Rodrigo Martinez

Neuritogenesis induced by thyroid hormone-treated astrocytes is mediated by epidermal growth factor/mitogen-activated protein kinase-phosphatidylinositol 3-kinase pathways and involves modulation of extracellular matrix proteins

Thyroid hormone (T3) plays a crucial role in several steps of cerebellar ontogenesis. By using a neuron-astrocyte coculture model, we have investigated the effects of T3-treated astrocytes on cerebellar neuronal differentiation in vitro. Neurons plated onto T3-astrocytes presented a 40–60% increase on the total neurite length and an increment in the number of neurites. Treatment of astrocytes with epidermal growth factor (EGF) yielded similar results, suggesting that this growth factor might mediate T3-induced neuritogenesis. EGF and T3 treatment increased fibronectin and laminin expression by astrocytes, suggesting that astrocyte neurite permissiveness induced by these treatments is mostly due to modulation of extracellular matrix (ECM) components. Such increase in ECM protein expression as well as astrocyte permissiveness to neurite outgrowth was reversed by the specific EGF receptor tyrosine kinase inhibitor, tyrphostin. Moreover, studies using selective inhibitors of several transduction-signaling cascades indicated that modulation of ECM proteins by EGF is mainly through a synergistic activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways. In this work, we provide evidence of a novel role of EGF as an intermediary factor of T3 action on cerebellar ontogenesis. By modulating the content of ECM proteins, EGF increases neurite outgrowth. Our data reveal an important role of astrocytes as mediators of T3-induced cerebellar development and partially elucidate the role of EGF and mitogen-activated protein kinase/phosphatidylinositol 3-kinase pathways on this process.

Neuro–glia interaction effects on GFAP gene: a novel role for transforming growth factor-β1

Central nervous system (CNS) development is highly guided by microenvironment cues specially provided by neuron–glia interactions. By using a transgenic mouse bearing part of the gene promoter of the astrocytic maturation marker GFAP (glial fibrillary acidic protein) linked to the β‐galactosidase (β‐Gal) reporter gene, we previously demonstrated that cerebral cortical neurons increase transgenic β‐Gal astrocyte number and activate GFAP gene promoter by secretion of soluble factors in vitro. Here, we identified TGF‐β1 as the major mediator of this event. Identification of TGF‐β1 in neuronal and astrocyte extracts revealed that both cell types might synthesize this factor, however, addition of neurons to astrocyte monolayers greatly increased TGF‐β1 synthesis and secretion by astrocytes. Further, by exploiting the advantages of cell culture system we investigated the influence of neuron and astrocyte developmental stage on such interaction. We demonstrated that younger neurons derived from 14 embryonic days wild‐type mice were more efficient in promoting astrocyte differentiation than those derived from 18 embryonic days mice. Similarly, astrocytes also exhibited timed‐schedule developed responsiveness to neuronal influence with embryonic astrocytes being more responsive to neurons than newborn and late postnatal astrocytes. RT‐PCR assays identified TGF‐β1 transcripts in young but not in old neurons, suggesting that inability to induce astrocyte differentiation is related to TGF‐β1 synthesis and secretion. Our work reveals an important role for neuron–glia interactions in astrocyte development and strongly implicates the involvement of TGF‐β1 in this event.

Soluble Factors Released by Toxoplasma gondii-Infected Astrocytes Down-Modulate Nitric Oxide Production by Gamma Interferon-Activated Microglia and Prevent Neuronal Degeneration

ABSTRACT The maintenance of a benign chronic Toxoplasma gondii infection is mainly dependent on the persistent presence of gamma interferon (IFN-γ) in the central nervous system (CNS). However, IFN-γ-activated microglia are paradoxically involved in parasitism control and in tissue damage during a broad range of CNS pathologies. In this way, nitric oxide (NO), the main toxic metabolite produced by IFN-γ-activated microglia, may cause neuronal injury during T. gondii infection. Despite the potential NO toxicity, neurodegeneration is not a common finding during chronic T. gondii infection. In this work, we describe a significant down-modulation of NO production by IFN-γ-activated microglia in the presence of conditioned medium of T. gondii-infected astrocytes (CMi). The inhibition of NO production was paralleled with recovery of neurite outgrowth when neurons were cocultured with IFN-γ-activated microglia in the presence of CMi. Moreover, the modulation of NO secretion and the neuroprotective effect were shown to be dependent on prostaglandin E2 (PGE2) production by T. gondii-infected astrocytes and autocrine secretion of interleukin-10 (IL-10) by microglia. These events were partially eliminated when infected astrocytes were treated with aspirin and cocultures were treated with anti-IL-10 neutralizing antibodies and RP-8-Br cyclic AMP (cAMP), a protein kinase A inhibitor. Further, the modulatory effects of CMi were mimicked by the presence of exogenous PGE2 and by forskolin, an adenylate cyclase activator. Altogether, these data point to a T. gondii-triggered regulatory mechanism involving PGE2 secretion by astrocytes and cAMP-dependent IL-10 secretion by microglia. This may reduce host tissue inflammation, thus avoiding neuron damage during an established Th1 protective immune response.

Cross-talk between neurons and glia: highlights on soluble factors.

The development of the nervous system is guided by a balanced action between intrinsic factors represented by the genetic program and epigenetic factors characterized by cell-cell interactions which neural cells might perform throughout nervous system morphogenesis. Highly relevant among them are neuron-glia interactions. Several soluble factors secreted by either glial or neuronal cells have been implicated in the mutual influence these cells exert on each other. In this review, we will focus our attention on recent advances in the understanding of the role of glial and neuronal trophic factors in nervous system development. We will argue that the functional architecture of the brain depends on an intimate neuron-glia partnership.

Toxoplasma gondii Prevents Neuron Degeneration by Interferon-γ-Activated Microglia in a Mechanism Involving Inhibition of Inducible Nitric Oxide Synthase and Transforming Growth Factor-β1 Production by Infected Microglia

Interferon (IFN)-gamma, the main cytokine responsible for immunological defense against Toxoplasma gondii, is essential in all infected tissues, including the central nervous system. However, IFN-gamma-activated microglia may cause tissue injury through production of toxic metabolites such as nitric oxide (NO), a potent inducer of central nervous system pathologies related to inflammatory neuronal disturbances. Despite potential NO toxicity, neurodegeneration is not commonly found during chronic T. gondii infection. In this study, we describe decreased NO production by IFN-gamma-activated microglial cells infected by T. gondii. This effect involved strong inhibition of iNOS expression in IFN-gamma-activated, infected microglia but not in uninfected neighboring cells. The inhibition of NO production and iNOS expression were parallel with recovery of neurite outgrowth when neurons were co-cultured with T. gondii-infected, IFN-gamma-activated microglia. In the presence of transforming growth factor (TGF)-beta1-neutralizing antibodies, the beneficial effect of the parasite on neurons was abrogated, and NO production reverted to levels similar to IFN-gamma-activated uninfected co-cultures. In addition, we observed Smad-2 nuclear translocation, a hallmark of TGF-beta1 downstream signaling, in infected microglial cultures, emphasizing an autocrine effect restricted to infected cells. Together, these data may explain a neuropreservation pattern observed during immunocompetent host infection that is dependent on T. gondii-triggered TGF-beta1 secretion by infected microglia.

Proliferation of cerebellar neurons induced by astrocytes treated with thyroid hormone is mediated by a cooperation between cell contact and soluble factors and involves the epidermal growth factor-protein kinase a pathway.

Cerebellar development is fully dependent on thyroid hormone (T3) levels. We have previously demonstrated a glia‐mediated effect of T3 on cerebellar neurons. We have reported that cerebellar astrocytes treated with thyroid hormone secrete epidermal growth factor (EGF), which directly induces neuronal proliferation and, indirectly, by increasing synthesis of extracellular matrix proteins, induces neurite outgrowth in vitro. Here, by using a neuron–astrocyte coculture model, we investigated the involvement of cell contact on neuronal proliferation. Culturing of cerebellar neurons on T3‐treated astrocyte carpets or conditioned medium derived from them (T3CM) yielded similar results, revealed by a 60% increase in cell population. However, the absolute number of neurons in coculture assays was greatly enhanced in comparison with that in CM assays (3.5–4‐fold). Bromodeoxyuridine (BrdU) incorporation assays revealed that such an increase was due mainly to proliferation of precursors cells. BrdU incorporation was three times higher in cell carpet (31%) than in CM (13%). Treatment of astrocytes by T3 increased neuronal proliferation either by T3CM (2.5 times) or by contact with T3‐treated astrocytes (1.5 times). Neuronal death was not affected by T3 treatment of astrocytes as revealed by either trypan blue viability assays or active caspase‐3 labeling. Treatment of astrocytes by EGF mimicked T3 effects on neuronal proliferation. Addition of the EGF receptor tyrosine kinase inhibitor genistein and the protein kinase A (PKA) inhibitor KT5720 to cocultutres and T3CM completely reversed neuronal proliferation. Our results implicate EGF and the PKA pathway in the proliferation induced by T3‐treated astrocytes. Furthermore, the fact that cocultures potentiate the effect of T3 on neuronal proliferation suggests that neuron–astrocyte contact may cooperate with astrocyte soluble factors to enhance neuronal population. Our data reveal an important role of astrocytes as mediators of T3‐induced cerebellar development and partially elucidate the role of cell contact and soluble factors on this process.

Thyroid hormone induces cerebellar neuronal migration and Bergmann glia differentiation through epidermal growth factor/mitogen-activated protein kinase pathway

Cerebellar development in the postnatal period is mainly characterized by an intense cellular proliferation in the external granular layer, followed by migration of granular cells in the molecular layer along the Bergmann glia (BG) fibers. Cerebellar ontogenesis undergoes dramatic modulation by thyroid hormones (THs), although their mechanism of action in this organ is still largely unknown. We previously demonstrated that THs induce astrocytes to secrete epidermal growth factor (EGF), which thus promotes cerebellar neuronal proliferation and extracellular matrix remodeling in vitro. In the present study, we investigated the effect of the TH/EGF pathway on granule neuronal migration. By taking advantage of rat explant and dissociated culture assays, we showed that cerebellar astrocytes treated with TH promote granule cell migration. The addition of neutralizing antibodies against EGF or the pharmacological inhibitor of EGF signaling, bis‐tyrphostin, completely inhibited TH‐astrocyte‐induced migration. Likewise, the addition of EGF itself greatly increased neuronal migration. Treatment of BG‐dissociated cultures by EGF dramatically induced an alteration in cell morphology, characterized by an elongation in the glial process. Both neuronal migration and BG elongation were inhibited by the mitogen‐activated protein kinase pathway inhibitor PD98059, suggesting that these events might be associated. Together, our results suggest that, by inducing EGF secretion, THs promote neuronal migration through BG elongation. Our data provide new clues to the molecular mechanism of THs in cerebellar development, and may contribute to a better understanding of some neuroendocrine disorders associated with migration deficits.

Effects of Transforming Growth Factor Beta 1 in Cerebellar Development: Role in Synapse Formation

Granule cells (GC) are the most numerous glutamatergic neurons in the cerebellar cortex and represent almost half of the neurons of the central nervous system. Despite recent advances, the mechanisms of how the glutamatergic synapses are formed in the cerebellum remain unclear. Among the TGF-β family, TGF-beta 1 (TGF-β1) has been described as a synaptogenic molecule in invertebrates and in the vertebrate peripheral nervous system. A recent paper from our group demonstrated that TGF-β1 increases the excitatory synapse formation in cortical neurons. Here, we investigated the role of TGF-β1 in glutamatergic cerebellar neurons. We showed that the expression profile of TGF-β1 and its receptor, TβRII, in the cerebellum is consistent with a role in synapse formation in vitro and in vivo. It is low in the early postnatal days (P1–P9), increases after postnatal day 12 (P12), and remains high until adulthood (P30). We also found that granule neurons express the TGF-β receptor mRNA and protein, suggesting that they may be responsive to the synaptogenic effect of TGF-β1. Treatment of granular cell cultures with TGF-β1 increased the number of glutamatergic excitatory synapses by 100%, as shown by immunocytochemistry assays for presynaptic (synaptophysin) and post-synaptic (PSD-95) proteins. This effect was dependent on TβRI activation because addition of a pharmacological inhibitor of TGF-β, SB-431542, impaired the formation of synapses between granular neurons. Together, these findings suggest that TGF-β1 has a specific key function in the cerebellum through regulation of excitatory synapse formation between granule neurons.

Pancreatite necro-hemorrágica: atualização e momento de operar

BACKGROUND: Necrotizing pancreatitis represents the most severe form of presentation from the clinical spectra of acute pancreatitis. Although known for many centuries, many questions remain open about this entity and a great number of articles were published about this matter in the last few years. METHOD: A throughout research in the literature, with special attention to the articles published in the last three years and indexed to the PubMed was performed. The following headings were used: pancreatitis, surgical procedures, necrosis. The initial research rendered about 13 000 articles, and the ones published in the last three years were evaluated. Some older, but remarkable articles were also included given their importance to this matter. CONCLUSION: The treatment of acute pancreatitis involves a great number of questions, among which the most important are the ones related to the use of antibiotics, type of diet employed and the questions related to the manangement of the infected necrosis. There were many radical changes instituted in the last years on all these topics and a constant updating must be necessarily done by the ones involved on the treatment of this disease.

Avaliação da proteína acídica fibrilar glial como marcador da injúria por isquemia-reperfusão hepática

OBJECTIVE To evaluate the expression of Glial Fibrillary Acidic Protein (GFAP) after ischemia-reperfusion injury. METHODS twenty four rats were divided into four groups: Control, submitted to anesthesia and liver biopsy; Sham, receiving injection of heparin through the vena cava and hepatic pedicle dissection, with liver biopsy after 24 hours; Ischemia-30, the same as Sham group, plus hepatic pedicle clamping for 30 minutes; and Ischemia-90, the same procedure of Ischemia-30 group, but with clamping period of 90 minutes. After 24 hours of observation, the animals underwent laparotomy and we evaluated their livers macroscopically, microscopically by hematoxylin-eosin (HE) and analyzed the expression of GFAP by Western Blotting. RESULTS There was no difference in the gross appearance of the livers between the different experimental groups, all having demonstrated normal morphology. HE analysis showed no significant differences with respect to lobule morphology. On the other hand, in the ischemia groups we observed neutrophilic infiltrates and small areas of necrosis. GFAP expression was similar in all groups, either qualitatively and quantitatively. CONCLUSION The expression of Glial Fibrillary Acidic Protein did not change in our model of ischemia-reperfusion.