the Subventricular zone (SVZ) and on early cortical development.

Methods: Using a modified Narishige stereotactic frame we have optimised and validated a mouse model of Papille Grade II/III GMH. A thymidine labelling strategy on day 1, combined with fluorescent immunohistochemistry at day 4 and day 21 has been used to determine the impact of GMH on postnatal cell division. CD133 MACS sorting at P4 in combination with RNA extraction, amplification and qPCR array has been used to define the impact of GMH on the molecular expression of the neural stem cells within the subventricular zone. Finally in-situ hybridisation has been used to validate targets identified through RNA analysis.

Results: 1. Stereotactic injection of autologous blood into the SVZ of newborn mice recapitulates key features of the human condition, namely failure to thrive (weight: control 3.101g 0.06, IVH 2.775g 0.08, p=0.002) and hydrocephalus (ventricular volume: control 0.054cm3 0.007, blood injected 0.078cm3 0.005, p=0.02)
2. GMH leads to an increase in the number of EdU+ve cells within the subventricular zone (cell count: control 59.73 5.53, IVH 160.1 15.18, p=<0.0001) and transcallosal pathway (cell count: control 26.98 1.98 IVH 43.22 3.38 p=0.0004) in association with increased markers of transient amplifying cells (MASH1 (p=0.0009), GFAP (p=0.00002), NG2 (p=0.006, DCX (p=0.003)) consistent with activation of proliferation.
3. RNA analysis from the NSPC (ie CD133+ve cells) at P4 reveals that IVH downregulates Notch expression >25 fold (p=0.01), a finding confirmed by Hes5 ISH.

Conclusion: Notch down regulation may represent a final common pathway following premature birth as such quantification of Notch expression may prove a useful prognostic indicator and raises the possibility that activation of Notch signalling could be a therapeutic strategy for GMH.

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