| 1. |
- Agudo, Marta, et al.
(author)
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Immediate Upregulation of Proteins Belonging to Different Branches of the Apoptotic Cascade in the Retina after Optic Nerve Transection and Optic Nerve Crush
- 2009
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In: Investigative Ophthalmology and Visual Science. - : Association for Research in Vision and Ophthalmology (ARVO). - 0146-0404 .- 1552-5783. ; 50:1, s. 424-431
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Journal article (peer-reviewed)abstract
- Purpose: To further investigate the molecular signals underlying optic nerve (ON) injury we have analyzed in adult control, ON transected and ON crushed retinas, the expression pattern and time-course regulation of the following proteins, all of which are linked to apoptosis through different pathways: Stat 1, Caspase 11 (inflammation and death), Cathepsins C and B (lysosomal death pathway), Calpain 1 (endoplasmic reticulum stress), Calreticulin (apoptosis marker), Jun (early response) and Ahr (cell cycle arrest). Methods: Adult female rats were subjected to either intraorbital optic nerve transection (IONT) or intraorbital optic nerve crush (IONC). Protein from naive and ON injured adult rat retinas was extracted at increasing time-points post-lesion and western blotting experiments carried out. For immnuhistofluorescence analyses, retinal ganglion cells (RGCs) were retrogradelly identified with fluorogold applied to the superior colliculi one week before injury. Results: Western blotting analyses revealed up-regulation of all the analyzed proteins as soon as 12 hours post-lesion (hpl) peaking at 48hpl, in agreement with our previous RNA studies1. Furthermore, immunohistofluorescence to radial sections show that all of them, but Stat1, are expressed by the primarily injured neurons, the RGCs, as seen by colocalization with FG. Conclusions: All analyzed proteins were up-regulated in the retina after IONT or IONC as soon as 12hpl, indicating that ON injury regulates several branches of the apoptotic cascade and suggesting that commitment to death might be an earlier event than previously anticipated.
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| 2. |
- Ahmadi, Mahboobah, et al.
(author)
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Human extraocular muscles in ALS
- 2010
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In: Investigative Ophthalmology and Visual Science. - : Association for Research in Vision and Ophthalmology (ARVO). - 0146-0404 .- 1552-5783. ; 51:7, s. 3494-3501
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Journal article (peer-reviewed)abstract
- PURPOSE. To investigate the general morphology, fiber type content, and myosin heavy chain (MyHC) composition of extraocular muscles (EOMs) from postmortem donors with amyotrophic lateral sclerosis (ALS) and to evaluate whether EOMs are affected or truly spared in this disease. METHODS. EOM and limb muscle samples obtained at autopsy from ALS donors and EOM samples from four control donors were processed for immunohistochemistry with monoclonal antibodies against distinct MyHC isoforms and analyzed by SDS-PAGE. In addition, hematoxylin and eosin staining and nicotinamide tetrazolium reductase (NADH-TR) activity were studied. RESULTS. Wide heterogeneity was observed in the appearance of the different EOMs from each single donor and between donors, irrespective of ALS type or onset. Pathologic morphologic findings in ALS EOMs included presence of atrophic and hypertrophic fibers, either clustered in groups or scattered; increased amounts of connective tissue; and areas of fatty replacement. The population of fibers stained with anti-MyHCslow tonic was smaller than that of MyHCIpositive fibers and was mostly located in the orbital layer in most of the ALS EOM samples, whereas an identical staining pattern for both fiber populations was observed in the control specimens. MyHCembryonic was notably absent from the ALS EOMs. CONCLUSIONS. The EOMs showed signs of involvement with altered fiber type composition, contractile protein content, and cellular architecture. However, when compared to the limb muscles, the EOMs were remarkably preserved. EOMs are a useful model for the study of the pathophysiology of ALS.
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| 3. |
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| 4. |
- Ahuja, Satpal
(author)
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Lectin microarray profiling and relative quantification of glycome associated with proteins of neonatal wt and rd1 mice retinae.
- 2013
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In: Investigative Ophthalmology & Visual Science. - : Association for Research in Vision and Ophthalmology (ARVO). - 1552-5783. ; 54:5, s. 3272-3280
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Journal article (peer-reviewed)abstract
- PURPOSE: To compare progressive dynamic, relative quantitative changes in glycans associated with retinal proteins of wild type (wt) and retinal degeneration 1 (rd1) mice during neonatal development and degeneration of retinae. METHODS: Proteins extracted from retinae of postnatal day 2 (PN2), PN7, PN14 wt and rd1 mice were labeled with Cy3-fluorescent dye. Glycome of these proteins was quantified relatively by lectin microarray technique. Net fluorescence emitted by individual complexes formed between forty five lectins and Cy3-labeled proteins was measured by evanescent-field-fluorescence-assisted microarray reader. RESULTS: GlcNAcβ1-oligomer and high-mannose/Manα1-6Man were major glycans associated with the proteins of PN2, PN7, PN14 wt and rd1 mice retinae. Gal/GalNAc/Man3-core-bi-/tri-antennary-complex; Sia2-3Galβ1-4GlcNAc and high-mannose glycans were mainly conjugated to proteins from PN7 rd1 and PN14 wt retinae, respectively. With increasing neonatal age, mannosylated, GlcNAcβ, and sialylated (minor component) glycans were increased and fucosylated GlcNAc/Galβ glycans were decreased significantly in wt retinal proteins. This trend was less evident in PN14 rd1 retinal proteins. Mouse retina was almost devoid of Siaα2-6 (except WGA bound Sia), Fucα1-2 and Gal/GalNAc containing glycans. STL reacting GlcNAc oligomers were high in PN2 rd1 retinae. CONCLUSIONS: Quantitative dynamic, relative variation in high-mannose and GlcNAc glycans, Siaα2-3Galβ1-4GlcNAc associated with proteins from PN2, PN7, PN14 wt and rd1 mice retinae suggested that these glycans participate in retinal development and degeneration and may be used as markers for retinal electrophysiological integrity during transplantation/therapy studies; Siaα2-3Galβ1-4GlcNAc specific Agrocybe cylindracea lectin and other lectins may be used to enrich/purify retinal ribbon synapse glycoproteins and rhodopsin. Further investigations are required.
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| 5. |
- Ahuja, Sat pal, et al.
(author)
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Glutathione S-transferase µ(GST) modifies activities of proteases and levels of cystatin C secreted by mouse retinal explants
- 2004
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In: Investigative Ophthalmology & Visual Science. - 1552-5783. ; 45, s. 352-352
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Conference paper (peer-reviewed)abstract
- Purpose: In one form of human autosomal recessive retinitis pigmentosa and in retinal degeneration (rd1) mouse, mutation occurs in the genes encoding ß subunit of rod photoreceptor cGMP phosphodiesterase. Therefore, rd1 mutant mouse is an appropriate model for human inherited retinal degeneration studies. Retinal explants are successfully cultured in serum free chemically defined R16 medium to evaluate effects of various rescue factors and retinal conditioned medium (RCM) for secreted molecules like proteases and their inhibitors. Cysteine protease inhibitor cystatin C has recently been identified in rodent neuroretina and RPE. RCM of explants treated with GST were analyzed for proteases and cystatin C to explain, in part, mode of action of GST in protection of degenerating retina. Methods: Postnatal day 2 (PN2) and PN7 control (wt) and rd1 were cultured with (10 ng / ml GST) and without GST in R16 medium, respectively, for 26 and 21 days in vitro (div). Retinal extracts (RE) and RCM were analyzed by fluorometry using casein green fluorescent labeled with BODIPY–FL (Molecular Probes) for total proteases; Z–Phe–Arg–NMec or Z–Arg–Arg–NMec for cysteine proteases and by ELISA for cystatin C, respectively, for levels and secretion of proteases and cystatin C. The protein content of RE was measured. Results: Protein content (µg) of RE from wt and rd1 retinal extracts respectively increased and decreased with age. Cystatin C (ng/ml RCM) content in wt and rd1 RE increased with age (was always higher in wt) up to PN14 and then decreased but was higher than that at PN2. Progressive secretion of cystatin C by PN2 explants was lower than that by PN7 explants; and that by rd1 PN2 and PN7 explants was initially lower up to in vitro age of PN19 and subsequently it was higher than that by wt explants. Secretion of total cystatin C by PN2 and PN7 wt and rd1 explants was similar and was increased by GST. During initial stage of culture total protease activity ({Delta} F / 100 µl RCM) in RCM of rd1 PN2 and PN7 explants was higher and was decreased in GST treated explants. Conclusions: Cystatin C content and secretion by wt RE is always higher and that of proteases is lower than that of rd1. Treatment with GST increases content of cystatin C and consequently decreases that of proteases especially cysteine proteases.
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| 6. |
- Ahuja, Sat pal, et al.
(author)
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rd1 Mouse retina shows an imbalance in the activity of cysteine protease cathepsins and their endogenous inhibitor cystatin C.
- 2008
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In: Investigative Ophthalmology & Visual Science. - : Association for Research in Vision and Ophthalmology (ARVO). - 1552-5783. ; 49:3, s. 1089-1096
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Journal article (peer-reviewed)abstract
- PURPOSE: To compare in vivo levels, spatial localization, and in vitro secretion of cysteine protease cathepsins and cystatin C (cysC) in the retinal degeneration 1 (rd1) mouse model of retinitis pigmentosa and control (wt) mouse retinas. METHODS: The spatial localization, protein contents, cysC levels and cathepsin-B, -S, and -L activities in wt and rd1 retinas at postnatal (PN) days 2, 7, 14, 21, and 28 were analyzed by immunostaining, spectrophotometry, ELISA, and fluorescence spectrophotometry. The in vitro secretion of cysC and cysteine proteases by PN7 retinal explants into the conditioned medium (RCM) was quantified. RESULTS: The pigment epithelium, photoreceptors, and inner retinal and ganglion cell layers of both wt and rd1 retinas showed cysC and cathepsin-B labeling. CysC immunostaining was extensive in the optic nerve head fibers. The rd1 explants secreted higher amounts of cysteine protease into the RCM. The protein content in wt and rd1 retinal extracts increased up to PN14, then decreased in rd1 but not in wt. In rd1 extracts at PN14 to -28, cathepsin activity was higher and increased with age, but the cysC level was higher and constant. The ratios of cathepsin activity to cysC (cathepsin-L at PN2 and total, -B, and -L at PN14 to -28) were higher in rd1 extracts. CONCLUSIONS: Similar localization of both cathepsin-B and cysC in wt and rd1 retinas along with lower proteins and higher cathepsin activity in rd1 retinal extracts and RCM are consistent with their localization in extracellular matrix and a role in physiopathologic remodeling in wt and rd1 retinas.
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| 7. |
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| 8. |
- Ali, Sara, et al.
(author)
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Ocular Fundus Morphology and Visual Function in Adolescents Born Moderate-to-Late Preterm
- 2023
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In: Investigative Ophthalmology and Visual Science. - : Association for Research in Vision and Ophthalmology. - 0146-0404 .- 1552-5783. ; 64:8
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Journal article (other academic/artistic)abstract
- Purpose: Previous studies have mostly focused on ophthalmological complications associated with being born extremely preterm despite that moderate-to-late preterm (MLP) account for 85% of all preterm births. The aim was to examine fundus morphology and visual function in adolescents born MLP, in comparison with controls born full-term.Methods: A prospective population-based cohort study of 247 MLP individuals (110 girls, gestational age 32-36 weeks) with no syndromes or history of retinopathy of prematurity participated in a neonatal study in 2002-2004. Later on, they have been included in ophthalmological follow-up studies at age 8 (n=50) and 12 (n=22). In the present study, 50 adolescents (26 girls; mean age 16.5 years) were examined regarding best corrected visual acuity (BCVA), refraction, and ocular morphology, measured by optical coherence tomography (Topcon, Japan). A group of 50 adolescents (30 girls, mean age 16.7 years) born full-term served as controls. Participants with refraction outside +/-6 diopters were excluded. T-test was used for statistical analysis.Results: The MLP-group (n=48) showed a thinner macular retinal nerve fibre layer (RNFL) inner mean in right eye (RE) (26.4±1.5 vs 27.1±1.7 μm; p=0.029) and in left eye (LE) (26.3±1.5 vs 27.0±1.5 μm; p=0.022) compared with controls. A thinner macular RNFL outer mean was found both in RE (40.2±4.4 vs 42.6±4.2 μm; p=0.011) and LE (40.3±4.0 vs 42.1±4.3 μm; p=0.034) (Fig.1A-B). A thicker central macular retinal thickness (MRT) (249.3±20.9 vs 239.9±16.4 μm; p=0.016) and a thinner total peripapillary (pp)RNFL (104.8±8.8 vs 109.1±8.3 μm; p=0.027) were found in RE. The BCVA in best eye was lower in the MLP-group (n=50) compared with controls (-0.09±0.08 vs -0.12±0.09 logMAR; p=0.022). At age 8, MLP births showed a thinner total macular volume and a thicker foveal minimum, central MRT, and central macular RNFL in RE. At age 12, a thicker foveal minimum and thinner outer macular RNFL were found in LE.Conclusions: MLP-birth may be associated with ophthalmological macular and ppRNFL changes as well as lower BCVA in adolescence. Similar morphology findings have been shown at younger ages, thus the fundus results persist into young adulthood.
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| 9. |
- Ali, Zaheer, et al.
(author)
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Photoreceptor Degeneration Accompanies Vascular Changes in a Zebrafish Model of Diabetic Retinopathy
- 2020
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In: Investigative Ophthalmology and Visual Science. - : ASSOC RESEARCH VISION OPHTHALMOLOGY INC. - 0146-0404 .- 1552-5783. ; 61:2
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Journal article (peer-reviewed)abstract
- PURPOSE. Diabetic retinopathy (DR) is a leading cause of vision impairment and blindness worldwide in the working-age population, and the incidence is rising. Until now it has been difficult to define initiating events and disease progression at the molecular level, as available diabetic rodent models do not present the full spectrum of neural and vascular pathologies. Zebrafish harboring a homozygous mutation in the pancreatic transcription factor pdx1 were previously shown to display a diabetic phenotype from larval stages through adulthood. In this study, pdx1 mutants were examined for retinal vascular and neuronal pathology to demonstrate suitability of these fish for modeling DR. METHODS. Vessel morphology was examined in pdx1 mutant and control fish expressing the fli1a:EGFP transgene. We further characterized vascular and retinal phenotypes in mutants and controls using immunohistochemistry, histology, and electron microscopy. Retinal function was assessed using electroretinography. RESULTS. Pdx1 mutants exhibit clear vascular phenotypes at 2 months of age, and disease progression, including arterial vasculopenia, capillary tortuosity, and hypersprouting, could be detected at stages extending over more than 1 year. Neural-retinal pathologies are consistent with photoreceptor dysfunction and loss, but do not progress to blindness. CONCLUSIONS. This study highlights pdx1 mutant zebrafish as a valuable complement to rodent and other mammalian models of DR, in particular for research into the mechanistic interplay of diabetes with vascular and neuroretinal disease. They are furthermore suited for molecular studies to identify new targets for treatment of early as well as late DR.
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| 10. |
- All-Ericsson, Charlotta, et al.
(author)
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c-Kit-dependent growth of uveal melanoma cells : a potential therapeutic target?
- 2004
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In: Investigative Ophthalmology and Visual Science. - : Association for Research in Vision and Ophthalmology (ARVO). - 0146-0404 .- 1552-5783. ; 45:7, s. 2075-82
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Journal article (peer-reviewed)abstract
- PURPOSE: This study was conducted to investigate the expression and functional impact of the proto-oncogene c-kit in uveal melanoma. METHODS: Based on immunohistochemical (IHC) study of paraffin-embedded specimens from 134 uveal melanomas and Western blot analysis on eight fresh-frozen samples the expression of c-kit in uveal melanoma was studied. Furthermore, the phosphorylation of c-kit and the impact of the tyrosine kinase inhibitor STI571 was examined in the three uveal melanoma cell lines OCM-1, OCM-3, and 92-1. RESULTS: Eighty-four of 134 paraffin-embedded samples and six of eight fresh-frozen samples expressed c-kit. c-Kit was strongly expressed and tyrosine phosphorylated in cultured uveal melanoma cells compared with cutaneous melanoma cells. Moreover, in contrast to cutaneous melanoma cell lines c-kit maintained a high phosphorylation level in serum-depleted uveal melanoma cells. No activation-related mutations in exon 11 of the KIT gene were found. On the contrary, expression of the stem cell growth factor (c-kit ligand) was detected in all three uveal melanoma cell lines, suggesting the presence of autocrine (paracrine) stimulation pathways. Treatment of uveal melanoma cell lines with STI571, which blocks c-kit autophosphorylation, resulted in cell death. The IC(50) of the inhibitory effects on c-kit phosphorylation and cell proliferation was of equal size and less than 2.5 microM. CONCLUSIONS: The results confirm that c-kit is vastly expressed in uveal melanoma, suggest that the c-kit molecular pathway may be important in uveal melanoma growth, and point to its use as a target for therapy with STI571.
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