Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of brain from mice with ischemia of the right common carotid artery or sham treated and then exposed to hypoxic conditions

ABSTRACT: Animals were anaesthetized with halothane (4% in 70% N2O: 30% O2). A small incision was made in the neck and the right common carotid artery was exposed and ligated with 4-0 surgical silk in three separate locations. The incision was closed, and the animal allowed to recover with access to food and water for 3 hours. Prior to exposure to hypoxia, mice were placed in a 35.5 C water bath for 10 minutes followed by hypoxic exposure which comprised 10 min (6% O2 / 94%N2) and 25 min (8% O2 /92%N2). At the end of the hypoxic interval, animals were allowed to recover in room air for 30 min and then return to their cage with free access to food and water for 24 hours. Animals were re-anaesthetized with Halothane, transcardially perfused with HBSS, decapitated, and brains were frozen on dry ice.

INSTRUMENT(S): G2505A DNA microarray scanner [Agilent]

ORGANISM(S): Mus musculus

DISEASE(S): sham treated

SUBMITTER: Oliver Wafzig

PROVIDER: E-MEXP-1253 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:In pregnancies involving preeclampsia (PE), there is evidence that the fetal-placental unit is under oxidative stress. Here we examined primary cell lines generated from umbilical cords (UC) delivered by mothers who had either a normal pregnancy or experienced early onset PE to determine whether the two had distinguishable phenotypes. While all UC provided outgrowths when established in 4 % O2, success was less assured for PE cords under ambient (20 % O2) conditions (P < 0.05). Moreover, proliferation rates of established PE lines, although similar to controls in 4 % O2, were significantly lower in 20 % O2. PE lines grown in 4 % O2 were also more susceptible to the pro-oxidant diethylmaleate than control lines, and unlike controls, were not protected by glutathione. Transcriptome profiling revealed only a few differentially regulated genes between PE lines and controls in 4 % O2 conditions, but confirmed the more severely stressed phenotype of the PE lines under 20 % O2. We conclude that the primary UC cell lines generated from PE births maintain a susceptibility to oxidative stress that is stable over many cell divisions, but whether the basis of this vulnerability is genetic or epigenetic remains unclear. RNA was isolated from early passages (< p5) of UC fibroblast lines (15 PE and 9 CTL lines, grown in T25 flasks) when they reached ~90% confluency in 4 % O2 conditions. In order to collect RNA from cells under 20 % O2, cell lines at either p 4, 5, or 6 were switched from 4 % O2 conditions to 20 % O2 conditions when they were approximately 20 % confluent. When they reached ~90% confluency (generally 2 days in 4 % O2 and 3-4 days in 20 % O2 conditions), medium was removed and RNA STAT60 (I ml; Tel-Test, Friendswood, TX) was immediately added to each T25 flask and RNA extracted by following the manufacturer’s instructions. The samples of RNA were submitted to University Texas Southwestern Medical Center Microarray Core Facility (https://microarray.swmed.edu/) and microarray analysis performed with Illumina HumanHT-12 v4 expression BeadChips. Raw intensity data were background subtracted by using BeadStudio software and analyzed further by GeneSpring 12.6 software (Agilent Technologies Inc., Santa Clara CA), according to the advanced workflow protocol: percentile shift and filter by flags (detected).

Project description:We have compared the gene-expression in cerebral arteries from rats after experimental SAH with the expression in cerebral vessels from sham operated animals. The model is well characterised (Prunell et al. 2003; Prunell et al. 2004) regarding the physiological changes but not regarding the gene expression pattern. Male Sprague-Dawley rats (350-400 g) were anaesthetized using 5% halothane (Halocarbon Laboratories, River Edge, New Jersey) in N2O/O2 (30:70). The rat was intubated and artificially ventilated with inhalation of 0.5-1.5% halothane in N2O/O2 (70:30) during the surgical procedure. The depth of anaesthesia was carefully monitored and the respiration checked by regularly withdrawing arterial blood samples for blood gas analysis (Radiometer, Copenhagen, Denmark). An arterial catheter to measure blood pressure was placed in the tail artery and a catheter to monitor intracranial pressure (ICP) was placed in the subocciptal membrane. At either side of the skull, 3 mm from the midline and 4 mm anteriorly from the bregma, holes were drilled through the skull bone down to dura mater (without perforation) allowing the placement of two laser-Doppler flow probes to measure CBF. Finally, a 27G blunt canula with side hole was introduced stereotactically 6.5 mm anterior to bregma in the midline at an angle of 30 degrees to the vertical. After 30 minutes of equilibration 250 ul blood was withdrawn from the tail catheter and injected intracranially at a pressure equal to the mean arterial blood pressure (80-100 mmHg). Subsequently the rat was kept under anaesthesia for another 60 minutes to allow recovery from the cerebral insult after which catheters were removed and the incisions closed. The rat was then revitalized and extubated. A subcutaneous injection of carprofen (4.0 mg/kg) (Pfizer, Denmark) was administered and the rat was hydrated subcutaneously using 40 ml isotonic sodium chloride at the end of the operation and at day one. During the period of observation the rat was monitored regularly, and if showing severe distress the animal was prematurely killed. In addition, a series of sham-operated rats were prepared. They went through the same procedure with the exception that no blood was injected intracisternally.

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Transcription profiling of brain from mice with ischemia of the right common carotid artery or sham treated and then exposed to hypoxic conditions

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