Retinal ganglion cell (RGC) death occurs after optic nerve injury due to acute trauma or chronic degenerative conditions such as optic neuropathies (e.g., glaucoma). Currently, there are no effective therapies to prevent permanent vision loss resulting from RGC death, underlining the need for research on the pathogenesis of RGC disorders. Modeling human RGC/optic nerve diseases in non-human primates is ideal because of their similarity to humans, but has practical limitations including high cost and ethical considerations. In addition, many retinal degenerative disorders are age-related making the study in primate models prohibitively slow. For these reasons, mice and rats are commonly used to model RGC injuries. However, as nocturnal mammals, these rodents have retinal structures that differ from primates - possessing less than one-tenth of the RGCs found in the primate retina. Here we report the diurnal thirteen-lined ground squirrel (TLGS) as an alternative model. Compared to other rodent models, the number and distribution of RGCs in the TLGS retina are closer to primates. The TLGS retina possesses ~600,000 RGCs with the highest density along the equatorial retina matching the location of the highest cone density (visual streak). TLGS and primate retinas also share a similar interlocking pattern between RGC axons and astrocyte processes in the retina nerve fiber layer (RNFL). In addition, using TLGS we establish a new partial optic nerve injury model that precisely controls the extent of injury while sparing a portion of the retina as an ideal internal control for investigating the pathophysiology of axon degeneration and RGC death. Moreover, in vivo optical coherence tomography (OCT) imaging and ex vivo microscopic examinations of the retina in optic nerve injured TLGS confirm RGC loss precedes proximal axon degeneration, recapitulating human pathology. Thus, the TLGS retina is an excellent model, for translational research in neurodegeneration and therapeutic neuroprotection.
Subscribe to Journal
Get full journal access for 1 year
only $9.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Data availability statement
The data that supports the findings of this study are available within the article and its supplementary material. Raw or additional data are available from the corresponding author upon reasonable request.
Chen S-K, Badea TC, Hattar S. Photoentrainment and pupillary light reflex are mediated by distinct populations of ipRGCs. Nature. 2011;476:92–5.
Dogiel AS. Die Retina der Vögel. Archiv f mikrosk Anat. 1895;44:622–48.
Dräger UC, Olsen JF. Origins of crossed and uncrossed retinal projections in pigmented and albino mice. J Comp Neurol. 1980;191:383–412.
Doi M, Imatani H, Sasoh M, Uji Y, Yamamura H. Displaced retinal ganglion cells in the Chinese hamster. Jpn J Ophthalmol. 1994;38:139–43.
Nadal-Nicolás FM, Salinas-Navarro M, Jiménez-López M, Sobrado-Calvo P, Villegas-Pérez MP, Vidal-Sanz M, et al. Displaced retinal ganglion cells in albino and pigmented rats. Front Neuroanat. 2014;8:99.
Ramón y Cajal S. The Structure of the retina (compiled and translated by Thorpe SA, Glickstein M). Springfield, Ill. C.C. Thomas, 1972.
Case LC, Tessier-Lavigne M. Regeneration of the adult central nervous system. Curr Biol. 2005;15:749–53.
Salinas-Navarro M, Alarcón-Martínez L, Valiente-Soriano FJ, Jiménez-López M, Mayor-Torroglosa S, Avilés-Trigueros M, et al. Ocular hypertension impairs optic nerve axonal transport leading to progressive retinal ganglion cell degeneration. Exp Eye Res. 2010;90:168–83.
Vidal-Sanz M, Salinas-Navarro M, Nadal-Nicolás FM, Alarcón-Martínez L, Valiente-Soriano FJ, de Imperial JM, et al. Understanding glaucomatous damage: anatomical and functional data from ocular hypertensive rodent retinas. Prog Retin Eye Res. 2012;31:1–27.
Ortín-Martínez A, Salinas-Navarro M, Nadal-Nicolás FM, Jiménez-López M, Valiente-Soriano FJ, García-Ayuso D, et al. Laser-induced ocular hypertension in adult rats does not affect non-RGC neurons in the ganglion cell layer but results in protracted severe loss of cone-photoreceptors. Exp Eye Res. 2015;132:17–33.
García-Ayuso D, Salinas-Navarro M, Nadal-Nicolás FM, Ortín-Martínez A, Agudo-Barriuso M, Vidal-Sanz M, et al. Sectorial loss of retinal ganglion cells in inherited photoreceptor degeneration is due to RGC death. Br J Ophthalmol. 2014;98:396–401.
Zhang J, Li L, Huang H, Fang F, Webber HC, Zhuang P, et al. Silicone oil-induced ocular hypertension and glaucomatous neurodegeneration in mouse. Elife. 2019;8:45881.
Sappington RM, Carlson BJ, Crish SD, Calkins DJ. The microbead occlusion model: a paradigm for induced ocular hypertension in rats and mice. Invest Ophthalmol Vis Sci. 2010;51:207–16.
Misantone LJ, Gershenbaum M, Murray M. Viability of retinal ganglion cells after optic nerve crush in adult rats. J Neurocytol. 1984;13:449–65.
Villegas-Pérez MP, Vidal-Sanz M, Rasminsky M, Bray GM, Aguayo AJ. Rapid and protracted phases of retinal ganglion cell loss follow axotomy in the optic nerve of adult rats. J Neurobiol. 1993;24:23–36.
Parrilla-Reverter G, Agudo M, Nadal-Nicolás F, Alarcón-Martínez L, Jiménez-López M, Salinas-Navarro M, et al. Time-course of the retinal nerve fibre layer degeneration after complete intra-orbital optic nerve transection or crush: a comparative study. Vision Res. 2009;49:2808–25.
Galindo-Romero C, Avilés-Trigueros M, Jiménez-López M, Valiente-Soriano FJ, Salinas-Navarro M, Nadal-Nicolás F, et al. Axotomy-induced retinal ganglion cell death in adult mice: quantitative and topographic time course analyses. Exp Eye Res. 2011;92:377–87.
Nadal-Nicolás FM, Jiménez-López M, Sobrado-Calvo P, Nieto-López L, Cánovas-Martínez I, Salinas-Navarro M, et al. Brn3a as a marker of retinal ganglion cells: qualitative and quantitative time course studies in naive and optic nerve-injured retinas. Invest Ophthalmol Vis Sci. 2009;50:3860–8.
Nadal-Nicolás FM, Sobrado-Calvo P, Jiménez-López M, Vidal-Sanz M, Agudo-Barriuso M. Long-term effect of optic nerve axotomy on the retinal ganglion cell layer. Invest Ophthalmol Vis Sci. 2015;56:6095–112.
Nadal-Nicolás FM, Jiménez-López M, Salinas-Navarro M, Sobrado-Calvo P, Vidal-Sanz M, Agudo-Barriuso M. Microglial dynamics after axotomy-induced retinal ganglion cell death. J Neuroinflammation. 2017;14:218.
Choe TE, Abbott CJ, Piper C, Wang L, Fortune B. Comparison of longitudinal in vivo measurements of retinal nerve fiber layer thickness and retinal ganglion cell density after optic nerve transection in rat. PLoS ONE. 2014;9:e113011.
Liu Y, McDowell CM, Zhang Z, Tebow HE, Wordinger RJ, Clark AF. Monitoring retinal morphologic and functional changes in mice following optic nerve crush. Invest Ophthalmol Vis Sci. 2014;55:3766–74.
Munguba GC, Galeb S, Liu Y, Landy DC, Lam D, Camp A, et al. Nerve fiber layer thinning lags retinal ganglion cell density following crush axonopathy. Invest Ophthalmol Vis Sci. 2014;55:6505–13.
Rovere G, Nadal-Nicolás FM, Agudo-Barriuso M, Sobrado-Calvo P, Nieto-López L, Nucci C, et al. Comparison of retinal nerve fiber layer thinning and retinal ganglion cell loss after optic nerve transection in adult albino rats. Invest Ophthalmol Vis Sci. 2015;56:4487–98.
Nadal-Nicolás FM, Vidal-Sanz M, Agudo-Barriuso M. The aging rat retina: from function to anatomy. Neurobiol Aging. 2018;61:146–68.
Jeon CJ, Strettoi E, Masland RH. The major cell populations of the mouse retina. J Neurosci. 1998;18:8936–46.
Szél A, Röhlich P. Two cone types of rat retina detected by anti-visual pigment antibodies. Exp Eye Res. 1992;55:47–52.
Cull G, Cioffi GA, Dong J, Homer L, Wang L. Estimating normal optic nerve axon numbers in non-human primate eyes. J Glaucoma. 2003;12:301–6.
Kim CB, Tom BW, Spear PD. Effects of aging on the densities, numbers, and sizes of retinal ganglion cells in rhesus monkey. Neurobiol Aging. 1996;17:431–8.
Jonas JB, Müller-Bergh JA, Schlötzer-Schrehardt UM, Naumann GO. Histomorphometry of the human optic nerve. Invest Ophthalmol Vis Sci. 1990;31:736–44.
Kryger Z, Galli-Resta L, Jacobs GH, Reese BE. The topography of rod and cone photoreceptors in the retina of the ground squirrel. Vis Neurosci. 1998;15:685–91.
Merriman DK, Sajdak BS, Li W, Jones BW. Seasonal and post-trauma remodeling in cone-dominant ground squirrel retina. Exp Eye Res. 2016;150:90–105.
Li W. Ground squirrel - A cool model for a bright vision. Semin Cell Dev Biol. 2020;106:127–34.
Jacobs GH. The distribution and nature of colour vision among the mammals. Biol Rev Camb Philos Soc. 1993;68:413–71.
Marshak DW, Mills SL. Short-wavelength cone-opponent retinal ganglion cells in mammals. Vis Neurosci. 2014;31:165–75.
Nadal-Nicolás FM, Kunze VP, Ball JM, Peng BT, Krishnan A, Zhou G, et al. True S-cones are concentrated in the ventral mouse retina and wired for color detection in the upper visual field. Elife. 2020;9:56840.
Ortín-Martínez A, Jiménez-López M, Nadal-Nicolás FM, Salinas-Navarro M, Alarcón-Martínez L, Sauvé Y, et al. Automated quantification and topographical distribution of the whole population of S- and L-cones in adult albino and pigmented rats. Invest Ophthalmol Vis Sci. 2010;51:3171–83.
Ortín-Martínez A, Nadal-Nicolás FM, Jiménez-López M, Alburquerque-Béjar JJ, Nieto-López L, García-Ayuso D, et al. Number and distribution of mouse retinal cone photoreceptors: differences between an albino (Swiss) and a pigmented (C57/BL6) strain. PLoS ONE. 2014;9:e102392.
Jacobs GH, Neitz M, Deegan JF, Neitz J. Trichromatic colour vision in New World monkeys. Nature. 1996;382:156–8.
Nathans J, Thomas D, Hogness DS. Molecular genetics of human color vision: the genes encoding blue, green, and red pigments. Science. 1986;232:193–202.
Yokoyama S, Yokoyama R. Molecular evolution of human visual pigment genes. Mol Biol Evol. 1989;6:186–97.
Finlay BL, Franco ECS, Yamada ES, Crowley JC, Parsons M, Muniz JAPC, et al. Number and topography of cones, rods and optic nerve axons in New and Old World primates. Vis Neurosci. 2008;25:289–99.
Van Hooser SD, Nelson SB. The squirrel as a rodent model of the human visual system. Vis Neurosci. 2006;23:765–78.
Andrews MT. Advances in molecular biology of hibernation in mammals. Bioessays. 2007;29:431–40.
Staples JF. Metabolic flexibility: hibernation, torpor, and estivation. Compr Physiol. 2016;6:737–71.
Ou J, Ball JM, Luan Y, Zhao T, Miyagishima KJ, Xu Y, et al. iPSCs from a Hibernator provide a platform for studying cold adaptation and its potential medical applications. Cell. 2018;173:851–63.
Luan Y, Ou J, Kunze VP, Qiao F, Wang Y, Wei L, et al. Integrated transcriptomic and metabolomic analysis reveals adaptive changes of hibernating retinas. J Cell Physiol. 2018;233:1434–45.
Long KO, Fisher SK. The distributions of photoreceptors and ganglion cells in the California ground squirrel, Spermophilus beecheyi. J Comp Neurol. 1983;221:329–40.
Johnson PT, Geller SF, Reese BE. Distribution, size and number of axons in the optic pathway of ground squirrels. Exp Brain Res. 1998;118:93–104.
Merriman DK, Lahvis G, Jooss M, Gesicki JA, Schill K. Current practices in a captive breeding colony of 13-lined ground squirrels (Ictidomys tridecemlineatus). Lab Anim (NY). 2012;41:315–25.
Nadal-Nicolás FM, Salinas-Navarro M, Vidal-Sanz M, Agudo-Barriuso M. Two methods to trace retinal ganglion cells with fluorogold: from the intact optic nerve or by stereotactic injection into the optic tract. Exp Eye Res. 2015;131:12–19.
Jospeh SA, Knigge KA, Kalejs LM, Hoffman RA, Reid P. A stereotaxic atlas of the brain of the 13-line ground squirrel (citellus tridecemlineatus). Rochester Univ, NY: Dept of Anatomy; 1966.
Vaidya PG. A comparative study of the visual system in the diurnal ground squirrel, Citellus tridecemlineatus tridecemlineatus and in the nocturnal guinea pig, Cavia cobaya. Z Anat Entwicklungsgesch. 1965;124:505–21.
Kwong JMK, Caprioli J, Piri N. RNA binding protein with multiple splicing: a new marker for retinal ganglion cells. Invest Ophthalmol Vis Sci. 2010;51:1052–8.
Rodriguez AR, de Sevilla Müller LP, Brecha NC. The RNA binding protein RBPMS is a selective marker of ganglion cells in the mammalian retina. J Comp Neurol. 2014;522:1411–43.
Famiglietti EV, Sharpe SJ. Regional topography of rod and immunocytochemically characterized “blue” and “green” cone photoreceptors in rabbit retina. Vis Neurosci. 1995;12:1151–75.
Sánchez-Migallón MC, Valiente-Soriano FJ, Salinas-Navarro M, Nadal-Nicolás FM, Jiménez-López M, Vidal-Sanz M, et al. Nerve fibre layer degeneration and retinal ganglion cell loss long term after optic nerve crush or transection in adult mice. Exp Eye Res. 2018;170:40–50.
Sánchez-Migallón MC, Valiente-Soriano FJ, Nadal-Nicolás FM, Vidal-Sanz M, Agudo-Barriuso M. Apoptotic retinal ganglion cell death after optic nerve transection or crush in mice: delayed RGC loss with BDNF or a Caspase 3 Inhibitor. Invest Ophthalmol Vis Sci. 2016;57:81–93.
Rodriguez-Ramos Fernandez J, Dubielzig RR. Ocular comparative anatomy of the family Rodentia. Vet Ophthalmol. 2013;16:94–9.
Pasteels B, Rogers J, Blachier F, Pochet R. Calbindin and calretinin localization in retina from different species. Vis Neurosci. 1990;5:1–16.
Sun D, Lye-Barthel M, Masland RH, Jakobs TC. The morphology and spatial arrangement of astrocytes in the optic nerve head of the mouse. J Comp Neurol. 2009;516:1–19.
Nadal-Nicolás FM, Jiménez-López M, Salinas-Navarro M, Sobrado-Calvo P, Alburquerque-Béjar JJ, Vidal-Sanz M, et al. Whole number, distribution and co-expression of brn3 transcription factors in retinal ganglion cells of adult albino and pigmented rats. PLoS ONE. 2012;7:e49830.
Zhang Y, Zhang S, Xia Y, Ji Y, Jiang W, Liet M, et al. In vivo evaluation of retinal ganglion cells and optic nerve’s integrity in large animals by multi-modality analysis. Exp Eye Res. 2020;197:108117.
Levkovitch-Verbin H, Quigley HA, Kerrigan-Baumrind LA, D’Anna SA, Kerrigan D, Pease ME. Optic nerve transection in monkeys may result in secondary degeneration of retinal ganglion cells. Invest Ophthalmol Vis Sci. 2001;42:975–82.
Abreu M, Kicliter E, Lugo-Garcia N. Displaced amacrine cells in the ganglion cell layer of the ground squirrel retina. P R Health Sci J. 1993;12:137–41.
Wässle H, Grünert U, Röhrenbeck J, Boycott BB. Cortical magnification factor and the ganglion cell density of the primate retina. Nature. 1989;341:643–6.
Wässle H, Grünert U, Röhrenbeck J, Boycott BB. Retinal ganglion cell density and cortical magnification factor in the primate. Vision Res. 1990;30:1897–911.
Curcio CA, Allen KA. Topography of ganglion cells in human retina. J Comp Neurol. 1990;300:5–25.
Mariani AP, Hersh LB. Synaptic organization of cholinergic amacrine cells in the rhesus monkey retina. J Comp Neurol. 1988;267:269–80.
Cepko CL, Austin CP, Yang X, Alexiades M, Ezzeddine D. Cell fate determination in the vertebrate retina. Proc Natl Acad Sci U.S.A. 1996;93:589–95.
Livesey FJ, Cepko CL. Vertebrate neural cell-fate determination: lessons from the retina. Nat Rev Neurosci. 2001;2:109–18.
Kisseleff E, Vigouroux RJ, Hottin C, Lourdel S, Shah P, Chédotal A, et al. Glycogen Synthase Kinase 3 Regulates the Genesis of the Rare Displaced Ganglion Cell Retinal Subtype. Preprint at https://doi.org/10.1101/2021.01.06.425300 (2021).
Johnson KP, Fitzpatrick MJ, Zhao L, Wang B, McCracken S, Williams PR, et al. Cell-type-specific binocular vision guides predation in mice. Neuron. 2021;109:1527–39.
Levkovitch-Verbin H, Quigley HA, Martin KRG, Zack DJ, Pease ME, Valenta DF. A model to study differences between primary and secondary degeneration of retinal ganglion cells in rats by partial optic nerve transection. Invest Ophthalmol Vis Sci. 2003;44:3388–93.
Khakh BS, Sofroniew MV. Diversity of astrocyte functions and phenotypes in neural circuits. Nat Neurosci. 2015;18:942–52.
Zai LJ, Wrathall JR. Cell proliferation and replacement following contusive spinal cord injury. Glia. 2005;50:247–57.
Yang T, Dai Y, Chen G, Cui S. Dissecting the dual role of the glial scar and scar-forming astrocytes in spinal cord injury. Front Cell Neurosci. 2020;14:78.
Greenhalgh AD, David S. Differences in the phagocytic response of microglia and peripheral macrophages after spinal cord injury and its effects on cell death. J Neurosci. 2014;34:6316–22.
Shinozaki Y, Shibata K, Yoshida K, Shigetomi E, Gachet C, Ikenaka K, et al. Transformation of astrocytes to a neuroprotective phenotype by microglia via P2Y1 receptor downregulation. Cell Rep. 2017;19:1151–64.
Anderson MA, Burda JE, Ren Y, Ao Y, O’Shea TM, Kawaguchiet R, et al. Astrocyte scar formation aids central nervous system axon regeneration. Nature. 2016;532:195–200.
Gu Y, Cheng X, Huang X, Yuan Y, Qin S, Tan Z, et al. Conditional ablation of reactive astrocytes to dissect their roles in spinal cord injury and repair. Brain Behav Immun. 2019;80:394–405.
Sugar O, Gerard RW. SPINAL CORD REGENERATION IN THE RAT. J Neurophysiol. 1940;3:1–19.
Clemente CD, Windle WF. Regeneration of severed nerve fibers in the spinal cord of the adult cat. J Comp Neurol. 1954;101:691–731.
Yiu G, He Z. Glial inhibition of CNS axon regeneration. Nat Rev Neurosci. 2006;7:617–27.
Ramírez AI, Salazar JJ, de Hoz R, Rojas B, Gallego BI, Salinas-Navarro M, et al. Quantification of the effect of different levels of IOP in the astroglia of the rat retina ipsilateral and contralateral to experimental glaucoma. Invest Ophthalmol Vis Sci. 2010;51:5690–6.
de Hoz R, Gallego BI, Ramírez AI, Rojas B, Salazar JJ, Valiente-Soriano FJ, et al. Rod-like microglia are restricted to eyes with laser-induced ocular hypertension but absent from the microglial changes in the contralateral untreated eye. PLoS ONE. 2013;8:e83733.
Rojas B, Gallego BI, Ramírez AI, Salazar JJ, de Hoz R, Valiente-Soriano FJ, et al. Microglia in mouse retina contralateral to experimental glaucoma exhibit multiple signs of activation in all retinal layers. J Neuroinflammation. 2014;11:133.
Osborne NN, Li G-Y, Ji D, Mortiboys HJ, Jackson S. Light affects mitochondria to cause apoptosis to cultured cells: possible relevance to ganglion cell death in certain optic neuropathies. J Neurochem. 2008;105:2013–28.
Osborne NN, Núñez-Álvarez C, Del Olmo-Aguado S, Merrayo-Lloves J. Visual light effects on mitochondria: the potential implications in relation to glaucoma. Mitochondrion. 2017;36:29–35.
Guttenplan KA, Stafford BK, El-Danaf RN, et al. Neurotoxic reactive astrocytes drive neuronal death after retinal injury. Cell Rep. 2020;31:107776.
We would like to thank the Animal Care team, especially Dr. Ginger Tansey, Amber Lopez, Denise Parker, Irina Bunea, and Kristi Creel for taking care of our TLGS colony. We also express our appreciation to Dr. Haohua Quiao and Dr. Yichao Li of the NIH Visual Function Core for providing training in the use of the SD-OCT. We would also like to thank Dr. David Pow (University of Queensland, Brisbane, Australia) for providing the glycine antibody. We also would like to thank Dr. Tao Sun (National Institute of Neurological Disorders and Stroke) for providing postmortem rat specimens, and Dr. Lauren Brinster (Diagnostic and research services branch-NIH), Dr. Mark A. Eldridge (National Institute of Mental Health) and, Dr. Julie Mattison and Dr. Kielee Jennings (Nonhuman Primate Core of the National Institute on Aging) for providing postmortem monkey specimens.
This research was supported by Intramural Research Program of the National Eye Institute, National Institutes of Health and, by the Office of the Assistant Secretary of Defense for Health Affairs and the Defense Health Agency J9, Research and Development Directorate, through the Vision Research Program under Award No. CDMRPL-18-0-VR180205. Opinions, interpretations, conclusions, and recommendations are those of the author and are not necessarily endorsed by the Department of Defense.
Ethics approval and consent to participate
Each species was treated and maintained according to their unique protocols (mouse, ASP#606; TLGS: ASP#595) approved by the National Institutes of Health guidelines for Animal Care and Use Committee in research and by the Ethical and Animal Studies Committee of the National Eye Institute. All animal studies conformed to the Statement for the Use of Animals in Ophthalmic and Vision research of the Association for Research in Vision and Ophthalmology (ARVO). In conducting this research, we adhered to the laws of the United States and regulations of the US Department of Agriculture.
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Xiao, X., Zhao, T., Miyagishima, K.J. et al. Establishing the ground squirrel as a superb model for retinal ganglion cell disorders and optic neuropathies. Lab Invest 101, 1289–1303 (2021). https://doi.org/10.1038/s41374-021-00637-y