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Retinal pigment epithelial (RPE) transplantation onto Bruch's membrane in organ culture
Friday, 5:30--7:OO P.M., Sep 25, 1992 Palazzo Dei Congressi
X ICER Abstracts 885
61
64
EPITHRLI~ (RPE) T~SPL~TATION ONTO BRUCH’S MEMBRANE IN ORGAN CULTURE. Lucian V. Del Priore and Russell Hornbeck. Washis University, St. Louis, MO RETINAL
PIGMENT
88%
ARGON LASER DEGENERATION
PHOTO~OAGULATION DELAYS FHOTO~~~~OR IN THE RCS RAT. r. C.. Chu. Y. m WARP Research Centre, Lions Eye Institute, QEII Medical Centre. Nedlands, Western Australia. Both intraocular injections of bFGF and subretinal needle damage have recently been shown to transiently delay the loss of pho~receptors in the RCS rat. We have investigated the effect of argon laser photocoagulation at the same time at which the above factors had an effect. The retina superior to the optic disc was irradiated with a grid of 40-60 50pm argon laser lesions of 250-350 mW in post-natal day 23 (P23) pink eyed RCS rats. At 7, 14, 30 and 60 days after irradiation the eyes were fixed and embedded in epon-araldite. Vertical sections through the optic nerve were stained with toluidine blue and analysed. High levels of irradiation destroyed the photoreceptors and pigment cells within the grid area. However, on the lesion flanks there were less pyknotic cells at 7 and 14 days and more surviving cells at 30 days. This correlated with a reduced debris layer along the flanking regions. Lower levels of irradiation did not destroy the photor~epto~ but appeared to activate the pigment cells to enter the debris layer. By 7 days most pyknotic cells had been removed leaving several rows of healthy cells. These cells were still present at 30 days after lesion (P53) but had degenerated by 60 days (P83). These results indicate that laser irradiation affects the retina in such a way that dying cells are removed more rapidly but the remaining cells survive for longer. The mechanisms of these effects are being examined.
We have developed an in vitro system for studying the transplantation of RPE cells onto Bruch’s membrane IBM) by modifying our previous organ culture system (Arch Ophth 106:1286>. Briefly, 6 mm buttons of eyewall containing BM. choroid, and sclera are trephined from freshly enucleated pig eyes, frozen at -20 C for at least 30 minutes, and thawed prior to use. Porcine passage 3 RPE cells are harvested from tissue culture flasks and plated onto BM in organ culture (150,000 cells/O.1 cc). RPE attach to BH and begin to flatten along BM within 2 hours of transplantation. One week after transplantation, BM is completely covered by a layer of transplanted RPE. The ability to transplant RPE cells harvested from tissue culture flasks & vitro is important in understanding the factors responsible for regulating RPE attachment to BM and RPE differentiation.
88%
65
889
RETINITJS PIGMENTOSA: PJJOTORECEmOR MORPHOLOGY AND A QUESTJON OF CJLJAJZY DlZECJS. Pi& J. Szezesny, institute of Ophthalmol~, Lcndon. CurrentIy: Au~n~i~k US& IUimistr 166, Ziirieh. Retinitis pigmentosa (RP) is a heterogeneous group of hereditary diseases of unknown etiology which lead to progressive degeneration of photoreceptors and the retinal pigment epithelium. A generalized structural defect of the cilia in various tissues, including photoreceptor connecting cilium, has been postulated as occurring in the X-linked form of RP (Arden and Fox 1979). However the literature on retinal pa~olo~ in RP reveals contradictor results on that issue. In this study the mo~holo~ of photoreceptors and the connecting cilium from 17 RP donors including X-linked RP, X chromosome carrier state, autosomal dominant and recessive RP was examined in electron microscopy. Photoreceptor preservation was commonly observed in some locations even in most advanced disease. These included perimacular area but not the macula itsele the area proximal to the optic nerve; and the far periphery. ~hotore~ptor cells showed two major pattern of changes associated with the stage of disease and regardless of genetic type of RI? I. wide spread autophagy at early stages of degeneration; II. contraction and migration of the photoreceptor inner segments into deeper retinal layers associated with proliferation and compression by Muller cells at the level of the outer limiting membrane in more advanced RP. Primary ciliary defects (eg additional or missing microtubul~) were found in none of the samples. Comparison of photoreceptors in normal and RP tissue showed thinner and longer cilia and longer centrioles in RP cells but no defects in the microtubule arrangements within the connecting cilia. The changes in the photoreceptor cytoskeleton appear to be associated with cell migration rather than the primary structural defect leading to cell degeneration in RI’. 63
66
887
890
MUTATIONS OF RHODOPSIN GENE FOUND IN JAPANESE FAMILIES WITH AUTOSOMAL DOMINANT RETINITIS PIGMENTQSA (ADRP) Sakuma. H., Hotta, Y.. Hayakawa, M.. Nakaiima. A., Fuiiki, K.. Kanai. A. (Department of Ophthalmology, Juntendo University, Tokyo 113 Japan) Shiono. T.. Nero, hL, S&uma. T., Tam&. M. (Department of Ophthalmology, Tohoku University, Sendai 980 Japan 1
VITREOUS BODY FLUORESCENCE CHANGES AFTER RETINAL LASER COAGULATION IN A RABBIT IN VIVO MODEL Libondi, T., Ragone, R., Romano, A.. Colonna G, and Auricchio, G. of Biochemistry and Biophysics, Eye Clinic and Dept. University of Naples, Italy Retinal photocoagulation induces a transient We used a rabbit in viva model inflammatory response. to assess whether retinal laser treatment affects vitreus spectrofluorimetric behaviour. Pigmented inbred underwent scattered retinal rabbit retinas photocoagulation (500 spots, 488 nm light, 50 micron, The burns were made similar to the 120 mw, 0,2 set). one which are observed in human diabetic retina laser treatment. 24 hrs lacer vitreous body was dissected, homogenized 1:l with BHT solution to avoid further oxidation, and treated with 0.15% SDS. The excitation spectra of the vitreous solution showed a peak at approx 350 nm. The emission spectra (excitation at 350 nm) showed the presence of a peak centered at 420 nm. The obtained fluorimetric results are suggestive for the presence of iminopropene bonds, which could be derived from the reaction of toxic aldehydes with free aminogroups of vitreous proteins, mainly collagens. This fluorescence behaviour can be appreciated even 7 represent days after laser treatment, and could longstanding changes of native conformation of vitreal structural proteins after laser retinal treatment.
Retinitis pigmentosa (RF) is a hereditary degenerative d~scasf !\‘l?lch causes blindness. RF is clinically and genetically heterogeneox. Smcc l31,!‘ia et aL(1990) found the mutations of codon 23, 58 and 347 in an allclc of the rhodopsin gene on chromosome 3 in ADRP patients, several mutations and deletions in the rhodopsin gene in ADRP patients were identified. WC have already reported a Japanese family with codon 347 mutation (Hotta et al. Acta Sot ~h~aImoI Jpn, 1992). We have analyzed 20 unrelated Japanese families including 29 patients with ADRP. High molecular weight DNAS were isolated from peripheral white blood cell. Four DNA fragments of exon 1.2,3/4 and 5 were amplified by means of polymerase chain reaction @CR) amplification. PCR prodticts of DNA were cut by restriction enzyme Nci I and Msp I to detect the codon 347 mutation and Dde I to detect the codon 58 mutation of the ~‘Iiodogsir~ gene. SSCP (Single Strand Conformation Polymorphism) and dot blot hybridization methods were employed to detect the additional mutations. We present here two different mutations including a family with codon 347 mutation which was the transition from CCG to CTG (Pro - 347 - Leu) in rhodopsin gene of 4 ADRP patients in a family, in which the mutation had been transmitted from proband’s mother to the proband and his two daughters. Their clinical findings Were most likely type I. The other \z’as a patient with codon 17 mutation which was the transition from ACG to ATG (Thr - 17 -Met) in rhodopsin gene. The patient was most likely t)‘Pc il. None of the codon 23 mutation was found in any patients we chcckcrl. Clinical findings of Japanese cases with the rhodopsin gene mutation :!I‘? similar to Caucasasian families which have been already reportccl.
S.252
X ICER Abstracts 885
61
64
EPITHRLI~ (RPE) T~SPL~TATION ONTO BRUCH’S MEMBRANE IN ORGAN CULTURE. Lucian V. Del Priore and Russell Hornbeck. Washis University, St. Louis, MO RETINAL
PIGMENT
88%
ARGON LASER DEGENERATION
PHOTO~OAGULATION DELAYS FHOTO~~~~OR IN THE RCS RAT. r. C.. Chu. Y. m WARP Research Centre, Lions Eye Institute, QEII Medical Centre. Nedlands, Western Australia. Both intraocular injections of bFGF and subretinal needle damage have recently been shown to transiently delay the loss of pho~receptors in the RCS rat. We have investigated the effect of argon laser photocoagulation at the same time at which the above factors had an effect. The retina superior to the optic disc was irradiated with a grid of 40-60 50pm argon laser lesions of 250-350 mW in post-natal day 23 (P23) pink eyed RCS rats. At 7, 14, 30 and 60 days after irradiation the eyes were fixed and embedded in epon-araldite. Vertical sections through the optic nerve were stained with toluidine blue and analysed. High levels of irradiation destroyed the photoreceptors and pigment cells within the grid area. However, on the lesion flanks there were less pyknotic cells at 7 and 14 days and more surviving cells at 30 days. This correlated with a reduced debris layer along the flanking regions. Lower levels of irradiation did not destroy the photor~epto~ but appeared to activate the pigment cells to enter the debris layer. By 7 days most pyknotic cells had been removed leaving several rows of healthy cells. These cells were still present at 30 days after lesion (P53) but had degenerated by 60 days (P83). These results indicate that laser irradiation affects the retina in such a way that dying cells are removed more rapidly but the remaining cells survive for longer. The mechanisms of these effects are being examined.
We have developed an in vitro system for studying the transplantation of RPE cells onto Bruch’s membrane IBM) by modifying our previous organ culture system (Arch Ophth 106:1286>. Briefly, 6 mm buttons of eyewall containing BM. choroid, and sclera are trephined from freshly enucleated pig eyes, frozen at -20 C for at least 30 minutes, and thawed prior to use. Porcine passage 3 RPE cells are harvested from tissue culture flasks and plated onto BM in organ culture (150,000 cells/O.1 cc). RPE attach to BH and begin to flatten along BM within 2 hours of transplantation. One week after transplantation, BM is completely covered by a layer of transplanted RPE. The ability to transplant RPE cells harvested from tissue culture flasks & vitro is important in understanding the factors responsible for regulating RPE attachment to BM and RPE differentiation.
88%
65
889
RETINITJS PIGMENTOSA: PJJOTORECEmOR MORPHOLOGY AND A QUESTJON OF CJLJAJZY DlZECJS. Pi& J. Szezesny, institute of Ophthalmol~, Lcndon. CurrentIy: Au~n~i~k US& IUimistr 166, Ziirieh. Retinitis pigmentosa (RP) is a heterogeneous group of hereditary diseases of unknown etiology which lead to progressive degeneration of photoreceptors and the retinal pigment epithelium. A generalized structural defect of the cilia in various tissues, including photoreceptor connecting cilium, has been postulated as occurring in the X-linked form of RP (Arden and Fox 1979). However the literature on retinal pa~olo~ in RP reveals contradictor results on that issue. In this study the mo~holo~ of photoreceptors and the connecting cilium from 17 RP donors including X-linked RP, X chromosome carrier state, autosomal dominant and recessive RP was examined in electron microscopy. Photoreceptor preservation was commonly observed in some locations even in most advanced disease. These included perimacular area but not the macula itsele the area proximal to the optic nerve; and the far periphery. ~hotore~ptor cells showed two major pattern of changes associated with the stage of disease and regardless of genetic type of RI? I. wide spread autophagy at early stages of degeneration; II. contraction and migration of the photoreceptor inner segments into deeper retinal layers associated with proliferation and compression by Muller cells at the level of the outer limiting membrane in more advanced RP. Primary ciliary defects (eg additional or missing microtubul~) were found in none of the samples. Comparison of photoreceptors in normal and RP tissue showed thinner and longer cilia and longer centrioles in RP cells but no defects in the microtubule arrangements within the connecting cilia. The changes in the photoreceptor cytoskeleton appear to be associated with cell migration rather than the primary structural defect leading to cell degeneration in RI’. 63
66
887
890
MUTATIONS OF RHODOPSIN GENE FOUND IN JAPANESE FAMILIES WITH AUTOSOMAL DOMINANT RETINITIS PIGMENTQSA (ADRP) Sakuma. H., Hotta, Y.. Hayakawa, M.. Nakaiima. A., Fuiiki, K.. Kanai. A. (Department of Ophthalmology, Juntendo University, Tokyo 113 Japan) Shiono. T.. Nero, hL, S&uma. T., Tam&. M. (Department of Ophthalmology, Tohoku University, Sendai 980 Japan 1
VITREOUS BODY FLUORESCENCE CHANGES AFTER RETINAL LASER COAGULATION IN A RABBIT IN VIVO MODEL Libondi, T., Ragone, R., Romano, A.. Colonna G, and Auricchio, G. of Biochemistry and Biophysics, Eye Clinic and Dept. University of Naples, Italy Retinal photocoagulation induces a transient We used a rabbit in viva model inflammatory response. to assess whether retinal laser treatment affects vitreus spectrofluorimetric behaviour. Pigmented inbred underwent scattered retinal rabbit retinas photocoagulation (500 spots, 488 nm light, 50 micron, The burns were made similar to the 120 mw, 0,2 set). one which are observed in human diabetic retina laser treatment. 24 hrs lacer vitreous body was dissected, homogenized 1:l with BHT solution to avoid further oxidation, and treated with 0.15% SDS. The excitation spectra of the vitreous solution showed a peak at approx 350 nm. The emission spectra (excitation at 350 nm) showed the presence of a peak centered at 420 nm. The obtained fluorimetric results are suggestive for the presence of iminopropene bonds, which could be derived from the reaction of toxic aldehydes with free aminogroups of vitreous proteins, mainly collagens. This fluorescence behaviour can be appreciated even 7 represent days after laser treatment, and could longstanding changes of native conformation of vitreal structural proteins after laser retinal treatment.
Retinitis pigmentosa (RF) is a hereditary degenerative d~scasf !\‘l?lch causes blindness. RF is clinically and genetically heterogeneox. Smcc l31,!‘ia et aL(1990) found the mutations of codon 23, 58 and 347 in an allclc of the rhodopsin gene on chromosome 3 in ADRP patients, several mutations and deletions in the rhodopsin gene in ADRP patients were identified. WC have already reported a Japanese family with codon 347 mutation (Hotta et al. Acta Sot ~h~aImoI Jpn, 1992). We have analyzed 20 unrelated Japanese families including 29 patients with ADRP. High molecular weight DNAS were isolated from peripheral white blood cell. Four DNA fragments of exon 1.2,3/4 and 5 were amplified by means of polymerase chain reaction @CR) amplification. PCR prodticts of DNA were cut by restriction enzyme Nci I and Msp I to detect the codon 347 mutation and Dde I to detect the codon 58 mutation of the ~‘Iiodogsir~ gene. SSCP (Single Strand Conformation Polymorphism) and dot blot hybridization methods were employed to detect the additional mutations. We present here two different mutations including a family with codon 347 mutation which was the transition from CCG to CTG (Pro - 347 - Leu) in rhodopsin gene of 4 ADRP patients in a family, in which the mutation had been transmitted from proband’s mother to the proband and his two daughters. Their clinical findings Were most likely type I. The other \z’as a patient with codon 17 mutation which was the transition from ACG to ATG (Thr - 17 -Met) in rhodopsin gene. The patient was most likely t)‘Pc il. None of the codon 23 mutation was found in any patients we chcckcrl. Clinical findings of Japanese cases with the rhodopsin gene mutation :!I‘? similar to Caucasasian families which have been already reportccl.
S.252