Increased expression of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in reactive astrocytes following ischemia.

Astrocytes respond to ischemic brain injury by proliferation, the increased expression of intermediate filaments and hypertrophy, which results in glial scar formation. In addition, they alter the expression of ion channels, receptors and transporters that maintain ionic/neurotransmitter homeostasis. Here, we aimed to demonstrate the expression of Hcn1-4 genes encoding hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in reactive astrocytes following focal cerebral ischemia (FCI) or global cerebral ischemia (GCI) and to characterize their functional properties. A permanent occlusion of the middle cerebral artery (MCAo) was employed to induce FCI in adult GFAP/EGFP mice, while GCI was induced by transient bilateral common carotid artery occlusion combined with hypoxia in adult rats. Using FACS, we isolated astrocytes from non-injured or ischemic brains and performed gene expression profiling using single-cell RT-qPCR. We showed that 2 weeks after ischemia reactive astrocytes express high levels of Hcn1-4 transcripts, while immunohistochemical analyses confirmed the presence of HCN1-3 channels in reactive astrocytes 5 weeks after ischemia. Electrophysiological recordings revealed that post-ischemic astrocytes are significantly depolarized, and compared with astrocytes from non-injured brains, they display large hyperpolarization-activated inward currents, the density of which increased 2-3-fold in response to ischemia. Their activation was facilitated by cAMP and their amplitudes were decreased by ZD7288 or low extracellular Na(+) concentration, suggesting that they may belong to the family of HCN channels. Collectively, our results demonstrate that regardless of the type of ischemic injury, reactive astrocytes express HCN channels, which could therefore be an important therapeutic target in poststroke therapy.

Evaluation of tumor suppressor gene expressions and aberrant methylation in the colon of cancer-induced rats: a pilot study.

Altered expression and methylation pattern of tumor suppressor and DNA repair genes, in particular involved in mismatch repair (MMR) pathway, frequently occur in primary colorectal (CRC) tumors. However, little is known about (epi)genetic changes of these genes in precancerous and early stages of CRC. The aim of this pilot study was to analyze expression profile and promoter methylation status of important tumor suppressor and DNA repair genes in the early stages of experimentally induced colorectal carcinogenesis. Rats were treated with azoxymethane (AOM), dextran sodium sulphate (DSS) or with their combination, and sacrificed 1 or 4 months post-treatment period. The down-regulation of Apc expression in left colon, detectable in animals treated with DSS-AOM and sacrificed 1 month after the end of treatment, represents most early marker of the experimental colorectal carcinogenesis. Significantly reduced gene expressions were also found in 5 out of 7 studied MMR genes (Mlh1, Mlh3, Msh3 Pms1, Pms2), regarding the sequential administration of DSS-AOM at 4 months since the treatment. Strong down-regulation was also discovered for Apc, Apex1, Mgmt and TP53. Tumors developed in rectum-sigmoid region displayed significantly lower Apc and Pms2 expressions. The decreased expression of studied genes was not in any case associated with aberrant methylation of promoter region. Present data suggest that down-regulation of Apc and MMR genes are prerequisite for the development of CRC. In this study we addressed for the first time early functional alterations of tumor suppressor genes with underlying epigenetic mechanisms in experimentally induced CRC in rats.

Heterogeneity of astrocytes: from development to injury - single cell gene expression.

Astrocytes perform control and regulatory functions in the central nervous system; heterogeneity among them is still a matter of debate due to limited knowledge of their gene expression profiles and functional diversity. To unravel astrocyte heterogeneity during postnatal development and after focal cerebral ischemia, we employed single-cell gene expression profiling in acutely isolated cortical GFAP/EGFP-positive cells. Using a microfluidic qPCR platform, we profiled 47 genes encoding glial markers and ion channels/transporters/receptors participating in maintaining K(+) and glutamate homeostasis per cell. Self-organizing maps and principal component analyses revealed three subpopulations within 10-50 days of postnatal development (P10-P50). The first subpopulation, mainly immature glia from P10, was characterized by high transcriptional activity of all studied genes, including polydendrocytic markers. The second subpopulation (mostly from P20) was characterized by low gene transcript levels, while the third subpopulation encompassed mature astrocytes (mainly from P30, P50). Within 14 days after ischemia (D3, D7, D14), additional astrocytic subpopulations were identified: resting glia (mostly from P50 and D3), transcriptionally active early reactive glia (mainly from D7) and permanent reactive glia (solely from D14). Following focal cerebral ischemia, reactive astrocytes underwent pronounced changes in the expression of aquaporins, nonspecific cationic and potassium channels, glutamate receptors and reactive astrocyte markers.

The added value of single-cell gene expression profiling.

Cells are the basic unit of life and they have remarkable abilities to respond individually as well as in concert to internal and external stimuli in a specific manner. Studying complex tissues and whole organs requires understanding of cell heterogeneity and responses to stimuli at the single-cell level. In this review, we discuss the potential of single-cell gene expression profiling, focusing on data analysis and biological interpretation. We exemplify several aspects of the added value of single-cell analysis by comparing the same experimental data at both single-cell and cell population level. Data normalization and handling of missing data are two important steps in data analysis that are performed differently at single-cell level compared with cell population level. Furthermore, we discuss how single-cell gene expression data can be viewed and how subpopulations of cells can be identified and characterized.

RT-qPCR work-flow for single-cell data analysis.

Individual cells represent the basic unit in tissues and organisms and are in many aspects unique in their properties. The introduction of new and sensitive techniques to study single-cells opens up new avenues to understand fundamental biological processes. Well established statistical tools and recommendations exist for gene expression data based on traditional cell population measurements. However, these workflows are not suitable, and some steps are even inappropriate, to apply on single-cell data. Here, we present a simple and practical workflow for preprocessing of single-cell data generated by reverse transcription quantitative real-time PCR. The approach is demonstrated on a data set based on profiling of 41 genes in 303 single-cells. For some pre-processing steps we present options and also recommendations. In particular, we demonstrate and discuss different strategies for handling missing data and scaling data for downstream multivariate analysis. The aim of this workflow is provide guide to the rapidly growing community studying single-cells by means of reverse transcription quantitative real-time PCR profiling.

The increased activity of TRPV4 channel in the astrocytes of the adult rat hippocampus after cerebral hypoxia/ischemia.

The polymodal transient receptor potential vanilloid 4 (TRPV4) channel, a member of the TRP channel family, is a calcium-permeable cationic channel that is gated by various stimuli such as cell swelling, low pH and high temperature. Therefore, TRPV4-mediated calcium entry may be involved in neuronal and glia pathophysiology associated with various disorders of the central nervous system, such as ischemia. The TRPV4 channel has been recently found in adult rat cortical and hippocampal astrocytes; however, its role in astrocyte pathophysiology is still not defined. In the present study, we examined the impact of cerebral hypoxia/ischemia (H/I) on the functional expression of astrocytic TRPV4 channels in the adult rat hippocampal CA1 region employing immunohistochemical analyses, the patch-clamp technique and microfluorimetric intracellular calcium imaging on astrocytes in slices as well as on those isolated from sham-operated or ischemic hippocampi. Hypoxia/ischemia was induced by a bilateral 15-minute occlusion of the common carotids combined with hypoxic conditions. Our immunohistochemical analyses revealed that 7 days after H/I, the expression of TRPV4 is markedly enhanced in hippocampal astrocytes of the CA1 region and that the increasing TRPV4 expression coincides with the development of astrogliosis. Additionally, adult hippocampal astrocytes in slices or cultured hippocampal astrocytes respond to the TRPV4 activator 4-alpha-phorbol-12,-13-didecanoate (4αPDD) by an increase in intracellular calcium and the activation of a cationic current, both of which are abolished by the removal of extracellular calcium or exposure to TRP antagonists, such as Ruthenium Red or RN1734. Following hypoxic/ischemic injury, the responses of astrocytes to 4αPDD are significantly augmented. Collectively, we show that TRPV4 channels are involved in ischemia-induced calcium entry in reactive astrocytes and thus, might participate in the pathogenic mechanisms of astroglial reactivity following ischemic insult.

Paternal benzo[a]pyrene exposure affects gene expression in the early developing mouse embryo.

The health of the offspring depends on the genetic constitution of the parental germ cells. The paternal genome appears to be important; e.g., de novo mutations in some genes seem to arise mostly from the father, whereas epigenetic modifications of DNA and histones are frequent in the paternal gonads. Environmental contaminants which may affect the integrity of the germ cells comprise the polycyclic aromatic hydrocarbon, benzo[a]pyrene (B[a]P). B[a]P has received much attention due to its ubiquitous distribution, its carcinogenic and mutagenic potential, and also effects on reproduction. We conducted an in vitro fertilization (IVF) experiment using sperm cells from B[a]P-exposed male mice to study effects of paternal B[a]P exposure on early gene expression in the developing mouse embryo. Male mice were exposed to a single acute dose of B[a]P (150 mg/kg, ip) 4 days prior to isolation of cauda sperm, followed by IVF of oocytes from unexposed superovulated mice. Gene expression in fertilized zygotes/embryos was determined using reverse transcription-qPCR at the 1-, 2-, 4-, 8-, and blastocyst cell stages of embryo development. We found that paternal B[a]P exposure altered the expression of numerous genes in the developing embryo especially at the blastocyst stage. Some genes were also affected at earlier developmental stages. Embryonic gene expression studies seem useful to identify perturbations of signaling pathways resulting from exposure to contaminants, and can be used to address mechanisms of paternal effects on embryo development.

Distinct expression/function of potassium and chloride channels contributes to the diverse volume regulation in cortical astrocytes of GFAP/EGFP mice.

Recently, we have identified two astrocytic subpopulations in the cortex of GFAP-EGFP mice, in which the astrocytes are visualized by the enhanced green-fluorescent protein (EGFP) under the control of the human glial fibrillary acidic protein (GFAP) promotor. These astrocytic subpopulations, termed high response- (HR-) and low response- (LR-) astrocytes, differed in the extent of their swelling during oxygen-glucose deprivation (OGD). In the present study we focused on identifying the ion channels or transporters that might underlie the different capabilities of these two astrocytic subpopulations to regulate their volume during OGD. Using three-dimensional confocal morphometry, which enables quantification of the total astrocytic volume, the effects of selected inhibitors of K⁺ and Cl⁻ channels/transporters or glutamate transporters on astrocyte volume changes were determined during 20 minute-OGD in situ. The inhibition of volume regulated anion channels (VRACs) and two-pore domain potassium channels (K(2P)) highlighted their distinct contributions to volume regulation in HR-/LR-astrocytes. While the inhibition of VRACs or K(2P) channels revealed their contribution to the swelling of HR-astrocytes, in LR-astrocytes they were both involved in anion/K⁺ effluxes. Additionally, the inhibition of Na⁺-K⁺-Cl⁻ co-transporters in HR-astrocytes led to a reduction of cell swelling, but it had no effect on LR-astrocyte volume. Moreover, employing real-time single-cell quantitative polymerase chain reaction (PCR), we characterized the expression profiles of EGFP-positive astrocytes with a focus on those ion channels and transporters participating in astrocyte swelling and volume regulation. The PCR data revealed the existence of two astrocytic subpopulations markedly differing in their gene expression levels for inwardly rectifying K⁺ channels (Kir4.1), K(2P) channels (TREK-1 and TWIK-1) and Cl⁻ channels (ClC2). Thus, we propose that the diverse volume changes displayed by cortical astrocytes during OGD mainly result from their distinct expression patterns of ClC2 and K(2P) channels.

Detection of immune cell response to M. tuberculosis-specific antigens by quantitative polymerase chain reaction.

One third of the world's population is latently infected with Mycobacterium tuberculosis (Mtb) and up to 10% of infected individuals develop active tuberculosis (TB) in their lifetime. Among the major challenges in the control of TB is the implementation of sensitive methods for detection of latent tuberculosis infection (LTBI). Currently, in vitro interferon gamma release assays, yielding single value readout, are used as an alternative to the traditional tuberculin skin test for the diagnosis of LTBI. More complex characterization of immune status of LTBI individuals, however, is desirable for indication of LTBI subjects for preventative chemotherapy. Here we describe a quantitative polymerase chain reaction (qPCR) for determination of expression levels of 14 genes, additional to interferon gamma, which was applied for comparison of the specific Mtb-antigen immune response of blood cells from healthy, latently infected, and TB individuals. With the use of principal component analysis and discriminant analysis, a pattern of mRNA levels of 6 genes was identified, allowing discrimination of healthy individuals from active TB and LTBI subjects. These results open the way to development of multimarker qPCR for the detection of LTBI.

Preconceptional paternal glycidamide exposure affects embryonic gene expression: single embryo gene expression study following in vitro fertilization.

Recognition of early determinants of disease onset has sparked an interest in paternally transmitted factors and their impact on the developing embryo. Acrylamide (AA), a widely distributed xenobiotic compound, is converted to its active metabolite glycidamide (GA) by the CYP2E1 enzyme. Based on its capacity to induce dominant lethal mutations, we hypothesized that paternal GA exposure would have a negative impact on embryonic genome activation, via GA-DNA and protamine adducts persisting in the fertilizing sperm. Using a combination of in vitro fertilization (IVF) techniques and RT-qPCR single embryo gene expression (SEGE), we studied the expression of key DNA repair genes and genes important for embryo development, at the 1-, 2-, 4- and 8-cell stage of the developing mouse embryo. Compared to controls paternal GA-exposure gave rise to an altered pattern of embryonic gene expression, with an initial reduced expression at early stages followed by increased expression at the 8-cell stage.

Gene expression profiling--Clusters of possibilities.

Advances in qPCR technology allow studies of increasingly large systems comprising many genes and samples. The increasing data sizes allow expression profiling both in the gene and the samples dimension while also putting higher demands on sound statistical analysis and expertise to handle and interpret its results. We distinguish between exploratory and confirmatory statistical studies. In this paper we demonstrate several techniques available for exploratory studies on a system of Xenopus laevis development from egg to tadpole. Techniques include hierarchical clustering, heatmap, principal component analysis and self-organizing maps. We stress that even though exploratory studies are excellent for generating hypotheses, results have not been proven statistically significant until an independent confirmatory study has been performed. An exploratory study may certainly be valuable in its own right, and there are often not enough resources to report both an exploratory and a confirmatory study at the same time. However, exploratory and confirmatory studies are intimately connected and we would like to raise that awareness among qPCR practitioners. We suggest that scientific reports should always have a hypothesis focus. Reports are either hypothesis generating, from an exploratory study, or hypothesis validating, from a confirmatory study, or both. In either case, we suggest the generated or validated hypotheses be specifically stated.

Analysis of in vitro and in vivo characteristics of human embryonic stem cell-derived neural precursors.

During the last decade, much progress has been made in developing protocols for the differentiation of human embryonic stem cells (hESCs) into a neural phenotype. The appropriate agent for cell therapy is neural precursors (NPs). Here, we demonstrate the derivation of highly enriched and expandable populations of proliferating NPs from the CCTL14 line of hESCs. These NPs could differentiate in vitro into functionally active neurons, as confirmed by immunohistochemical staining and electrophysiological analysis. Neural cells differentiated in vitro from hESCs exhibit broad cellular heterogeneity with respect to developmental stage and lineage specification. To analyze the population of the derived NPs, we used fluorescence-activated cell sorting (FACS) and characterized the expression of several pluripotent and neural markers, such as Nanog, SSEA-4, SSEA-1, TRA-1-60, CD24, CD133, CD56 (NCAM), beta-III-tubulin, NF70, nestin, CD271 (NGFR), CD29, CD73, and CD105 during long-term propagation. The analyzed cells were used for transplantation into the injured rodent brain; the tumorigenicity of the transplanted cells was apparently eliminated following long-term culture. These results complete the characterization of the CCTL14 line of hESCs and provide a framework for developing cell selection strategies for neural cell-based therapies.