Project description:To address whether hypoxia contributes to muscle patho-physiology, normal, adult C57 mice were exposed to chronic, normobaric and hypobaric hypoxic environments for 2 weeks in order to simulate levels of hypoxaemia reported in DMD patients with advanced respiratory insufficiency. Control mice were maintained under normoxic conditions. Control and experimental mice were studied. Experiment Overall Design: Adult male C57Bl/10 mice were used in experiments. At the beginning of the experiment mice were divided into two groups, control (room condition) and hypoxic (hypoxic condition), over a period of 2 weeks. The hypoxic group was gradually exposed to lower levels of hypoxia in an especially designed and hermetically closed hypoxic chamber. A Pegas 4000 MF(Columbus Instruments) gas blending system was used. The oxygen level was gradually decreased from 21% to 8% over one week and animals were kept at 8% oxygen for another 7 days. Animalâs weights were monitored daily. Food and water were changed daily for the time of the experiment. After two weeks animals were euthanized using CO2, Extensor digitorum longus (EDL) and Soleus muscles were dissected for physiological studies. Prior to physiological analysis each group was divided into two smaller groups (subgroups). In the first subgroup the primary focus on physiological muscle analysis was on (EDL) while in the other subgroup it was on Soleus. This division allowed us that both muscles, EDL and Soleus were analyzed at the same time after euthanization. Experiment Overall Design: RNA isolation Experiment Overall Design: Total RNA was isolated from each QF muscle by Tri-Reagent (Ambion, Austin, TX) as described by manufacturer. Isolated total RNA were cleaned up by RNeasy mini kit (Qiagen, Valencia, CA) as described by manufacturer. The purity and concentration of total RNA were determined by measurement of absorbance at 260 and 280nm using a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). To satisfy our purity criteria, we discarded all RNA samples that did not have a 260/280 ratio between 1.8 and 2.1. To satisfy our criteria for integrity, we required that all RNA used in our experiments have single peaks for the 18S and 28S bands as determined by the Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Palo Alto, CA). Experiment Overall Design: Linear amplification and cRNA labeling Experiment Overall Design: Three microliter total RNA was used for each sample to obtain linearly amplified labeled cRNA by using GeneChipÂ® One-Cycle Target Labeling kit (Affymetrix, Santa Clara, CA) as manufacturer described. Briefly; total RNA was used to generate double-stranded cDNA with the T7-oligo (dT) primer. This double-stranded cDNA was used in vitro transcription and biotin labeling steps. Labeled cRNA yield and purity were determined by measuring the absorbance at 260 and 280nm. All the cRNA 260/280 ratios were between 1.9 and 2.1. Quality control (QC) of the labeled cRNA products was assessed by performing 1Âµg labeled cRNA on 2% agarose gel to see similar RNA smear type. Experiment Overall Design: Fragmentation and microarray hybridization Experiment Overall Design: Fifteen micrograms labeled cRNA of hypoxic or normoxic QF muscle was fragmented, and 10Î¼g was hybridized to AffymetrixÂ® Mouse 430 ver 2.0 GeneChip arrays for 18â24h (Affymetrix Inc.). Each microarray was washed and stained with streptavidinâphycoerythrin and scanned at a 6-Î¼m resolution with Agilent model G2500A GeneArray scanner A visual quality control measurement was performed to ensure proper hybridization after each chip was scanned.

Project description:Reduced oxygenation of photoreceptors and the RPE in the ageing eye may be a risk factor for the development of both neovascular and dry AMD. Chronic activation of the molecular response to hypoxia in RPE or photoreceptors leads to retinal degeneration that depends on hypoxia inducible transcription factors. For approaching the identification of accessible markers characteristic for photoreceptors with an activated hypoxic response, we used a proteomics approach to determine the protein composition of the vitreous humor in mice. To discriminate between rod and cone-specific effects we used genetically modified mice that had the hypoxic response activated specifically in rods of a rod-dominant retina (rodΔVhl) or a genetically engineered all-cone retina (coneΔVhl). For comparison, we used wild-type mice exposed for 6 hours to acute hypoxia. We identified 1,357 unique proteins in the vitreous of mice after acute hypoxia, 1,624 in rodΔVhl and 1,895 in coneΔVhl mice. Of these, 1,043 proteins were common to all three types of mice. Of the identified proteins, 257 were significantly regulated by a factor of 1.5 or more in hypoxic mice, 258 in rodΔVhl and 356 in coneΔVhl mice in at least one of the three analyzed time points. Only 51 of the significantly regulated proteins were common to the vitreous of rodΔVhl and coneΔVhl mice, suggesting different consequences of the activated hypoxic response for rods and cones. Guanylate binding protein 2 (GBP2) was found at increased levels in the vitreous of both rodΔVhl and coneΔVhl mice at all time points tested. This was also reflected by increased gene expression in the retina. Although retinal expression of the AMD-associated gene alpha-2 macroglobulin (A2M) appeared increased in both types of mice, the protein was only found elevated in the vitreous of rodΔVhl mice. Other proteins found increased included Serpina3n, synaptosome associated protein 25 (SNAP25) and others. The distinct protein compositions present at early and late time points, suggest a well-regulated process in our models. We hypothesize that some of the proteins identified at early time points may potentially be used as markers for the chronic hypoxic response of photoreceptors.

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Comparison of the Human Transcriptional Response to Three Hypoxic Environments

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