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BackgroundNeonatal stroke is common and causes life-long motor and cognitive sequelae. Because neonates with stroke are not diagnosed until days-months after the injury, chronic targets for repair are needed. We evaluated oligodendrocyte maturity and myelination and assessed oligodendrocyte gene expression changes using single cell RNA sequencing (scRNA seq) at chronic timepoints in a mouse model of neonatal arterial ischemic stroke.MethodsMice underwent sixty minutes of transient right middle cerebral artery occlusion (MCAO) on postnatal day 10 (p10) and received 5-ethynyl-2’-deoxyuridine (EdU) on post-MCAO days 3-7 to label dividing cells. Animals were sacrificed 14 and 28-30 days post-MCAO for immunohistochemistry and electron microscopy. Oligodendrocytes were isolated from striatum 14 days post-MCAO for scRNA seq and differential gene expression analysis.ResultsThe density of Olig2+EdU+cells was significantly increased in ipsilateral striatum 14 days post-MCAO and the majority of oligodendrocytes were immature. Density of Olig2+EdU+cells declined significantly between 14 and 28 days post-MCAO without a concurrent increase in mature Olig2+EdU+cells. By 28 days post-MCAO there were significantly fewer myelinated axons in ipsilateral striatum. scRNA seq identified a cluster of “disease associated oligodendrocytes (DOLs)” specific to the ischemic striatum, with increased expression of MHC class I genes. Gene ontology analysis suggested decreased enrichment of pathways involved in myelin production in the reactive cluster.ConclusionsOligodendrocytes proliferate 3-7 days post-MCAO and persist at 14 days, but fail to mature by 28 days. MCAO induces a subset of oligodendrocytes with reactive phenotype, which may be a therapeutic target to promote white matter repair.

Background: Cognitive deficits are common long-term sequelae after neonatal stroke, occurring in to 70% of school aged children. Despite this, chronic cognitive deficits have not been evaluated in animal models of neonatal stroke. Objective: To determine whether neonatal mice have behavioral memory and hippocampal cellular and plasticity changes after transient middle cerebral artery occlusion (tMCAO). Methods: C57/BL6 mice underwent 60 minutes of right tMCAO using the intraluminal filament model, or sham surgery, followed by reperfusion on postnatal day 10 (p10). Mice underwent contextual fear conditioning (CFC) testing to evaluate spatial memory 14 days after tMCAO (p24, juvenile equivalent). A separate set of animals were sacrificed at the same timepoint for Crestly Violet staining and stereology of CA1 neurons, or for LTP recordings. Increase in field excitatory post-synaptic potential (fEPSP) slope 60 min after theta-burst stimulation (TBS) was analyzed as a measurement of synaptic plasticity (LTP). Results: Animals had a significant decrease in % time freezing in the CFC paradigm 14 days after p10 tMCAO compared to sham surgery animals, indicating behavioral spatial memory impairment (Figure 1A). Animals who underwent p10 tMCAO also had a deficit in LTP after TBS in ipsilateral compared to contralateral CA1 (figure 1B). Animals had a decrease density of CA1 hippocampal neurons in the ipsilateral hippocampus compared to contralateral hippocampus 14 days after p10 tMCAO (Figure 1C). Conclusion: Our results show that neonatal stroke causes CA1 hippocampal pyramidal cell death and synaptic plasticity deficits which contribute to chronic memory impairment.

Background: Chronic white matter changes after neonatal stroke have not been well studied. Histologically, we see a robust increase in oligodendrocytes (OLs) in injured striatum 14 days post-middle cerebral artery occlusion (MCAO) in neonatal mice. The contribution of these cells to chronic white matter injury and repair has not been evaluated. Objective: Evaluate changes in striatal OL cell gene expression after neonatal MCAO. Methods: Mice underwent 60 minutes of MCAO at postnatal day 10 using the filament model and sacrificed 14 days later for fluorescent antibody cell sorting and single cell RNA sequencing. Single cell suspensions from Injured (ipsilateral) and uninjured (contralateral) striata were incubated with antibodies to immature and mature OLs. Cells expressing OL markers were collected and captured using 10x Genomics Chromium with V3.1 chemistry and analyzed in Seurat V3.1. Results: We captured a total of 4598 cells, with ~250,000 reads per cell. Our data set was comprised of 2399 oligodendrocytes (915 Contralateral, 1484 Ipsilateral). Feature plots of OL markers demonstrate that the entire lineage is present in our cell population (Fig 1A). Unbiased clustering identified 10 sub-populations of oligodendrocytes (Fig 1B). In ipsilateral striatum there was a significant decrease in the proportion of cells in cluster 8 (p Conclusions: At 14 days after neonatal stroke in mice scSEQ reveals a depletion of an OPC sub-population and an increase in sub-mature clusters of oligodendrocytes in ipsilateral striatum. Ongoing analysis of differential gene expression will reveal new insights into these cells and potential targets to promote white matter repair after neonatal stroke.

The incidence of stroke in children is 2.4 per 100,000 person-years and results in long-term motor and cognitive disability. In ischemic stroke, white matter (WM) is frequently injured, but is relatively understudied compared to grey matter injury. Previous research suggests that the cellular response to WM ischemic injury is different at different ages. Little is known about whether WM repair mechanisms differ in children and adults. We utilized a model of focal ischemic WM injury to determine the oligodendrocyte (OL) response to focal WM ischemic injury in juvenile and adult mice. Methods: Juvenile (21–25 days of age) versus adult (2–3 months of age) mice underwent stereotaxic injection of the potent vasoconstrictor N5-(1-iminoethyhl)-L-ornithine (L-NIO) into the lateral corpus callosum (CC). Animals were sacrificed on postoperative day 3 (acute) or 21 (chronic). Cell birth-dating was performed acutely after WM stroke with 5-ethynyl-2-deoxyuridine (EdU) injected intraperitoneally. Immunohistochemistry was performed, as well as stereology, to measure injury volume. The acute oligodendrocyte progenitor cell (OPC) proliferation and the chronic OL cell fate were determined with immunohistochemistry. Compound action potentials were measured in the CC at acute and chronic time points. Results: Acutely WM injury volume was smaller in juveniles. There was significantly greater OPC proliferation in juvenile animals (acute) compared to adults, but newly born OLs did not survive and mature into myelinating cells at chronic time points. In addition, juveniles did not have improved histological or functional recovery when compared to adults. Protecting newly born OPCs is a potential therapeutic target in children with ischemic stroke.

Introduction: Neonatal stroke is a common cause of lifelong neurologic disability. White matter repair after neonatal stroke has been understudied. Objective: Characterize acute myelin injury within striatal white matter and determine if endogenous remyelination occurs chronically. Methods: Postnatal day 10 (p10) mice underwent MCAO for 60 minutes, followed by reperfusion, and animals were sacrificed on post-op day (POD) 3, 14 or 28. Immunohistochemistry (IHC) was used to assess oligodendrocyte maturation, and white matter integrity. Gait was assessed on POD 14 or 30. Results: On POD3 there is a significant decrease in neuronal density in the ipsilateral striatum compared to contralateral. There is also a significant reduction in mature oligodendrocytes density. At this timepoint, axons are preserved (measured as %SMI34 + pixels), but there is significant myelin loss (measured as %MBP + pixels) in the ipsilateral striatum (fig 1A-D). On POD 14 there is persistently decreased myelin density in ipsilateral striatum compared to contralateral, and the proportion of oligodendrocytes with a mature phenotype (Olig2 + CC1 + /Olig2 + ) is significantly lower. Both myelin density and maturational index of oligodendrocytes recover by POD 28. At fourteen days after MCAO there is a significant reduction in gait length on the left side, which recovers by 28 days (fig1 E-G). Conclusions: 60 minute MCAO in neonatal mice produces striatal injury with oligodendrocyte and myelin loss but preservation of axons, providing a substrate for repair. Myelin deficit persists at 14 days, and there is an oligodendrocyte maturational delay at this same time. Myelination and oligodendrocyte maturation recover between 14 and 28 days, corresponding to recovery of motor function. Future studies will focus on whether interventions that accelerate oligodendrocyte maturation and re-myelination can improve early functional outcome.