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212 Reports combination of unilateral SO palsy and heterotopic pulleys can produce a clinical pattern that resembles bilateral SO palsy, with excessive excyclotorsion compared with a unilateral SO palsy in the setting of normal EOM pulley position. References 1. Demer JL, Miller JM, Poukens V, Vinters HV, Glasgow BJ. Evidence for fibromuscular pulleys of the recti extraocular muscles. Invest Opbthalmol Vis Sd. 1995;36:1125-1136. 2. Miller JM. Functional anatomy of normal human rectus muscles. Vision Res. 1989;2S>:223-240. 3. Clark RA, Miller JM, Demer JD. Location and stability of rectus muscle pulleys: muscle paths as a function of gaze. Invest Ophthalmol Vis Sci. 1997;38:227-240. 4. Demer JL, Miller JM, Rosenbaum AL. Effect of transposition surgery on rectus muscle paths by magnetic resonance imaging. Ophthalmology. 1993;100:475-487. Establishment and Characterization of a Retinal Muller Cell Line Vijay P. Sarthy,1 Sevan J. Brodjian,1 Kamla Dutt,2 Breandan N. Kennedy? Randall P. French,1 and John W. Crabb5 Primary cultures of Miiller cells have proven useful in cell biologic, developmental, and electrophysiological studies of Miiller cells. However, the limited lifetime of the primary cultures and contamination from non-neural cells have restricted the utility of these cultures. The aim of this study was to obtain an immortalized cell line that exhibits characteristics of Muller cells. PURPOSE. METHODS. Primary Muller cell cultures were prepared from retinas of rats exposed to 2 weeks of constant light. Cells were immortalized by transfection with simian virus 40. Single clones were obtained by repeatedly passaging cells using cloning wells. Immunocytochemical and immunoblotting studies were carried out with glial nbrillary acidic protein (GFAP)-specific and cellular retinaldehyde-binding protein (CRALBP)-specinc antibodies. Transient transfections with CRALBP-luciferase constructs were performed by electroporation. Oncogene transformation resulted in the establishment of a permanent cell line that could be readily propagated. Immunocytochemical and immunoblotting RESULTS. From the Department of Ophthalmology, Northwestern University Medical School, Chicago, Illinois; the department of Pathology, and Cell Biology and Anatomy, Morehouse Medical School, Atlanta, Georgia; and the 3W. Alton Jones Cell Center, Lake Placid, New York. Supported by National Eye Institute grants EY-03523 and EY06603 and by an unrestricted award from Research to Prevent Blindness Inc. Submitted for publication January 14, 1997; revised June 10, 1997; accepted September 19, 1997. Proprietary interest category: N. Reprint requests: Vijay Sarthy, Department of Ophthalmology, Tarry 5-715, Northwestern University Medical School W113, 300 E. Superior St., Chicago, IL 60611. IOVS, January 1998, Vol 39, No. 1 5. Clark RA, Miller JM, Rosenbaum AL, Demer JL. Heterotopic muscie pulleys or oblique muscle dysfunction? J Am Assoc Pediatr Ophthalmol. Stabismus. 1998. In press. 6. Guyton DL, Weingarten PE. Sensory torsion as the cause of primary oblique muscle overaction/underaction and A- and Vpattern strabismus. Binoc Vis Eye Muscle Surg Q. 1992;9:209236. 7. Demer JL, Miller JM. Magnetic resonance imaging of the functional anatomy of the superior oblique. Invest Ophthalmol Vis Sci. 1995; 36:906-913. 8. Miller JM, Robinson DA. A model of the mechanics of binocular alignment. Comput Biomed Res. 1984; 17:436-470. 9. Cheng H, Burdon MA, Shun-Shin GA, Czypionka S. Dissociated eye movements in craniosynostosis: a hypothesis revived. Br J Ophthalmol. 1993;77:563-568. 10. Krzizok T, Wagner D, Kaufmann H. Elucidation of restrictive motility in high myopia by magnetic resonance imaging. Arch Ophthalmol. 1996;115:1019-1027. studies demonstrated that the Muller cell line, rMC-1, expressed both GFAP, a marker for reactive gliosis in Muller cells, and CRALBP, a marker for Muller cells in the adult retina. Transient transfection assays showed that promoter-proximal sequences of the CRALBP gene were able to stimulate reporter gene expression in rMC-1. CONCLUSIONS. Viral oncogene transformation has been successfully used to isolate a permanent cell line that expresses Muller cell phenotype. The rMC-1 cells continue to express both induced and basal markers found in primary Muller cell cultures as well as in the retina. The availability of rMC-1 should facilitate gene expression studies in Muller cells and improve our understanding of Miiller cell-neuron interactions. (Invest Ophthalmol Vis Sci. 1998;39:212-2l6) M uller cells are the most abundant non-neuronal cells in the vertebrate retina, and they perform diverse functions that support the activity of retinal neurons, hi recent years, the availability of dissociated cell preparations and primary Miiller cell cultures has greatly facilitated cell biologic, biochemical, developmental, and electrophysiological studies of Muller cells. Miiller cell cultures have been obtained from neonatal and adult retinas, and from retinas with inherited dystrophy or constant light damage.1"8 However, primary Muller cell cultures have certain problems that limit their utility: the cells have a limited life span and undergo senescence with passage; the cultures are usually contaminated with astrocytes and microglia3'9; unless a large number of eyes is used, only a small number of cultures can be obtained3; and the small culture size restricts their use to morphologic, immunocytochemical, and electrophysiological studies. Some problems associated with primary cultures can be overcome by establishing permanent cell lines through immortalization of primary cells with viral oncogenes. During gene regulation studies using transfection assays, we found that the small number of cells and the low transfection efficiency in primary Muller cell cultures were serious drawbacks.10 This motivated us to establish a Muller cell line. This report describes the isolation and immunochemical identification of a Muller cell line (rMC-1) from adult rat retina. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933426/ on 05/03/2017 Reports IOVS, January 1998, Vol 39, No. 1 METHODS Primary Miiller cell cultures used for immortalization were obtained from rats exposed to constant light to induce photoreceptor loss.3 In previous work, it has been reported that cell cultures prepared from light-damaged retinas consist predominantly of Miiller cells.3 Briefly, Sprague-Dawley rats were exposed to constant light for 2 weeks. The retinas were isolated, treated with trypsin, dissociated, and plated into 75-mm2 flasks containing Dulbecco's minimal essential medium supplemented with 10% fetal bovine serum and glutamine. After a single passage, primary Miiller cell cultures were transfected by a modified calcium phosphate method."" 1 3 Ten micrograms of simian virus 40 (SV40) DNA was mixed with 0.5 ml of 2 X Hepes buffer, pH 7.05, and an equal volume of 0.4 M CaCl2 in 10 mM Tris, pH 7.6, and 1 mM ethylenediaminetetraacetic acid. The mixture was applied to Miiller cells in 75-mm2 flasks (approximately 50% confluency). After 12 hours, cells were treated with 15% glycerol for 90 seconds at room temperature and maintained in regular growth medium. Clones began to appear by 2 to 3 weeks and were expanded by repeatedly passaging cells using cloning wells as described earlier.13 This protocol has been successfully used to obtain immortalized pigment epidielium and retinal cell lines.13 Because our primary interest was to obtain a cell line suitable for glial fibrillary acidic protein (GFAP) gene expression studies, seven clones were immunostained with a GFAP antibody, and one positive clone, rMC-1, was chosen for additional characterization. Immunocytochemical and immunoblotting studies were carried out as described previously.314 Animals were used according to the tenets of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. RESULTS Figure la shows die morphology of cells from a primary culture of Miiller cells. The majority of cells in these cultures are the large, flat cells, which have been previously shown to be Miiller cells.3 A small number of astrocytes, endodielial cells, or microglia are also usually present.3'9 The Miiller cell line, rMC-1, was derived by transforming primary cultures with SV40 DNA. Figure lb presents die morphologic features of rMC-1. Unlike the primary culaires, all cells in the rMC-1 cultures have die same morphology—the cells are more com- TABLE 1. Luciferase Levels in rMC-1 and 3T3 Cells Transfected with a pCRALBP-/wc Construct pCRALBP-/«c pGL2B-/wc rMC-1 3T3 180 ± 48 13.2 ± 4.1 9.8 ± 0.27 8.3 ± 0.6 Miiller cells (rMC-1) and fibroblasts (3T3) were grown to 50% to 70% confluency and cotransfected with 20 ixg of DNA per 100-mm2 culture. The construct pCRALBP-/«c contains a 0.3-kb, 5' fragment of human CRALBP gene cloned into the promoterless vector, pGL2B-/wc. Cotransfection with pSVlac vector was used to normalize transfection assays. Forty-eight hours following electroporation, cells were harvested and assayed for luciferase and j3-galactosidase using a commercially available kit. Luciferase activities were expressed as photon counts per second X 103 and were normalized to /3-gal activities in each case to control for variations in transfection efficiency. All values are expressed as mean ± SEM for three separate determinations. 213 pact and elongated widi a less prominent nucleus. The cells grow rapidly with a doubling time of approximately 48 to 52 hours. To demonstrate that rMC-1 is derived from Miiller cells, we carried out immunocytochemical studies using antibodies to GFAP and cellular retinaldehyde-binding protein (CRALBP). In primary cultures, GFAP is a useful marker for reactive Miiller cells (Fig. lc), and astrocytes, whereas endothelial cells and microglia do not express this marker.15 CRALBP is known to be expressed by Miiller cells but not by astrocytes in die adult mammalian retina.16 As shown in Figures Id, le, If, rMC-1 cells were imniunoreactive for both GFAP (Fig. Id) and CRALBP (Figs, le, If). Moreover, immunostaining was seen widi both N-temiinal (Fig. le) and C-terminal (Fig. If) CRALBP antibodies. These results demonstrate that rMC-1 expresses antigens specifically associated with Miiller cells in the adult retina. An intriguing feature was that immunostaining was often perinuclear. We do not know the reason for this pattern of immunostaining. Although rMC-1 expressed GFAP, the intensity of immunostaining was considerably weaker than that observed with primary cultures (compare Figs, lc, Id). Finally, when rMC-1 cells were stained widi a monoclonal antibody to SV40-T antigen, all nuclei were strongly reactive, showing that rMC-1 cells contained SV40 (Fig. Ig). Omission of the primary antibody resulted in no immunostaining (Fig. In). In addition to immunocytochemistry, we performed immunoblotting to further characterize the antigen recognized by GFAP and CRALBP antibodies. Cell extracts were separated by polyacrylamide gel electrophoresis and blotted onto nitrocellulose. The blots were stained with a monospecific, polyclonal GFAP antibody or with antipeptide, CRALBP antibodies specific to N-terminal and C-terminal regions. Extracts from primary Miiller cells or bovine retina were used as controls. As shown in Figure 2, a single band of ~ 5 0 kDa was GFAP immunoreactive in rMC-1 and primary Muller cell extracts, demonstrating that rMC-1 expressed GFAP.14 In extracts from primary cultures, there were several lower size bands that were likely to be GFAP degradation products. Immunoblots stained for three different CRALBP antibodies showed that, in rat retina and rMC-1 extracts, the antibodies recognized a single protein of about the same size as CRALBP (36 kDa). l7 An additional band, slightly smaller in size, was noted in the cell extracts (Fig. 2). We do not know the origin of the smaller band; it might be a degradation product of CRALBP. Finally, we have used rMC-1 cells to test their utility for transfection assays. For this purpose, a construct containing the proximal (0.3 kb), 5' sequences of the human CRALBP gene was transfected into rMC-1 and NIH 3T3 cells, a fibroblast cell line, which served as a control. In addition, transfections were also carried out with a vector, pGL2B, that lacked the CRALBP promoter. As shown in Table 1, in rMC-1, pGL2 with a 0.3-kb CRALBP promoter stimulated luciferase expression 14-fold compared with a promoterless vector (Table 1). Moreover, the stimulation in reporter expression was cell typespecific, because luciferase activity was relatively lower in 3T3 cells. These data show that the immediate, 5'-flanking sequences of human CRALBP gene contain DNA sequences that stimulate CRALBP gene expression in Miiller cells. Therefore, the rMC-1 cell line allowed us to extend gene regulation studies of CRALBP to Miiller cells, in addition to RPE cells. 1 8 1 9 DISCUSSION The present report describes the isolation and characterization of an oncogene-transformed retinal cell line that exhib- Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933426/ on 05/03/2017 214 Report IOVS. Tanuarv 1998. Vol 39, No. 1 1. Morphology and immunocytochemical characterization of Miiller cells, (a) Primary culture of Miiller cells, (b) Miiller cell line, rMC-1. (c) Primary Miiller cell culture stained with glial fibrillary acidic protein (GFAP) antibody, (d) rMC-1 reacted with GFAP antibody. (e; f) rMC-1 stained with N-terminal and C-terminal cellular retinaldehyde-binding protein antibodies, respectively, (g) rMC-1 reacted with a monoclonal antibody to SV4O T-antigen. (h) rMC-1, immunocytochemistry with no primary antibody added. Magnifications: 160X (a, b); 960X (c, d); 56OX (e); and 48OX (f-h). FIGURE its phenotypic characteristics of retinal Miiller cells. This study shows that viral oncogene transformation can be successfully used to isolate permanent cell lines from primary Miiller cell cultures. Our findings are consistent with a recent report in which a disabled viral construct carrying human papillomavirus-l6 E6/E7 genes was used to transform Miiller cells.20 The availability of a permanently established MiiUer cell line should facilitate a variety of studies such as identification of Miiller cell-specific antigens, Miiller cellneuron and Muller cell-endothelial cell interactions, char- Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933426/ on 05/03/2017 Reports 215 IOVS, January 1998, Vol 39, No. 1 *• f GFAP S17L K25Q * $ Coomassie Blue Is S31F FIGURE 2. Western blot analysis of glial fibrillary acidic protein (GFAP) and cellular retinaldehyde-binding protein (CRALBP) expression in rMC-1 cells. Extracts of rMC-1, primary Miiller cell cultures (pMC; —100 jutg), or rat retina (—100 /xg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose, and the blots were reacted with a polyclonal, bovine GFAP antibody (Dako, Carpinteria, CA) orpolyclonal antipeptide antibodies to bovine CRALBP. The antibodies used for blotting are indicated underneath the blots. Closed arrow, GFAP band; open arrow, CRALBP band in the retina. The peptide antibodies, purified immunoglobulin Gs were used at a concentration of 1 jag/ml, and recognize N-terminal CRALBP residues 1-17 (S17L), residues 46-70 (K25Q), and Gterminal residues 286-316 (S31F). Detections were carried out with an Enhanced Chemi-Luminescence Kit (Amersham Life Science, Arlington Heights, IL). The Coomassie Blue-stained gel following the transfer to nitrocellulose is also shown. acterization of growth factors and cytokines secreted by Miiller cells, and regulation of gene expression in Miiller cells. Moreover, these studies should lead to a better understanding of Miiller cell functions in the mammalian retina. A cknowledgment The authors thank Kathleen Rundell, Northwestern University, for the monoclonal antibody to SV40-T antigen. References 1. Adler RP, Magistretti J, Hyndman AG, Shoemaker WJ. Purification and cytochemical identification of neuronal and non-neuronal cells in chick retina cultures. Dev JVeurosci. 1982;5:27-39. 2. Burke JM, Foster SJ. Culture of adult rabbit retinal glial cells: methods and cellular origin of explunt outgrowth. Curr Eye Res. 1984;3:1169-1178. 3. Sarthy PV. Establishment of Miiller cell cultures from adult rat retina. Brain Res, 1985:337:138-141. 4. Roberge FG, Caspi RR, Chan CC, Kuwabara T, Nussenblatt RB. Long-term culture ol Miiller cells from adult rats in the presence of activated lymphocytes/monocytes products. Curr Eye Res. 1985; 4:975-982. 5. Wakakura M, Foulds WS. Immunocytochemical characteristics of Miiller cell cultures from adult rabbit retina. Invest Ophtbalmol VisSci. 1988;29:892-900. 6. Lewis GP, Kaska DD, Vaughn DK, Fisher SK. An immunocytochemical study of cat retinal Miiller cells in culture. Exp Eye Res. 1988;47:855-868. 7. Hicks D, Courtois Y. The growth and behavior of rat retinal Miiller cells in vitro. 1. An improved method for isolation and culture. Exp Eye Res. 1990;51:119-129. 8. Aotaki-Keen AE, Harvey AK, dejuan E, Hjelmekmd LM. Primary culture of human retinal glia. Invest Ophthalmol Vis Sci. 1991 ;32: 1733-1738. 9. Roque RS, Caldwell RB. Isolation and culture of retinal microglia. Curr Eye Res. 1993; 12:285-291. 10. French R, Sarthy V. Glial fibriUary acidic protein (GFAP) gene regulation in transfected Miiller cell cultures. [ARVO Abstract] Invest Ophtbalmol Vis Sci. 1996;37:S337. 11. Graham FL, van der Eb. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973;52:456467. 12. Srinivasan A, Reddy EP, Aaronson, SA. Abelson murine leukemia virus: molecular cloning of infectious integrated proviral DNA. Proc Natl Acad Sci USA. 1981;78:2077-2081. 13. Dutt K, Scott M, Del Monte M, et al. Establishment of human retinal pigment epithelial cell lines by oncogenes. Oncogene. 1990;5: 195-200. 14. Einsenfeld AJ, Bunt-Milam AH, Sarthy PV. Miiller cell expression of glial fibrillary acidic protein after genetic and experimental photoreceptor degeneration in the rat retina. Invest Ophthalmol Vis Sci. 1984;25:1321-1328, Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933426/ on 05/03/2017 216 Reports 15. Sarthy V. Reactive gliosis in retinal degeneration. In: Hollyfield JE, Anderson RE, LaVail MM, eds. Retinal Degenerations. Boca Raton, FL: CRC Press; 1991:109-116. 16. Saari JC. Retinoids in photosensitive systems. In: Sporn MB, Roberts, AB, Goodman DS, eds. The Retinoids: Biology, Chemistry and Medicine. New York: Raven Press; 1994:351- 385. 17. Crabb JW, Gaur VP, Garwin GG, et al. Topological and epitope mapping of the cellular retinaldehyde binding protein from retina. J Biol Chem. 1991 ;266:16674-16683. IOVS, January 1998, Vol 39, No. 1 18. Kennedy BN, Goldflam S, Chang MA, et al. Promoter analysis of the human CRALBP gene. [ARVO Abstract.] Invest Ophthalmol Vis Sci. 1995;36:S124. 19. Kennedy BN, Chang MA, Campochiaro P, Zack DJ, Crabb JW. Potential regulators of CRALBP gene expression. [ARVO Abstract.] Invest Ophthalmol Vis Sci. 1996;37:S336. 20. Roque RS, Agarwal N, Wordinger RJ, et al. Human papillomavirus-16 E6/E7 transfected retinal cell line expresses the Miiller cell phenotype. Exp Eye Res. 1997;64:519-527. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933426/ on 05/03/2017