Variable functional recovery and minor cell loss in the ganglion cell layer of the lizard Gallotia galloti after optic nerve axotomy E. Santos a, b, * , M.M. Romero-Alemán b , M. Monzón-Mayor b , C. Yanes a a Departamento de Microbiología y Biología Celular, Universidad de La Laguna, 38206 Tenerife (Canary Islands), Spain b Departamento de Morfología (Biología Celular), Universidad de Las Palmas de Gran Canaria, 35016 Gran Canaria (Canary Islands), Spain article info Article history: Received 6 June 2013 Accepted in revised form 26 September 2013 Available online 31 October 2013 Keywords: regeneration axon regrowth vision pupillary light reflex reptile abstract The lizard Gallotia galloti shows spontaneous and slow axon regrowth through a permissive glial scar after optic nerve axotomy. Although much of the expression pattern of glial, neuronal and extracellular matrix markers have been analyzed by our group, an estimation of the cell loss in the ganglion cell layer (GCL) and the degree of visual function recovery remained unresolved. Thus, we performed a series of tests indicative of effective visual function (pupillary light reflex, accommodation, visually elicited behavior) in 18 lizards at 3, 6, 9 and 12 months post-axotomy which were then processed for immu- nohistochemistry for the neuronal markers SMI-31 (neurofilaments), Tuj1 (beta-III tubulin) and SV2 (synaptic vesicles) at the last timepoint. Separately, cell loss in the GCL was estimated by comparative quantitation of DAPI þ nuclei in control and 12 months experimental lizards. Additionally,15 lizards were processed for electron microscopy to monitor relevant ultrastructural changes in the GCL, optic nerve and optic tract throughout regeneration. Hypertrophy of RGCs was persistent, morphology of the re- generated nerves varied from narrow to neuroma-like features and larger regenerated axons underwent remyelination by 9 months. The estimated cell loss in the GCL was 27% and two-third of the animals recovered the pupillary light reflex which involves the pretectum. Strikingly, visually elicited behavior involving the tectum was only restored in two specimens, presumably due to the higher complexity of this pathway. These preliminary results indicate that limited functional regeneration occurs spontane- ously in the severely injured visual system of the lacertid G. galloti. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction Retinal ganglion cells (RGCs) can regenerate their axons in fish, amphibians, and certain species of reptiles, but not in mammals. The axon regeneration process in the lizard Gallotia galloti occurs despite a mammalian-like environment represented by (1) persistent glial scar (Lang et al., 2002, 2008; Romero-Alemán et al., 2013), (2) upregulation of axon growth inhibitors such as the myelin-associated inhibitors (Lang et al., 1998), tenascin-R (Lang et al., 2008) and (3) the absence of proliferating cells in the con- trol and experimental retina as a source of new-born neurons (Casañas et al., 2011; Romero-Alemán et al., 2013). In contrast, the regeneration process in anamniotes (e.g. fish and frogs) occurs in a highly supportive microenvironment represented by (1) transitory gliosis (Stafford et al., 1990), (2) continuous adult retinal neuro- genesis, (3) lack, dysfunctionality or downregulation of axon growth inhibitors (Becker et al., 1999, 2004), (4) upregulation of axon growth promoters (e.g., neurotrophins, Caminos et al., 1999; myelin protein zero, Schweitzer et al., 2003; invading Schwann cells, Nona et al., 2000). These differences may account for the successful functional regeneration in fish and amphibians as compared to the limited functional recovery in the lizard Cteno- phorus ornatus. Interestingly, visual training in C. ornatus restored vision in all animals, but to varying extents, and always less than in normal animals (Beazley et al., 2003). To date, the agamid lizard C. ornatus, the lacertid G. galloti, and the snake Vipera aspis are the only reptilian species in which abundant RGC axons regrow after crush or transection reaching the superficial optic tectum and other visual areas (Rio et al., 1989; Beazley et al., 1997; Lang et al., 1998; Dunlop et al., 2000). How- ever, in two Australian species of geckos and a turtle, axons show Abbreviations: CNS, central nervous system; GCL, ganglion cell layer; NFL, nerve fiber layer; OCh, optic chiasm; OT, optic tectum; OTr, optic tract; Pia, piamater; RGCs, retinal ganglion cells; sfgs, stratum fibrosum et griseum superficialis; so, stratum opticum. * Corresponding author. Departamento de Microbiología y Biología Celular, Uni- versidad de La Laguna, 38206 Tenerife (Canary Islands), Spain. Tel.: þ34 922318416; fax: þ34 922318311. E-mail address: esantos@ull.es (E. Santos). Contents lists available at ScienceDirect Experimental Eye Research journal homepage: www.elsevier.com/locate/yexer 0014-4835/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.exer.2013.09.020 Experimental Eye Research 118 (2014) 89e99