C3a Receptor Signaling Inhibits Neurodegeneration Induced by Neonatal Hypoxic-Ischemic Brain Injury

Hypoxic-ischemic neonatal encephalopathy due to perinatal asphyxia is the leading cause of brain injury in newborns. Clinical data suggest that brain inflammation induced by perinatal insults can persist for years. We previously showed that signaling through the receptor for complement peptide C3a (C3aR) protects against cognitive impairment induced by experimental perinatal asphyxia. To investigate the long-term neuropathological effects of hypoxic-ischemic injury to the developing brain and the role of C3aR signaling therein, we subjected wildtype mice, C3aR deficient mice, and mice expressing biologically active C3a in the CNS to mild hypoxic-ischemic brain injury on postnatal day 9. We found that such injury triggers neurodegeneration and pronounced reactive gliosis in the ipsilesional hippocampus both of which persist long into adulthood. Transgenic expression of C3a in reactive astrocytes reduced hippocampal neurodegeneration and reactive gliosis. In contrast, neurodegeneration and microglial cell density increased in mice lacking C3aR. Intranasal administration of C3a for 3 days starting 1 h after induction of hypoxia-ischemia reduced neurodegeneration and reactive gliosis in the hippocampus of wildtype mice. We conclude that neonatal hypoxic-ischemic brain injury leads to long-lasting neurodegeneration. This neurodegeneration is substantially reduced by treatment with C3aR agonists, conceivably through modulation of reactive gliosis.

Stroke subtype-dependent synapse elimination by reactive gliosis in mice

The pathological role of reactive gliosis in CNS repair remains controversial. In this study, using murine ischemic and hemorrhagic stroke models, we demonstrated that microglia/macrophages and astrocytes are differentially involved in engulfing synapses in the reactive gliosis region. By specifically deleting MEGF10 and MERTK phagocytic receptors, we determined that inhibiting phagocytosis of microglia/macrophages or astrocytes in ischemic stroke improved neurobehavioral outcomes and attenuated brain damage. In hemorrhagic stroke, inhibiting phagocytosis of microglia/macrophages but not astrocytes improved neurobehavioral outcomes. Single-cell RNA sequencing revealed that phagocytosis related biological processes and pathways were downregulated in astrocytes of the hemorrhagic brain compared to the ischemic brain. Together, these findings suggest that reactive microgliosis and astrogliosis play individual roles in mediating synapse engulfment in pathologically distinct murine stroke models and preventing this process could rescue synapse loss.

Changes in Brain Matrix Glycan Sulfation Associate With Reactive Gliosis and Motor Coordination in Mice With Head Trauma

Perineuronal nets (PNNs) are extracellular matrix (ECM) structures that enmesh and regulate neurocircuits involved in motor and sensory function. Maladaptive changes to the composition and/or abundance of PNNs have been implicated in preclinical models of neuroinflammation and neurocircuit destabilization. The central nervous system (CNS) is limited in its capacity to repair and reorganize neural networks following traumatic brain injury (TBI) and little is known about mechanisms of ECM repair in the adult brain after TBI. In this study, adult male C57BL/6 mice were subjected to a TBI via a controlled cortical impact (CCI) to the right motor and somatosensory cortices. At 7 days following CCI, histological analysis revealed a loss of Wisteria floribunda agglutinin (WFA) positive PNN matrices in the ipsilateral cortex. PNNs are comprised of chondroitin sulfate (CS) and dermatan sulfate (DS)-glycosaminoglycans (GAGs), the composition of which are known to influence neuronal integrity and repair. Using an innovative liquid chromatography tandem mass spectrometry (LC-MS/MS) method, we analyzed the relative abundance of six specific CS/DS-GAG isomers (Δ4S-, Δ6S-, Δ4S6S-, Δ2S6S-, Δ0S-CS, and Δ2S4S-DS) from fixed-brain sections after CCI injury. We report a significant shift in CS/DS-GAG sulfation patterns within the rostro-caudal extent of the injury site from mice exposed to CCI at 7 days, but not at 1 day, post-CCI. In the ipsilateral thalamus, the appearance of WFA+ puncta occurred in tandem with gliosis at 7 days post-CCI, but weakly colocalized with markers of gliosis. Thalamic WFA+ puncta showed moderate colocalization with neuronal ubiquitin C-terminal hydrolase L1 (UCHL1), a clinical biomarker for TBI injury. A shift in CS/DS-GAG sulfation was also present in the thalamus including an increase of 6S-CS, which is a specific isomer that associates with the presence of glial scarring. Upregulation of the 6S-CS-specific sulfotransferase (CHST3) gene expression was accompanied by reactive gliosis in both the ipsilateral cortex and thalamus. Moreover, changes in 6S-CS extracted from the thalamus positively correlated with deficits in motor coordination after CCI. Collectively, these data argue that CCI alters CS/DS-GAG sulfation in association with the spatiotemporal progression of neurorepair. Therapeutic interventions targeting restoration of CS/DS-GAG sulfation patterns may improve outcomes from TBI.

The TGFβ/Notch axis facilitates Müller cell-to-epithelial transition to ultimately form a chronic glial scar

Background Contrasting with zebrafish, retinal regeneration from Müller cells (MCs) is largely limited in mammals, where they undergo reactive gliosis that consist of a hypertrophic response and ultimately results in vision loss. Transforming growth factor β (TGFβ) is essential for wound healing, including both scar formation and regeneration. However, targeting TGFβ may affect other physiological mechanisms, owing its pleiotropic nature. The regulation of various cellular activities by TGFβ relies on its interaction with other pathways including Notch. Here, we explore the interplay of TGFβ with Notch and how this regulates MC response to injury in zebrafish and mice. Furthermore, we aimed to characterize potential similarities between murine and human MCs during chronic reactive gliosis. Methods Focal damage to photoreceptors was induced with a 532 nm diode laser in TgBAC (gfap:gfap-GFP) zebrafish (ZF) and B6-Tg (Rlbp1-GFP) mice. Transcriptomics, immunofluorescence, and flow cytometry were employed for a comparative analysis of MC response to laser-induced injury between ZF and mouse. The laser-induced injury was paired with pharmacological treatments to inhibit either Notch (DAPT) or TGFβ (Pirfenidone) or TGFβ/Notch interplay (SIS3). To determine if the murine laser-induced injury model translates to the human system, we compared the ensuing MC response to human donors with early retinal degeneration. Results Investigations into injury-induced changes in murine MCs revealed TGFβ/Notch interplay during reactive gliosis. We found that TGFβ1/2 and Notch1/2 interact via Smad3 to reprogram murine MCs towards an epithelial lineage and ultimately to form a glial scar. Similar to what we observed in mice, we confirmed the epithelial phenotype of human Müller cells during gliotic response. Conclusion The study indicates a pivotal role for TGFβ/Notch interplay in tuning MC stemness during injury response and provides novel insights into the remodeling mechanism during retinal degenerative diseases. Graphical abstract

Harnessing Astrocytes and Müller Glial Cells in the Retina for Survival and Regeneration of Retinal Ganglion Cells

Astrocytes have been associated with the failure of axon regeneration in the central nervous system (CNS), as it undergoes reactive gliosis in response to damages to the CNS and functions as a chemical and physical barrier to axon regeneration. However, beneficial roles of astrocytes have been extensively studied in the spinal cord over the years, and a growing body of evidence now suggests that inducing astrocytes to become more growth-supportive can promote axon regeneration after spinal cord injury (SCI). In retina, astrocytes and Müller cells are known to undergo reactive gliosis after damage to retina and/or optic nerve and are hypothesized to be either detrimental or beneficial to survival and axon regeneration of retinal ganglion cells (RGCs). Whether they can be induced to become more growth-supportive after retinal and optic nerve injury has yet to be determined. In this review, we pinpoint the potential molecular pathways involved in the induction of growth-supportive astrocytes in the spinal cord and suggest that stimulating the activation of these pathways in the retina could represent a new therapeutic approach to promoting survival and axon regeneration of RGCs in retinal degenerative diseases.

Minocycline Promotes Functional Recovery in Ischemic Stroke by Modulating Microglia Polarization Through STAT1/STAT6 Pathways

BackgroundIncreasing evidence suggests that microglia experience two distinct phenotypes after acute ischemic stroke (AIS): a deleterious M1 phenotype and a neuroprotective M2 phenotype. Promoting the phenotype shift of M1 microglia to M2 microglia is thought to improve functional recovery after AIS. Minocycline, a tetracycline antibiotic, can improve functional recovery after cerebral ischemia in pre-clinical and clinical research. However, the role and mechanisms of minocycline in microglia polarization is unclear.MethodsUsing the transient middle cerebral artery occlusion - reperfusion (MCAO/R) model, we treated mice with saline or different minocycline concentration (10, 25, or 50 mg/kg, i.p., daily for 2 wk) at 24 h after reperfusion. Neurobehavioral evaluation, rotarod test, and corner turning test were carried out on day 14 after reperfusion. Then, neuronal injury, reactive gliosis, and microglia polarization were performed on day 7 following MCAO/R. Finally, we treated primary microglial cultures with LPS (Lipopolysaccharide; 100 ng/mL) plus IFN-γ (20 ng/mL) 24 h to induce M1 phenotype and observed the effects of minocycline on the M1/M2-related mRNAs and the STAT1/STAT6 pathway.ResultsWe found that a 14-day treatment with minocycline increased the survival rate and promoted functional outcomes evaluated with neurobehavioral evaluation, rotarod test, and corner turning test. Meanwhile, minocycline reduced the brain infarct volume, alleviated neuronal injury, and suppressed reactive gliosis on day 7 following MCAO/R. Moreover, we observed an additive effect of minocycline on microglia polarization to the M1 and M2 phenotypes in vivo and in vitro. In the primary microglia, we further found that minocycline prevented neurons from OGD/R-induced cell death in neuron-microglia co-cultures via regulating M1/M2 microglia polarization through the STAT1/STAT6 pathway. ConclusionMinocycline promoted microglial M2 polarization and inhibited M1 polarization, leading to neuronal survival and neurological functional recovery. The findings deepen our understanding of the mechanisms underlying minocycline-mediated neuroprotection in AIS.