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Research Letter |

Alteration of Vitreal Retinoschisin Level in Human Primary Retinal Detachment FREE

Sten Kjellström, MD, PhD1; Fredrik Ghosh, MD, PhD1; Camasamudram Vijayasarathy, PhD2; Sten Andréasson, MD, PhD1
[+] Author Affiliations
1Department of Ophthalmology, University of Lund, Lund, Sweden
2National Institute of Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, Maryland
JAMA Ophthalmol. 2014;132(3):353-354. doi:10.1001/jamaophthalmol.2013.6306.
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Published online

Retinoschisin (RS1), the product of RS1 located on the X chromosome, is expressed mainly in retina.1 The 24-kDa RS1 encodes a highly conserved sequence motif termed the discoidin domain, which is a critical determinant of the structural and functional integrity of the retina.2 Mutations in RS1 that lead to either complete loss of RS1 expression or its accumulation as a nonfunctional misfolded form are the underlying causes of X-linked retinoschisis (XLRS).3 This disorder, seen exclusively in young males, is characterized by splitting, or schisis, affecting all retinal layers. The precise molecular mechanism by which RS1 functions is still undefined. During development, there is a wave of RS1 expression beginning at the retinal surface and spreading progressively in more distal retinal layers as they differentiate.4 In adult mice, RS1 is expressed in all retinal neurons except horizontal cells,4 with a predominance in photoreceptor inner segments and bipolar cells.

In patients with XLRS, the retina is more prone to retinal detachment (RD) compared with the general population (10% vs 0.01%, respectively).5 These detachments are difficult to surgically reattach, making the postoperative outcome unfavorable.6,7 To assess the potential role of RS1 in retinal response to detachment, we analyzed RS1 levels in vitreous samples derived from patients’ eyes with or without RD.

The research procedures were in accordance with institutional guidelines and the Declaration of Helsinki. Approval for the study was obtained from the Committee of Ethics, University Hospital, Lund, Sweden.

Surgery

Fourteen patients (mean age, 59 years) with primary RD of less than 7 days’ duration and with no previous retinal surgery or intravitreal treatment were included in this study. After explanation of the planned operation and answering the questions, written informed consent was obtained directly from each participant. As healthy control participants, 12 patients (mean age, 68 years) with epiretinal membrane and no previous retinal surgery were included. All patients underwent a standard 3-port pars plana vitrectomy including a biopsy of 1.0 to 1.5 mL of undiluted central vitreous obtained under air infusion via suction through the vitrectomy probe (Innovit and Constellation; Alcon Laboratories). The sample was thereafter kept at −80°C for later examination.

Immunoblot Analysis

The protein concentrations of the vitreous samples were determined by bicinchoninic acid assay (Thermo Scientific). Equal amounts of vitreous protein from different samples (10 µg each) were resolved on a gradient 4% to 12% nonreducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electrotransferred to a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Inc). After blocking the membrane in 5% nonfat milk in phosphate-buffered saline/Tween (PBST) solution (3.2mM disodium hydrogen phosphate, 0.5mM monopotassium phosphate, 1.3mM potassium chloride, 135mM sodium chloride, 0.1% Tween 20, pH 7.4) for 1 hour at room temperature, the blot was incubated overnight at 4°C with anti-RS1 antibody produced in rabbit (titer, 1:1000).8 The membrane was then subjected to three 5-minute rinses in a PBST solution. The membrane was then incubated with a horseradish peroxidase–conjugated goat antirabbit antibody (titer, 1:10 000; GE Healthcare Life Sciences) for 1 hour at room temperature. After three 5-minute rinses in a PBST solution, the blot was developed with SuperSignal West Dura Chemiluminescent Substrate (Thermo Scientific). The chemiluminescent images were acquired by imaging the membrane on a Kodak image station (Image Station 2000R; Eastman Kodak Co), and the intensity of each approximately 200-kDa octameric RS1 band was analyzed using the spot density analysis software provided. The relative protein levels were calculated from the band intensity of each protein normalized to the intensity of recombinant histidine-tagged RS1 (purified from baculovirus/insect cell expression system). Western blotting and quantification analysis were performed in at least 3 biological replications.

Vitreous concentrations, measured as intensity units, of RS1 were significantly lower in patients with newly detached retinas compared with control vitreous samples from nondetached retinas (epiretinal membrane). The mean (SE) RS1 vitreous concentration was 49 609 (7192) intensity units in detached retinas and 80 540 (12 379) intensity units in nondetached retinas (Mann-Whitney U test, P = .04) (Figure).

Place holder to copy figure label and caption
Figure.
Quantitation of Retinoschisin in Vitreous From Newly Detached and Nondetached Retinas

The nondetached retina uses the epiretinal membrane for analysis. RS1 indicates retinoschisin; center horizontal lines, mean; and error bars, standard error.

Graphic Jump Location

Like most previous studies, we have used data from samples of vitreous collected from patients undergoing pars plana vitrectomy for epiretinal membranes as a reference for considering the changes in vitreous.9 To our knowledge, this is the first study showing decreased levels of RS1 in vitreous of patients with primary RD in comparison with control samples collected from patients undergoing peeling of epiretinal membranes. The decreased levels of RS1 in patients with RD could possibly be explained by depletion of RS1 in the detachment phase.

The potential role of RS1 in patients with RD is still unknown. In a mouse model of RD, Farjo et al10 reported upregulation of RS1 messenger RNA within 2 hours of RD, which is consistent with the potential role of RS1 in retinal cell adhesions and extracellular remodeling. Furthermore, gene therapy in a mouse model of XLRS has also demonstrated a decrease in schisis cavities and improved retinal signaling evaluated by electroretinography.8,11 If RS1 has a key role in retinal remodeling after RD, the lack of RS1 could be one reason why treatment of RD in patients with XLRS would be particularly challenging. Further studies are needed to clarify the role of RS1 in the remodeling process following RD.

Corresponding Author: Sten Kjellström, MD, PhD, Department of Ophthalmology, University of Lund, S 221 85, Lund, Sweden (sten.kjellstrom@med.lu.se).

Published Online: December 26, 2013. doi:10.1001/jamaophthalmol.2013.6306.

Author Contributions: Dr Kjellström had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study concept and design: Kjellström, Ghosh, Andréasson.

Acquisition of data: Kjellström, Vijayasarathy.

Analysis and interpretation of data: All authors.

Drafting of the manuscript: All authors.

Critical revision of the manuscript for important intellectual content: Kjellström, Vijayasarathy.

Statistical analysis: Kjellström.

Obtained funding: Kjellström, Ghosh, Andréasson.

Administrative, technical, or material support: All authors.

Study supervision: Kjellström, Ghosh, Andréasson.

Conflict of Interest Disclosures: None reported.

Funding/Support: This study was supported by grants from the Faculty of Medicine at Lund University, Stiftelsen Synfrämjandets Forskningsfond, Fiundation Olle Engkvist, and the Swedish Medical Research Council.

Role of the Sponsor: The funding organizations had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

Sauer  CG, Gehrig  A, Warneke-Wittstock  R,  et al.  Positional cloning of the gene associated with X-linked juvenile retinoschisis. Nat Genet. 1997;17(2):164-170.
PubMed   |  Link to Article
Vijayasarathy  C, Ziccardi  L, Sieving  PA.  Biology of retinoschisin. Adv Exp Med Biol. 2012;723:513-518.
PubMed
Molday  RS, Kellner  U, Weber  BH.  X-linked juvenile retinoschisis: clinical diagnosis, genetic analysis, and molecular mechanisms. Prog Retin Eye Res. 2012;31(3):195-212.
PubMed   |  Link to Article
Takada  Y, Fariss  RN, Tanikawa  A,  et al.  A retinal neuronal developmental wave of retinoschisin expression begins in ganglion cells during layer formation. Invest Ophthalmol Vis Sci. 2004;45(9):3302-3312.
PubMed   |  Link to Article
Sasaki  K, Ideta  H, Yonemoto  J, Tanaka  S, Hirose  A, Oka  C.  Epidemiologic characteristics of rhegmatogenous retinal detachment in Kumamoto, Japan. Graefes Arch Clin Exp Ophthalmol. 1995;233(12):772-776.
PubMed   |  Link to Article
Regillo  CD, Tasman  WS, Brown  GC.  Surgical management of complications associated with X-linked retinoschisis. Arch Ophthalmol. 1993;111(8):1080-1086.
PubMed   |  Link to Article
Rosenfeld  PJ, Flynn  HW  Jr, McDonald  HR,  et al.  Outcomes of vitreoretinal surgery in patients with X-linked retinoschisis. Ophthalmic Surg Lasers. 1998;29(3):190-197.
PubMed
Zeng  Y, Takada  Y, Kjellstrom  S,  et al.  RS-1 gene delivery to an adult Rs1h knockout mouse model restores ERG b-Wave with reversal of the electronegative waveform of X-linked retinoschisis. Invest Ophthalmol Vis Sci. 2004;45(9):3279-3285.
PubMed   |  Link to Article
Yoshida  S, Ishikawa  K, Matsumoto  T, Yoshida  A, Ishibashi  T, Kono  T.  Reduced concentrations of angiogenesis-related factors in vitreous after vitrectomy in patients with proliferative diabetic retinopathy. Graefes Arch Clin Exp Ophthalmol. 2010;248(6):799-804.
PubMed   |  Link to Article
Farjo  R, Peterson  WM, Naash  MI.  Expression profiling after retinal detachment and reattachment: a possible role for aquaporin-0. Invest Ophthalmol Vis Sci. 2008;49(2):511-521.
PubMed   |  Link to Article
Kjellstrom  S, Bush  RA, Zeng  Y, Takada  Y, Sieving  PA.  Retinoschisin gene therapy and natural history in the Rs1h-KO mouse: long-term rescue from retinal degeneration. Invest Ophthalmol Vis Sci. 2007;48(8):3837-3845.
PubMed   |  Link to Article

Figures

Place holder to copy figure label and caption
Figure.
Quantitation of Retinoschisin in Vitreous From Newly Detached and Nondetached Retinas

The nondetached retina uses the epiretinal membrane for analysis. RS1 indicates retinoschisin; center horizontal lines, mean; and error bars, standard error.

Graphic Jump Location

Tables

References

Sauer  CG, Gehrig  A, Warneke-Wittstock  R,  et al.  Positional cloning of the gene associated with X-linked juvenile retinoschisis. Nat Genet. 1997;17(2):164-170.
PubMed   |  Link to Article
Vijayasarathy  C, Ziccardi  L, Sieving  PA.  Biology of retinoschisin. Adv Exp Med Biol. 2012;723:513-518.
PubMed
Molday  RS, Kellner  U, Weber  BH.  X-linked juvenile retinoschisis: clinical diagnosis, genetic analysis, and molecular mechanisms. Prog Retin Eye Res. 2012;31(3):195-212.
PubMed   |  Link to Article
Takada  Y, Fariss  RN, Tanikawa  A,  et al.  A retinal neuronal developmental wave of retinoschisin expression begins in ganglion cells during layer formation. Invest Ophthalmol Vis Sci. 2004;45(9):3302-3312.
PubMed   |  Link to Article
Sasaki  K, Ideta  H, Yonemoto  J, Tanaka  S, Hirose  A, Oka  C.  Epidemiologic characteristics of rhegmatogenous retinal detachment in Kumamoto, Japan. Graefes Arch Clin Exp Ophthalmol. 1995;233(12):772-776.
PubMed   |  Link to Article
Regillo  CD, Tasman  WS, Brown  GC.  Surgical management of complications associated with X-linked retinoschisis. Arch Ophthalmol. 1993;111(8):1080-1086.
PubMed   |  Link to Article
Rosenfeld  PJ, Flynn  HW  Jr, McDonald  HR,  et al.  Outcomes of vitreoretinal surgery in patients with X-linked retinoschisis. Ophthalmic Surg Lasers. 1998;29(3):190-197.
PubMed
Zeng  Y, Takada  Y, Kjellstrom  S,  et al.  RS-1 gene delivery to an adult Rs1h knockout mouse model restores ERG b-Wave with reversal of the electronegative waveform of X-linked retinoschisis. Invest Ophthalmol Vis Sci. 2004;45(9):3279-3285.
PubMed   |  Link to Article
Yoshida  S, Ishikawa  K, Matsumoto  T, Yoshida  A, Ishibashi  T, Kono  T.  Reduced concentrations of angiogenesis-related factors in vitreous after vitrectomy in patients with proliferative diabetic retinopathy. Graefes Arch Clin Exp Ophthalmol. 2010;248(6):799-804.
PubMed   |  Link to Article
Farjo  R, Peterson  WM, Naash  MI.  Expression profiling after retinal detachment and reattachment: a possible role for aquaporin-0. Invest Ophthalmol Vis Sci. 2008;49(2):511-521.
PubMed   |  Link to Article
Kjellstrom  S, Bush  RA, Zeng  Y, Takada  Y, Sieving  PA.  Retinoschisin gene therapy and natural history in the Rs1h-KO mouse: long-term rescue from retinal degeneration. Invest Ophthalmol Vis Sci. 2007;48(8):3837-3845.
PubMed   |  Link to Article

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