Activation of the Kynurenine Pathway and Production of Inflammatory Cytokines by Astrocytes and Microglia Infected With Neospora caninum.

In the central nervous system, astrocytes and microglia contribute to homeostasis, regulating the immune response to infectious agents. Neospora caninum is an obligate intracellular protozoan that infects different animal species and it is encysted in their nervous tissue while triggering an immune response modulated by glia. This study aimed to evaluate the infection of primary cultures of rat glial cells by N. caninum through the catabolites of tryptophan, the expression of inflammatory mediators and the integrity of neural tissue. Infection with this coccidium resulted in morphological and functional changes, particularly astrogliosis and microgliosis, and increased the expression of the inflammatory mediators TNF, IL1ß, IL-10, and arginase, as well as mRNA for CCL5 and CCL2, molecules involved in the CNS chemotaxis. The infection with N. caninum in glial cells also triggered the activation of the tryptophan pathway, characterized by increased kynurenine 2,3 monooxygenase (KMO) mRNA expression, and by the production of the excitotoxin quinolinic acid (QUIN). Moreover, glia-neuron co-cultures, when exposed to the secretome derived from N. caninum infected glial cells, presented greater neurons distribution and formation of neurite extensions, associated to morphological changes in astrocytes compatible with neuro-preservation. Considering that the tryptophan catabolism is associated to immune response, these findings suggest that glial activation in N. caninum infection should be responsible for modulating the inflammatory status in an attempt to restore the nervous system homeostasis, since excessive inflammatory response can cause irreversible damage to tissue preservation.

Neurological Infection, Kynurenine Pathway, and Parasitic Infection by Neospora caninum.

Neuroinflammation is one of the most frequently studied topics of neurosciences as it is a common feature in almost all neurological disorders. Although the primary function of neuroinflammation is to protect the nervous system from an insult, the complex and sequential response of activated glial cells can lead to neurological damage. Depending on the type of insults and the time post-insult, the inflammatory response can be neuroprotective, neurotoxic, or, depending on the glial cell types, both. There are multiple pathways activated and many bioactive intermediates are released during neuroinflammation. One of the most common one is the kynurenine pathway, catabolizing tryptophan, which is involved in immune regulation, neuroprotection, and neurotoxicity. Different models have been used to study the kynurenine pathway metabolites to understand their involvements in the development and maintenance of the inflammatory processes triggered by infections. Among them, the parasitic infection Neospora caninum could be used as a relevant model to study the role of the kynurenine pathway in the neuroinflammatory response and the subset of cells involved.

JM-20 Treatment After Mild Traumatic Brain Injury Reduces Glial Cell Pro-inflammatory Signaling and Behavioral and Cognitive Deficits by Increasing Neurotrophin Expression.

Traumatic brain injury (TBI) is considered a public health problem and is often related to motor and cognitive disabilities, besides behavioral and emotional changes that may remain for the rest of the subject's life. Resident astrocytes and microglia are the first cell types to start the inflammatory cascades following TBI. It is widely known that continuous or excessive neuroinflammation may trigger many neuropathologies. Despite the large numbers of TBI cases, there is no effective pharmacological treatment available. This study aimed to investigate the effects of the new hybrid molecule 3-ethoxycarbonyl-2-methyl-4-(2-nitrophenyl)-4,11-dihydro1H-pyrido[2,3-b][1,5]benzodiazepine (JM-20) on TBI outcomes. Male Wistar rats were submitted to a weight drop model of mild TBI and treated with a single dose of JM-20 (8 mg/kg). Twenty-four hours after TBI, JM-20-treated animals showed improvements on locomotor and exploratory activities, and short-term memory deficits induced by TBI improved as well. Brain edema was present in TBI animals and the JM-20 treatment was able to prevent this change. JM-20 was also able to attenuate neuroinflammation cascades by preventing glial cells-microglia and astrocytes-from exacerbated activation, consequently reducing pro-inflammatory cytokine levels (TNF-α and IL-1ß). BDNF mRNA level was decreased 24 h after TBI because of neuroinflammation cascades; however, JM-20 restored the levels. JM-20 also increased GDNF and NGF levels. These results support the JM-20 neuroprotective role to treat mild TBI by reducing the initial damage and limiting long-term secondary degeneration after TBI.

Saponin-rich fraction from Agave sisalana: effect against malignant astrocytic cells and its chemical characterisation by ESI-MS/MS.

Astrocytic tumour cells derived from human (GL-15) and rat (C6) gliomas, as well as non-tumoural astrocytic cells, were exposed to the saponin-rich fraction (SF) from Agave sisalana waste and the cytotoxic effects were evaluated. Cytotoxicity assays revealed a reduction of cell viability that was more intensive in glioma than in non-tumoural cells. The SF induced morphological changes in C6 cells. They were characterised by cytoplasmic vacuole formation associated with increase in the formation of acidic lysosomes. The SF was subjected to purification on Sephadex LH-20, which characterised three probable steroidal saponins (sisalins) by electrospray ionisation mass spectrometry multistage (ESI-MSn). Sisalins from sisal may be responsible for the cytotoxicity, which involves cytoplasmatic vacuole formation and selective action for glioma cells.

Neuroprotection During Neospora caninum Infection Is Related To the Release of Neurotrophic Factors BDNF and NGF.

Neospora caninum is a parasite that infects many animal species and has tropism for various tissues, particularly the nervous system, where it generally remains in cysts. Under N. caninum infection, glial cells activate immune responses by a Th2 profile, suggesting an immunologically privileged environment that controls parasite proliferation, with neuronal preservation. In this study, we investigated the role of soluble neurotrophic factors released by glial cells on neuronal integrity during N. caninum infection in vitro. Primary cultures of rat glial cells enriched in astrocytes were infected with N. caninum tachyzoites (1:1) for 24 hr. Neuron-glia co-cultures were cultured for 24 hr with conditioned medium from glial cells infected with N. caninum (CMNc) and from uninfected cultures (control). Cell viability was determined through a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test; astrocyte morphology and reactivity were determined through immunocytochemistry for glial fibrillar acid protein (GFAP) and the integrity of neurons through immunocytochemistry for ß-tubulin III. Expression of inflammatory cytokines and neurotrophic factors was determined through RT-qPCR. The MTT test demonstrated that 1:1 was the best parasite/host cell ratio, considering that it was enough to increase metabolism of glial cells when compared with control cultures and was not cytotoxic after 48 hr infection. N. caninum-infected glial cultures responded with astrogliosis characterized by an increase in GFAP expression and increase in IL-10 (2-fold), BDNF (1.6-fold), and NGF (1.7-fold) gene expression. In the neuron/glia co-cultures, it was observed that treatment with CMNc induced neuritis outgrowth without toxicity. Together, these results show that modulatory mechanisms by neurotrophic factors derived from glial cells, primarily astrocytes during the N. caninum infection, can favor neuroprotection.