- Email: [email protected]
Rat retinal ganglion cells in culture
Exp. Eye Res. (3991) 53, 565-572
Rat Retinal NAOTO Glaucoma
Service,
(Received
Ganglion
TAKAHASHI,
DEAN
Yale University Science, 330 13 July
Cells
CUMMINS
AND
in Culture JOSEPH
CAPRIOLI’
School of Medicine, Department of Ophthalmology Cedar Street, New Haven, CT 06510, U.S.A.
1990
and accepted
in revised
form
19 December
and
Visual
7990)
A stablecell culture systemof identifiedretinal ganglioncellswould facilitate the investigationof cellular mechanismsof damagefrom glaucomaand other disorders.We have developeda reliabletechniqueto culture retinal ganglion cellson a glial cell monolayer which extendsviability and promotesextensive neurite outgrowth. Dissociated retinal cellsfrom 5-7-day-old Sprague-Dawleyrats werecultured on glial monolayersderivedfrom rat cerebralhemispheres. Retinal ganglioncellswere labeledwith retrograde fluorescentmarkersinjectedinto the superiorcolliculusor in culture with monoclonalantibody to Thy1 antigen. SinceThy-l antigen is not entirely specificfor retinal ganglioncells,and fluorescentmarkers fadein oldercultures,the identity of Thy- 1 markedcellswasconfirmedwith whole-cellelectrophysiologic recordings.Labeled,physiologicallyintact retinal ganglion cellswere identified for at least 31 days in culture. Many retinal ganglioncellsshowedneurite elongationof 2 mm or more and developedcomplex intercellular networks. This cell culture systemmay be usedto form the basisfor future studiesof the electrophysiologyand transport propertiesof retinal ganglioncellsunder normal culture conditionsand under adverseconditionssuch as thosethat mimic ischemiaor mechanicaldeformation. Key words:retinal ganglion cells: cell culture; glaucoma:Thy-l antigen: monoclonalantibodies.
1,
2.
Introduction
The cellular and molecular mechanisms of damage to retinal ganglion cells (RGCs)from glaucoma and other disorders might be investigated if a convenient cell culture system of identified RGCs with extended viability was available. The morphologic and electrophysiologic characterization of these cells in culture could provide a platform from which to launch studies of ischemia and other mechanisms of damage. Dispersed RGCs from enzymatically digested retina have been labeled with fluorescent dyes (Sarthy, Curtis and Catterall, 1983) or with specific antibodies (Barnstable and Drager, 1984), but extended viability of cultured cells with long neurites and dendritic trees is difficult to achieve. Two studies which have demonstrated extended viability are those of Montague and Friedlander (1989) on cultures of cat retinal neurons, and MacLeish and Townes-Anderson (1988) on cultured salamander retinal neurons. The cat retinal cells survived up to 8 weeks and the salamander neurons survived for several months. We report the behavior of labeled RGCsfrom the neonatal rat in culture on glial cell monolayers in which extensive neurite outgrowth occurs, and long-term viability of RGCs is demonstrated electrophysiologitally.
* For reprint requests at: Yale University Department of Ophthalmology and Visual Street. New Haven, CT 06510, U.S.A.
00144835/91/l
School of Medicine, Science. 330 Cedar
10565+08 $03.00/O
Materials and Methods
Retrogradelabeling The use of animals in this work was performed in accordance with the Association for Research in Vision and Ophthalmology Resolution on the Humane Use of Animals in research. Five- to seven-day-old Sprague-Dawley rats were anesthetized by chilling in ice for 10 min. One microliter of a 1 y0 suspension of granular blue in physiological saline was injected into the superior colliculi. Injections were performed with a l-p1 Hamilton syringe fitted with a glass capillary tip drawn to a fine point and were performed under an operating microscope. Injections were made under direct observation after developing a small skull flap just posterior to the transverse sinus (Barnstable and Drager, 1984). Preparation of Retinal Cell Suspensions Retinal cells were dissociated according to the method described by Leifer et al. (1984). Two days after injection of the fluorescent dye, rats were killed by decapitation. After gentle enucleation, the eyes were immersed in Hanks’ balanced salt solution (Gibco, Grand Island, NY) containing 100 U ml-’ penicillin and 100 ,ug ml-’ streptomycin. The corneas were excised and the lens and vitreous were removed with fine forceps under an operating microscope. The retinas were gently isolated with blunt dissection. Four retinas were incubated at 36 “C for 40 min in 10 ml Hanks’ balanced salt solution containing bovine serum albumin (0.2 mg ml-‘), m-cysteine 0 1991 AcademicPressLimited
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(0.2 mg ml-‘), and papain (26 U ml-‘) (Sigma, St Louis, MO). The tissue was rinsed with Hanks’ and replaced with 4 ml culture medium and the tissue triturated through a l-ml pipette. After complete dissociation of the retina into a fine suspension, 0.2 ml of the supernatant suspension was placed on a glial cell monolayer in a dish containing 1.5 ml medium. Approximately 3 x lo6 retinal cells were plated per dish. The culture medium consisted of Eagle’s minimum essential medium (Gibco), glutamine (2 mM), glucose ( 16 mM), and rat serum (5 %). Cultures were maintained in a humidified atmosphere of 5 % CO, and 9 5 % air at 3 7 “C. The medium was changed every 3 days.
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was discarded and the monolayer rinsed twice with Hanks’. Trypsin (0.1%) in Hanks’ was added to the flask and incubated until the cells detached from the base, usually in 5-10 min. The cell suspension was centrifuged and the trypsin solution replaced with medium to approximate a cell density of 1.2-2.0 x 10” cells ml-‘. One milliliter of this cell suspension was placed on a prepared Petri dish. Approximately 5 days after subculture, the glia spread over the cover slip in such a way as to maintain spaces between glial ‘islands ’ and were then used for subsequent retinal cultures. Staining with Thy-l Antibody
The base of a 35-mm plastic Petri dish (Corning, Medfield, MA) is too thick to observe cells with the high-magnification objective of an inverted microscope. To enable appropriate microscopic examination, the base of the Petri dish was replaced with a cover slip (Bray, 1970). A 2-cm diameter hole was drilled through the center of the dish and the cover slip was attached with Sylgard (a silicone elastomer, Dow Corning Corporation). After the silicone set, the Petri dish was rinsed with 9 5 % ethanol followed by distilled water and was sterilized with ultraviolet light. In experiments with laminin-coated dishes, a lOOpg ml-l solution of Cr-Laminin (Collaborative Research Incorporated, Bedford, MA) was prepared with culture medium. Petri dishes containing 0.5 ml CrLaminin solution were maintained at room temperature for 45 min. The solution was then aspirated and the retinal cell suspensions plated.
Thy-l antibody was supplied by Dr Colin Barnstable (Yale University School of Medicine, Department of Ophthalmology and Visual Science). It is a mouse monoclonal antibody (IgG2a) raised against rat cerebral cortical Thy-l antigen (Barnstable and Drager, 1984). Dissociated cells were incubated in HBSS with 5% goat serum for 30 min at room temperature with IgG2s (ascites fluid, 1: lOOO), washed by centrifugation and labeled for 30 min with fluorescein-conjugated goat anti-mouse IgG (Cappell. 1: 100). For in vitro staining, cultures were rinsed three times with 5% goat serum in HBSS, incubated with IgG2a (ascites fluid, 1: 1000) for 30 min at 3 7 “C. After washing three times with HBSS, cultures were incubated with fluorescein-conjugated goat antimouse IgG (Cappell, 1:100-l :200) for 30 min at 37 “C, washed again three times and viewed with an inverted microscope equipped with epifluorescence. As a control, mouse serum was used with the same protocol (Barnstable and Drager, 1984).
Preparation of Glial Cell Monolayers
Electrophysiologic
Glial cultures were prepared from the cerebral hemispheres of neonatal rats, l-2 days old, according to the method of McCaffery, Raju and Bennett (1984). After decapitation, the cerebral hemispheres were dissected and placed into Hanks’ balanced salt solution containing 100 U ml-’ penicillin and 100 ,ug ml-l streptomycin. Meninges were stripped off under an operating microscope and the hemisphere incubated for 15 min at 3 7 ‘C with 0.2 5 % trypsin in Hanks’. The tissue was transferred to culture media and triturated through an 18-gauge needle. Cell suspensions were diluted such that one hemisphere was placed in a total of 10 ml: this solution was dispersed in a 75-cm tissue culture flask and maintained in a humidified atmosphere of 5% CO, and 95% air at 37 “C. The medium was changed twice a week. The culture medium consisted of Eagle’s minimum essential medium, glutamine (2 mM), and fetal bovine serum ( 10 %). When the cells formed a confluent monolayer about S-10 days after primary culture, subcultures to Petri dishes or other flasks were prepared. Medium
Patch electrodes were made from 1.5 mm diameter capillary glass pulled on a patch-clamp puller and flrepolished. Recording electrodes were filled with a salt solution consisting of 150 mM KC1 with 10 mM Hepes buffer and had a resistance range of 4-6 mR. The reference electrode was a chlorided silver pellet coated on the sides with teflon and inserted into a 1.5mm diameter capillary glass tube. Recording and reference electrodes were connected to a Hx. 01 impedancematching headstage that delivered signals to an Axoclamp IIA patch-clamp amplifier (Axon Instruments, Foster City, CA). The recordings were viewed in ‘real time ’ on a Hitachi VC602 5 digital storage oscilloscope and a Graphtec WR3101 four channel chart recorder. Recordings were also stored on a Dagan DAS 900 VCR with a built-in pulse code modulator. Currents were injected by driving the current clamp output of the Axoclamp IIA amplifier with an S44 Grass stimulator. Retinal ganglion cells cultured on a glial cell monolayer in Petri dishes were viewed with a Zeiss IM-
Preparation of Culture Dishes
Characterization of Cells
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3 5 phase-contrast fluorescence microscope. The cultures were continuously perfused during recording with oxygenated HBSS buffered with 10 mM Hepes and maintained at 37 “C with a Leiden LU-CBl microincubation system driven by a TC-102 temperature controller (Medical Systems Corporation). The volume of fluid in the Petri dish was 1.5 ml and the rate of perfusion was O-7 ml min’. Fluid was perfused with a two-channel peristaltic pump (Sigma). Perfusion solutions were made with HBSS without Mg”’ and contained either 2 mM CoCl, or 2 mM MgCl, (Sigma). Solutions were also injected with a Sage microperfusion pump via a 20-pm diameter glass pipette positioned approximately 100 pm from the cell body. The recording micropipette was positioned with a Narashige remote three-dimensional hydraulic micromanipulator. 3.
Results
Two substrates were employed for retinal culture. Laminin was the least effective substrate for neuronal attachment and survival. Some retinal neurons extended neurites on the laminin-coated cover slip 1 day after plating ; these cells were not generally maintained for more than 2 to 3 days. Miiller cells began to spread over the cover slip l-2 days after plating, and in some instances the overlying retinal neurons survived for up to 2 weeks. Subsequent experiments employed a glial cell monolayer derived from neonatal rat cerebral cortex as a substrate. This glial population was less mobile than the Miiller cell population and could successfully maintain retinal neurons for periods of up to 2 months. In such cases an extensive intercellular network of retinal neurites formed. Freshly dissociated RGCs were identified with the cytoplasmic retrograde-label granular blue or with fluorescein-conjugated Thy-l antibody on the cell membrane (Fig. 1). RGCs labeled with Thy-l antibody showed an irregular surface fluorescence. There was no significant extension of neurites for the first 6 hr after plating. After 1 day in culture, some RGCs showed neurite extension. The underlying glia, surrounding cells, and complex neurite network which subsequently developed often made it difficult to visualize the entire length of neurites. When the neurites contained granular blue, their extensions could be seen with transmission light microscopy and high magnification (Fig. 2). Figure 3 demonstrates the decrease with time of RGCs identified with granular blue. Only rarely could RGCs be identified in culture after 1 week with this labeling technique. Thy- l-stained neurons showed a well-defined surface fluorescence with good staining of neurites (Fig. 4). By 15 days after plating, RGCs were frequently observed with individual neurites more than 2 mm long. The Thy- 1 fluorescence decreased gradually several days after staining and the diffuse fluorescence of the cell membrane gradually became spotty and
FIG. 1. Retinal ganglion cells just after dissociation. Retinal ganglion cells were labeled by retrograde transport of granular blue from the superior colliculus. After dissociation, they were also labeled by fluorescein-conjugated antibody to Thy-l antigen. A retinal ganglion ceil is shown with phase optics (A), granular blue fluorescence (B) and fluorescein-conjugated antibody staining to Thy-l antigen
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FIG. 2. Ketinal ganglion cells labeled with granular blue in culture. A, Retinal ganglion cells 3 days after plating on glia viewed with a combination of epifluorescence and transmitted light. The neurites of these retinal ganglion cells contained granular blue and enabled visualization of their extensions. B. Retinal ganglion cells 4 days after plating viewed on glia viewed with a combination of epifluorescence and phase optics.
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FIG. 3. The numberof retinal ganglioncellsidentifiedwith granular blue in culture on a glial monolayer. Thirty fields werecountedper dishat x 400 and six disheswerecounted at 6. 24. 48. 72 and 96 hr.
eventually disappeared. Thy-l staining was not entirely specific for RGCs, since occasionally other cells, probably glia, also stained (Fig. 4). Thy-l-stained neurons with extensive neurite networks were identified for up to 48 days in culture. Electrophysiologic recordings from fast blue or Thy7-labeled cells revealed resting potentials which averaged ( &SD.) 59 + 5.1 mV (n = 58). Spontaneous synaptic activity was recorded from retinal ganglion cells marked by fast blue or Thy-l after 3-5 days in culture. This consisted of summations of excitatory depolarizing currents with action potentials and inhibitory hyperpolarizing currents (Fig. 5). Synaptic activity was reversibly blocked by 2 mM Co2+ or 2 mM Mg2+. Both agents are potent inhibitors of chemically mediated synaptic transmission (Miller et al., 198 1). Such recordings, typical of physiologically viable RGCs, were obtained from cells for up to 31 days in culture. Recordings from cells older than 3 1 days have not yet been attempted. Many recordings that were characteristic of RGCs were obtained from cells marked with Thy-l which were not labeled with granular blue. Since retrograde marking does not till all the RGCs plated in culture, this was particularly evident in older cultures (more than 15 days) where no granular blue-labeled cells were present. 4.
Discussion
Neuronal cultures often require embryonic or neonatal tissue to exhibit viability and growth. Several investigators have successfully cultured RGCsfrom 2to 3-day-old neonatal rats (Raff et a1.,1979; Sarthy, Curtis and Catterall, 1983). The number of viable RGCs decreasesrapidly. with an 80% reduction of
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initial density by 48 hr. Media conditioned with chick optic tectum and rat superior colliculus extracts have maintained greater numbers of RGCs (Sarthy et al., 1983; McCaffery et al., 1984). McCaffery and coworkers (1984) reported enhanced survival of RGCs on a glial monolayer compared to culture techniques which employed artificial substrates such as laminin (Cohen et al., 198 6). Greatly enhanced neurite outgrowth was also observed in these co-cultures. Leifer et al. (1984) used 5-12-day-old rats as a source of RGCs, and monoclonal antibodies to Thy-l were used as a substrate to enhance regeneration of retinal ganglion cell neurites in culture. These relatively mature RGCswere maintained for over 2 weeks in the absence of superior colliculus derivatives. Recently, several laboratories have demonstrated extended survival of retinal cell cultures. MacLeish and Townes-Anderson (1988) cultured salamander retinal neurons ; viability was demonstrated for 7 months by morphologic and electrophysiologic criteria. Montague and Friedlander (1989 1 cultured cat retinal neurons and demonstrated survival for 8 weeks. We have shown that neonatal rat RGCssurvive in culture for at least 31 days, as demonstrated by Thy-l labeling and typical electrophysiologic responses. The proper identification of RGCs in culture is important to mechanistic studies of cellular damage. Retrograde markers are reliable, since the RGCs are the only retinal neurons that project ‘to the brain, However, retrograde markers may also be taken up by glia (Bentivoglio et al., 1979, 1980; Aschoff and Hollander, 1982). We have observed granular blue in phagocytic cells shortly after dissociation. Barres et al. (1988) observed the retrograde marker fast blue in cells other than RGCs after dissociation. Fortunately, phagocytic cells are usually easily differentiated from neurons by their appearance. In the normal development of the rat, substantial RGC death has been observed during the first 10 postnatal days (Cunningham, Mohler and Giordane, 1982 ; Potts, Dreher and Bennett, 1982 ; Dreher, Potts and Bennett, 1983). We used 5-7-day-old rats as a cell source; some of the degenerated RGCs may have been programmed to die and were phagocytized by macrophages after plating. Hume, Perry and Gordon (1983) used antibody against the macrophage-specific antigen F4-80 to show that macrophages from the general circulation invade the developing mouse retina during times of programmed neuronal death. Thy-l antigen is a glycoprotein found on the surface of a variety of rodent cell types. The highest concentration of rat Thy-l antigen has been found in the brain and thymus. Barnstable and Drager (1984) described Thy-l antigen as a ganglion cell-specific marker in the rodent retina. Double-labeling experiments showed that many more cells were successfully labeled by antibody to Thy-l than with intracellular fluorescent markers. However, virtually all neurons
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inward membrane currents are associatedwith neurite regeneration in RGCs and that blockade of electrical activity promotes degeneration. Electrophysiologic studies could be extended to investigate mechanisms of damage. Two broad theories have been advanced to explain the pathophysiology of glaucomatous damage to retinal ganglion cells: (1) mechanical distortion at the level of the lamina cribrosa blocks axonal transport: and (2) increased intraocular pressure and other factors decreaseperfusion of the anterior optic nerve head and cause ischemic damage to RGCs. Measurements of axonal flow and electrophysiology in individual cells in culture under various conditions of oxygen deprivation and mechanical distortion may provide information about the cellular and molecular mechanisms of retinal ganglion cell dysfunction and death from glaucoma and other disorders. Acknowledgements
FIG. 5. A, Spontaneous synaptic activity recorded from a Thy-l marked retinal ganglion cell after 3 1 days in culture. Resting membrane potential was -62 mV. B, Reversible cobalt (2 mM) blockade of spontaneous synaptic activity. This recording was made after 16 days in culture. The resting membrane potential was - 6 5 mV.
marked with granular blues were also marked by the cell-surface monoclonal Thy-l antibody. There may be a subpopulation of cells (perhaps amacrine cells) that are also marked by Thy-l antibody. Therefore, whole-cell electrophysiologic recordings were also obtained from cells marked with Thy-l. All cells demonstrated the typical electrophysiologic characteristics of retinal ganglion cells. Resting membrane potentials averaged 59 + 5.1 mV (n = 58). Cells displayed the characteristic spiking behavior of retinal ganglion cells, with action-potential amplitudes of 40-60 mV riding on small generator potentials of 2-5 mV. This spontaneous synaptic activity was reversibly blocked by 2 mM Co2+or 2 mM Mg”. This study demonstrates that viability of cultured retinal ganglion cells can be achieved for at least 4 weeks and probably longer by employing a glial monolayer substrate combined with staining living ganglion cells with monoclonal antibody to Thy-l antigen. This technique presents the possibility of studying the effects of hypoxic, anoxic, or hyperbaric damage to retinal ganglion cells over an extended time period in culture. Lipton ( 1986, 198 7) employed patch-clamp electrophysiologic recordings on freshly dissociatedcells to demonstrate that calcium-mediated
The authors would like to thank Dr Colin Barnstable for his valuable suggestions during the course of this work. This study was supported in part by grants from the U.S. Public Health Service and EY-07648 and EY-09114 (Dr Caprioli), EY-00785 (Yale Center Grant), Research to Prevent Blindness, Inc., Foresight, Inc., and the Connecticut Lions Eye Research Foundation. Inc.
References Aschoff, A. and Hollander. H. (1982). Fluorescent compounds as retrograde tracers compared with horseradish peroxidase (HRP). I. A parametric study in the central visual system of the albino rat. I. Neurosci. Methods 6, 179-9 7. Barnstable. C. J. and Drager, V. C. (1984). Thy-l antigen: A ganglion cell specific marker in rodent retina. Neuroscience 11, 847-55. Barres, B. A., Silverstein, B. E., Corey, D. E’. and Chun. L. L. Y. (1988). Immunological, morphological, and electrophysiological variation among retinal ganglion cells purified by panning. Neuron 1, 791-803. Bentivoglio. M.. Kuypers, H. G. J. M., Catsman-Berrevoets, C. E. and Dann, 0. (1979). Fluorescent retrograde neuronal labelling in rat by means of substances binding specifically to adenine-thymine rich DNA. Neuro-sci. Letts 12. 23540. Bentivoglio, M., Kuypers. H. G. J. M., Catsman-Berrevoets, C. E. and Dann, 0. (1980). Two new fluorescent retrograde neuronal tracers which are transported over long distances. Neurosci. Letts 18, 25-30.
Bray, D. (1970). Surfacemovementsduring the growth of single explanted 65. 905-10.
neurons.
Proc. Natl. Arad. Sci. U.S.A.
Cohen,J.. Burne, J. F., Winter, J. and Bartlett, P. (1986). Retinal ganglion cells lose responseto laminin with maturation. Nature 322, 465-7. Cunningham,T. J.. Mohler, I. M. and Giordane,D. L. (1982). Naturally occurring neuron death in the ganglion cell layer of the neonatal rat. Morphology and evidence for
FIG. 4. Retinal ganglion cells 7 days after plating on glia. This cell was double-labeled by granular blue and antibody to Thy-l antigen and was viewed with phase optics (A), granular blue fluorescence (B) and Thy-l label fluorescence (C). The arrow indicates an underlying glial cell which was labeled with antibody to Thy-l antigen.
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regional correspondence with neuron death in colliculus. Dev. Bruin Res. 2, 203-215. Dreher, B., Potts, R. A. and Bennett, M. R. ( 1983). Evidence that the early postnatal reduction in the number of rat retinal ganglion cells is due to a wave of ganglion cell death. Neurosci. Letts 36, 225-60. Hume, D. A., Perry, V. H. and Gordon, S. (1983). Immunohistochemical localization of a macrophage-specific antigen in developing mouse retina: phagocytosis of dying neurons and differentiation of microglial cells to form a regular array in the plexiform layers. 7. Cell Biol. 97,253-7.
Liefer. D., Lipton, S. A., Barnstable. C. J. and Masland. R. H. (1984). Monoclonal antibody to Thy-l enhances regeneration of processes by rat retinal ganglion cells in culture. Science 224, 303-6. Lipton. S. A. (1986). Blocking of electrical activity promotes the death of mammalian retinal ganglion cells in culture. Proc. Natl. Ad. Sci. U.S.A. 83, 9774-8. Lipton, S. A. (1987). Bursting of calcium-activated cationselective channels is associated with neurite regeneration in a mammalian central neuron. Neurosci. Letts 982,
MacLeish.
21-28.
P. R. and Townes-Anderson,
E. (1988).
Growth
ET AL.
and synapse formation among major classes of adult salamander retinal neurons in vitro. Neurone 1, 7 5 l-60. McCaffery, C. A., Raju. T. R. and Bennett, M. R. (1984). Effect of cultured astroglia on the survival of neonatal rat retinal ganglion cells in vitro. Dev. Biol. 104.441-8. Miller, R.. Frumkes, T.. Slaughter, M., and Dacheaux. R. ( 198 1). Physiological and pharmacological basis of GABA and glycine action on neurons of mudpuppy retina. II. Amacrine and ganglion cells. J. Neurophysiol. 45.
764-82.
Montague, P. R. and Friedlander. M. J. ( 1989). Expression ot an instinsic growth strategy by mammalian retinal neurons. Proc. Nafl. Ad. Sci. U.S.A. 86. 7223-7. Potts, R. A., Dreher. B. and Bennett, M. R. (1982). The loss of ganglion cells in the developing retina of the rat. Dev. Brain Rc~s.3. 481-h. Raff, M.. Fields, K., Sen-Itiroh. H.. Mirsky, R.. Pruss. R. and Winter, J. ( 1979). Cell-type specific markers for distinguishing and studying neurons and the major classes of glial cells in culture. Brain Res. 174. 283-308. Sarthy, P. V.. Curtis, B. M. and Catterall. W. A. (1983). Retrograde labelling, enrichment, and characterization of retinal ganglion cells from the neonatal rat. 1. Ntwmsci.
3. 2532-44.
Rat Retinal NAOTO Glaucoma
Service,
(Received
Ganglion
TAKAHASHI,
DEAN
Yale University Science, 330 13 July
Cells
CUMMINS
AND
in Culture JOSEPH
CAPRIOLI’
School of Medicine, Department of Ophthalmology Cedar Street, New Haven, CT 06510, U.S.A.
1990
and accepted
in revised
form
19 December
and
Visual
7990)
A stablecell culture systemof identifiedretinal ganglioncellswould facilitate the investigationof cellular mechanismsof damagefrom glaucomaand other disorders.We have developeda reliabletechniqueto culture retinal ganglion cellson a glial cell monolayer which extendsviability and promotesextensive neurite outgrowth. Dissociated retinal cellsfrom 5-7-day-old Sprague-Dawleyrats werecultured on glial monolayersderivedfrom rat cerebralhemispheres. Retinal ganglioncellswere labeledwith retrograde fluorescentmarkersinjectedinto the superiorcolliculusor in culture with monoclonalantibody to Thy1 antigen. SinceThy-l antigen is not entirely specificfor retinal ganglioncells,and fluorescentmarkers fadein oldercultures,the identity of Thy- 1 markedcellswasconfirmedwith whole-cellelectrophysiologic recordings.Labeled,physiologicallyintact retinal ganglion cellswere identified for at least 31 days in culture. Many retinal ganglioncellsshowedneurite elongationof 2 mm or more and developedcomplex intercellular networks. This cell culture systemmay be usedto form the basisfor future studiesof the electrophysiologyand transport propertiesof retinal ganglioncellsunder normal culture conditionsand under adverseconditionssuch as thosethat mimic ischemiaor mechanicaldeformation. Key words:retinal ganglion cells: cell culture; glaucoma:Thy-l antigen: monoclonalantibodies.
1,
2.
Introduction
The cellular and molecular mechanisms of damage to retinal ganglion cells (RGCs)from glaucoma and other disorders might be investigated if a convenient cell culture system of identified RGCs with extended viability was available. The morphologic and electrophysiologic characterization of these cells in culture could provide a platform from which to launch studies of ischemia and other mechanisms of damage. Dispersed RGCs from enzymatically digested retina have been labeled with fluorescent dyes (Sarthy, Curtis and Catterall, 1983) or with specific antibodies (Barnstable and Drager, 1984), but extended viability of cultured cells with long neurites and dendritic trees is difficult to achieve. Two studies which have demonstrated extended viability are those of Montague and Friedlander (1989) on cultures of cat retinal neurons, and MacLeish and Townes-Anderson (1988) on cultured salamander retinal neurons. The cat retinal cells survived up to 8 weeks and the salamander neurons survived for several months. We report the behavior of labeled RGCsfrom the neonatal rat in culture on glial cell monolayers in which extensive neurite outgrowth occurs, and long-term viability of RGCs is demonstrated electrophysiologitally.
* For reprint requests at: Yale University Department of Ophthalmology and Visual Street. New Haven, CT 06510, U.S.A.
00144835/91/l
School of Medicine, Science. 330 Cedar
10565+08 $03.00/O
Materials and Methods
Retrogradelabeling The use of animals in this work was performed in accordance with the Association for Research in Vision and Ophthalmology Resolution on the Humane Use of Animals in research. Five- to seven-day-old Sprague-Dawley rats were anesthetized by chilling in ice for 10 min. One microliter of a 1 y0 suspension of granular blue in physiological saline was injected into the superior colliculi. Injections were performed with a l-p1 Hamilton syringe fitted with a glass capillary tip drawn to a fine point and were performed under an operating microscope. Injections were made under direct observation after developing a small skull flap just posterior to the transverse sinus (Barnstable and Drager, 1984). Preparation of Retinal Cell Suspensions Retinal cells were dissociated according to the method described by Leifer et al. (1984). Two days after injection of the fluorescent dye, rats were killed by decapitation. After gentle enucleation, the eyes were immersed in Hanks’ balanced salt solution (Gibco, Grand Island, NY) containing 100 U ml-’ penicillin and 100 ,ug ml-’ streptomycin. The corneas were excised and the lens and vitreous were removed with fine forceps under an operating microscope. The retinas were gently isolated with blunt dissection. Four retinas were incubated at 36 “C for 40 min in 10 ml Hanks’ balanced salt solution containing bovine serum albumin (0.2 mg ml-‘), m-cysteine 0 1991 AcademicPressLimited
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(0.2 mg ml-‘), and papain (26 U ml-‘) (Sigma, St Louis, MO). The tissue was rinsed with Hanks’ and replaced with 4 ml culture medium and the tissue triturated through a l-ml pipette. After complete dissociation of the retina into a fine suspension, 0.2 ml of the supernatant suspension was placed on a glial cell monolayer in a dish containing 1.5 ml medium. Approximately 3 x lo6 retinal cells were plated per dish. The culture medium consisted of Eagle’s minimum essential medium (Gibco), glutamine (2 mM), glucose ( 16 mM), and rat serum (5 %). Cultures were maintained in a humidified atmosphere of 5 % CO, and 9 5 % air at 3 7 “C. The medium was changed every 3 days.
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was discarded and the monolayer rinsed twice with Hanks’. Trypsin (0.1%) in Hanks’ was added to the flask and incubated until the cells detached from the base, usually in 5-10 min. The cell suspension was centrifuged and the trypsin solution replaced with medium to approximate a cell density of 1.2-2.0 x 10” cells ml-‘. One milliliter of this cell suspension was placed on a prepared Petri dish. Approximately 5 days after subculture, the glia spread over the cover slip in such a way as to maintain spaces between glial ‘islands ’ and were then used for subsequent retinal cultures. Staining with Thy-l Antibody
The base of a 35-mm plastic Petri dish (Corning, Medfield, MA) is too thick to observe cells with the high-magnification objective of an inverted microscope. To enable appropriate microscopic examination, the base of the Petri dish was replaced with a cover slip (Bray, 1970). A 2-cm diameter hole was drilled through the center of the dish and the cover slip was attached with Sylgard (a silicone elastomer, Dow Corning Corporation). After the silicone set, the Petri dish was rinsed with 9 5 % ethanol followed by distilled water and was sterilized with ultraviolet light. In experiments with laminin-coated dishes, a lOOpg ml-l solution of Cr-Laminin (Collaborative Research Incorporated, Bedford, MA) was prepared with culture medium. Petri dishes containing 0.5 ml CrLaminin solution were maintained at room temperature for 45 min. The solution was then aspirated and the retinal cell suspensions plated.
Thy-l antibody was supplied by Dr Colin Barnstable (Yale University School of Medicine, Department of Ophthalmology and Visual Science). It is a mouse monoclonal antibody (IgG2a) raised against rat cerebral cortical Thy-l antigen (Barnstable and Drager, 1984). Dissociated cells were incubated in HBSS with 5% goat serum for 30 min at room temperature with IgG2s (ascites fluid, 1: lOOO), washed by centrifugation and labeled for 30 min with fluorescein-conjugated goat anti-mouse IgG (Cappell. 1: 100). For in vitro staining, cultures were rinsed three times with 5% goat serum in HBSS, incubated with IgG2a (ascites fluid, 1: 1000) for 30 min at 3 7 “C. After washing three times with HBSS, cultures were incubated with fluorescein-conjugated goat antimouse IgG (Cappell, 1:100-l :200) for 30 min at 37 “C, washed again three times and viewed with an inverted microscope equipped with epifluorescence. As a control, mouse serum was used with the same protocol (Barnstable and Drager, 1984).
Preparation of Glial Cell Monolayers
Electrophysiologic
Glial cultures were prepared from the cerebral hemispheres of neonatal rats, l-2 days old, according to the method of McCaffery, Raju and Bennett (1984). After decapitation, the cerebral hemispheres were dissected and placed into Hanks’ balanced salt solution containing 100 U ml-’ penicillin and 100 ,ug ml-l streptomycin. Meninges were stripped off under an operating microscope and the hemisphere incubated for 15 min at 3 7 ‘C with 0.2 5 % trypsin in Hanks’. The tissue was transferred to culture media and triturated through an 18-gauge needle. Cell suspensions were diluted such that one hemisphere was placed in a total of 10 ml: this solution was dispersed in a 75-cm tissue culture flask and maintained in a humidified atmosphere of 5% CO, and 95% air at 37 “C. The medium was changed twice a week. The culture medium consisted of Eagle’s minimum essential medium, glutamine (2 mM), and fetal bovine serum ( 10 %). When the cells formed a confluent monolayer about S-10 days after primary culture, subcultures to Petri dishes or other flasks were prepared. Medium
Patch electrodes were made from 1.5 mm diameter capillary glass pulled on a patch-clamp puller and flrepolished. Recording electrodes were filled with a salt solution consisting of 150 mM KC1 with 10 mM Hepes buffer and had a resistance range of 4-6 mR. The reference electrode was a chlorided silver pellet coated on the sides with teflon and inserted into a 1.5mm diameter capillary glass tube. Recording and reference electrodes were connected to a Hx. 01 impedancematching headstage that delivered signals to an Axoclamp IIA patch-clamp amplifier (Axon Instruments, Foster City, CA). The recordings were viewed in ‘real time ’ on a Hitachi VC602 5 digital storage oscilloscope and a Graphtec WR3101 four channel chart recorder. Recordings were also stored on a Dagan DAS 900 VCR with a built-in pulse code modulator. Currents were injected by driving the current clamp output of the Axoclamp IIA amplifier with an S44 Grass stimulator. Retinal ganglion cells cultured on a glial cell monolayer in Petri dishes were viewed with a Zeiss IM-
Preparation of Culture Dishes
Characterization of Cells
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3 5 phase-contrast fluorescence microscope. The cultures were continuously perfused during recording with oxygenated HBSS buffered with 10 mM Hepes and maintained at 37 “C with a Leiden LU-CBl microincubation system driven by a TC-102 temperature controller (Medical Systems Corporation). The volume of fluid in the Petri dish was 1.5 ml and the rate of perfusion was O-7 ml min’. Fluid was perfused with a two-channel peristaltic pump (Sigma). Perfusion solutions were made with HBSS without Mg”’ and contained either 2 mM CoCl, or 2 mM MgCl, (Sigma). Solutions were also injected with a Sage microperfusion pump via a 20-pm diameter glass pipette positioned approximately 100 pm from the cell body. The recording micropipette was positioned with a Narashige remote three-dimensional hydraulic micromanipulator. 3.
Results
Two substrates were employed for retinal culture. Laminin was the least effective substrate for neuronal attachment and survival. Some retinal neurons extended neurites on the laminin-coated cover slip 1 day after plating ; these cells were not generally maintained for more than 2 to 3 days. Miiller cells began to spread over the cover slip l-2 days after plating, and in some instances the overlying retinal neurons survived for up to 2 weeks. Subsequent experiments employed a glial cell monolayer derived from neonatal rat cerebral cortex as a substrate. This glial population was less mobile than the Miiller cell population and could successfully maintain retinal neurons for periods of up to 2 months. In such cases an extensive intercellular network of retinal neurites formed. Freshly dissociated RGCs were identified with the cytoplasmic retrograde-label granular blue or with fluorescein-conjugated Thy-l antibody on the cell membrane (Fig. 1). RGCs labeled with Thy-l antibody showed an irregular surface fluorescence. There was no significant extension of neurites for the first 6 hr after plating. After 1 day in culture, some RGCs showed neurite extension. The underlying glia, surrounding cells, and complex neurite network which subsequently developed often made it difficult to visualize the entire length of neurites. When the neurites contained granular blue, their extensions could be seen with transmission light microscopy and high magnification (Fig. 2). Figure 3 demonstrates the decrease with time of RGCs identified with granular blue. Only rarely could RGCs be identified in culture after 1 week with this labeling technique. Thy- l-stained neurons showed a well-defined surface fluorescence with good staining of neurites (Fig. 4). By 15 days after plating, RGCs were frequently observed with individual neurites more than 2 mm long. The Thy- 1 fluorescence decreased gradually several days after staining and the diffuse fluorescence of the cell membrane gradually became spotty and
FIG. 1. Retinal ganglion cells just after dissociation. Retinal ganglion cells were labeled by retrograde transport of granular blue from the superior colliculus. After dissociation, they were also labeled by fluorescein-conjugated antibody to Thy-l antigen. A retinal ganglion ceil is shown with phase optics (A), granular blue fluorescence (B) and fluorescein-conjugated antibody staining to Thy-l antigen
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FIG. 2. Ketinal ganglion cells labeled with granular blue in culture. A, Retinal ganglion cells 3 days after plating on glia viewed with a combination of epifluorescence and transmitted light. The neurites of these retinal ganglion cells contained granular blue and enabled visualization of their extensions. B. Retinal ganglion cells 4 days after plating viewed on glia viewed with a combination of epifluorescence and phase optics.
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FIG. 3. The numberof retinal ganglioncellsidentifiedwith granular blue in culture on a glial monolayer. Thirty fields werecountedper dishat x 400 and six disheswerecounted at 6. 24. 48. 72 and 96 hr.
eventually disappeared. Thy-l staining was not entirely specific for RGCs, since occasionally other cells, probably glia, also stained (Fig. 4). Thy-l-stained neurons with extensive neurite networks were identified for up to 48 days in culture. Electrophysiologic recordings from fast blue or Thy7-labeled cells revealed resting potentials which averaged ( &SD.) 59 + 5.1 mV (n = 58). Spontaneous synaptic activity was recorded from retinal ganglion cells marked by fast blue or Thy-l after 3-5 days in culture. This consisted of summations of excitatory depolarizing currents with action potentials and inhibitory hyperpolarizing currents (Fig. 5). Synaptic activity was reversibly blocked by 2 mM Co2+ or 2 mM Mg2+. Both agents are potent inhibitors of chemically mediated synaptic transmission (Miller et al., 198 1). Such recordings, typical of physiologically viable RGCs, were obtained from cells for up to 31 days in culture. Recordings from cells older than 3 1 days have not yet been attempted. Many recordings that were characteristic of RGCs were obtained from cells marked with Thy-l which were not labeled with granular blue. Since retrograde marking does not till all the RGCs plated in culture, this was particularly evident in older cultures (more than 15 days) where no granular blue-labeled cells were present. 4.
Discussion
Neuronal cultures often require embryonic or neonatal tissue to exhibit viability and growth. Several investigators have successfully cultured RGCsfrom 2to 3-day-old neonatal rats (Raff et a1.,1979; Sarthy, Curtis and Catterall, 1983). The number of viable RGCs decreasesrapidly. with an 80% reduction of
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initial density by 48 hr. Media conditioned with chick optic tectum and rat superior colliculus extracts have maintained greater numbers of RGCs (Sarthy et al., 1983; McCaffery et al., 1984). McCaffery and coworkers (1984) reported enhanced survival of RGCs on a glial monolayer compared to culture techniques which employed artificial substrates such as laminin (Cohen et al., 198 6). Greatly enhanced neurite outgrowth was also observed in these co-cultures. Leifer et al. (1984) used 5-12-day-old rats as a source of RGCs, and monoclonal antibodies to Thy-l were used as a substrate to enhance regeneration of retinal ganglion cell neurites in culture. These relatively mature RGCswere maintained for over 2 weeks in the absence of superior colliculus derivatives. Recently, several laboratories have demonstrated extended survival of retinal cell cultures. MacLeish and Townes-Anderson (1988) cultured salamander retinal neurons ; viability was demonstrated for 7 months by morphologic and electrophysiologic criteria. Montague and Friedlander (1989 1 cultured cat retinal neurons and demonstrated survival for 8 weeks. We have shown that neonatal rat RGCssurvive in culture for at least 31 days, as demonstrated by Thy-l labeling and typical electrophysiologic responses. The proper identification of RGCs in culture is important to mechanistic studies of cellular damage. Retrograde markers are reliable, since the RGCs are the only retinal neurons that project ‘to the brain, However, retrograde markers may also be taken up by glia (Bentivoglio et al., 1979, 1980; Aschoff and Hollander, 1982). We have observed granular blue in phagocytic cells shortly after dissociation. Barres et al. (1988) observed the retrograde marker fast blue in cells other than RGCs after dissociation. Fortunately, phagocytic cells are usually easily differentiated from neurons by their appearance. In the normal development of the rat, substantial RGC death has been observed during the first 10 postnatal days (Cunningham, Mohler and Giordane, 1982 ; Potts, Dreher and Bennett, 1982 ; Dreher, Potts and Bennett, 1983). We used 5-7-day-old rats as a cell source; some of the degenerated RGCs may have been programmed to die and were phagocytized by macrophages after plating. Hume, Perry and Gordon (1983) used antibody against the macrophage-specific antigen F4-80 to show that macrophages from the general circulation invade the developing mouse retina during times of programmed neuronal death. Thy-l antigen is a glycoprotein found on the surface of a variety of rodent cell types. The highest concentration of rat Thy-l antigen has been found in the brain and thymus. Barnstable and Drager (1984) described Thy-l antigen as a ganglion cell-specific marker in the rodent retina. Double-labeling experiments showed that many more cells were successfully labeled by antibody to Thy-l than with intracellular fluorescent markers. However, virtually all neurons
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inward membrane currents are associatedwith neurite regeneration in RGCs and that blockade of electrical activity promotes degeneration. Electrophysiologic studies could be extended to investigate mechanisms of damage. Two broad theories have been advanced to explain the pathophysiology of glaucomatous damage to retinal ganglion cells: (1) mechanical distortion at the level of the lamina cribrosa blocks axonal transport: and (2) increased intraocular pressure and other factors decreaseperfusion of the anterior optic nerve head and cause ischemic damage to RGCs. Measurements of axonal flow and electrophysiology in individual cells in culture under various conditions of oxygen deprivation and mechanical distortion may provide information about the cellular and molecular mechanisms of retinal ganglion cell dysfunction and death from glaucoma and other disorders. Acknowledgements
FIG. 5. A, Spontaneous synaptic activity recorded from a Thy-l marked retinal ganglion cell after 3 1 days in culture. Resting membrane potential was -62 mV. B, Reversible cobalt (2 mM) blockade of spontaneous synaptic activity. This recording was made after 16 days in culture. The resting membrane potential was - 6 5 mV.
marked with granular blues were also marked by the cell-surface monoclonal Thy-l antibody. There may be a subpopulation of cells (perhaps amacrine cells) that are also marked by Thy-l antibody. Therefore, whole-cell electrophysiologic recordings were also obtained from cells marked with Thy-l. All cells demonstrated the typical electrophysiologic characteristics of retinal ganglion cells. Resting membrane potentials averaged 59 + 5.1 mV (n = 58). Cells displayed the characteristic spiking behavior of retinal ganglion cells, with action-potential amplitudes of 40-60 mV riding on small generator potentials of 2-5 mV. This spontaneous synaptic activity was reversibly blocked by 2 mM Co2+or 2 mM Mg”. This study demonstrates that viability of cultured retinal ganglion cells can be achieved for at least 4 weeks and probably longer by employing a glial monolayer substrate combined with staining living ganglion cells with monoclonal antibody to Thy-l antigen. This technique presents the possibility of studying the effects of hypoxic, anoxic, or hyperbaric damage to retinal ganglion cells over an extended time period in culture. Lipton ( 1986, 198 7) employed patch-clamp electrophysiologic recordings on freshly dissociatedcells to demonstrate that calcium-mediated
The authors would like to thank Dr Colin Barnstable for his valuable suggestions during the course of this work. This study was supported in part by grants from the U.S. Public Health Service and EY-07648 and EY-09114 (Dr Caprioli), EY-00785 (Yale Center Grant), Research to Prevent Blindness, Inc., Foresight, Inc., and the Connecticut Lions Eye Research Foundation. Inc.
References Aschoff, A. and Hollander. H. (1982). Fluorescent compounds as retrograde tracers compared with horseradish peroxidase (HRP). I. A parametric study in the central visual system of the albino rat. I. Neurosci. Methods 6, 179-9 7. Barnstable. C. J. and Drager, V. C. (1984). Thy-l antigen: A ganglion cell specific marker in rodent retina. Neuroscience 11, 847-55. Barres, B. A., Silverstein, B. E., Corey, D. E’. and Chun. L. L. Y. (1988). Immunological, morphological, and electrophysiological variation among retinal ganglion cells purified by panning. Neuron 1, 791-803. Bentivoglio. M.. Kuypers, H. G. J. M., Catsman-Berrevoets, C. E. and Dann, 0. (1979). Fluorescent retrograde neuronal labelling in rat by means of substances binding specifically to adenine-thymine rich DNA. Neuro-sci. Letts 12. 23540. Bentivoglio, M., Kuypers. H. G. J. M., Catsman-Berrevoets, C. E. and Dann, 0. (1980). Two new fluorescent retrograde neuronal tracers which are transported over long distances. Neurosci. Letts 18, 25-30.
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FIG. 4. Retinal ganglion cells 7 days after plating on glia. This cell was double-labeled by granular blue and antibody to Thy-l antigen and was viewed with phase optics (A), granular blue fluorescence (B) and Thy-l label fluorescence (C). The arrow indicates an underlying glial cell which was labeled with antibody to Thy-l antigen.
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regional correspondence with neuron death in colliculus. Dev. Bruin Res. 2, 203-215. Dreher, B., Potts, R. A. and Bennett, M. R. ( 1983). Evidence that the early postnatal reduction in the number of rat retinal ganglion cells is due to a wave of ganglion cell death. Neurosci. Letts 36, 225-60. Hume, D. A., Perry, V. H. and Gordon, S. (1983). Immunohistochemical localization of a macrophage-specific antigen in developing mouse retina: phagocytosis of dying neurons and differentiation of microglial cells to form a regular array in the plexiform layers. 7. Cell Biol. 97,253-7.
Liefer. D., Lipton, S. A., Barnstable. C. J. and Masland. R. H. (1984). Monoclonal antibody to Thy-l enhances regeneration of processes by rat retinal ganglion cells in culture. Science 224, 303-6. Lipton. S. A. (1986). Blocking of electrical activity promotes the death of mammalian retinal ganglion cells in culture. Proc. Natl. Ad. Sci. U.S.A. 83, 9774-8. Lipton, S. A. (1987). Bursting of calcium-activated cationselective channels is associated with neurite regeneration in a mammalian central neuron. Neurosci. Letts 982,
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Montague, P. R. and Friedlander. M. J. ( 1989). Expression ot an instinsic growth strategy by mammalian retinal neurons. Proc. Nafl. Ad. Sci. U.S.A. 86. 7223-7. Potts, R. A., Dreher. B. and Bennett, M. R. (1982). The loss of ganglion cells in the developing retina of the rat. Dev. Brain Rc~s.3. 481-h. Raff, M.. Fields, K., Sen-Itiroh. H.. Mirsky, R.. Pruss. R. and Winter, J. ( 1979). Cell-type specific markers for distinguishing and studying neurons and the major classes of glial cells in culture. Brain Res. 174. 283-308. Sarthy, P. V.. Curtis, B. M. and Catterall. W. A. (1983). Retrograde labelling, enrichment, and characterization of retinal ganglion cells from the neonatal rat. 1. Ntwmsci.
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