Stem cells in endometrium and their role in the pathogenesis of endometriosis

The human endometrium is a dynamic tissue that undergoes cycles of growth and regression with each menstrual cycle. Adult progenitor stem cells are likely responsible for this remarkable regenerative capacity; these same progenitor stem cells may also have an enhanced capacity to generate endometriosis if shed in a retrograde fashion. The progenitor stem cells reside in the uterus; however, less-committed mesenchymal stem cells may also travel from other tissues such as bone marrow to repopulate the progenitor population. Mesenchymal stem cells are also involved in the pathogenesis of endometriosis and may be the principle source of endometriosis outside of the peritoneal cavity when they differentiate into endometriosis in ectopic locations. Finally, besides progenitor stem cells, recent publications have identified multipotent stem cells in the endometrium. These multipotent stem cells are a readily available source of cells that are useful in tissue engineering and regenerative medicine. Endometrial stem cells have been used to generate chondrocytes, myocytes, neurons, and adiposites in vitro as well as to replace dopaminergic neurons in a murine model of Parkinson`s disease.

DNA polymorphisms as tools for spinal cord injury research

Study Design: Data mining of single nucleotide polymorphisms (SNPs) in gene pathways related to spinal cord injury (SCI). Objectives: To identify gene polymorphisms putatively implicated with neuronal damage evolution pathways, potentially useful to SCI study. Setting: Departments of Psychiatry and Orthopedics, Faculdade de Medicina, Universidade de Sao Paulo, Brazil. Methods: Genes involved with processes related to SCI, such as apoptosis, inflammatory response, axonogenesis, peripheral nervous system development and axon ensheathment, were determined by evaluating the `Biological Process` annotation of Gene Ontology (GO). Each gene of these pathways was mapped using MapViewer, and gene coordinates were used to identify their polymorphisms in the SNP database. As a proof of concept, the frequency of subset of SNPs, located in four genes (ALOX12, APOE, BDNF and NINJ1) was evaluated in the DNA of a group of 28 SCI patients and 38 individuals with no SC lesions. Results: We could identify a total of 95 276 SNPs in a set of 588 genes associated with the selected GO terms, including 3912 nucleotide alterations located in coding regions of genes. The five non-synonymous SNPs genotyped in our small group of patients, showed a significant frequency, reinforcing their potential use for the investigation of SCI evolution. Conclusion: Despite the importance of SNPs in many aspects of gene expression and protein activity, these gene alterations have not been explored in SCI research. Here we describe a set of potentially useful SNPs, some of which could underlie the genetic mechanisms involved in the post trauma spinal cord damage.

Stereologic investigation of the posterior part of the hippocampus in schizophrenia

Structural magnetic resonance imaging and postmortem studies showed volume loss in the hippocampus in schizophrenia. The noted tissue reduction in the posterior section suggests that some cellular subfractions within this structure might be reduced in schizophrenia. To address this, we investigated numbers and densities of neurons, oligodendrocytes and astrocytes in the posterior hippocampal subregions in postmortem brains from ten patients with schizophrenia and ten matched controls using design-based stereology performed on Nissl-stained sections. Compared to the controls, the patients with schizophrenia showed a significant decrease in the mean number of oligodendrocytes in the left and right CA4. This is the first finding of reduced numbers of oligodendrocytes in CA4 of the posterior part of the hippocampus in schizophrenia. Our results are in line with earlier findings in the literature concerning decreased numbers of oligodendrocytes in the prefrontal cortex in schizophrenia. Our results may indicate disturbed connectivity of the CA4 of the posterior part of the hippocampus in schizophrenia and, thus, contribute to the growing number of studies showing the involvement of posterior hippocampal pathology in the pathophysiology of schizophrenia.

In situ apoptosis of adaptive immune cells and the cellular escape of rabies virus in CNS from patients with human rabies transmitted by Desmodus rotundus

The aim of the current study was to investigate the apoptosis of neurons, astrocytes and immune cells from human patients that were infected with rabies virus by vampire bats bite. Apoptotic neurons were identified by their morphology and immune cells were identified using double immunostaining. There were very few apoptotic neurons present in infected tissue samples, but there was an increase of apoptotic infiltrating CD4+ and TCD8+ adaptive immune cells in the rabies infected tissue. No apoptosis was present in NK, macrophage and astrocytes. The dissemination of the human rabies virus within an infected host may be mediated by viral escape of the virus from an infected cell and may involve an anti-apoptotic mechanism, which does not kill the neuron or pro-apoptosis of TCD4+ and TCD8+ lymphocytes and which allows for increased proliferation of the virus within the CNS by attenuation of the adaptive immune response. (C) 2011 Elsevier B.V. All rights reserved.

Equivalent Neurogenic Potential of Wild-Type and GFP-Labeled Fetal-Derived Neural Progenitor Cells Before and After Transplantation Into the Rodent Hippocampus

Introduction. The hippocampal formation is a specific structure in the brain where neurogenesis occurs throughout adulthood and in which the neuronal cell loss causes various demential states. The main goal of this study was to verify whether fetal neural progenitor cells (NPCs) from transgenic rats expressing green fluorescent protein (GFP) retain the ability to differentiate into neuronal cells and to integrate into the hippocampal circuitry after transplantation. Methods. NPCs were isolated from E14 (gestational age: 14 days postconception) transgenic-Lewis and wild-type Sprague-Dawley rat embryos. Wild-type and transgenic cells were expanded and induced to differentiate into a neuronal lineage in vitro. Immunocytochemical and electrophysiological analysis were performed in both groups. GFP-expressing cells were implanted into the hippocampus and recorded electrophysiologically 3 months thereafter. Immunohistochemical analysis confirmed neuronal differentiation, and the yield of neuronal cells was determined stereologically. Results. NPCs derived from wild-type and transgenic animals are similar regarding their ability to generate neuronal cells in vitro. Neuronal maturity was confirmed by immunocytochemistry and electrophysiology, with demonstration of voltage-gated ionic currents, firing activity, and spontaneous synaptic currents. GFP-NPCs were also able to differentiate into mature neurons after implantation into the hippocampus, where they formed functional synaptic contacts. Conclusions. GFP-transgenic cells represent an important tool in transplantation studies. Herein, we demonstrate their ability to generate functional neurons both in vitro and in vivo conditions. Neurons derived from fetal NPCs were able to integrate into the normal hippocampal circuitry. The high yield of mature neurons generated render these cells important candidates for restorative approaches based on cell therapy.

Circulating levels of brain-derived neurotrophic factor: correlation with mood, cognition and motor function

Brain-derived neurotrophic factor (BDNF) is the most widely distributed neurotrophin in the CNS, where it plays several pivotal roles in synaptic plasticity and neuronal survival. As a consequence, BDNF has become a key target in the physiopathology of several neurological and psychiatric diseases. Recent studies have consistently reported altered levels of BDNF in the circulation (i.e., serum or plasma) of patients with major depression, bipolar disorder, Alzheimer`s disease, Huntington`s disease and Parkinson`s disease. Correlations between serum BDNF levels and affective, cognitive and motor symptoms have also been described. BDNF appears to be an unspecific biomarker of neuropsychiatric disorders characterized by neurodegenerative changes.

Inhibition of phospholipase A(2) in rat brain decreases the levels of total Tau protein

The microtubule-associated protein Tau promotes the assembly and stability of microtubules in neuronal cells. Six Tau isoforms are expressed in adult human brain. All six isoforms become abnormally hyperphosphorylated and form neurofibrillary tangles in Alzheimer disease (AD) brains. In AD, reduced activity of phospholipase A(2) (PLA(2)), specifically of calcium-dependent cytosolic PLA(2) (cPLA(2)) and calcium-independent intracellular PLA(2) (iPLA(2)), was reported in the cerebral cortex and hippocampus, which positively correlated with the density of neurofibrillary tangles. We previously demonstrated that treatment of cultured neurons with a dual cPLA(2) and iPLA(2) inhibitor, methyl arachidonyl fluorophosphonate (MAFP), decreased total Tau levels and increased Tau phosphorylation at Ser(214) site. The aim of this study was to conduct a preliminary investigation into the effects of in vivo infusion of MAFP into rat brain on PLA(2) activity and total Tau levels in the postmortem frontal cortex and dorsal hippocampus. PLA(2) activity was measured by radioenzymatic assay and Tau levels were determined by Western blotting using the anti-Tau 6 isoforms antibody. MAFP significantly inhibited PLA(2) activity in the frontal cortex and hippocampus. The reactivity to the antibody revealed three Tau protein bands with apparent molecular weight of close to 40, 43 and 46 kDa in both brain areas. MAFP decreased the 46 kDa band intensity in the frontal cortex, and the 43 and 46 kDa band intensities in the hippocampus. The results indicate that in vivo PLA(2) inhibition in rat brain decreases the levels of total (nonphosphorylated plus phosphorylated) Tau protein and corroborate our previous in vitro findings.

Inhibition of motor cortex excitability with 15 Hz transcranial alternating current stimulation (tACS)

There remains a lack of solid evidence showing whether transcranial stimulation with weak alternating current (transcranial alternating current stimulation, tACS) can in fact induce significant neurophysiological effects. Previously, a study in which tACS was applied for 2 and 5 min with current density = 0.16-0.25 A/m(2) was unable to show robust effects on cortical excitability. Here we applied tACS at a significantly higher current density (0.80 A/m(2)) for a considerably longer duration (20 min) and were indeed able to demonstrate measurable changes to cortical excitability. Our results show that active 15 Hz tACS of the motor cortex (electrodes placed at C3 and C4) significantly diminished the amplitude of motor evoked potentials and decreased intracortical facilitation (ICF) as compared to baseline and sham stimulation. In addition, we show that our method of sham tACS is a reliable control condition. These results support the notion that AC stimulation with weak currents can induce significant changes in brain excitability; in this case, 15 Hz tACS led to a pattern of inhibition of cortical excitability. We propose that tACS may have a dampening effect on cortical networks and perhaps interfere with the temporal and spatial summation of weak subthreshold electric potentials. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Individual vulnerability to escalated aggressive behavior by a low dose of alcohol: decreased serotonin receptor mRNA in the prefrontal cortex of male mice

Low to moderate doses of alcohol consumption induce heightened aggressive behavior in some, but not all individuals. Individual vulnerability for this nonadaptive behavior may be determined by an interaction of genetic and environmental factors with the sensitivity of alcohol`s effects on brain and behavior. We used a previously established protocol for alcohol oral self-administration and characterized alcohol-heightened aggressive (AHA) mice as compared with alcohol non-heightened (ANA) counterparts. A week later, we quantified mRNA steady state levels of several candidate genes in the serotonin [5-hydroxytryptamine (5-HT)] system in different brain areas. We report a regionally selective and significant reduction of all 5-HT receptor subtype transcripts, except for 5-HT(3), in the prefrontal cortex of AHA mice. Comparable gene expression profile was previously observed in aggressive mice induced by social isolation or by an anabolic androgenic steroid. Additional change in the 5-HT(1B) receptor transcripts was seen in the amygdala and hypothalamus of AHA mice. In both these areas, 5-HT(1B) mRNA was elevated when compared with ANA mice. In the hypothalamus, AHA mice also showed increased transcripts for 5-HT(2A) receptor. In the midbrain, 5-HT synthetic enzyme, 5-HT transporter and 5-HT receptors mRNA levels were similar between groups. Our results emphasize a role for postsynaptic over presynaptic 5-HT receptors in mice which showed escalated aggression after the consumption of a moderate dose of alcohol. This gene expression profile of 5-HT neurotransmission components in the brain of mice may suggest a vulnerability trait for alcohol-heightened aggression.

Equal Numbers of Neuronal and Nonneuronal Cells Make the Human Brain an Isometrically Scaled-Up Primate Brain

The human brain is often considered to be the most cognitively capable among mammalian brains and to be much larger than expected for a mammal of our body size. Although the number of neurons is generally assumed to be a determinant of computational power, and despite the widespread quotes that the human brain contains 100 billion neurons and ten times more glial cells, the absolute number of neurons and glial cells in the human brain remains unknown. Here we determine these numbers by using the isotropic fractionator and compare them with the expected values for a human-sized primate. We find that the adult male human brain contains on average 86.1 +/- 8.1 billion NeuN-positive cells (""neurons"") and 84.6 +/- 9.8 billion NeuN-negative (""nonneuronal"") cells. With only 19% of all neurons located in the cerebral cortex, greater cortical size (representing 82% of total brain mass) in humans compared with other primates does not reflect an increased relative number of cortical neurons. The ratios between glial cells and neurons in the human brain structures are similar to those found in other primates, and their numbers of cells match those expected for a primate of human proportions. These findings challenge the common view that humans stand out from other primates in their brain composition and indicate that, with regard to numbers of neuronal and nonneuronal cells, the human brain is an isometrically scaled-up primate brain. J. Comp. Neurol. 513:532-541, 2009. (c) 2009 Wiley-Liss, Inc.

Mouse Leydig cells express multiple P2X receptor subunits

ATP acts on cellular membranes by interacting with P2X (ionotropic) and P2Y (metabotropic) receptors. Seven homomeric P2X receptors (P2X(1)-P2X(7)) and seven heteromeric receptors (P2X(1/2), P2X(1/4), P2X(1/5), P2X(2/3), P2X(2/6), P2X(4/6), P2X(4/7)) have been described. ATP treatment of Leydig cells leads to an increase in [Ca(2+)](i) and testosterone secretion, supporting the hypothesis that Ca(2+) signaling through purinergic receptors contributes to the process of testosterone secretion in these cells. Mouse Leydig cells have P2X receptors with a pharmacological and biophysical profile resembling P2X(2). In this work, we describe the presence of several P2X receptor subunits in mouse Leydig cells. Western blot experiments showed the presence of P2X(2), P2X(4), P2X(6), and P2X(7) subunits. These results were confirmed by immunofluorescence. Functional results support the hypothesis that heteromeric receptors are present in these cells since 0.5 mu M ivermectin induced an increase (131.2 +/- 5.9%) and 3 mu M ivermectin a decrease (64.2 +/- 4.8%) in the whole-cell currents evoked by ATP. These results indicate the presence of functional P2X(4) subunits. P2X(7) receptors were also present, but they were non-functional under the present conditions because dye uptake experiments with Lucifer yellow and ethidium bromide were negative. We conclude that a heteromeric channel, possibly P2X(2/4/6), is present in Leydig cells, but with an electrophysiological and pharmacological phenotype characteristic of the P2X(2) subunit.

Descriptive findings on the morphology of dendritic spines in the rat medial amygdala

The rat posterodorsal medial amygdala (MePD) is a brain area in which gonadal hormones induce notable plastic effects in the density of dendritic spines. Dendritic spines are post-synaptic specializations whose shape and spacing change neuronal excitability. Our aim was to obtain new data on the dendritic spines morphology and density from MePD neurons using the carbocyanine dye Dil under confocal microscopy. In adult male rats, the dendritic spine density of the medial branches of the left MePD (mean +/- SD) was 1.15 +/- 0.67 spines/dendritic mu m. From the total sampled, approximately 53% of the spines were classified as thin, 22.5% as ""mushroom-like"", and 21.5% as stubby/wide. Other spine shapes (3%) included those ramified, with a filopodium-like or a gemule appearance, and others with a protruding spinule. Additional experiment joining Dil and synaptophysin (a pre-synaptic protein) labeling suggested synaptic sites on dendritic shafts and spines. Dendritic spines showed synaptophysin puncta close to their head and neck, although some spines had no evident labeled puncta on them or, conversely, multiple puncta appeared upon one spine. These results advance previous light microscopy results by revealing features and complexities of the dendritic spines at the same time that give new insight on the possible synaptic organization of the adult rat MePD. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

The ""single-section"" Golgi method adapted for formalin-fixed human brain and light microscopy

The Golgi method has been used for over a century to describe the general morphology of neurons in the nervous system of different species. The ""single-section"" Golgi method of Gabbott and Somogyi (1984) and the modifications made by Izzo et al. (1987) are able to produce consistent results. Here, we describe procedures to show cortical and subcortical neurons of human brains immersed in formalin for months or even years. The tissue was sliced with a vibratome, post-fixed in a combination of paraformaldehyde and picric acid in phosphate buffer, followed by osmium tetroxide and potassium dicromate, ""sandwiched"" between cover slips, and immersed in silver nitrate. The whole procedure takes between 5 and 11 days to achieve good results. The Golgi method has its characteristic pitfalls but, with this procedure, neurons and glia appear well-impregnated, allowing qualitative and quantitative studies under light microscopy. This contribution adds to the basic techniques for the study of human nervous tissue with the same advantages described for the ""single-section"" Golgi method in other species; it is easy and fast, requires minimal equipment, and provides consistent results. (C) 2010 Elsevier B.V. All rights reserved.

NOVEL 65 kDa RNA-BINDING PROTEIN IN SQUID PRESYNAPTIC TERMINALS

A polyclonal antibody (C4), raised against the head domain of chicken myosin Va, reacted strongly towards a 65 kDa polypeptide (p65) on Western blots of extracts from squid optic lobes but did not recognize the heavy chain of squid myosin V. This peptide was not recognized by other myosin Va antibodies, nor by an antibody specific for squid myosin V. In an attempt to identify it, p65 was purified from optic lobes of Loligo plei by cationic exchange and reverse phase chromatography. Several peptide sequences were obtained by mass spectroscopy from p65 cut from sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gels. BLAST analysis and partial matching with expressed sequence tags (ESTs) from a Loligo pealei data bank indicated that p65 contains consensus signatures for the heterogeneous nuclear ribonucleoprotein (hnRNP) A/B family of RNA-binding proteins. Centrifugation of post mitochondrial extracts from optic lobes on sucrose gradients after treatment with RNase gave biochemical evidence that p65 associates with cytoplasmic RNP complexes in an RNA-dependent manner. Immunohistochemistry and immunofluorescence studies using the C4 antibody showed partial co-labeling with an antibody against squid synaptotagmin in bands within the outer plexiform layer of the optic lobes and at the presynaptic zone of the stellate ganglion. Also, punctate labeling by the C4 antibody was observed within isolated optic lobe synaptosomes. The data indicate that p65 is a novel RNA-binding protein located to the presynaptic terminal within squid neurons and may have a role in synaptic localization of RNA and its translation or processing. (C) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

Synaptic transmission block by presynaptic injection of oligomeric amyloid beta

Early Alzheimer`s disease (AD) pathophysiology is characterized by synaptic changes induced by degradation products of amyloid precursor protein (APP). The exact mechanisms of such modulation are unknown. Here, we report that nanomolar concentrations of intraaxonal oligomeric (o)A beta 42, but not oA beta 40 or extracellular oA beta 42, acutely inhibited synaptic transmission at the squid giant synapse. Further characterization of this phenotype demonstrated that presynaptic calcium currents were unaffected. However, electron microscopy experiments revealed diminished docked synaptic vesicles in oA beta 42-microinjected terminals, without affecting clathrin-coated vesicles. The molecular events of this modulation involved casein kinase 2 and the synaptic vesicle rapid endocytosis pathway. These findings open the possibility of a new therapeutic target aimed at ameliorating synaptic dysfunction in AD.