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HCC formation in rat

ABSTRACT: A total of 10 male Fischer 344 rats, 5 weeks old, were purchased from Charles River Japan, Incorporated, Atsugi, Kanagawa, Japan. Rats were divided into 2 groups each consisting of 5 animals housed in a plastic cage with white flake bedding in an air-conditioned room. Rats were used for the experimentation after a 1-week acclimation on a basal diet (CRF-1, Oriental Yeast Corporation, Itabashi, Tokyo, Japan) and allowed free access to food and tap water throughout the acclimation and experimental periods. Body weight, food consumption, and water intake were monitored weekly. The CDAA diet was obtained from Dyets, Bethlehem, PA. After acclimation, group 1 rats were administered the basal diet, while group 2 rats were placed on the CDAA diet for 70 weeks and then sacrificed. The livers were taken and macroscopically examined. Portions of group 1 livers, as well as the macroscopic tumors and their surrounding, non-cancerous tissues of group 2 livers were fixed in 10% neutrally-buffered-formalin for 24 hours, embedded in paraffin, processed for the routine hematoxylin-and-eosin staining procedure, and histologically examined. Remaining portions of these 3 types of liver tissues were immediately frozen in liquid nitrogen and stored at -80C. Group 1 liver tissues, group 2 liver tissues from non-cancerous areas, and group 2 liver tissues from tumors (after histologically diagnosed as HCCs; see the results section) were used as normal liver samples, surrounding non-cancerous liver samples, and HCC samples, hereafter referred to as CON, NC, and CA, respectively. The microarray and RT-PCR experiments and the subsequent data analysis were performed using 5 CON samples from individual group 1 rats and 5 matched pairs of NC and CA samples from individual group 2 animals. RNA Isolation and Probe Labeling. Total RNA from liver tissues was isolated using RNeasy Midi kits (QIAGEN, Hilden, Germany). The integrity of the RNA was checked by electrophoresis on 1% agarose-formaldehyde gels. Five micrograms of total RNA from individual samples were labeled with Cy3 (Amersham Biosciences, Uppsara, Sweden) using BD Atlas Powerscript Fluorescent Labeling kits (BD Biosciences Clontech, Palo Alto, CA) according to the manufacturer’s protocols. Hybridization, Scanning, and Quantification. Cy3-labeled probes were hybridized to Atlas Rat 3.8 I microarrays (BD Biosciences Clontech) containing 3757 genes for 16 hours at 50C. After hybridization, microarrays were washed, dried, and scanned using a GMS 418 confocal laser scanner (Genetic MicroSystems, Woburn, MA). Fluorescence intensities of the Cy3 channels were quantified using ImaGene 4.0 software (BioDiscovery, El Segundo, CA). Data analysis was performed using the GeneSpring software, version 5.1 (Silicon Genetics, Redwood City, CA), including appropriate statistics. Dividing by the median calculated from all of the signal intensities for the Cy3 in a given sample normalized the fluorescence signal for each gene. The VALUEs for each gene was then calculated by dividing the normalized signal by the median of each gene to offset differences in expression levels between genes. Keywords: parallel sample

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE1946 | GEO | 2005/02/01

SECONDARY ACCESSION(S): PRJNA90445

REPOSITORIES: GEO

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Project description:In our previous study, we reported that a diet rich in antioxidants such as coral calcium hydride (CCH) increased the endogenous antioxidant ability in the hippocampus of rats. We conducted this study to test the hypothesis that diet supplementation with CCH would change the gene expression in rats and to understand how CCH enhances antioxidant ability. We used a DNA array to compare the expression levels in the hippocampus of rats fed with CCH for 2 weeks with those of rats fed a normal diet. Immune response-related genes were down-regulated, while nuclear respiratory factor 2 and aldehyde dehydrogenase 3A were up-regulated. Our findings about the changes in the mRNA levels of these genes well explain the physiological finding of enhanced antioxidant ability in rat brain. CCH was obtained from ICB, Ltd., Sendai, Japan, and coral calcium (CC) was purchased from Coralbio, Okinawa, Japan. Male Wistar rats were acquired from Kyudo, Co., Ltd. and maintained at the Experimental Animal Center of the University of Miyazaki at a controlled ambient temperature of 23 ¬¨¬± 1 ¬¨‚àûC and 50 ¬¨¬± 10% relative humidity. The Committee for Ethics on Animal Experiments, Faculty of Medicine, University of Miyazaki, Japan, reviewed and approved the experimental design. Six-week-old male Wistar rats (n = 8) were assigned to 2 groups: standard diet-fed group (CE-2, Clea Japan, Inc., Tokyo, Japan) and CCH-fed group. The CCH diet was standard CE-2 feed supplemented with 0.1% CCH powder. Inhibition of accelerated aging and an increase in the in vivo antioxidant ability was observed in SAM/P-8 mice fed a diet supplemented with 0.1% CCH. In accordance with these reports, the CCH concentration used in our study was set at 0.1%.The animals were killed by cervical dislocation at the age of 8 weeks. They were decapitated and the hippocampi were removed and rapidly frozen in liquid nitrogen. The hippocampi were then homogenized with a conventional rotor-stator homogenizer. Total RNA was then extracted from the tissues by using the RNeasy Lipid Tissue Mini Kit (Qiagen, Valencia, CA).

Project description:The clinical results obtained with organ allografts from marginal or extended donors are less satisfactory over both the short-term and long-term than the outcome of grafting from living donors. Early postoperative graft function depends on several pretransplant factors, with the major donor influences being brain death (BD) and ischemia/reperfusion(I/R) injury. The effects of BD and I/R injury not only reduce the number of functioning nephrons, but also trigger a host immune response to the grafted kidney. It has been hypothesized that allografts from marginal sources may not be biologically inert at the time of surgery, but may already be programmed to initiate or amplify a host response. To exclude the effects of allogenicity, we established a rat renal isograft model using dead donors and compared the results with those obtained after grafting from living donors. In this study, we performed an analysis of the changes of gene expression in rat kidney isografts using samples obtained at 0(pre-transplant) and 1 hour time points since transplantation procedure. The results enhanced our insight into the pathways and cascades that are activated or down-regulated by BD and/or I/R injury. Better knowledge of the key components of BD or I/R injury will provide clues to the factors triggering progression that leads to a decline of organ viability, as well as ideas on how to overcome such graft failures. Such data may also allow us to identify novel biomarkers as predictors of adverse outcomes. Experiment Overall Design: Inbred male Lewis rats were used for these experiments as recipients and donors. The left kidney was transplanted orthotopically by end-to-end anastomosis. The contralateral right kidney was removed at the time of transplantation. The time of operative ischemia for transplantation was 30 min, which did not vary between animals. Donor animals were divided into two groups. The experimental group received kidneys from BD rats, while kidneys from living donors were used in the control group. Brain death was produced by gradually increasing the intracranial pressure that led to brain stem herniation. The rats were mechanically ventilated for a period of 6 hours. We compared the gene expression profiles of renal isografts from BD donors and grafts from living donors using a high-density oligonucleotide microarray that contained approximately 20,500 genes. Experiment Overall Design: For DNA microarray experiments, 200 ng aliquots of total RNA were labeled using the Agilent Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies Product) according to the manufacturerâ??s instructions. RNA purified from each kidney graft was used for microarray analysis (Cy3-labeled), with pooled RNA derived from normal kidneys as a template control (Cy5-labeled). After checking the labeling efficiency, 1ï?g aliquots of Cy5-labeled normal control RNA and Cy3-labeled RNA from individual grafts (control 0 hour, 1 hour, BD 0 hour, BD 1 hour, n=3/group) were mixed, and then hybridized to Agilent Rat Oligo Microarrays (Product No. G4130A). After washing, the microarray slides were analyzed with an Agilent Microarray scanner and software (scanner model G2565BA). Data analysis was performed using Agilent Feature Extraction software (Ver. A.7.1.1), and Excel 2003 (Microsoft). The data were imported into GeneSpring 7.0 (Silicon Genetics, Redwood City, CA), with per spot, per chip, and intensity dependent (lowess) normalization being applied for each array. The ratio of the normalized channels (Cy3/Cy5) was used to assess the level of expression.

Project description:Messenger RNA of C trachomatis infected monocytes of the three healthy donors was compared with the corresponding mock-infected samples. For mock infection, SPG buffer was used instead of the C trachomatis suspension, all other procedures were performed identically. Cells were resuspended in solution D and total RNA was extracted by application of phenol:chloroform 5:1 (pH 4.5, Ambion, Austin, TX) followed by precipitation in isopropanol at -80°C for 1 hr and incubation with RQ1 DNase (Promega, Madison, WI) at 37 °C for 1 hr. The signal intensity of the G3PDH housekeeping gene on the microarray membrane was used as indirect quality marker for the RNA utilized as only experiments were included in the analysis that revealed a G3PDH signal intensity within 1.5 times the standard deviation of all membranes evaluated in our study. The entire microarray procedure and its analysis has recently been validated and reported in detail. Total RNA of all donors was pooled and for each microarray experiment 150 µg were reverse transcribed and amplified by SMART™-PCR (BD Biosciences Clontech, Palo Alto, CA), a technology that allows reverse transcription of small amounts of total RNA and subsequent amplification of the entire cDNA. Probes were labeled with 32P and subsequently over night hybridized to a filter-based nylon membrane containing immobilized cDNA-specific sequences from a total of 1,184 genes (Human Atlas Array 1.2, BD Biosciences Clontech, Palo Alto, CA). For analysis, we focused solely on the 159 cytokines, chemokines and their receptors as given by the manufacturer. Signal intensities were retrieved by a STORM 860 scanner (Molecular Dynamics, Sunnyvale, CA) in combination with the AtlasImage 2.0 software (BD Biosciences Clontech). Data from all signal intensities were then subtracted by the local background intensity measured around each gene. The local background intensities of all individual genes were subsequently averaged, resulting in the mean background intensity of a particular membrane. Gene expression in our experiments was determined by a spot intensity of a single transcript that exceeded twice the mean background. Normalisation to background and to the G3PDH housekeeping gene was achieved by the global (sum) normalization method. Signal intensity data are given as the ratio of C trachomatis infected vs mock infected probes. Keywords: expression analysis

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HCC formation in rat

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