Characterization of microglia behaviour in healthy and pathological conditions with image analysis tools.

Microglia are very sensitive to changes in the environment and respond through morphological, functional and metabolic adaptations. To depict the modifications microglia undergo under healthy and pathological conditions, we developed free access image analysis scripts to quantify microglia morphologies and phagocytosis. Neuron-glia cultures, in which microglia express the reporter tdTomato, were exposed to excitotoxicity or excitotoxicity + inflammation and analysed 8 h later. Neuronal death was assessed by SYTOX staining of nucleus debris and phagocytosis was measured through the engulfment of SYTOX+ particles in microglia. We identified seven morphologies: round, hypertrophic, fried egg, bipolar and three 'inflamed' morphologies. We generated a classifier able to separate them and assign one of the seven classes to each microglia in sample images. In control cultures, round and hypertrophic morphologies were predominant. Excitotoxicity had a limited effect on the composition of the populations. By contrast, excitotoxicity + inflammation promoted an enrichment in inflamed morphologies and increased the percentage of phagocytosing microglia. Our data suggest that inflammation is critical to promote phenotypical changes in microglia. We also validated our tools for the segmentation of microglia in brain slices and performed morphometry with the obtained mask. Our method is versatile and useful to correlate microglia sub-populations and behaviour with environmental changes.

Clemastine Induces an Impairment in Developmental Myelination.

Abnormalities in myelination are associated to behavioral and cognitive dysfunction in neurodevelopmental psychiatric disorders. Thus, therapies to promote or accelerate myelination could potentially ameliorate symptoms in autism. Clemastine, a histamine H1 antagonist with anticholinergic properties against muscarinic M1 receptor, is the most promising drug with promyelinating properties. Clemastine penetrates the blood brain barrier efficiently and promotes remyelination in different animal models of neurodegeneration including multiple sclerosis, ischemia and Alzheimer's disease. However, its role in myelination during development is unknown. We showed that clemastine treatment during development increased oligodendrocyte differentiation in both white and gray matter. However, despite the increase in the number of oligodendrocytes, conduction velocity of myelinated fibers of corpus callosum decreased in clemastine treated mice. Confocal and electron microscopy showed a reduction in the number of myelinated axons and nodes of Ranvier and a reduction of myelin thickness in corpus callosum. To understand the mechanisms leading to myelin formation impairment in the presence of an excess of myelinating oligodendrocytes, we focused on microglial cells that also express muscarinic M1 receptors. Importantly, the population of CD11c+ microglia cells, necessary for myelination, as well as the levels of insulin growth factor-1 decrease in clemastine-treated mice. Altogether, these data suggest that clemastine impact on myelin development is more complex than previously thought and could be dependent on microglia-oligodendrocyte crosstalk. Further studies are needed to clarify the role of microglia cells on developmental myelination.

Dendritic Cells and Microglia Have Non-redundant Functions in the Inflamed Brain with Protective Effects of Type 1 cDCs.

Brain CD11c+ cells share features with microglia and dendritic cells (DCs). Sterile inflammation increases brain CD11c+ cells, but their phenotype, origin, and functions remain largely unknown. We report that, after cerebral ischemia, microglia attract DCs to the inflamed brain, and astroglia produce Flt3 ligand, supporting development and expansion of CD11c+ cells. CD11c+ cells in the inflamed brain are a complex population derived from proliferating microglia and infiltrating DCs, including a major subset of OX40L+ conventional cDC2, and also cDC1, plasmacytoid, and monocyte-derived DCs. Despite sharing certain morphological features and markers, CD11c+ microglia and DCs display differential expression of pattern recognition receptors and chemokine receptors. DCs excel CD11c- and CD11c+ microglia in the capacity to present antigen through MHCI and MHCII. Of note, cDC1s protect from brain injury after ischemia. We thus reveal aspects of the dynamics and functions of brain DCs in the regulation of inflammation and immunity.

CCR2 deficiency in monocytes impairs angiogenesis and functional recovery after ischemic stroke in mice.

Inflammatory Ly6ChiCCR2+ monocytes infiltrate the brain after stroke but their functions are not entirely clear. We report that CCR2+ monocytes and CCR2+ lymphocytes infiltrate the brain after permanent ischemia. To underscore the role of CCR2+ monocytes, we generated mice with selective CCR2 deletion in monocytes. One day post-ischemia, these mice showed less infiltrating monocytes and reduced expression of pro-inflammatory cytokines, markers of alternatively macrophage activation, and angiogenesis. Accordingly, Ly6Chi monocytes sorted from the brain of wild type mice 24 h post-ischemia expressed pro-inflammatory genes, M2 genes, and pro-angiogenic genes. Flow cytometry showed heterogeneous phenotypes within the infiltrating Ly6ChiCCR2+ monocytes, including a subgroup of Arginase-1+ cells. Mice with CCR2-deficient monocytes displayed a delayed inflammatory rebound 15 days post-ischemia that was not found in wild type mice. Furthermore, they showed reduced angiogenesis and worse behavioral performance. Administration of CCR2+/+ bone-marrow monocytes to mice with CCR2-deficient monocytes did not improve the behavioral performance suggesting that immature bone-marrow monocytes lack pro-reparative functions. The results show that CCR2+ monocytes contribute to acute post-ischemic inflammation and participate in functional recovery. The study unravels heterogeneity in the population of CCR2+ monocytes infiltrating the ischemic brain and suggests that pro-reparative monocyte subsets promote functional recovery after ischemic stroke.

Location of Neutrophils in Different Compartments of the Damaged Mouse Brain After Severe Ischemia/Reperfusion.

Background and Purpose- Ischemia attracts neutrophils to the injured brain. However, neutrophil location and access to the damaged brain tissue is not yet entirely understood. We aimed to investigate neutrophil location in a mouse model of cerebral ischemia/reperfusion. Methods- Adult male C57BL/6 mice (n=52) received 45-minute intraluminal middle cerebral artery occlusion followed by 14, 24, 48, or 96 hours of reperfusion. Sham-operated mice (n=9) were subjected to the entire surgical procedure. We used wild-type mice and CatchupIVM mice expressing a red fluorescent protein in neutrophils. In addition, fluorescent neutrophils obtained from reporter DsRed (discosoma red fluorescent protein) mice were transferred intravenously to wild-type mice after ischemia. Mice received transcardial paraformaldehyde perfusion, the brain was cryoprotected, frozen, and cryostat sections were studied by immunofluorescence and confocal microscopy. Results- Ischemia induced a time-dependent increase in brain neutrophil numbers versus sham operation. We detected neutrophils in the leptomeninges, ventricles, capillary lumen, perivascular spaces, and parenchyma within the infarcted core. Most ischemic mice showed neutrophils in the leptomeninges and perivascular spaces, whereas the presence and number of neutrophils in the parenchyma was variable among ischemic mice. During the first 24 hours, only a few mice showed parenchymal neutrophils, but the frequency of mice showing neutrophils in the parenchyma and neutrophil numbers increased at 48 and 96 hours. We also detected signs of basement membrane disruption and hints of occasional neutrophil degranulation and formation of neutrophil extracellular traps. Conclusions- After ischemia/reperfusion, neutrophils accumulate in the leptomeninges and perivascular spaces, and eventually can reach the infarcted brain parenchyma.

CD69 Plays a Beneficial Role in Ischemic Stroke by Dampening Endothelial Activation.

RATIONALE: CD69 is an immunomodulatory molecule induced during lymphocyte activation. Following stroke, T-lymphocytes upregulate CD69 but its function is unknown. OBJECTIVE: We investigated whether CD69 was involved in brain damage following an ischemic stroke. METHODS AND RESULTS: We used adult male mice on the C57BL/6 or BALB/c backgrounds, including wild-type mice and CD69-/- mice, and CD69+/+ and CD69-/- lymphocyte-deficient Rag2-/- mice, and generated chimeric mice. We induced ischemia by transient or permanent middle cerebral artery occlusion. We measured infarct volume, assessed neurological function, and studied CD69 expression, as well as platelet function, fibrin(ogen) deposition, and VWF (von Willebrand factor) expression in brain vessels and VWF content and activity in plasma, and performed the tail-vein bleeding test and the carotid artery ferric chloride-induced thrombosis model. We also performed primary glial cell cultures and sorted brain CD45-CD11b-CD31+ endothelial cells for mRNA expression studies. We blocked VWF by intravenous administration of anti-VWF antibodies. CD69-/- mice showed larger infarct volumes and worse neurological deficits than the wild-type mice after ischemia. This worsening effect was not attributable to lymphocytes or other hematopoietic cells. CD69 deficiency lowered the time to thrombosis in the carotid artery despite platelet function not being affected. Ischemia upregulated Cd69 mRNA expression in brain endothelial cells. CD69-deficiency increased fibrin(ogen) accumulation in the ischemic tissue, and plasma VWF content and activity, and VWF expression in brain vessels. Blocking VWF reduced infarct volume and reverted the detrimental effect of CD69-/- deficiency. CONCLUSIONS: CD69 deficiency promotes a prothrombotic phenotype characterized by increased VWF and worse brain damage after ischemic stroke. The results suggest that CD69 acts as a downregulator of endothelial activation.

Microglial cell loss after ischemic stroke favors brain neutrophil accumulation.

Stroke attracts neutrophils to the injured brain tissue where they can damage the integrity of the blood-brain barrier and exacerbate the lesion. However, the mechanisms involved in neutrophil transmigration, location and accumulation in the ischemic brain are not fully elucidated. Neutrophils can reach the perivascular spaces of brain vessels after crossing the endothelial cell layer and endothelial basal lamina of post-capillary venules, or migrating from the leptomeninges following pial vessel extravasation and/or a suggested translocation from the skull bone marrow. Based on previous observations of microglia phagocytosing neutrophils recruited to the ischemic brain lesion, we hypothesized that microglial cells might control neutrophil accumulation in the injured brain. We studied a model of permanent occlusion of the middle cerebral artery in mice, including microglia- and neutrophil-reporter mice. Using various in vitro and in vivo strategies to impair microglial function or to eliminate microglia by targeting colony stimulating factor 1 receptor (CSF1R), this study demonstrates that microglial phagocytosis of neutrophils has fundamental consequences for the ischemic tissue. We found that reactive microglia engulf neutrophils at the periphery of the ischemic lesion, whereas local microglial cell loss and dystrophy occurring in the ischemic core are associated with the accumulation of neutrophils first in perivascular spaces and later in the parenchyma. Accordingly, microglia depletion by long-term treatment with a CSF1R inhibitor increased the numbers of neutrophils and enlarged the ischemic lesion. Hence, microglial phagocytic function sets a critical line of defense against the vascular and tissue damaging capacity of neutrophils in brain ischemia.

Differential expression of E-type prostanoid receptors 2 and 4 in microglia stimulated with lipopolysaccharide.

BACKGROUND: Cyclooxygenase-2 (COX-2) is induced under inflammatory conditions, and prostaglandin E2 (PGE2) is one of the products of COX activity. PGE2 has pleiotropic actions depending on the activation of specific E-type prostanoid EP1-4 receptors. We investigated the involvement of PGE2 and EP receptors in glial activation in response to an inflammatory challenge induced by LPS. METHODS: Cultures of mouse microglia or astroglia cells were treated with LPS in the presence or absence of COX-2 inhibitors, and the production of PGE2 was measured by ELISA. Cells were treated with PGE2, and the effect on LPS-induced expression of TNF-α messenger RNA (mRNA) and protein was studied in the presence or absence of drug antagonists of the four EP receptors. EP receptor expression and the effects of EP2 and EP4 agonists and antagonists were studied at different time points after LPS. RESULTS: PGE2 production after LPS was COX-2-dependent. PGE2 reduced the glial production of TNF-α after LPS. Microglia expressed higher levels of EP4 and EP2 mRNA than astroglia. Activation of EP4 or EP2 receptors with selective drug agonists attenuated LPS-induced TNF-α in microglia. However, only antagonizing EP4 prevented the PGE2 effect demonstrating that EP4 was the main target of PGE2 in naïve microglia. Moreover, the relative expression of EP receptors changed during the course of classical microglial activation since EP4 expression was strongly depressed while EP2 increased 24 h after LPS and was detected in nuclear/peri-nuclear locations. EP2 regulated the expression of iNOS, NADPH oxidase-2, and vascular endothelial growth factor. NADPH oxidase-2 and iNOS activities require the oxidation of NADPH, and the pentose phosphate pathway is a main source of NADPH. LPS increased the mRNA expression of the rate-limiting enzyme of the pentose pathway glucose-6-phosphate dehydrogenase, and EP2 activity was involved in this effect. CONCLUSIONS: These results show that while selective activation of EP4 or EP2 exerts anti-inflammatory actions, EP4 is the main target of PGE2 in naïve microglia. The level of EP receptor expression changes from naïve to primed microglia where the COX-2/PGE2/EP2 axis modulates important adaptive metabolic changes.

A case of bilateral macrodontia of mandibular second premolars from a Chalcolithic context in the Iberian Peninsula.

Isolated macrodontia, consisting of the gigantism of a single tooth, is an extremely rare condition. Only 16 cases of isolated macrodontia of mandibular second premolars have been reported to date. Although the aetiology of this phenomenon remains unknown, many authors have related it to the control of the apoptotic process, leading to the patterning and size of dental cusps. There is not a clear genetic inheritance pattern since only two of those 16 cases correspond to close relatives. To our knowledge, there have been no reports of isolated macrodontia of mandibular second premolars in archaeological remains. Cova del Pantà de Foix site is a Chalcolithic sepulchral cave situated in the North-East of the Iberian Peninsula in which the remains of at least 30 individuals were recovered. Most of these individuals show several signs of environmental stressors. The current study presents the first case of isolated bilateral macrodontia of mandibular premolars from an archaeological context, corresponding to a young male individual discovered in this site. This condition could be the last consequence of environmental factors epigenetically affecting apoptosis processes in early tooth development and a possible genetic predisposition to show a shape-deviation in the dentition.