Neuroinflammation is associated with changes in glial mGluR5 expression and the development of neonatal excitotoxic lesions

It has been hypothesized that neuroinflammation triggered during brain development can alter brain functions later in life. We investigated the contribution of inflammation to the alteration of normal brain circuitries in the context of neuroexcitotoxicity following neonatal ventral hippocampal lesions in rats with ibotenic acid, an NMDA glutamate receptor agonist. Excitotoxic ibotenic acid lesions led to a significant and persistent astrogliosis and microglial activation, associated with the production of inflammatory mediators. This response was accompanied by a significant increase in metabotropic glutamate receptor type 5 (mGluR5) expression within two distinct neuroinflammatory cell types; astrocytes and microglia. The participation of inflammation to the neurotoxin-induced lesion was further supported by the prevention of hippocampal neuronal loss, glial mGluR5 expression and some of the behavioral perturbations associated to the excitotoxic lesion by concurrent anti-inflammatory treatment with minocycline. These results indicate that neuroinflammation significantly contributes to long-lasting excitotoxic effects of the neurotoxin and to some behavioral phenotypes associated with this model. Thus, the control of the inflammatory response may prevent the deleterious effects of excitotoxic processes that are triggered during brain development, limiting the risk to develop some of the behavioral manifestations related to these processes in adulthood.     

Interleukin 15 expression in the CNS: blockade of its activity prevents glial activation after an inflammatory injury

Although reactive glia formation after neuronal degeneration or traumatic damage is one of the hallmarks of central nervous system (CNS) injury, we have little information on the signals that direct activation of resting glia. IL-15, a pro-inflammatory cytokine involved in regulating the response of T and B cells, may be also key for the regulation of early inflammatory events in the nervous system. IL-15 was expressed in the CNS, most abundantly in cerebellum and hippocampus, mainly in astrocytes and in some projection neurons. Using a rodent model of acute inflammatory injury [lipopolysaccharide (LPS) injection], we found enhanced expression of IL-15 in both reactive astroglia and microglia, soon after CNS injury. Blockade of IL-15 activity with an antibody to the cytokine, reversed activation of both glial types, suggesting that IL-15 has a major role in the generation of gliotic tissue and in the regulation of neuroimmune responses. Because IL-15 appears to modulate the inflammatory environment acutely generated after CNS injury, regulating IL-15 expression seems a clear antiinflammatory therapy to improve the outcome of neurodegenerative diseases and CNS trauma.     

Clozapine reduces GLT-1 expression and glutamate uptake in astrocyte cultures

To test the hypothesis that clozapine-induced reduction of glutamate transporter-1 (GLT-1) expression is mediated by astrocytes, we studied the effects of clozapine on Glu transporters and Glu uptake in primary astrocyte cultures of the cerebral cortex. Astrocyte cultures treated for 48 h with clozapine exhibited a reduction in GLT-1 levels of about 50%, whereas glutamate-aspartate transporter (GLAST) levels remained unchanged. Glu uptake was also lowered, and this reduction was dose-dependent. Our findings indicate that clozapine reduces GLT-1 expression and function by a mechanism that directly involves astrocytes. A better understanding of the molecular events by which antipsychotics regulate Glu uptake can contribute to identify new targets for the treatment of schizophrenia.     

Astrocytic and microglia cells reactivity induced by neonatal administration of glutamate in cerebral cortex of the adult rats.

Recent studies confirm that astrocytes and neurons are associated with the synaptic transmission, particularly with the regulation of glutamate (Glu) levels. Therefore, they have the capacity to modulate the Glu released from neurons into the extracellular space. It has also been demonstrated an intense astrocytic and microglia response to physical or chemical lesions of the central nervous system. However, the persistence of the response of the glial cells in adult brain had not been previously reported, after the excitotoxic damage caused by neonatal dosage of monosodium glutamate (MSG) to newborn rats. In this study, 4 mg/g body weight of MSG were administered to newborn rats at 1, 3, 5, and 7 days after birth, at the age of 60 days the astrocytes and the microglia cells were analyzed with immunohistochemical methods in the fronto-parietal cortex. Double labeling to glial fibrillary acidic protein (GFAP) and BrdU, or isolectin-B(4) and BrdU identified astrocytes or microglia cells that proliferated; immunoblotting and immunoreactivity to vimentin served for assess immaturity of astrocytic intermediate filaments. The results show that the neonatal administration of MSG-induced reactivity of astrocytes and microglia cells in the fronto-parietal cortex, which was characterized by hyperplasia; an increased number of astrocytes and microglia cells that proliferated, hypertrophy; increased complexity of the cytoplasm extension of both glial cells and expression of RNAm to vimentin, with the presence of vimentin-positive astrocytes. This glial response to neuroexcitotoxic stimulus of Glu on the immature brain, which persisted to adulthood, suggests that the neurotransmitter Glu could trigger neuro-degenerative illnesses.     

Cytokine regulation of astrocyte function: in-vitro studies using cells from the human brain

Participation of astrocytes in central nervous system pathophysiology is likely to involve cytokines, both as stimulators and mediators of astrocyte function. We have used highly enriched human astrocyte cultures as an experimental tool to investigate the influence of cytokines on adhesion molecule expression and synthesis of mediators that are probably important in immune and inflammatory reactions involving the nervous system and in cerebral tissue repair. The response of astrocytes to interferon-γ mainly resulted in increased expression of major histocompatibility complex antigens and co-stimulatory molecules (intercellular adhesion molecule-1, LFA-1α) which mediate astrocyte-T-cell interactions. Another co-stimulatory molecule, B7, was neither expressed nor inducible by IFN-γ and other cytokines. TNF-α and IL-1β were more efficient in stimulating synthesis of immunoregulatory and proinflammatory cytokines (IL-6, IL-8 and colony-stimulating factors), cytokine antagonists (TNF-α soluble receptors), or cytokines with a possible neuroprotective role (leukemia inhibitory factor); they also increased expression of some co-stimulatory molecules (intercellular adhesion molecule-1 and vascular cell adhesion molecule-1). Transforming growth factor-β₁ was a strong inducer of leukemia inhibitory factor, but did not affect either major histocompatibility complex/co-stimulatory molecule expression or cytokine synthesis. Thus, different cytokines activate distinct functional programs in astrocytes, which may play a specific role in different brain diseases or at different stages of the same disease. It was additionally observed that the response of human astrocytes to cytokines (in particular the inducible synthesis of certain cytokines) varied greatly depending on the presence or absence of neurons in the culture system. This finding suggests that neuronal-glial interactions may be implicated in determining the activation threshold of astrocytes to inflammatory cytokines.     

Lack of NG2 exacerbates neurological outcome and modulates glial responses after traumatic brain injury

Traumatic brain injury (TBI) is a major cause of death and disability. The underlying pathophysiology is characterized by secondary processes including neuronal death and gliosis. To elucidate the role of the NG2 proteoglycan we investigated the response of NG2‐knockout mice (NG2‐KO) to TBI. Seven days after TBI behavioral analysis, brain damage volumetry and assessment of blood brain barrier integrity demonstrated an exacerbated response of NG2‐KO compared to wild‐type (WT) mice. Reactive astrocytes and expression of the reactive astrocyte and neurotoxicity marker Lcn2 (Lipocalin‐2) were increased in the perilesional brain tissue of NG2‐KO mice. In addition, microglia/macrophages with activated morphology were increased in number and mRNA expression of the M2 marker Arg1 (Arginase 1) was enhanced in NG2‐KO mice. While TBI‐induced expression of pro‐inflammatory cytokine genes was unchanged between genotypes, PCR array screening revealed a marked TBI‐induced up‐regulation of the C‐X‐C motif chemokine 13 gene Cxcl13 in NG2‐KO mice. CXCL13, known to attract immune cells to the inflamed brain, was expressed by activated perilesional microglia/macrophages seven days after TBI. Thirty days after TBI, NG2‐KO mice still exhibited more pronounced neurological deficits than WT mice, up‐regulation of Cxcl13, enhanced CD45+ leukocyte infiltration and a relative increase of activated Iba‐1+/CD45+ microglia/macrophages. Our study demonstrates that lack of NG2 exacerbates the neurological outcome after TBI and associates with abnormal activation of astrocytes, microglia/macrophages and increased leukocyte recruitment to the injured brain. These findings suggest that NG2 may counteract neurological deficits and adverse glial responses in TBI. GLIA 2016;64:507–523     

Neuroinflammation as modifier of genetically caused neurological disorders of the central nervous system: Understanding pathogenesis and chances for treatment

Genetically caused neurological disorders of the central nervous system (CNS) are usually orphan diseases with poor or even fatal clinical outcome and few or no treatments that will improve longevity or at least quality of life. Neuroinflammation is common to many of these disorders, despite the fact that a plethora of distinct mutations and molecular changes underlie the disorders. In this article, data from corresponding animal models are analyzed to define the roles of innate and adaptive inflammation as modifiers and amplifiers of disease. We describe both common and distinct patterns of neuroinflammation in genetically mediated CNS disorders and discuss the contrasting mechanisms that lead to adverse versus neuroprotective effects. Moreover, we identify the juxtaparanode as a neuroanatomical compartment commonly associated with inflammatory cells and ongoing axonopathic changes, in models of diverse diseases. The identification of key immunological effector pathways that amplify neuropathic features should lead to realistic possibilities for translatable therapeutic interventions using existing immunomodulators. Moreover, evidence emerges that neuroinflammation is not only able to modify primary neural damage‐related symptoms but also may lead to unexpected clinical outcomes such as neuropsychiatric syndromes.     

Early expression of glutamate transporter proteins in ramified microglia after controlled cortical impact injury in the rat

Traumatic brain injury is followed by increased extracellular glutamate concentration. Uptake of glutamate is mainly mediated by the glial glutamate transporters GLAST and GLT-1. Extent and distribution of GLAST and GLT-1 were studied in a rat model of controlled cortical impact injury (CCII). Western Blot analysis revealed lowest levels of GLAST and GLT-1 with a decrease by 40%-54% and 42%-49% between 24 and 72 h posttrauma. By 8 h after CCII, CSF glutamate levels were increased (10.5 microM vs. 2.56 microM in controls; P < 0.001), reaching maximum values by 48 h. A significant increase in de novo GLAST and GLT-1 expressing ramified microglia was observed within 4 h, reached a stable level by 48 h, and remained high up to 72 h after CCII. Furthermore, ramified microglia de novo expressed the neuronal glutamate transporter EAAC1 after CCII. Following CCII, GLAST/GLT-1 and GFAP coexpressing astrocytes were immediately reduced, reaching minimum levels within 8 h. This reduction of expression could be either due to protein downregulation or loss of astrocytes. At 72 h, a marked population of GLAST- and GLT-1-positive reactive astrocytes appeared. These results support the hypothesis that reduced astrocytic GLAST and GLT-1 protein levels following CCII contribute to evolving secondary injury. Microglia are capable of de novo expressing glutamate transporter proteins, indicating that the expression of glial and neuronal glutamate transporters is not restricted to a specific glial or neuronal lineage. Ramified microglia may play an important compensatory role in the early regulation of extracellular glutamate after CCII.