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Moderate-Grade Germinal Matrix Haemorrhage Activates Cell Division in the 1 Neonatal Mouse Subventricular Zone. 2 3 William J Da

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Moderate-Grade Germinal Matrix Haemorrhage Activates Cell Division in the 1 Neonatal Mouse Subventricular Zone. 2 3 William J Da 1 Moderate-grade germinal matrix haemorrhage activates cell division in the 2 neonatal mouse subventricular zone. 3 4 William J Dawes1*, Xinyu Zhang1, Stephen P.J. Fancy2, David Rowitch2 and Silvia 5 Marino1* 6 7 1 Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen 8 Mary University of London, 4 Newark Street, London E1 2AT, UK 9 2 Departments of Pediatrics and Neurosurgery, Eli and Edythe Broad Institute for 10 Stem Cell Research and Regeneration Medicine and Howard Hughes Medical 11 Institute, University of California San Francisco, 513 Parnassus Avenue, San 12 Francisco, CA, 94143, USA 13 * Corresponding authors 14 William J Dawes and Silvia Marino 15 Blizard Institute, 4 Newark Street, London E1 2AT 16 Tel +44 207 882 2585, 17 Fax +44 207 882 2180, 18 Email: [email protected] 19 20 Running title: Neural stem cells and neonatal brain haemorrhage 21 22 Abstract 23 Precise temporal and spatial control of the neural stem progenitor cells within the 24 subventricular zone germinal matrix of the brain is important for normal development 25 in the third trimester and early postnatal period. High metabolic demands of 26 proliferating germinal matrix precursors, coupled with the flimsy structure of the 27 germinal matrix cerebral vasculature, are thought to account for high rates of 28 haemorrhage in extremely- and very-low birth weight preterm infants. Germinal 29 matrix haemorrhage can commonly extend to intraventricular haemorrhage. Because 30 neural stem progenitor cells are sensitive to micro-environmental cues from the 31 ventricular, intermediate and basal domains within the germinal matrix, haemorrhage 32 has been postulated to impact neurological outcome through aberration of normal 33 neural stem/progenitor cells behaviour 34 35 We have developed an animal model of neonatal germinal matrix haemorrhage using 36 stereotactic injection of autologous blood into the mouse neonatal germinal matrix. 37 Pathological analysis at 4 days post injury shows high rates of intraventricular 38 extension and ventricular dilatation but low rates of parenchymal disruption outside 39 the germinal zone, recapitulating key features of human “Papile grade III” IVH. At 4 40 days post injury we observed proliferation in the wall of the lateral ventricle with 41 significantly increased numbers of transient amplifying cells within the subventricular 42 zone and corpus callosum. Analysis at 21 days post injury revealed that cortical 43 development was also affected with increased neuronal and concomitant reduced 44 oligodendroglial differentiation. 45 46 At the molecular level, we show down regulation of the expression of the 47 transmembrane receptor Notch2 in CD133+ve cells of the SVZ, raising the possibility 48 that the burst of precocious proliferation seen in our experimental mouse model and 49 the skewed differentiation could be mediated by down regulation of the Notch 50 pathway within the proximal / ventricular domain. These findings raise the possibility 51 that Notch regulation plays a critical role in mediating the response of the neonatal 52 SVZ to ischaemic and haemorrhagic insults. 53 54 55 Keywords 56 Neural stem/progenitor cells, postnatal gliogenesis, postnatal neurogenesis, germinal 57 matrix haemorrhage, mouse models 58 59 Introduction 60 61 Delayed primigravida (first pregnancy) and the use of in vitro fertilisation have 62 contributed to an increase in the incidence of premature birth in all developed 63 countries [1,2] and despite advances in perinatal care, haemorrhage within the 64 germinal matrix (GM) remains a commonly recognised complication seen in up to 65 45% of extremely premature babies weighing 500-750g [3]. The cause of brain injury 66 associated with premature birth is complex and multifactorial with ischaemia [4] and 67 inflammation [5] playing key roles. In addition to this outcome has been shown to 68 correlate with the severity of haemorrhage with extension into the ventricle and loss 69 of brain parenchyma secondary to porencephalic cyst formation being associated with 70 significant neurodevelopmental disabilities [6-8]. The prevention of premature birth 71 and reducing the incidence of haemorrhage remain key research goals. However, the 72 need for new modalities of treatment to limit neurodisability in this vulnerable patient 73 group is clearly evident. 74 75 In order for normal brain development to take place, the behaviour of the neural 76 stem/progenitor cells (NSPC) is tightly regulated in both a temporal and spatial 77 fashion. This occurs through the balance of the cell intrinsic mechanisms and micro- 78 environmental factors [9,10]. The microenvironment within the GM in which the 79 NSC reside can be conceptualised as consisting of three domains [10]; the proximal / 80 ventricular domain which responds to signalling within the CSF [11] and from 81 interaction with the ependymal cells [12], the intermediate zone in which the NSC 82 responds to cues arising from the intermediate progenitor cells [13,14] and 83 neurotransmitters released within the SVZ [15] and the distal / basal domain which is 84 under the influence of cues from the circulation and endothelial cells [16]. Plausible 85 mechanisms can be envisaged through which any and all of these micro- 86 environmental domains could be affected due to GMH. 87 88 Whilst GABAergic interneurons are known to arise from the SVZ in the final 89 trimester [17-19], lineage tracing experiments have highlighted the critical role that 90 the GM plays in the production of astrocytic and oligodendrocytic precursors [20] 91 with the majority of oligodendrocytes developing during late embryogenesis and early 92 postnatal life [21]. This surge in progenitor formation coincides with the peak 93 incidence of GMH (23-28 weeks) and given the critical role that the oligodendrocyte 94 lineage is likely to play in the encephalopathy of prematurity (EP) [22] we 95 hypothesised that GMH might be responsible for a primary stem cell disorder in an 96 otherwise developmentally normal brain (i.e. no underlying genetic / pathological 97 abnormality) making it an appealing target for therapeutic intervention. 98 99 GMH is recognised to cause both destructive and developmental impacts on the 100 developing brain of the premature neonate [23]. To date, published models of IVH 101 have focused on modelling severe haemorrhage with large parenchymal defects seen 102 [24-27]. This approach is likely to mask the more subtle developmental impact of 103 IVH on the NSPC within the SVZ; as such we endeavoured to produce a model with 104 minimal cortical disruption to uncover this subtle mechanism. 105 106 Combining stereotactic injection of autologous blood at P0 [28] with a thymidine 107 labelling strategy at P1 [29], we show that GMH caused an activation of proliferation 108 in the wall of the lateral ventricle, which eventually resulted in an altered cellular 109 composition of the cortex with an increased number of neuronal elements and 110 concomitant depletion of oligodendrocytes. Expression analysis of the 111 CD133/Prominin-positive cell fraction (a transmembrane glycoprotein expressed by 112 NSC and ependymal cells within the lateral ventricle during early postnatal 113 development [30], demonstrated down-regulation of the expression of Notch2, a well- 114 known regulator of NSPC function in the proximal / ventricular domain [9,31], in 115 these cells following GMH. 116 117 Material and Methods 118 119 Animals 120 All procedures had Home Office approval (Animals Scientific Procedures Act 1986, 121 PPL 70/7275). C57BL/6 mice were used throughout. Cages were checked daily and 122 intracranial autologous blood injections were undertaken on the afternoon of the first 123 day of life. 124 125 Stereotactic intracranial injection of autologous blood 126 To facilitate accurate and reproducible restraint of the P0 pup, modifications were 127 made to a Narishige stereotactic frame based on the work of Merkle et al [28] (Figure 128 1A&B). P0 pups were anaesthetised on ice for 3 minutes and 30 seconds prior to 129 being fixed into the frame and autologous blood, collected from the tail tip 130 (Microvette™ CB300 VWR) was injected via a customized 1cm 30-gauge needle; 131 1mm posterior & 1.5mm superior to the posterior border of the left eye with a forward 132 angulation of 24 degrees and a depth of 2mm (Figure S1). In the Sham group all 133 experimental conditions were equivalent with the exception that the mice underwent 134 needle injection only without blood injection. 135 136 EdU administration 137 Intraperitoneal injections of EdU (12.5mg/kg) (Life technologies™) were given on 138 day 1 of life, according to published protocols [29]. 139 140 Immunohistochemistry and histology 141 Mice were transcardially perfused under terminal anaesthesia with 0.9% saline 142 followed by 4% paraformaldehyde. (PFA). Brains were dissected and post-fixed for 2 143 hours in 4% PFA at 4°C. Tissue was cryoprotected with 30% sucrose overnight, 144 mounted in O.C.T. (VWR™) medium and sectioned at 10μm on a cryostat (Leica™). 145 146 For EdU staining samples were blocked with 3% BSA (Sigma™)/ Phosphate- 147 Buffered Saline (PBS)/0.1% Triton X100 and incubated for 30 minutes with 148 proprietary Click-iT® solutions (Life technologies™). For immunofluorescent double 149 staining the following antibodies were used: Rabbit anti-GFAP 1:400 (Dako™): 150 Rabbit anti-NG2 1:200 (Millipore™): Guinea Pig anti-Dcx 1:2000 151 (MerckMillipore™): Mouse anti-NeuN 1:100 (Millipore™): Mouse anti-MASH1 152 1:200 (BD Biosciences™): Rabbit anti-Iba1 1:100 (Wako™): Mouse anti-Olig2 153 1:500 (Charles-Stiles Lab). All incubations with primary antibody were undertaken 154 overnight. After washing with PBS, sections were incubated with appropriate Alexa 155 Fluor® secondary antibodies diluted 1:500 (Invitrogen™) for 2 h at room temperature, 156 washed in PBS and mounted in Fluoromount™ (Sigma Aldrich™) or Vectashield® 157 with DAPI (Vector Laboratories™). 158 159 Volumetric analysis using the Cavalieri probe 160 To quantify ventricular volume we adopted a stereological approach using the 161 Cavalieri estimator probe within Stereoinvestigator MBF Bioscience™.
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