Adrenalectomy enhances endotoxemia-induced hypophagia: higher activation of corticotrophin-releasing-factor and proopiomelanocortin hypothalamic neurons

Inflammatory and infectious processes evoke neuroendocrine and behavioral changes known as acute-phase response that includes activation of the hypothalamo-pituitary-adrenal (HPA) axis and reduction of food intake. Besides its action as the most important ACTH secretagogue, corticotrophin-releasing factor (CRF), synthesized in the paraventricular nucleus (PVN), is also involved in the control of food intake. Alpha-melanocyte stimulating hormone (alpha-MSH) in the arcuate nucleus also plays a role in the energy homeostasis, possessing anorexigenic effects. To investigate the participation of neuropeptides involved in the regulation of food intake during endotoxemia, we administrated lipopolysaccharide (LPS) in sham-operated and adrenalectomized (ADX) male Wistar rats to evaluate food intake, hormone responses and Fos-CRF and Fos-alpha-MSH immunoreactivity in the PVN and arcuate nucleus, as well as CRF and POW mRNA expression in these hypothalamic nuclei. In sham-operated rats, treatment with LPS (100 mu g/kg) showed lower food intake, higher plasma ACTH and corticosterone levels, as well as an increase in Fos-CRF double labeled neurons and CRF mRNA expression in the PVN, with no changes in Fos-alpha-MSH immunoreactivity and POW mRNA expression in the arcuate nucleus, compared to saline treated rats. After LPS treatment, ADX rats showed further increase in plasma ACTH levels, marked decrease of food intake, higher Fos-CRF immunoreactive neurons in the PVN and CRF mRNA expression, as well as an increase in Fos-alpha-MSH immunoreactivity and POW mRNA expression in the arcuate nucleus, compared to sham-operated rats treated with LPS. In conclusion, the present data indicate that the marked hypophagia during endotoxemia following ADX is associated with an increased activation of CRF and POW neurons in the hypothalamus and an increased mRNA expression of these neuropeptides. (C) 2008 Elsevier Inc. All rights reserved.

The A61 G EGF polymorphism is associated with development of extraaxial nervous system tumors but not with overall survival

Epidermal growth factor can activate several signaling pathways, leading to proliferation, differentiation, and tumorigenesis of epithelial tissues by binding with its receptor. The EGF protein is involved in nervous system development, and polymorphisms in the EGF gene on chromosome band 4q25 are associated with brain cancers. The purpose of this study was to investigate the association between the single-nucleotide polymorphism of EGF + 61 G/A and extraaxial brain tumors in a population of the southeast of Brazil. We analyzed the genotype distribution of this polymorphism in 90 patients and 100 healthy subjects, using the polymerase chain reaction restriction fragment length polymorphism technique. Comparison of genotype distribution revealed a significant difference between patients and control subjects (P < 0.001). The variant genotypes of A/G and G/G were associated with a significant increase of the risk of tumor development, compared with the homozygote A/A (P < 0.0001). When the analyses were stratified, we observed that the genotype GIG was more frequent in female patients (P = 0.021). The same genotype was observed more frequently in patients with low-grade tumors (P = 0.001). Overall survival rates did not show statistically significant differences. Our data suggest that the EGF A61 G polymorphism can be associated with susceptibility to development of these tumors. (C) 2010 Elsevier Inc. All rights reserved.

Meningiomas and schwannomas: Molecular subgroup classification found by expression arrays

Microarray gene expression profiling is a high-throughput system used to identify differentially expressed genes and regulation patterns, and to discover new tumor markers. As the molecular pathogenesis of meningiomas and schwannomas, characterized by NF2 gene alterations, remains unclear and suitable molecular targets need to be identified, we used low density cDNA microarrays to establish expression patterns of 96 cancer-related genes on 23 schwannomas, 42 meningiomas and 3 normal cerebral meninges. We also performed a mutational analysis of the NF2 gene (PCR, dHPLC, Sequencing and MLPA), a search for 22q LOH and an analysis of gene silencing by promoter hypermethylation (MS-MLPA). Results showed a high frequency of NF2 gene mutations (40%), increased 22q LOH as aggressiveness increased, frequent losses and gains by MLPA in benign meningiomas, and gene expression silencing by hypermethylation. Array analysis showed decreased expression of 7 genes in meningiomas. Unsupervised analyses identified 2 molecular subgroups for both meningiomas and schwannomas showing 38 and 20 differentially expressed genes, respectively, and 19 genes differentially expressed between the two tumor types. These findings provide a molecular subgroup classification for meningiomas and schwannomas with possible implications for clinical practice.

Relation between alcohol consumption and traffic violations and accidents in the region of Ribeirao Preto, Sao Paulo State

In recent years, alcohol consumption has been considered an important public health problem. Ethanol, the alcohol used in beverages, is a drug that affects the central nervous system (CNS) and impairs driving skills and co-ordination, increasing risk of deaths and injuries derived from crashes and road accidents. Consumption of alcoholic beverages is implicated with premature deaths, injuries and damages caused by motor vehicle crashes, which result in high costs to government and society. Considering that alcohol consumption is the main responsible factor for deaths and disabilities in young people, the aim of this work was to evaluate the prevalence of blood alcohol in offenders and/or fatal and non-fatal victims of traffic occurrences in the region of Ribeirao Preto, Sao Paulo State, from 2005 to 2007. The results revealed that in 2134 cases investigated, blood alcohol positivity was generally found in young adults, 25-45 years old and male. The study showed the high risk of drinking and driving and the importance in establishing actions of prevention and intervention to promote the reduction in the number of traffic occurrences related to consumption of alcoholic beverages. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Proteomic analysis of low- to high-grade astrocytomas reveals an alteration of the expression level of raf kinase inhibitor protein and nucleophosmin

Proteomic approaches have been useful for the identification of aberrantly expressed proteins in complex diseases such as cancer. These proteins are not only potential disease biomarkers, but also targets for therapy. The aim of this study was to identify differentially expressed proteins in diffuse astrocytoma grade II, anaplastic astrocytoma grade III and glioblastoma multiforme grade IV in human tumor samples and in non-neoplastic brain tissue as control using 2-DE and MS. Tumor and control brain tissue dissection was guided by histological hematoxylin/eosin tissue sections to provide more than 90% of tumor cells and astrocytes. Six proteins were detected as up-regulated in higher grade astrocytomas and the most important finding was nucleophosmin (NPM) (p < 0.05), whereas four proteins were down-regulated, among them raf kinase inhibitor protein (RKIP) (p < 0.05). We report here for the first time the alteration of NPM and RKIP expression in brain cancer. Our focus on these proteins was due to the fact that they are involved in the PI3K/AKT/mTOR and RAS/RAF/MAPK pathways, known for their contribution to the development and progression of gliomas. The proteomic data for NPM and RKIP were confirmed by Western blot, quantitative real-time PCR and immunohistochemistry. Due to the participation of NPM and RKIP in uncontrolled proliferation and evasion of apoptosis, these proteins are likely targets for drug development.

An Experimental Model of Meningoencephalomyelitis by Rocio Flavivirus in Balb/C Mice: Inflammatory Response, Cytokine Production, and Histopathology

Rocio virus (ROCV) is a flavivirus, probably transmitted by Culex mosquitoes and maintained in nature as a zoonosis of wild birds. Rocio virus caused a human epidemic of severe encephalitis that lasted from 1973 to 1980 in the Ribeira valley, in the southeastern coast of Brazil. After this outbreak, serologic evidence of ROCV circulation has been reported and public health authorities are concerned about a return of ROCV outbreaks in Brazil. We show here a study on the pathogenesis and the physiopathology of ROCV disease in the central nervous system of a Balb/C young adult mice experimental model. The animals were intraperitoneally infected by ROCV and followed from 0 to 9 days after infection, when all of them died. Nervous tissue samples were collected from infected animals for immunohistochemistry and molecular biology analysis. We observed the virus in the central nervous system, the inflammatory changes induced by Th1 and Th2 cytokines, and the final irreversible damage of nervous tissues by neuronal degeneration and apoptosis. These findings can help to better understand the pathogenesis and physiopathology of the human meningoencephalomyelitis by ROCV and other flaviviruses.

Immunolocalization of nitric oxide synthase isoforms in human archival and rat tissues, and cultured cells

Nitric oxide (NO) exerts important physiological and pathological roles in humans. The study of NO requires the immunolocalization of its synthesizing enzymes, neuronal, endothelial and inducible NO synthases (NOS). NOS are labile to formalin-fixation and paraffin-embedding, which are used to prepare human archival tissues. This lability has made NOS immunohistochemical studies difficult, and a detailed protocol is not yet available. We describe here a protocol for the immunolocalization of NOS isoforms in human archival cerebellum and non-nervous tissues, and in rat tissues and cultured cells. Neuronal NOS antigenicity in human archival and rat nervous tissue sections was microwave-retrieved in 50 mM Tris-HCl buffer, pH 9.5, for 20 min at 900W. Neuronal NOS was expressed in stellate, basket, Purkinje and granule cells in human and rat cerebellum. Archival and frozen human cerebellar sections showed the same neuronal NOS staining pattern. Archival cerebellar sections not subjected to antigen retrieval stained weakly. Antigenicity of inducible NOS in human lung was best retrieved in 10 mM sodium citrate buffer, pH 6.0, for 15 min at 900W. Inflammatory cells in a human lung tuberculoma were strongly stained by anti-inducible NOS antibody. Anti-endothelial NOS strongly stained kidney glomeruli. Cultured PC12 cells were strongly stained by anti-neuronal NOS without antigen retrieving. The present immunohistochemistry protocol is easy to perform, timeless, and suitable for the localization of NOS isoforms in nervous and non-nervous tissues, in human archival and rat tissues. It has been extensively used in our laboratory, and is also appropriate for other antigens. (C) 2011 Elsevier B.V. All rights reserved.

Drosophila melanogaster lipins are tissue-regulated and developmentally regulated and present specific subcellular distributions

Lipins constitute a novel family of Mg2+-dependent phosphatidate phosphatases that catalyze the dephosphorylation of phosphatidic acid to yield diacylglycerol, an important intermediate in lipid metabolism and cell signaling. Whereas a single lipin is detected in less complex organisms, in mammals there are distinct lipin isoforms and paralogs that are differentially expressed among tissues. Compatible with organism tissue complexity, we show that the single Drosophila Lpin1 ortholog (CG8709, here named DmLpin) expresses at least three isoforms (DmLpinA, DmLpinK and DmLpinJ) in a temporal and spatially regulated manner. The highest levels of lipin in the fat body, where DmLpinA and DmLpinK are expressed, correlate with the highest levels of triacylglycerol (TAG) measured in this tissue. DmLpinK is the most abundant isoform in the central nervous system, where TAG levels are significantly lower than in the fat body. In the testis, where TAG levels are even lower, DmLpinJ is the predominant isoform. Together, these data suggest that DmLpinA might be the isoform that is mainly involved in TAG production, and that DmLpinK and DmLpinJ could perform other cellular functions. In addition, we demonstrate by immunofluorescence that lipins are most strongly labeled in the perinuclear region of the fat body and ventral ganglion cells. In visceral muscles of the larval midgut and adult testis, lipins present a sarcomeric distribution. In the ovary chamber, the lipin signal is concentrated in the internal rim of the ring canal. These specific subcellular localizations of the Drosophila lipins provide the basis for future investigations on putative novel cellular functions of this protein family.

miR-29b and miR-125a Regulate Podoplanin and Suppress Invasion in Glioblastoma

Glioblastoma is the most frequent and malignant brain tumor, characterized by an elevated capacity for cellular proliferation and invasion. Recently, it was demonstrated that podoplanin membrane sialo-glycoprotein encoded by PDPN gene is over-expressed and related to cellular invasion in astrocytic tumors; however the mechanisms of regulation are still unknown. MicroRNAs are noncoding RNAs that regulate gene expression and several biological processes and diseases, including cancer. Nevertheless, their roles in invasion, proliferation, and apoptosis of glioblastoma are not completely understood. In this study, we focused on miR-29b and miR-125a, which were predicted to regulate PDPN, and demonstrated that these microRNAs directly target the 30 untranslated region of PDPN and inhibit invasion, apoptosis, and proliferation of glioblastomas. Furthermore, we report that miR-29b and miR-125a are downregulated in glioblastomas and also in CD133-positive cells. Taken together, these results suggest that miR-29b and miR-125a represent potential therapeutic targets in glioblastoma. (C) 2010 Wiley-Liss, Inc.

Co-Expression of Leptin and Oestrogen Receptors in the Preoptic-Hypothalamic Area

The interaction between the reproductive axis and energy balance suggests that leptin acts as a possible mediator. This hormone acts in the regulation of metabolism, feeding behaviour and reproduction. Animals homozygous for the gene `ob` (ob/ob) are obese and infertile, and these effects are reversed after systemic administration of leptin. Thus, the present study aimed to determine: (i) whether cells that express leptin also express oestrogen receptors of type-alpha (ER-alpha) or -beta (ER-beta) in the medial preoptic area (MPOA) and in the arcuate (ARC), dorsomedial (DMH) and ventromedial hypothalamic nucleus and (ii) whether there is change in the gene and protein expression of leptin in these brain areas in ovariectomised (OVX) animals when oestrogen-primed. Wistar female rats with normal oestrous cycles or ovariectomised oestrogen-primed or vehicle (oil)-primed were utilised. To determine whether there was a co-expression, immunofluorescence was utilised for double staining. Confocal microscopy was used to confirm the co-expression. The technique of real-time polymerase chain reaction and western blotting were employed to analyse gene and protein expression, respectively. The results obtained showed co-expression of leptin and ER-alpha in the MPOA and in the DMH, as well as leptin and ER-beta in the MPOA, DMH and ARC. However, we did not detect leptin in the MPOA, ARC and DMH using western blotting and there was no statistical difference in leptin gene expression in the MPOA, DMH, ARC, pituitary or adipose tissue between OVX rats treated with oestrogen or vehicle. In conclusion, the results obtained in the present study confirm that the brain is also a source of leptin and reveal co-expression of oestrogen receptors and leptin in the same cells from areas related to reproductive function and feeding behaviour. Although these data corroborate the previous evidence obtained concerning the interaction between the action of brain leptin and reproductive function, the physiological relevance of this interaction remains uncertain and additional studies are necessary to elucidate the exact role of central leptin.

The GABAergic system contributes to the anxiolytic-like effect of essential oil from Cymbopogon citratus (lemongrass)

Ethnopharmacological relevance: The essential oil (EO) from Cymbopogon citratus (DC) Stapf is reported to have a wide range of biological activities and is widely used in traditional medicine as an infusion or decoction. However, despite this widely use, there are few controlled studies confirming its biological activity in central nervous system. Materials and methods: The anxiolytic-like activity of the EO was investigated in light/dark box (LDB) and marble-burying test (MBT) and the antidepressant activity was investigated in forced-swimming test (FST) in mice. Flumazenil, a competitive antagonist of benzodiazepine binding and the selective 5-HT(1A) receptor antagonist WAY100635 was used in experimental procedures to determine the action mechanism of EO. To exclude any false positive results in experimental procedures, mice were submitted to the rota-rod test. We also quantified some neurotransmitters at specific brain regions after EO oral acute treatment. Results: The present work found anxiolytic-like activity of the EO at the dose of 10 mg/kg in a LDB. Flumazenil, but not WAY100635, was able to reverse the effect of the EO in the LOB, indicating that the EO activity occurs via the GABA(A) receptor-benzodiazepine complex. Only at higher doses did the EO potentiate diethyl-ether-induced sleeping time in mice. In the FST and MBT, EO showed no effect. Finally, the increase in time spent in the light chamber, demonstrated by concomitant treatment with ineffective doses of diazepam (DZP) and the EO, revealed a synergistic effect of the two compounds. The lack of activity after long-term treatment in the LDB test might be related to tolerance induction, even in the DZP-treated group. Furthermore, there were no significant differences between groups after either acute or repeated treatments with the EO in the rota-rod test. Neurochemical evaluation showed no amendments in neurotransmitter levels evaluated in cortex, striatum, pons, and hypothalamus. Conclusions: The results corroborate the use of Cymbopogon citratus in folk medicine and suggest that the anxiolytic-like effect of its EO is mediated by the GABA(A) receptor-benzodiazepine complex. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

Behavior: A Relevant Tool for Brain-immune System Interaction Studies

Neuroimmunomodulation describes the field focused on understanding the mechanisms by which the central nervous system interacts with the immune system, potentially leading to changes in animal behavior. Nonetheless, not many articles dealing with neuroimmunomodulation employ behavior as an analytical endpoint. Even fewer papers deal with social status as a possible modifier of neuroimmune phenomena. In the described sets of experiments, we tackle both, using a paradigm of social dominance and subordination. We first review data on the effects of different ranks within a stable hierarchical relationship. Submissive mice in this condition display more anxiety-like behaviors, have decreased innate immunity, and show a decreased resistance to implantation and development of melanoma metastases in their lungs. This suggests that even in a stable, social, hierarchical rank, submissive animals may be subjected to higher levels of stress, with putative biological relevance to host susceptibility to disease. Second, we review data on how dominant and submissive mice respond differentially to lipopolysaccharide (LPS), employing a motivational perspective to sickness behavior. Dominant animals display decreased number and frequency in several aspects of behavior, particularly agonistic social interaction, that is, directed toward the submissive cage mate. This was not observed in submissive mice that maintained the required behavior expected by its dominant mate. Expression of sickness behavior relies on motivational reorganization of priorities, which are different along different social ranks, leading to diverse outcomes. We suggest that in vitro assessment of neuroimmune phenomena can only be understood based on the behavioral context in which they occur.

The neuroprotective effect of dental pulp cells in models of Alzheimer`s and Parkinson`s disease

Aim of the present study was to investigate the neuroprotective effect of dental pulp cells (DPCs) in in vitro models of Alzheimer and Parkinson disease. Primary cultures of hippocampal and ventral mesencephalic neurons were treated for 24 h with amyloid beta (A beta(1-42)) peptide 1-42 and 6-OHDA, respectively. DPCs isolated from adult rat incisors were previously cultured in tissue culture inserts and added to the neuron cultures 2 days prior to neurotoxin treatment. Cell viability was assessed by the MTT assay. The co-culture with DPCs significantly attenuated 6-OHDA and A beta(1-42)-induced toxicity in primary cultures of mesencephalic and hippocampal neurons, and lead to an increase in neuronal viability in untreated cultures, suggesting a neurotrophic effect in both models. Furthermore, human dental pulp cells expressed a neuronal phenotype and produced the neurotrophic factors NGF, GDNF, BDNF, and BMP2 shown by microarray screening and antibody staining for the representative proteins. DPCs protected primary neurons in in vitro models of Alzheimer`s and Parkinson`s disease and can be viewed as possible candidates for studies on cell-based therapy.

Central NOS inhibition differentially affects vasopressin gene expression in hypothalamic nuclei in septic rats

Our aim was to investigate the effect of central NOS inhibition on hypothalamic arginine vasopressin (AVP) gene expression, hormone release and on the cardiovascular response during experimental sepsis. Male Wistar rats were intracerebroventricularly injected with the non-selective NO synthase (NOS) inhibitor (L-NAME) or aminoguanidine, a selective inhibitor of the inducible isoform (iNOS). After 30 min. sepsis was induced by cecal ligation and puncture (CLP) causing an increase in heart rate (HR), as well as a reduction in median arterial pressure (MAP) and AVP expression ratio (AVP(R)), mainly in the supraoptic nucleus. AVP plasma levels (AVP(P)) increased in the early but not in the late phase of sepsis. L-NAME pretreatment increased MAP but did not change HR. It also resulted in an increase in AVP(P) at all time points, except 24 h, when it returned to basal levels. AVP(R), however remained reduced in both nuclei. Aminoguanidine pretreatment resulted in increased MAP in the early phase and higher AVP(R) in the supraoptic, but not in the paraventricular nucleus, while AVP(P) remained elevated at all time points. We suggest that increased central NO production, mainly inducible NOS-derived, reduces AVP gene expression differentially in supraoptic and paraventricular nuclei, and that this may contribute to low AVP plasma levels and hypotension in the late phase of sepsis. (c) 2010 Elsevier B.V. All rights reserved.

Neonatal endotoxin exposure changes neuroendocrine, cardiovascular function and mortality during polymicrobial sepsis in adult rats

Our aim was to investigate whether neonatal LPS challenge may improve hormonal, cardiovascular response and mortality, this being a beneficial adaptation when adult rats are submitted to polymicrobial sepsis by cecal ligation and puncture (CLP). Fourteen days after birth, pups received an intraperitoneal injection of lipopolysaccharide (LPS; 100 mu g/kg) or saline. After 8-12 weeks, they were submitted to CLP, decapitated 4,6 or 24 h after surgery and blood was collected for vasopressin (AVP), corticosterone and nitrate measurement, while AVP contents were measured in neurohypophysis, supra-optic (SON) and paraventricular (PVN) nuclei. Moreover, rats had their mean arterial pressure (MAP) and heart rate (HR) evaluated, and mortality and bacteremia were determined at 24 h. Septic animals with neonatal LPS exposure had higher plasma AVP and corticosterone levels, and higher c-Fos expression in SON and PVN at 24 h after surgery when compared to saline treated rats. The LPS pretreated group showed increased AVP content in SON and PVN at 6 h, while we did not observe any change in neurohypophyseal AVP content. The nitrate levels were significantly reduced in plasma at 6 and 24 h after surgery, and in both hypothalamic nuclei only at 6 h. Septic animals with neonatal LPS exposure showed increase in MAP during the initial phase of sepsis, but HR was not different from the neonatal saline group. Furthermore, neonatally LPS exposed rats showed a significant decrease in mortality rate as well as in bacteremia. These data suggest that neonatal LPS challenge is able to promote beneficial effects on neuroendocrine and cardiovascular responses to polymicrobial sepsis in adulthood. (C) 2011 Elsevier B.V. All rights reserved.