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Glial fibrillary acidic (GFA) protein in Müller cells of the human retina
Friday, 5:30-7:00 P.M., Sep 25, 1992 Palazzo Dei Congressi
X ICER Abstracts
25
849
PHARMACOLOGICAL CHARACTERIZATION OF BOVINE RETINAL MICROVESSELS. Prasad S. Kulkarni. Andrew M. Roberts, Irvina G. Joshua Department of Ophthalmology and Visual Sciences and the Department of Physiology & Biophysics, University of Louisville, Louisville, Kentucky.
Astrocytes arc interconnected via gap junctions that permit the passage of ions and small molecules, thereby providing a basis for direct intercellular communication. The present study examined whether astmcytes form gap junctions with another type of glia, oligodendrocytes. Living rabbit retinae were incubated in the fluorescent dye Hoechst 38317, which labels the morphologically distinct nuclei of astrocytes and presumptive oligodendrocytes. Individual cells in the nerve fibre layer, near the edge of the medullary rays, were injected with Lucifer Yellow. This dye reveals the complete morphology of injected cells, and its low molecular weight enables it to pass through gap junctions. Each of the astrocytes injected (N=87) were dyecoupled to all astrocytes and oligodendrocytes within their dendritic field (20-200 cells; mean = 71). By contrast, none of the oligodendrocytes injected (N=51) were dye-coupled to other cells. These findings indicate that Lucifer Yellow passes bidirectionally through inter-astrocytic gap junctions but unidirectionally through astro-oligodendrocytic gap junctions. Unidirectional dye-coupling has not been shown previously in the vertebrate nervous system, although it has been reported between heterologous cells in vitro [Flagg-Newton & Loewenstein, Science 207,771 (1980)]. Our findings have two important implications: 1. Functions thought to be served exclusively by oligodendrocytes (e.g. myelination of CNS axons) may be influenced or modulated by astrocytes; 2. The unidirectional transfer of molecules provides a basis for a hierarchy of command similar to that seen among cells linked by chemical synapses.
Responses of retinal vasculature to various drugs has been studied using retinal arterial rings (180200im diameters). However, these studies determined reactivities of only larger microvessels. Therefore, we developed a method to observe a wide range of microvessels (lo-200pm diameters) in the bovine retina. Bovine retina was positioned over an optical port of a tissue bath containing physiological salt solution at 37-C and bubbled with 95% 0, and 5% CO,. Retinal microvessels were observed using closed-circuit television microscopy. Retinal vessels were classified according to the branching pattern with the major supplying arteriole being designated the first order arteriole (130-200fim) and subsequent branches designated 2nd order (30-80fium) and 3rd order (lo-20Fm). Arterioles were relatively unresponsive to catecholamines. Endothelin-1 caused prolonged arteriolar constriction while KC1 (O.Ol-12OmM) and PGF,, (O.Ol-1On.M) caused vasoconstriction of 1st and 2nd order arterioles. Supported by NE1 Grant EY-02861, School of Med., University of Louisville.
850
26
Do-smucE-ALvAscuLoG~sIs? ~0IogyandAuato~,ophtbalmoklgr The Medical
Interferon-7 (IFN7) induces prcductioo and expression of major histocoapatihility co+wlex class ll molecules on the human retinal glial cells. Rowever. knowledge concerning the regulation of this class II antigen expression is limited. In this study we examined the possibility that the function of certain ion channels in retinal glial cells loa.v be linked with the expression of class Il antigens induced by IFN-7. Cultured human retinal glial cells we prepared as described previously: cells with class Il antigens were detected with the use of specific mow.xlonal antibodies that were labeled with fluorescent probes.(Mano et al. 1991. Expt Eye Res. 53 603-606) After a threeday exposure to IFN-7 with or without various ion channels blockers, fluorescence of labeled cells were assayed by flow cytanetry. Results are shown below. Mean fl uoreseenee
intensity none IFN7 (lOOu/ml)
lFN7tnifedipine(lOtiM) lFN7tlTX(S#M) IFN7tamiloride(lOOfiM) IFNrtTEA(20flM)
24.23
1. 5
96. 6 96.6 97. 3 80.6
108.6!1
92. 6
of Georgia,
Augusta,
GA, USA
VesseL4
bum-vaebotbIaminlnandandAsAetsPcytic plxnxml~tba-~but~netlPetricted tbavessdwalla rntbaaomjuatparip~to~ FactorVIIIandlamhinarenoterpmmed. hmtmd,thteavasmlar’ anmhwahofflbronectin~tnand DollbleIabelane$ais~tbattbe aEamcybarablprecim
The data suggest that the induction of class ll antigens on human retinal glial cells by IFN7 as is partially affected by an amiloridesensitive Nat/H’ antiporter. tetraethylann~iua(TEA)-sensitive K channels, but not by nifedipine-sensitive ti channels or tetrodotoxin (TTX)-sensitive Na channels.
Supported 27
College
immunolocau~mv~oecma peripheraigradbntbehhdawaveof&ratingahmytes
x of class n positive cells
125.5?1 122.5?2 126.O?l 89.5?1
853
29
ROLE OF ION CRANNRLS IN INTRRFERON-7 INDUCED CLASS ll ANTIGEN EXPRESSION BY HUM4N RETINAL CLIAL CELLS. Tc&a Mano Shunji Kusaka. Masanori Tsujioka. Tane Memorial Eye Hospital.
Treatment
852
26
UNIDIRECTIONAL DYE-COUPLING BETWEEN RETINAL GLIA Robinson. S.R.. H~~~&~I.E.C.G.M.. Munro. M.N.. Vanev. D.I. Vision, Touch and Hearing Research Centre, University of Queensland, Brisbane, Australia, 4072.
851
by NlH-JZYO46l8
and MCGFU
854
30
GLIAL FIBRILLARY IICIDIC(GFA) PP@“CIN : 1 MC’LLER CELL5 OF TEI: HL’:lAII EETITJA SMIGEMIT3U, T., MPJINA,Y ., -.-.______ “Y&SAHAR&,n!. Deydrtmfnt of O~hthalrr:ology & *Pathology, Fujita Health University, Scl-001 of Iledicine, Toyoake-City, JAPAN
EVIDENCE REGULATION Zelmw, App/ied
AND IMPLICATION OF A LOCAL IN RETINAL CAPILLARY BEDS. Ft., ‘Ogum, Y., Gumn, T., Shahkli, M. and Kkyu, J..
Physics
Labomtofy,
Illinois, USA *curmnt~ at: Deperbnent
GFA Protein, r- protein originally isolated from fibrous neuroglja(fn< et al.,l?;l), is the chenical subilnit of astroglial s-ecific intermediate filaments. Lately we have exanined the GrAP reaction of Xhller cells in humn-~ The n eyes by the peroxidase-antiDeroxidase method. SUijJeCtS were two types of human eyes : the one was of B cadaver(died of adenocarcinoms of the stomach) with choroidnl metastasis,no metastasis, retjnoblastona, melanoma of the choroid and ocular injury, and the other was of J normal adult(four eyes). The GFAP-positive reaction was unexceptionally detected in the radial fibers of Milller cells in each subject tumorous and inju-while it was not detected in the eye with no rY eye, The antibody against metastasis and the normal eye. GFAP has been reported to idiosyncratically react to astroglia filaments which increase when the nervous tissue is in a pathological condition. It is guessed, th.erefore, that the GFAP staining of the ilClller cells in the pathological eye were positive in our examination because the astroglia filaments in the Iltiller cells weIn re caused to increase by a variety of factors. comparison with literatures Freviously issued on the GF AP reaction. our evarrination four.d that the normal reti na showed negative anti the pathological retina showed positive in the Miiller cells.
U/C Eye Center, of Ophfha/m/ogy,
Unhwsity
HEMODYNAMIC
of ////nois at Chicago,
Kyoto Uniwzsiiy,
Chicago,
Kyoto, Japan.
The examlwtion of the retinal macrwasculature has provided a wealth of information but little is known on the hemodynamics of the retinal mlcroclrculatlon. An understanding at this level Is important since In many vascular diseases the wiliest signs of damage appear at the capillary level. We have applied our technique of laser Targeted Dellvery 10 stwly. in monkeys, the microcirculatlon ai various macular kxallons. The method consists of creating well deffned bdl d dye and fdlcmring their course through the arterldes, capilfaries and venules to assess the regional blood flow and the capillary transl velocity. In one set of experlments the retkml hemodynamks were altered by hemcdilution. The blood flow in the arterlal bed lncreasad by 20% accompanied by a compambie change In transii vdocNy In the capfllary bed next to the fovea. However, this change gradually Increased up to 180% as the adw d the macula was probed. The results indicate the retinal mkrwascdature is lowfly regulated by rsdistrlbutfon of flow into fewer c~p#aries Independently d fhe regional arterial changes. The nature of the local regulation to stimuli other than hemodllutlon will be presanted. The role of this mechanism in the normal physidogy and the implications to vascular pathogenesis will bs discussed.
S.246
X ICER Abstracts
25
849
PHARMACOLOGICAL CHARACTERIZATION OF BOVINE RETINAL MICROVESSELS. Prasad S. Kulkarni. Andrew M. Roberts, Irvina G. Joshua Department of Ophthalmology and Visual Sciences and the Department of Physiology & Biophysics, University of Louisville, Louisville, Kentucky.
Astrocytes arc interconnected via gap junctions that permit the passage of ions and small molecules, thereby providing a basis for direct intercellular communication. The present study examined whether astmcytes form gap junctions with another type of glia, oligodendrocytes. Living rabbit retinae were incubated in the fluorescent dye Hoechst 38317, which labels the morphologically distinct nuclei of astrocytes and presumptive oligodendrocytes. Individual cells in the nerve fibre layer, near the edge of the medullary rays, were injected with Lucifer Yellow. This dye reveals the complete morphology of injected cells, and its low molecular weight enables it to pass through gap junctions. Each of the astrocytes injected (N=87) were dyecoupled to all astrocytes and oligodendrocytes within their dendritic field (20-200 cells; mean = 71). By contrast, none of the oligodendrocytes injected (N=51) were dye-coupled to other cells. These findings indicate that Lucifer Yellow passes bidirectionally through inter-astrocytic gap junctions but unidirectionally through astro-oligodendrocytic gap junctions. Unidirectional dye-coupling has not been shown previously in the vertebrate nervous system, although it has been reported between heterologous cells in vitro [Flagg-Newton & Loewenstein, Science 207,771 (1980)]. Our findings have two important implications: 1. Functions thought to be served exclusively by oligodendrocytes (e.g. myelination of CNS axons) may be influenced or modulated by astrocytes; 2. The unidirectional transfer of molecules provides a basis for a hierarchy of command similar to that seen among cells linked by chemical synapses.
Responses of retinal vasculature to various drugs has been studied using retinal arterial rings (180200im diameters). However, these studies determined reactivities of only larger microvessels. Therefore, we developed a method to observe a wide range of microvessels (lo-200pm diameters) in the bovine retina. Bovine retina was positioned over an optical port of a tissue bath containing physiological salt solution at 37-C and bubbled with 95% 0, and 5% CO,. Retinal microvessels were observed using closed-circuit television microscopy. Retinal vessels were classified according to the branching pattern with the major supplying arteriole being designated the first order arteriole (130-200fim) and subsequent branches designated 2nd order (30-80fium) and 3rd order (lo-20Fm). Arterioles were relatively unresponsive to catecholamines. Endothelin-1 caused prolonged arteriolar constriction while KC1 (O.Ol-12OmM) and PGF,, (O.Ol-1On.M) caused vasoconstriction of 1st and 2nd order arterioles. Supported by NE1 Grant EY-02861, School of Med., University of Louisville.
850
26
Do-smucE-ALvAscuLoG~sIs? ~0IogyandAuato~,ophtbalmoklgr The Medical
Interferon-7 (IFN7) induces prcductioo and expression of major histocoapatihility co+wlex class ll molecules on the human retinal glial cells. Rowever. knowledge concerning the regulation of this class II antigen expression is limited. In this study we examined the possibility that the function of certain ion channels in retinal glial cells loa.v be linked with the expression of class Il antigens induced by IFN-7. Cultured human retinal glial cells we prepared as described previously: cells with class Il antigens were detected with the use of specific mow.xlonal antibodies that were labeled with fluorescent probes.(Mano et al. 1991. Expt Eye Res. 53 603-606) After a threeday exposure to IFN-7 with or without various ion channels blockers, fluorescence of labeled cells were assayed by flow cytanetry. Results are shown below. Mean fl uoreseenee
intensity none IFN7 (lOOu/ml)
lFN7tnifedipine(lOtiM) lFN7tlTX(S#M) IFN7tamiloride(lOOfiM) IFNrtTEA(20flM)
24.23
1. 5
96. 6 96.6 97. 3 80.6
108.6!1
92. 6
of Georgia,
Augusta,
GA, USA
VesseL4
bum-vaebotbIaminlnandandAsAetsPcytic plxnxml~tba-~but~netlPetricted tbavessdwalla rntbaaomjuatparip~to~ FactorVIIIandlamhinarenoterpmmed. hmtmd,thteavasmlar’ anmhwahofflbronectin~tnand DollbleIabelane$ais~tbattbe aEamcybarablprecim
The data suggest that the induction of class ll antigens on human retinal glial cells by IFN7 as is partially affected by an amiloridesensitive Nat/H’ antiporter. tetraethylann~iua(TEA)-sensitive K channels, but not by nifedipine-sensitive ti channels or tetrodotoxin (TTX)-sensitive Na channels.
Supported 27
College
immunolocau~mv~oecma peripheraigradbntbehhdawaveof&ratingahmytes
x of class n positive cells
125.5?1 122.5?2 126.O?l 89.5?1
853
29
ROLE OF ION CRANNRLS IN INTRRFERON-7 INDUCED CLASS ll ANTIGEN EXPRESSION BY HUM4N RETINAL CLIAL CELLS. Tc&a Mano Shunji Kusaka. Masanori Tsujioka. Tane Memorial Eye Hospital.
Treatment
852
26
UNIDIRECTIONAL DYE-COUPLING BETWEEN RETINAL GLIA Robinson. S.R.. H~~~&~I.E.C.G.M.. Munro. M.N.. Vanev. D.I. Vision, Touch and Hearing Research Centre, University of Queensland, Brisbane, Australia, 4072.
851
by NlH-JZYO46l8
and MCGFU
854
30
GLIAL FIBRILLARY IICIDIC(GFA) PP@“CIN : 1 MC’LLER CELL5 OF TEI: HL’:lAII EETITJA SMIGEMIT3U, T., MPJINA,Y ., -.-.______ “Y&SAHAR&,n!. Deydrtmfnt of O~hthalrr:ology & *Pathology, Fujita Health University, Scl-001 of Iledicine, Toyoake-City, JAPAN
EVIDENCE REGULATION Zelmw, App/ied
AND IMPLICATION OF A LOCAL IN RETINAL CAPILLARY BEDS. Ft., ‘Ogum, Y., Gumn, T., Shahkli, M. and Kkyu, J..
Physics
Labomtofy,
Illinois, USA *curmnt~ at: Deperbnent
GFA Protein, r- protein originally isolated from fibrous neuroglja(fn< et al.,l?;l), is the chenical subilnit of astroglial s-ecific intermediate filaments. Lately we have exanined the GrAP reaction of Xhller cells in humn-~ The n eyes by the peroxidase-antiDeroxidase method. SUijJeCtS were two types of human eyes : the one was of B cadaver(died of adenocarcinoms of the stomach) with choroidnl metastasis,no metastasis, retjnoblastona, melanoma of the choroid and ocular injury, and the other was of J normal adult(four eyes). The GFAP-positive reaction was unexceptionally detected in the radial fibers of Milller cells in each subject tumorous and inju-while it was not detected in the eye with no rY eye, The antibody against metastasis and the normal eye. GFAP has been reported to idiosyncratically react to astroglia filaments which increase when the nervous tissue is in a pathological condition. It is guessed, th.erefore, that the GFAP staining of the ilClller cells in the pathological eye were positive in our examination because the astroglia filaments in the Iltiller cells weIn re caused to increase by a variety of factors. comparison with literatures Freviously issued on the GF AP reaction. our evarrination four.d that the normal reti na showed negative anti the pathological retina showed positive in the Miiller cells.
U/C Eye Center, of Ophfha/m/ogy,
Unhwsity
HEMODYNAMIC
of ////nois at Chicago,
Kyoto Uniwzsiiy,
Chicago,
Kyoto, Japan.
The examlwtion of the retinal macrwasculature has provided a wealth of information but little is known on the hemodynamics of the retinal mlcroclrculatlon. An understanding at this level Is important since In many vascular diseases the wiliest signs of damage appear at the capillary level. We have applied our technique of laser Targeted Dellvery 10 stwly. in monkeys, the microcirculatlon ai various macular kxallons. The method consists of creating well deffned bdl d dye and fdlcmring their course through the arterldes, capilfaries and venules to assess the regional blood flow and the capillary transl velocity. In one set of experlments the retkml hemodynamks were altered by hemcdilution. The blood flow in the arterlal bed lncreasad by 20% accompanied by a compambie change In transii vdocNy In the capfllary bed next to the fovea. However, this change gradually Increased up to 180% as the adw d the macula was probed. The results indicate the retinal mkrwascdature is lowfly regulated by rsdistrlbutfon of flow into fewer c~p#aries Independently d fhe regional arterial changes. The nature of the local regulation to stimuli other than hemodllutlon will be presanted. The role of this mechanism in the normal physidogy and the implications to vascular pathogenesis will bs discussed.
S.246