Microglial Cells Prevent Hemorrhage in Neonatal Focal Arterial Stroke

Perinatal stroke leads to significant morbidity and long-term neurological and cognitive deficits. The pathophysiological mechanisms of brain damage depend on brain maturation at the time of stroke. To understand whether microglial cells limit injury after neonatal stroke by preserving neurovascular integrity, we subjected postnatal day 7 (P7) rats depleted of microglial cells, rats with inhibited microglial TGFbr2/ALK5 signaling, and corresponding controls, to transient middle cerebral artery occlusion (tMCAO). Microglial depletion by intracerebral injection of liposome-encapsulated clodronate at P5 significantly reduced vessel coverage and triggered hemorrhages in injured regions 24 h after tMCAO. Lack of microglia did not alter expression or intracellular redistribution of several tight junction proteins, did not affect degradation of collagen IV induced by the tMCAO, but altered cell types producing TGFβ1 and the phosphorylation and intracellular distribution of SMAD2/3. Selective inhibition of TGFbr2/ALK5 signaling in microglia via intracerebral liposome-encapsulated SB-431542 delivery triggered hemorrhages after tMCAO, demonstrating that TGFβ1/TGFbr2/ALK5 signaling in microglia protects from hemorrhages. Consistent with observations in neonatal rats, depletion of microglia before tMCAO in P9 Cx3cr1(GFP/+)/Ccr2(RFP/+) mice exacerbated injury and induced hemorrhages at 24 h. The effects were independent of infiltration of Ccr2(RFP/+) monocytes into injured regions. Cumulatively, in two species, we show that microglial cells protect neonatal brain from hemorrhage after acute ischemic stroke. SIGNIFICANCE STATEMENT The pathophysiological mechanisms of brain damage depend on brain maturation at the time of stroke. We assessed whether microglial cells preserve neurovascular integrity after neonatal stroke. In neonatal rats, microglial depletion or pharmacological inhibition of TGFbr2/ALK5 signaling in microglia triggered hemorrhages in injured regions. The effect was not associated with additional changes in expression or intracellular redistribution of several tight junction proteins or collagen IV degradation induced by stroke. Consistent with observations in neonatal rats, microglial depletion in neonatal mice exacerbated stroke injury and induced hemorrhages. The effects were independent of infiltration of monocytes into injured regions. Thus, microglia protect neonatal brain from ischemia-induced hemorrhages, and this effect is consistent across two species.