Al whole-mounts of normotensive animals (n = 3) was 21126369 RGCs/mm2. IOP elevation (n = 3) significantly reduced the RGC density to 14886532 RGCs/mm2 (p,0.001) at 8 weeks after cauterization. These data are in agreement with previous RGC quantifications in glaucoma usingProtein Changes in NeurodegenerationTable 1. Retinal proteins identified by two-dimensional gel electrophoresis and subsequent matrix-assisted laser desorption ionization mass spectrometry.No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31Protein Glucose related-protein 78 HSP 70 isoform 2 Glucose related-protein 75 Glial fibrillary acidic protein Enolase 2 ATP synthase beta subunit Beta actin Retinaldehyde-binding protein Kinase 18334597 associated HSP 90 Crystalline mu Glucose-6-phosphatase isomerase Retinoidacid receptor responder protein Recoverin Class I beta tubulin Synthaxin 2 Phosphatydylethanolamin bindingprotein HSP 60 Enolase 1 Craniofacial dev. Protein 1, cyclin G1 Calmodulin Adaptor related protein complex3 Malate dehydrogenase Proteasome Peroxiredoxin 6 ATP synthase delta subunit Nucleoside diphosphate kinase B Carbonic anhydrase 1 Triose-phtosphat isomerase bb3 crystallin Phosphoglycerate mutase Bb2 crystallin Acetyl-coenzyme A dehydrogenasePotential function Cytosceleton Protein folding ATPase activity Molecular chaperone Cytosceleton Carbohydrate transport, metabolism ATP biosynthesis Structural protein,cytoskeleton Transport of retinalaldehyde Molecular chaperone Amino acid transport, metabolism Carbohydrate transport, metabolism Type 2 membran protein Regulation of rhodopsin Tubulin, cytoskeleton Epithelial morphogenesis Lipid and ATP binding Protein turnover, chaperonin Glycolysis, lyase Cytochrome, craniofacial development Calcium-binding protein Intracellular trafficking and secretion Energy production and conversion Inhibitor of apoptosis Thiol-specific antioxidant protein ATP biosynthesis Synthesis of nucleoside triphosphate Hydration of carbon dioxide Glycolysis Structural protein of the eye lens Glycolysis Structural protein of the eye lens Fatty acid, lipid metabolismMW/kDA 78 53 73,6 50 47 50,7 41,7 44,5 44,4 33,5 29 29 23 45 33,3 20,7 61 47 34 35 34 36 30 25 19 18 28 28 24 29 23The numbers in column 1 correspond to those given in Fig. 4. Column 4 lists the molecular mass of the respective protein. HSP = heat-shock protein. doi:10.1371/journal.pone.0049730.tMicroarrays and quantitative real-time polymerase chain reactionTo confirm that the change in the expression of crystallin is reflected at the mRNA level, microarray analysis 1407003 and additional qRT-PCR were performed on normotensive samples as well as on samples at 4 weeks after IOP elevation. Harvesting of the retinal samples 4 weeks after IOP elevation (before the initiation of antihypertensive therapy) enabled us to detect whether crystallin mRNAs were up-regulated due to IOP and down-regulated due to antihypertensive treatment. Microarray analysis and qRT-PCR were get 370-86-5 conducted after reverse transcription of isolated RNA for cryaA, cryaB, crybb1, ML-264 crybb2, crybb3, and crybb4. Compared with normotensive retinas, the gene activity in hypertensive retinas was up-regulated by four- to tenfold (Fig. 7). All data support the initial hypothesis that retinal crystallins, and in particular crybb2,are sensitive markers for detecting the pharmacological influences of drugs that are topically applied to reduce elevated IOP.DiscussionThere are three principal findings from this s.Al whole-mounts of normotensive animals (n = 3) was 21126369 RGCs/mm2. IOP elevation (n = 3) significantly reduced the RGC density to 14886532 RGCs/mm2 (p,0.001) at 8 weeks after cauterization. These data are in agreement with previous RGC quantifications in glaucoma usingProtein Changes in NeurodegenerationTable 1. Retinal proteins identified by two-dimensional gel electrophoresis and subsequent matrix-assisted laser desorption ionization mass spectrometry.No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31Protein Glucose related-protein 78 HSP 70 isoform 2 Glucose related-protein 75 Glial fibrillary acidic protein Enolase 2 ATP synthase beta subunit Beta actin Retinaldehyde-binding protein Kinase 18334597 associated HSP 90 Crystalline mu Glucose-6-phosphatase isomerase Retinoidacid receptor responder protein Recoverin Class I beta tubulin Synthaxin 2 Phosphatydylethanolamin bindingprotein HSP 60 Enolase 1 Craniofacial dev. Protein 1, cyclin G1 Calmodulin Adaptor related protein complex3 Malate dehydrogenase Proteasome Peroxiredoxin 6 ATP synthase delta subunit Nucleoside diphosphate kinase B Carbonic anhydrase 1 Triose-phtosphat isomerase bb3 crystallin Phosphoglycerate mutase Bb2 crystallin Acetyl-coenzyme A dehydrogenasePotential function Cytosceleton Protein folding ATPase activity Molecular chaperone Cytosceleton Carbohydrate transport, metabolism ATP biosynthesis Structural protein,cytoskeleton Transport of retinalaldehyde Molecular chaperone Amino acid transport, metabolism Carbohydrate transport, metabolism Type 2 membran protein Regulation of rhodopsin Tubulin, cytoskeleton Epithelial morphogenesis Lipid and ATP binding Protein turnover, chaperonin Glycolysis, lyase Cytochrome, craniofacial development Calcium-binding protein Intracellular trafficking and secretion Energy production and conversion Inhibitor of apoptosis Thiol-specific antioxidant protein ATP biosynthesis Synthesis of nucleoside triphosphate Hydration of carbon dioxide Glycolysis Structural protein of the eye lens Glycolysis Structural protein of the eye lens Fatty acid, lipid metabolismMW/kDA 78 53 73,6 50 47 50,7 41,7 44,5 44,4 33,5 29 29 23 45 33,3 20,7 61 47 34 35 34 36 30 25 19 18 28 28 24 29 23The numbers in column 1 correspond to those given in Fig. 4. Column 4 lists the molecular mass of the respective protein. HSP = heat-shock protein. doi:10.1371/journal.pone.0049730.tMicroarrays and quantitative real-time polymerase chain reactionTo confirm that the change in the expression of crystallin is reflected at the mRNA level, microarray analysis 1407003 and additional qRT-PCR were performed on normotensive samples as well as on samples at 4 weeks after IOP elevation. Harvesting of the retinal samples 4 weeks after IOP elevation (before the initiation of antihypertensive therapy) enabled us to detect whether crystallin mRNAs were up-regulated due to IOP and down-regulated due to antihypertensive treatment. Microarray analysis and qRT-PCR were conducted after reverse transcription of isolated RNA for cryaA, cryaB, crybb1, crybb2, crybb3, and crybb4. Compared with normotensive retinas, the gene activity in hypertensive retinas was up-regulated by four- to tenfold (Fig. 7). All data support the initial hypothesis that retinal crystallins, and in particular crybb2,are sensitive markers for detecting the pharmacological influences of drugs that are topically applied to reduce elevated IOP.DiscussionThere are three principal findings from this s.
