Inflammation modulates brain injury after perinatal arterial stroke. We previously demonstrated that microglial cells play an important role in protecting neonatal brain from acute stroke by phagocytosing dying neurons, attenuating cytokine accumulation and by protecting neurovascular integrity. Microglia may modulate injury in neonatal stroke via crosstalk between cells via other mechanisms, such as by releasing extracellular vesicles from microglia (MEV), including exosomes (MExo) and microvesicles (MMV). Aim: Elucidate the mechanisms of MEV communication with brain cells in injured neonatal brain and role of these vesicles in protection. Methods: Transient middle cerebral artery occlusion (tMCAO) was performed in postnatal day 9 (P9). Microglial cells were isolated by CD11b-conjugated beads from ischemic and contralateral cortex 24h after reperfusion and plated at same density for 96h. MEV were isolated by multi-step centrifugation (MMV) and ultra-centrifugation (MExo) and labeled with MiniClaret dye. Uptake of MEV from contralateral/ injured cortex by Iba1 + -cells from contralateral and injured regions was determined 10, 30 & 120min. Images co-stained with flotillin-1, which is strongly expressed in MMV but not in MExo, were analyzed using Volocity ® . Results: The uptake of MEV from injured cortex by microglia from injured cortex was significantly higher than uptake of contralateral-MEV by microglia from uninjured cortex (5-fold at 10min; p<0.0001) regardless of time. Uptake of MEV from injured regions by microglia from contralateral cortex and contralateral-MEV by microglia from injured cortex were low. While the number of ipsilateral and contralateral flotillin-1 + -MMV was similar, uptake of flotillin-1 - -MExo and ratio of ipsilateral MExo/MEV were significantly increase. Summary: Our data demonstrate selective enhancement of microglial communication with MEV from activated microglia after acute neonatal stroke as well as distinct MEV-subtype-dependent mechanisms of communication in injured brain. This mechanism could provide a better understanding of the role of microglia on the severity of neonatal stroke. Support: AHA17IRG33430004, RO1NS44025, RO1HL139685
Stroke is among the top 10 causes of death in children. The developmental stage of the brain is central to stroke pathophysiology. The incidence of childhood arterial ischemic stroke (CAIS) is lower than of perinatal arterial ischemic stroke but the rate of recurrence is strikingly high. Vascular inflammation is seen as major contributor to CAIS but the mechanisms that govern structural-functional basis of vascular abnormalities remain poorly understood. To identify the contribution of immune-neurovascular interactions to CAIS, we established stroke model in postnatal day 21 (P21) mice. We demonstrate acute functional deficits and histological injury and chronic MRI-identifiable injury, brain atrophy and marked derangements in the vascular network. In contrast to negligible albumin leakage and neutrophil infiltration following acute perinatal stroke, CAIS leads to significantly increased albumin leakage and neutrophil infiltration in injured regions of wild type mice and mice with functional CX3CR1-CCR2 receptors. In mice with dysfunctional CX3CR1-CCR2 signaling, extravascular albumin leakage is significantly attenuated, infiltration of injurious Ccr2 + -monocytes essentially aborted, accumulation of Ly6G+ neutrophils reduced and acute injury attenuated. Unique identifiers of microglia and monocytes revealed phenotypic changes in each cell subtype of the monocyte lineage after CAIS. Taken together, CX3CR1-CCR2-dependent microglia-monocyte signaling contributes to cerebrovascular leakage, inflammation and CAIS injury.
The scavenger receptor CD36 is injurious in acute experimental focal stroke and neurodegenerative diseases in the adult. We investigated the effects of genetic deletion of CD36 (CD36ko) on acute injury, and oxidative and inflammatory signaling after neonatal stroke.
This study examined dark microglia-a state linked to central nervous system pathology and neurodegeneration-during postnatal development in the mouse ventral hippocampus, finding that dark microglia interact with blood vessels and synapses and perform trogocytosis of pre-synaptic axon terminals. Furthermore, we found that dark microglia in development notably expressed C-type lectin domain family 7 member A (CLEC7a), lipoprotein lipase (LPL) and triggering receptor expressed on myeloid cells 2 (TREM2) and required TREM2, differently from other microglia, suggesting a link between their role in remodeling during development and central nervous system pathology. Together, these results point towards a previously under-appreciated role for dark microglia in synaptic pruning and plasticity during normal postnatal development.
Background: The developmental stage of the brain at the stroke onset plays a key role in the pathophysiology of injury, with neuroinflammation as a major injury modulator (Rayasam et al 2022; Hagberg et al. 2015). We reported that in the neonatal brain microglia secrete extracellular vesicles, exosomes and microvesicels, under physiological conditions and that microglial activation after transient MCA occlusion (tMCAO) in postnatal day 9 (P9) mice alters “cargo” of microglial-derived extracellular vesicles (MEV) and increases MEV uptake by microglia in vitro (Lecuyer et al., Neurobiol Dis. 2021). Here, we examined whether MEV administration protects the neonatal brain from stroke. Methods: P9 C57BL6 mice were subjected to a 3h tMCAO, microglia isolated from naïve mice and from injured (Inj) and contralateral (Contra) regions 24h after reperfusion using CD11b-conjugated beads, and cultured for 4 days in10% exosome-free FBS. MEV were purified from culture medium by multistep centrifugation [x300 g (Phase1, p1), x1,200 g (p2), x12,000 g (p3), and x100,000 g (p4)], concentration determined by Nanosight, MEV labelled with Claret-FR. MEV uptake by microglia from Inj and Contra regions was examined in vitro. 0.5 or 3.0 x 10 8 MEV were injected retro-orbital 2h after reperfusion. Behaviour outcomes (24h) and injury (at 4 days) were determined. Results: Post-vivo, uptake of p4C or p4I was increased by microglia from Inj, but not from Contra regions and reduced cell size and morphology within 30 min. Administration of p4-MEV from naïve (p4N-MEV), Contra (p4C-MEV) or Inj (p4I-MEV) regions significantly improved performance on Open Field test and reduced injury, whereas p3N-MEV did not affect inury. MEV increased survival (100%) compared to non-treated (77%) and vehicle-treated (83%) mice. We are defining p4 and p3 identity (exosomes Vs. microvesicles). Summary: p4-MEV but not p3-MEVprotect from neonatal stroke. Funding: R01 NS44025, R01 HL139685, R21 NS098514
Neonatal encephalopathy in human babies is a serious condition associated with permanent neurological deficits. Diffusion-weighted MRI (DWI) is increasingly used for early diagnosis of brain injury in human babies. The relationship between the presence of DWI abnormalities and cellular injury, including apoptosis, during the neonatal period are not well understood. We asked whether the extent of injury depicted on DWI can predict the presence of caspase-3 activation, a quantitative marker of apoptotic injury, after hypoxia-ischemia (H-I) in postnatal day 7 rats.Injury volume was determined by DWI at 2 hours, 24 hours, and 7 days after H-I and compared with histology. Caspase-3 activation and microgliosis were determined at 24 hours post-H-I.DWI-defined lesions (eg, decreased apparent diffusion coefficient) at 24 hours post-H-I correlated with a major increase in caspase-3 activity in the injured hemisphere and predicted injury. A modest but significant increase in caspase-3 activity occurred in the cortex of rats that had no apparent diffusion coefficient decrease in the injured hemisphere but had unilaterally enlarged regions of high apparent diffusion coefficient at the ipsilateral ventricle/white matter interface. Caspase-3 activity was similar in both hemispheres in pups with unchanged DWI.Abnormal DWI signal at 24 hours post-H-I is predictive of caspase-3 activation and can be used as an indicator that injury involving an apoptotic-like mechanism is present. Our data also suggest that the presence of an enlarged unilateral region with high apparent diffusion coefficient at the ventricle/white matter interface without significant apparent diffusion coefficient decrease in the cortex is a sign of modest caspase-3 activation after H-I.
Microglial cells support brain homeostasis under physiological conditions and modulate brain injury in a context-dependent and brain maturation-dependent manner. Microglial cells protect neonatal brain from acute stroke. While microglial signaling via direct cell-cell interaction and release of variety of molecules is intensely studied, less is known about microglial signaling via release and uptake of extracellular vesicles (EVs). We asked whether neonatal stroke alters release of microglial EVs (MEV) and MEV communication with activated microglia. We pulled down and plated microglia from ischemic-reperfused and contralateral cortex 24 h after transient middle cerebral artery occlusion (tMCAO) in postnatal day 9 mice, isolated and characterized microglia-derived microvesicles (P3-MEV) and exosomes (P4-MEV), and determined uptake of fluorescently labeled P3-MEV and P4-MEV by plated microglia derived from ischemic-reperfused and contralateral cortex. We then examined how reducing EVs release in neonatal brain—by intra-cortical injection of CRISPR-Cas9-Smpd3/KO (Smpd3/KD) to downregulate Smpd3 gene to disrupt neutral sphingomyelinase-2 (N-SMase2)—impacts P3-MEV and P4-MEV release and stroke injury. Both size and protein composition differed between P3-MEV and P4-MEV. tMCAO further altered protein composition of P3-MEV and P4-MEV and significantly, up to 5-fold, increased uptake of both vesicle subtypes by microglia from ischemic-reperfused regions. Under physiological conditions neurons were the predominant cell type expressing N-SMase-2, an enzyme involved in lipid signaling and EVs release. After tMCAO N-SMase-2 expression was diminished in injured neurons but increased in activated microglia/macrophages, leading to overall reduced N-SMase-2 activity. Compared to intracerebral injection of control plasmid, CRISPR-Cas9-Smpd3/Ct, Smpd3/KD injection further reduced N-SMase-2 activity and significantly reduced injury. Smpd3 downregulation decreased MEV release from injured regions, reduced Smpd3/KD-P3-MEV uptake and abolished Smpd3/KD-P4-MEV uptake by microglia from ischemic-reperfused region. Cumulatively, these data demonstrate that microglial cells release both microvesicles and exosomes in naïve neonatal brain, that the state of microglial activation determines both properties of released EVs and their recognition/uptake by microglia in ischemic-reperfused and control regions, suggesting a modulatory role of MEV in neonatal stroke, and that sphingosine/N-SMase-2 signaling contributes both to EVs release and uptake (predominantly P4-MEV) after neonatal stroke.
Neonatal stroke is associated with the N-methyl-D-aspartate receptor (NMDAR)-mediated excitotoxic brain injury. Src family kinases (SFKs) are considered to be the molecular hub for NMDAR regulation. We determined the relationship between SFKs activation and NMDAR tyrosine phosphorylation after neonatal hypoxia-ischemia (HI) and investigated the neuroprotective potential of a selective SFKs inhibitor, PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3, 4-d] pyramidine), against neonatal brain ischemic injury. The Rice-Vannucci model was adapted for neonatal HI injury in postnatal day 7 CD1 mice. SFKs activity in the postsynaptic densities was measured by Western blot. NMDAR tyrosine phosphorylation and their association with SFKs were determined by coimmunoprecipitation. Brains from animals treated with PP2 or its inactive analog, PP3, were examined histologically with cresyl violet and iron stain to assess the degree of damage. Neonatal HI resulted in a rapid and transient increase in tyrosine phosphorylation of NMDAR subunits NR2A and NR2B. This upregulation correlated with the enhanced association of Fyn and Src with NR2A and NR2B. SFKs were activated in the postsynaptic densities after HI. Inhibition of SFKs with PP2 attenuated brain injury after neonatal HI, whereas PP3 did not protect the brain from the HI insult. SFKs may play an important role in NMDAR-mediated excitotoxicity and downstream events leading to neuronal death after neonatal HI. Inhibition of SFKs may provide protection against neonatal stroke. Rather than blockade of NMDAR after HI in the developing brain, it may be safer and more beneficial to manipulate components of the NMDAR signaling complex at the postsynaptic density.
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