Maternal n-3 Polyunsaturated Fatty Acid Enriched Diet Commands Fatty Acid Composition in Postnatal Brain and Protects from Neonatal Arterial Focal Stroke

The fetus is strongly dependent on nutrients from the mother, including polyunsaturated fatty acids (PUFA). In adult animals, n-3 PUFA ameliorates stroke-mediated brain injury, but the modulatory effects of different PUFA content in maternal diet on focal arterial stroke in neonates are unknown. This study explored effects of maternal n-3 or n-6 enriched PUFA diets on neonatal stroke outcomes. Pregnant mice were assigned three isocaloric diets until offspring reached postnatal day (P) 10–13: standard, long-chain n-3 PUFA (n-3) or n-6 PUFA (n-6) enriched. Fatty acid profiles in plasma and brain of mothers and pups were determined by gas chromatography–mass spectrometry and cytokines/chemokines by multiplex protein analysis. Transient middle cerebral artery occlusion (tMCAO) was induced in P9-10 pups and cytokine and chemokine accumulation, caspase-3 and calpain-dependent spectrin cleavage and brain infarct volume were analyzed. The n-3 diet uniquely altered brain lipid profile in naïve pups. In contrast, cytokine and chemokine levels did not differ between n-3 and n-6 diet in naïve pups. tMCAO triggered accumulation of inflammatory cytokines and caspase-3-dependent and -independent cell death in ischemic-reperfused regions in pups regardless of diet, but magnitude of neuroinflammation and caspase-3 activation were attenuated in pups on n-3 diet, leading to protection against neonatal stroke. In conclusion, maternal/postnatal n-3 enriched diet markedly rearranges neonatal brain lipid composition and modulates the response to ischemia. While standard diet is sufficient to maintain low levels of inflammatory cytokines and chemokines under physiological conditions, n-3 PUFA enriched diet, but not standard diet, attenuates increases of inflammatory cytokines and chemokines in ischemic-reperfused regions and protects from neonatal stroke. Graphic Abstract

Neuroprotective effects of Sonic hedgehog agonist SAG in a rat model of neonatal stroke

Background Neonatal stroke affects 1 in 2800 live births and is a major cause of neurological injury. The Sonic hedgehog (Shh) signaling pathway is critical for central nervous system (CNS) development and has neuroprotective and reparative effects in different CNS injury models. Previous studies have demonstrated beneficial effects of small molecule Shh-Smoothened agonist (SAG) against neonatal cerebellar injury and it improves Down syndrome-related brain structural deficits in mice. Here we investigated SAG neuroprotection in rat models of neonatal ischemia–reperfusion (stroke) and adult focal white matter injury. Methods We used transient middle cerebral artery occlusion at P10 and ethidium bromide (EB) injection in adult rats to induce damage. Following surgery and SAG or vehicle treatment, we analyzed tissue loss, cell proliferation and fate, and behavioral outcome. Results We report that a single dose of SAG administered following neonatal stroke preserved brain volume, reduced gliosis, enhanced oligodendrocyte progenitor cell (OPC) and EC proliferation, and resulted in long-term cognitive improvement. Single-dose SAG also promoted proliferation of OPCs following focal demyelination in the adult rat. Conclusions These findings indicate benefit of one-time SAG treatment post insult in reducing brain injury and improving behavioral outcome after experimental neonatal stroke. Impact A one-time dose of small molecule Sonic hedgehog agonist protected against neonatal stroke and improved long-term behavioral outcomes in a rat model. This study extends the use of Sonic hedgehog in treating developing brain injury, previously shown in animal models of Down syndrome and cerebellar injury. Sonic hedgehog agonist is one of the most promising therapies in treating neonatal stroke thanks to its safety profile and low dosage.

Abstract 52: Changes in Oligodendrocyte Sub-Populations After Neonatal Stroke

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 <0.0001, proportions test, Fig 1C), which also express OL progenitor cell (OPC) markers. There was a significant increase in the proportion of cells in clusters 1 and 5. Pathway analysis suggest that both these clusters are comprised of pre-myelinating oligodendrocytes. 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.

Abstract P581: Neonatal Stroke is Not Associated With Birth Trauma

Acute neonatal stroke causes cerebral palsy, lifelong morbidity and mortality. Neonatal arterial ischemic stroke (NAIS) and hemorrhagic stroke (NHS) are most common. Pathophysiology is poorly understood and causation is often attributed to observed obstetrical factors such as instrumentation (forceps or vacuum) or operative delivery despite no empiric evidence supporting an association. We explored the relationship between birth trauma and neonatal stroke via population-based, prospectively collected registries in Southern Alberta, Canada. Consecutive cases of NAIS (n=59), NHS (n=20), and neonatal hypoxic-ischemic encephalopathy HIE with MRI-confirmed injury (HIE+, n=78)) were compared to neonates without injury (HIE-n=77). Cranial soft tissue swelling was objectively quantified as a trauma score from T1-weighted images using a semi-automatic segmentation method performed by two blinded investigators. Maternal, obstetrical, perinatal, and outcome variables were obtained from medical records. Multinomial regression modeling evaluated the relationship between diagnosis and birth trauma as measured by total soft tissue swelling score and diagnosis (HIE- as controls). Across the 234 infants studied, mean age at MRI (4.1+/-1.3 days) and sex (54% male) were comparable. Measurable scalp trauma was present in 93(40%), the proportion of which did not differ across groups. On univariate analysis, mean trauma scores did not differ between groups, were not associated with NHS or HIE+, and were lower for the NAIS group compared to HIE- (controls). Multinomial modeling revealed no relationship between scalp trauma and outcome. We conclude that the leading forms of acquired neonatal brain injury are not associated with objectively measured birth trauma. The term “birth trauma” should be removed from the perinatal stroke vernacular to help counsel traumatized parents and advance studies of genuine pathophysiology.

The neuroprotective effects of Sonic hedgehog pathway agonist SAG in a rat model of neonatal stroke

ABSTRACTObjectiveNeonatal stroke affects 1 in 2800 live births and is a major cause of neurological injury. The Sonic Hedgehog (Shh) signaling pathway is critical for central nervous system (CNS) development and has neuroprotective and reparative effects in different CNS injury models. Previous studies have demonstrated beneficial effects of small molecule Shh-Smoothened-agonist (SAG) against neonatal cerebellar injury and it improves Down syndrome-related brain structural deficits in mice. Here, we investigated SAG neuroprotection in rat models of neonatal ischemia-reperfusion (stroke) and adult focal white matter injury.MethodsWe used transient middle cerebral artery occlusion at P10 and ethidium bromide injection in adult rats to induce damage. Following surgery and SAG or vehicle treatment we analyzed tissue loss, cell proliferation and fate, and behavioral outcome.ResultsWe report that a single dose of SAG administered following neonatal stroke preserved brain volume, reduced inflammation, enhanced oligodendrocyte progenitor cell (OPC) and EC proliferation, and resulted in long-term cognitive improvement. Single-dose SAG also promoted proliferation of OPCs following focal demyelination in the adult rat.ConclusionThese findings indicate benefit of one-time SAG treatment post-insult in reducing brain injury and improving behavioral outcome after experimental neonatal stroke.