Microglial NLRP3 Inflammasome Induces Excitatory Synaptic Loss Through IL-1ß-Enriched Microvesicle Release: Implications for Sepsis-Associated Encephalopathy.

Acute cerebral dysfunction is a pathological state common in severe infections and a pivotal determinant of long-term cognitive outcomes. Current evidence indicates that a loss of synaptic contacts orchestrated by microglial activation is central in sepsis-associated encephalopathy. However, the upstream signals that lead to microglial activation and the mechanism involved in microglial-mediated synapse dysfunction in sepsis are poorly understood. This study investigated the involvement of the NLRP3 inflammasome in microglial activation and synaptic loss related to sepsis. We demonstrated that septic insult using the cecal ligation and puncture (CLP) model induced the expression of NLRP3 inflammasome components in the brain, such as NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3), apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC), caspase-1, and IL-1ß. Immunostaining techniques revealed increased expression of the NLRP3 inflammasome in microglial cells in the hippocampus of septic mice. Meanwhile, an in vitro model of primary microglia stimulated with LPS exhibited an increase in mitochondrial reactive oxygen species (ROS) production, NLRP3 complex recruitment, and IL-1ß release. Pharmacological inhibition of NLRP3, caspase-1, and mitochondrial ROS all decreased IL-1ß secretion by microglial cells. Furthermore, we found that microglial NLRP3 activation is the main pathway for IL-1ß-enriched microvesicle (MV) release, which is caspase-1-dependent. MV released from LPS-activated microglia induced neurite suppression and excitatory synaptic loss in neuronal cultures. Moreover, microglial caspase-1 inhibition prevented neurite damage and attenuated synaptic deficits induced by the activated microglial MV. These results suggest that microglial NLRP3 inflammasome activation is the mechanism of IL-1ß-enriched MV release and potentially synaptic impairment in sepsis.

Neuroinflammation in Sepsis: Molecular Pathways of Microglia Activation.

Frequently underestimated, encephalopathy or delirium are common neurological manifestations associated with sepsis. Brain dysfunction occurs in up to 80% of cases and is directly associated with increased mortality and long-term neurocognitive consequences. Although the central nervous system (CNS) has been classically viewed as an immune-privileged system, neuroinflammation is emerging as a central mechanism of brain dysfunction in sepsis. Microglial cells are major players in this setting. Here, we aimed to discuss the current knowledge on how the brain is affected by peripheral immune activation in sepsis and the role of microglia in these processes. This review focused on the molecular pathways of microglial activity in sepsis, its regulatory mechanisms, and their interaction with other CNS cells, especially with neuronal cells and circuits.

Lymphatic vessels in human adipose tissue.

Despite being considered present in most vascularised tissues, lymphatic vessels have not been properly shown in human adipose tissue (AT). Our goal in this study is to investigate an unanswered question in AT biology, regarding lymphatic network presence in tissue parenchyma. Using human subcutaneous (S-) and visceral (V-) AT samples with whole mount staining for lymphatic specific markers and three-dimensional imaging, we showed lymphatic capillaries and larger lymphatic vessels in the human VAT. Conversely, in the human SAT, microcirculatory lymphatic vascular structures were rarely detected and no initial lymphatics were found.

Superparamagnetic iron oxide nanoparticles as a tool to track mouse neural stem cells in vivo.

Cell transplantation offers a promising approach in many neurological disorders. Neural stem (NS) cells are potential candidates for cell therapy. The ability to track the grafted cells in the host tissue will refine this therapy. Superparamagnetic iron oxide nanoparticles (SPION) have been suggested as a feasible method, but there is no consensus about its safety. Here we investigated the feasibility of label NS cells with SPION and track by MRI after transplantation into mouse striatum with SPION cells and its therapeutic effects by grafting the cells into mouse striatum. We demonstrated that SPION-labeled NS cells display normal patterns of cellular processes including proliferation, migration, differentiation and neurosphere formation. Transmission electron microscopy reveals SPION in the cytoplasm of the cells, which was confirmed by microanalysis. Neurons and astrocytes generated from SPION-labeled NS cells were able to carry nanoparticles after 7 days under differentiation. SPION-labeled NS cells transplanted into striatum of mice were detected by magnetic resonance imaging (MRI) and microscopy 51 days later. In agreement with others reports, we demonstrated that NS cells are able to incorporate SPION in vitro without altering the stemness, and can survive and be tracked by MRI after they have been grafted into mice striatum.

CD60b: Enriching Neural Stem/Progenitor Cells from Rat Development into Adulthood.

CD60b antigens are highly expressed during development in the rat nervous system, while in the adult their expression is restricted to a few regions, including the subventricular zone (SVZ) around the lateral ventricles-a neurogenic niche in the adult brain. For this reason, we investigated whether the expression of C60b is associated with neural stem/progenitor cells in the SVZ, from development into adulthood. We performed in vitro and in vivo analyses of CD60b expression at different stages and identified the presence of these antigens in neural stem/progenitor cells. We also observed that CD60b could be used to purify and enrich a population of neurosphere-forming cells from the developing and adult brain. We showed that CD60b antigens (mainly corresponding to ganglioside 9-O-acetyl GD3, a well-known molecule expressed during central nervous system development and mainly associated with neuronal migration) are also present in less mature cells and could be used to identify and isolate neural stem/progenitor cells during development and in the adult brain. A better understanding of molecules associated with neurogenesis may contribute not only to improve the knowledge about the physiology of the mammalian central nervous system, but also to find new treatments for regenerating tissue after disease or brain injury.

Sofosbuvir protects Zika virus-infected mice from mortality, preventing short- and long-term sequelae.

Zika virus (ZIKV) causes significant public health concerns because of its association with congenital malformations, neurological disorders in adults, and, more recently, death. Considering the necessity to mitigate ZIKV-associated diseases, antiviral interventions are an urgent necessity. Sofosbuvir, a drug in clinical use against hepatitis C virus (HCV), is among the FDA-approved substances endowed with anti-ZIKV activity. In this work, we further investigated the in vivo activity of sofosbuvir against ZIKV. Neonatal Swiss mice were infected with ZIKV (2 × 107 PFU) and treated with sofosbuvir at 20 mg/kg/day, a concentration compatible with pre-clinical development of this drug. We found that sofosbuvir reduced acute levels of ZIKV from 60 to 90% in different anatomical compartments, such as the blood plasma, spleen, kidney, and brain. Early treatment with sofosbuvir doubled the percentage and time of survival of ZIKV-infected animals. Sofosbuvir also prevented the acute neuromotor impairment triggered by ZIKV. In the long-term behavioural analysis of ZIKV-associated sequelae, sofosbuvir prevented loss of hippocampal- and amygdala-dependent memory. Our results indicate that sofosbuvir inhibits ZIKV replication in vivo, which is consistent with the prospective necessity of antiviral drugs to treat ZIKV-infected individuals.

Intraspinal bone-marrow cell therapy at pre- and symptomatic phases in a mouse model of amyotrophic lateral sclerosis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a progressive neurological disease that selectively affects the motor neurons. The details of the mechanisms of selective motor-neuron death remain unknown and no effective therapy has been developed. We investigated the therapy with bone-marrow mononuclear cells (BMMC) in a mouse model of ALS (SOD1(G93A) mice). METHODS: We injected 10(6) BMMC into the lumbar portion of the spinal cord of SOD1(G93A) mice in presymptomatic (9 weeks old) and symptomatic (14 weeks old) phases. In each condition, we analyzed the progression of disease and the lifespan of the animals. RESULTS: We observed a mild transitory delay in the disease progression in the animals injected with BMMC in the presymptomatic phase. However, we observed no increase in the lifespan. When we injected BMMC in the symptomatic phase, we observed no difference in the animals' lifespan or in the disease progression. Immunohistochemistry for NeuN showed a decrease in the number of motor neurons during the course of the disease, and this decrease was not affected by either treatment. Using different strategies to track the BMMC, we noted that few cells remained in the spinal cord after transplantation. This observation could explain why the BMMC therapy had only a transitory effect. CONCLUSION: This is the first report of intraspinal BMMC therapy in a mouse model of ALS. We conclude this cellular therapy has only a mild transitory effect when performed in the presymptomatic phase of the disease.

Bone Marrow-Derived Cells as a Therapeutic Approach to Optic Nerve Diseases.

Following optic nerve injury associated with acute or progressive diseases, retinal ganglion cells (RGCs) of adult mammals degenerate and undergo apoptosis. These diseases have limited therapeutic options, due to the low inherent capacity of RGCs to regenerate and due to the inhibitory milieu of the central nervous system. Among the numerous treatment approaches investigated to stimulate neuronal survival and axonal extension, cell transplantation emerges as a promising option. This review focuses on cell therapies with bone marrow mononuclear cells and bone marrow-derived mesenchymal stem cells, which have shown positive therapeutic effects in animal models of optic neuropathies. Different aspects of available preclinical studies are analyzed, including cell distribution, potential doses, routes of administration, and mechanisms of action. Finally, published and ongoing clinical trials are summarized.

Distribution of mesenchymal stem cells and effects on neuronal survival and axon regeneration after optic nerve crush and cell therapy.

Bone marrow-derived cells have been used in different animal models of neurological diseases. We investigated the therapeutic potential of mesenchymal stem cells (MSC) injected into the vitreous body in a model of optic nerve injury. Adult (3-5 months old) Lister Hooded rats underwent unilateral optic nerve crush followed by injection of MSC or the vehicle into the vitreous body. Before they were injected, MSC were labeled with a fluorescent dye or with superparamagnetic iron oxide nanoparticles, which allowed us to track the cells in vivo by magnetic resonance imaging. Sixteen and 28 days after injury, the survival of retinal ganglion cells was evaluated by assessing the number of Tuj1- or Brn3a-positive cells in flat-mounted retinas, and optic nerve regeneration was investigated after anterograde labeling of the optic axons with cholera toxin B conjugated to Alexa 488. Transplanted MSC remained in the vitreous body and were found in the eye for several weeks. Cell therapy significantly increased the number of Tuj1- and Brn3a-positive cells in the retina and the number of axons distal to the crush site at 16 and 28 days after optic nerve crush, although the RGC number decreased over time. MSC therapy was associated with an increase in the FGF-2 expression in the retinal ganglion cells layer, suggesting a beneficial outcome mediated by trophic factors. Interleukin-1ß expression was also increased by MSC transplantation. In summary, MSC protected RGC and stimulated axon regeneration after optic nerve crush. The long period when the transplanted cells remained in the eye may account for the effect observed. However, further studies are needed to overcome eventually undesirable consequences of MSC transplantation and to potentiate the beneficial ones in order to sustain the neuroprotective effect overtime.

Sustained effect of bone marrow mononuclear cell therapy in axonal regeneration in a model of optic nerve crush.

In adult mammals, the regeneration of the optic nerve is very limited and at the moment there are several groups trying different approaches to increase retinal ganglion cell (RGC) survival and axonal outgrowth. One promising approach is cell therapy. In previous work, we performed intravitreal transplantation of bone-marrow mononuclear cells (BMMCs) after optic nerve crush in adult rats and we demonstrated an increase in RGC survival and axon outgrowth 14 days after injury. In the present work, we investigated if these results could be sustained for a longer period of time. Optic nerve crush was performed in Lister-hooded adult rats and BMMC or saline injections were performed shortly after injury. Neuronal survival and regeneration were evaluated in rats׳ retina and optic nerve after 28 days. We demonstrated an increase of 5.2 fold in the axon outgrowth 28 days after lesion, but the BMMCs had no effect on RGC survival. In an attempt to prolong RGC survival, we established a new protocol with two BMMC injections, the second one 7 days after the injury. Untreated animals received two injections of saline. We observed that although the axonal outgrowth was still increased after the second BMMC injection, the RGC survival was not significantly different from untreated animals. These results demonstrate that BMMCs transplantation promotes neuroregeneration at least until 28 days after injury. However, the effects on RGC survival previously observed by us at 14 days were not sustained at 28 days and could not be prolonged with a second dose of BMMC.

The role of cigarette smoking and liver enzymes polymorphisms in anti-tuberculosis drug-induced hepatotoxicity in Brazilian patients.

Tuberculosis (TB) is still a major health concern and side-effects related to the treatment, especially drug-induced hepatotoxicity (DIH), should be better investigated. In the present study, a possible association between anti-TB DIH and cigarette smoking, N-acetyltransferase 2 (NAT2), Cytochrome P450 2E1 (CYP2E1) and Cytochrome P450 3A4 (CYP3A4) genotypes was studied in 131 TB Brazilian patients. The NAT2 and CYP3A4 genetic polymorphisms were determined using a polymerase chain reaction (PCR) direct sequencing approach and genetic polymorphisms of CYP2E1 gene were determined by restriction fragment length polymorphism (RFLP). The risk of anti-TB DIH was lower in rapid/intermediate acetylators when compared to slow acetylators (OR: 0.34, CI 95: 0.16-0.71; p < 0.01). A decreased risk of developing anti-TB DIH was also observed in active smokers when compared to non-smokers (OR: 0.28, 95 CI: 0.11-0.64; p < 0.01). Significant association between CYP3A4 genotypes and hepatotoxicity was not observed, as well as between CYP2E1 genotype and hepatotoxicity, whose frequency of patients with wild homozygous was more prevalent. The anti-TB drugs interactions with smoking on hepatotoxicity, as well as the NAT2 phenotype, may require to adjust therapeutic regimen dosages or alarm in case of adverse event developments.

Bone-marrow cell therapy induces differentiation of radial glia-like cells and rescues the number of oligodendrocyte progenitors in the subventricular zone after global cerebral ischemia.

The subventricular zone (SVZ) is recognized as one of the neurogenic regions in the adult mammalian central nervous system and the presence of cells that share similar characteristics with developmental radial glia, the radial glia-like cells (RGLCs) has been demonstrated in this region. In this study, we investigated whether and how SVZ cells respond to global ischemia and/or to the intravenous transplant of bone-marrow mononuclear cells (BMMCs). Adult rats were subjected to bilateral common carotid ligation (BCCL) and after 1 day 2×10(7) BMMCs or saline injection. The BMMC transplant stimulated a transitory increase in the proliferation of SVZ cells in the BCCL group. We observed a significant increase in the number of RGLCs 3days after ischemia, in both BCCL and BCCL+BMMC groups. However, this increase persisted in the subsequent days only in BCCL animals that received the transplant. BMMC transplantation also inhibits the reduction of NG2-positive oligodendrocyte progenitors in the SVZ observed in the BCCL group. Interestingly, brain-derived neurotrophic factor (BDNF) expression was up-regulated in the SVZ in the treated animals, but not in the other groups. These data thus suggest that BMMC transplantation modulates the phenotype of RGLCs/progenitors in the SVZ and could have a protective role after ischemia.

Cell therapy modulates expression of Tax1-binding protein 1 and synaptotagmin IV in a model of optic nerve lesion.

PURPOSE: Bone marrow mononuclear cells (BMMCs) have been used with considerable success to improve regeneration and/or functional recovery in animal models of neurologic diseases. Injected into the host, they migrate to the damaged areas and release cytokines and/or trophic factors, which are capable of altering the genetic program of the injured tissue cells. In this study, there was a search for genes with altered expression in a model of optic nerve crush and cell therapy. METHODS: Optic nerve crush was followed by an intravitreous injection of BMMCs or vehicle in adult rats. After 14 days, we obtained a transcriptome screening of the retinas using differential display and automatic sequencing, followed by q-PCR, Western blot, and immunohistochemistry of selected genes and proteins. RESULTS: Among the differentially displayed genes, transcription of the antiapoptotic Tax1-binding protein 1 (Tax1BP1) and Synaptotagmin IV (Syt IV), an immediate early gene, is increased in the treated group. Tax1BP1 expression is robust in the ganglion cell layer and is significantly increased by cell therapy. Syt IV is expressed by activated Müller cells and astrocytes in the retina and optic nerve, without changes in protein levels among the groups. CONCLUSIONS: Tax1BP1 and Syt IV transcription and/or expression are differently modulated by optic nerve crush and BMMC treatment, and might be related to neuronal damage and cell-therapy effects in the retina. The increased expression of Tax1BP1 in the treated eyes could be involved in the neuroprotective effects of BMMCs that were described previously by our group.

Bone marrow mononuclear cells increase retinal ganglion cell survival and axon regeneration in the adult rat.

The central nervous system (CNS) of adult mammals generally does not regenerate, and many studies have attempted to identify factors that could increase neuroprotection and/or axonal outgrowth after CNS lesions. Using the optic nerve crush of rats as a model for CNS injury, we investigated the effect of intravitreal transplantation of syngeneic bone-marrow mononuclear cells (BMMCs) on the survival of retinal ganglion cells (RGC) and on the regeneration of optic axons. Control animals received intravitreal saline injections after lesion. Injections of BMMCs resulted in a 1.6-fold increase in the number of RGCs surviving 14 days after injury. The BMMC-treated animals also had increased numbers of axons, which grew up to 1.5 mm from the crush site, and also had reduced Müller glia activation. Analysis of mRNAs in all conditions revealed an increase in levels of fibroblast growth factor 2 (FGF-2) mRNA in treated animals 14 days after injury. To investigate whether the regenerated axons could reach the brain, we retrograde labeled the RGCs by injecting a lipophilic tracer into the superior colliculus. We also analyzed the expression of NGFI-A in the superficial layers of the superior colliculus as a possible marker of synaptic input from RGC axons. We found evidence that more RGCs were able to reach the brain after treatment and we showed that NGFI-A expression was higher in the treated animals 60 days after injury. These results demonstrate that transplant of BMMCs can increase neuroprotection and neuroregeneration after injury in a model of optic nerve crush, and these effects could be mediated by FGF-2.

Oncomodulin links inflammation to optic nerve regeneration.

The inflammatory response that accompanies central nervous system (CNS) injury can affect neurological outcome in both positive and negative ways. In the optic nerve, a CNS pathway that normally fails to regenerate when damaged, intraocular inflammation causes retinal ganglion cells (RGCs) to switch into an active growth state and extend lengthy axons down the nerve. The molecular basis of this phenomenon is uncertain. A prior study showed that oncomodulin (Ocm), a Ca(2+)-binding protein secreted by a macrophage cell line, is a potent axon-promoting factor for RGCs. However, it is not known whether Ocm contributes to the physiological effects of intraocular inflammation in vivo, and there are conflicting reports in the literature regarding its expression and significance. We show here that intraocular inflammation causes infiltrative cells of the innate immune system to secrete high levels of Ocm, and that agents that prevent Ocm from binding to its receptor suppress axon regeneration. These results were verified in different strains, species, and experimental models, and establish Ocm as a potent growth-promoting signal between the innate immune system and neurons in vivo.

Radial glia-like cells persist in the adult rat brain.

During development, radial glia cells contribute to neuronal migration and neurogenesis, and differentiate into astrocytes by the end of the developmental period. Recently, it was demonstrated that during development, radial glia cells, in addition to their role in migration, also give rise to neuroblasts. Furthermore, radial glial cells remain in the adult brain as adult neural stem cells (NSC) in the subventricular zone (SVZ) around the lateral ventricles (LVs), and generate new neurons continuously throughout adulthood. In this study, we used immunohistochemical and morphological methods to investigate the presence of radial glia-like cells around the LVs during the postnatal development period until adulthood in rats. In all ages of rats studied, we identified cells with morphological and immunocytochemical features that are similar to the radial glia cells found in the embryonic brain. Similarly to the radial glia, these cells express nestin and vimentin, and have a radial morphology, extending perpendicularly as processes from the ventricle wall. These cells also express GFAP, GLAST, and Pax6, and proliferate. In the brains of adult rats, we identified cells with relatively long processes (up to 600 mum) in close apposition with migrating neuroblasts. Our results showed that the radial glia-like cells present in the adult rat brain share several morphological and functional characteristics with the embryonic radial glia. We suggest that the embryonic radial glia cells located around the LV walls do not complete their transformation into astrocytes, but rather persist in adulthood.