The Effect of Retinal Ganglion Cell Injury on Light-Induced Photoreceptor Degeneration Robert J. Casson,1 Glyn Chidlow,1 John P. M. Wood,1 Manuel Vidal-Sanz,2 and Neville N. Osborne1 PURPOSE. To determine the effect of optic nerve transection photoreceptors against light-induced injury. An unusual aspect (ONT) and excitotoxic retinal ganglion cell (RGC) injury on of the ONT-induced photoreceptor protection is that it specif- light-induced photoreceptor degeneration. ically affects the retinal ganglion cells (RGCs), yet subsequently METHODS. Age- and sex-matched rats underwent unilateral ONT protects the outer retina. This phenomenon implies the exis- D tence of retrograde communication systems within the retina, or received intravitreal injections of N-methyl- -aspartate 5,6 (NMDA). The fellow eye received sham treatment, and 7 or 21 possibly involving Mu¨ller cells and FGF-2, but does not days later each eye was subjected to an intense photic injury. exclude the possibility that the effect is specific to ONT. A Maximum a- and b-wave amplitudes of the flash electroretino- nonspecific effect would suggest that similar responses might gram (ERG) were measured at baseline, after the RGC insult, be occurring in a wide range of optic neuropathies. We hy- and 5 days after the photic injury. Semiquantitative reverse pothesized that the protective effect of ONT may be a gener- transcription-polymerase chain reaction analysis and immuno- alizable effect and that other forms of inner retinal injury such blot analysis were used to assess rod opsin mRNA and rhodop- as excitotoxic injury may also protect against LIPD. Further- sin kinase protein levels and to measure defined trophic factors more, although FGF-2 has been implicated as the agent respon- 7 or 21 days after ONT or injection of NMDA. Structural sible for the ONT effect, other trophic factors may be involved. changes after the insults were determined histologically and For example, intraocular injection of brain-derived neurotro- immunohistochemically. phic factor (BDNF), neurotrophin (NT)-3, ciliary neurotrophic factor (CNTF), and glial-derived neurotrophic factor (GDNF) RESULTS. ONT caused time-dependent reductions in the mean a- rescue photoreceptors in animal models of retinal degenera- and b-wave amplitudes. Seven days after intravitreal NMDA the tion.7–10 In addition, the temporal relationship between ONT b-wave amplitude was reduced, but the a-wave was unaffected. and the degree of protection against LIPD has not been re- ONT and NMDA injection attenuated the light-induced reduc- ported. tions in the a- and b-wave. Rod opsin mRNA levels and rho- Hence, the aims of the present study were (1) to confirm dopsin kinase protein levels were also significantly greater in that ONT protects against LIPD and to examine the time- the axotomized and NMDA-treated eyes compared with the dependent nature of this effect, (2) to correlate the degree of sham-treated fellow eyes after the photic injury. Structural protection with changes in retinal trophic factor levels, (3) to protection in the RGC-injured eyes was also evident histologi- determine the effect of NMDA-induced retinal injury on LIPD, cally. Fibroblast growth factor (FGF)-2, ciliary neurotrophic and (4) to compare changes in retinal trophic factor levels after factor (CNTF), and glial fibrillary acidic protein (GFAP) were NMDA-induced excitotoxicity with changes after ONT. significantly upregulated after ONT and NMDA. CONCLUSIONS. ONT and intravitreal injection of NMDA protect against subsequent photic injury. This protection may relate to METHODS the activation of retinal glial cells and the possible action of trophic factors such as FGF-2 and CNTF. (Invest Ophthalmol Treatment of Animals Vis Sci. 2004;45:685–693) DOI:10.1167/iovs.03-0674 Procedures used in this study conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were n recent years, various insults (mechanical injury, bright approved by the Home Office in the United Kingdom. Adult female light, and ischemia)1–3 have been shown to protect the I Wistar rats (200–250 g) housed in a 12-hour light–dark cycle were retina against subsequent light-induced photoreceptor degen- used for all experiments. Food and water were provided ad libitum. eration (LIPD). A particularly curious form of protective insult Anesthesia was achieved with a combination of intramuscular Hyp- was reported by Bush and Williams,4 who found that optic norm 0.4 mL/kg (fentanyl citrate 0.315 mg/mL and fluanisone 10 nerve transection (ONT) affords histologic protection to the mg/mL; Janssen Pharmaceutica, Beerse, Belgium) and diazepam 0.4 mL/kg. Animals were killed by an overdose of intraperitoneal pento- barbitone. From the 1Nuffield Laboratory of Ophthalmology, Oxford Univer- sity, Oxford, United Kingdom; and the 2Department of Ophthalmol- Optic Nerve Transection ogy, University of Mercia, Spain. Supported by the European Community (E.C. PRO-AGE-RET Pro- A superior conjunctival approach to the optic nerve was chosen gram) QLK6-CT-2001-00385. because it minimized operating time (Ͻ5 minutes per eye) and globe Submitted for publication July 1, 2003; revised September 22, manipulation. Anesthetized rats underwent a lateral canthotomy, the 2003; accepted October 22, 2003. superior lid was retracted with a 5-0 silk traction suture, and a superior Disclosure: R.J. Casson, None; G. Chidlow, None; J.P.M. Wood, fornix-based conjunctival flap was created. The superior muscle com- None; , None; , None M. Vidal-Sanz N.N. Osborne plex was divided (episcleral vortex veins were not transected) and the The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertise- optic nerve exposed by blunt dissection. The posterior ciliary artery ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. was identified on the ventromedial aspect of the nerve sheath and the Corresponding author: Neville N. Osborne, Nuffield Laboratory of optic nerve sheath was incised longitudinally, exposing the optic Ophthalmology, Walton St, Oxford, OX2 6AW UK; nerve. The nerve was then transected 3 mm from the globe, and the [email protected]. fundus was checked to ensure that retinal blood flow had not been Investigative Ophthalmology & Visual Science, February 2004, Vol. 45 No. 2 Copyright © Association for Research in Vision and Ophthalmology 685 Downloaded from iovs.arvojournals.org on 09/28/2021 686 Casson et al. IOVS, February 2004, Vol. 45 No. 2 TABLE 1. Primer Sequences Annealing Product Size Temperature Accession mRNA Primer Sequences (bp) (°C) Number GAPDH 5Ј-CATCAAGAAGGTGGTGAAGCAGG-3Ј 206 52 AF106860 5Ј-CCACCACCCTGTTGCTGTAGCCA-3Ј Rhodopsin 5Ј-CAGTGTTCATGTGGGATTGACT-3Ј 365 52 Z46957 5Ј-ATGATTGGGTTGTAGATGGAGG-3Ј Thy-1 5Ј-CGCTTTATCAAGGTCCTTACTC-3Ј 344 52 X03150 5Ј-GCGTTTTGAGATATTTGAAGGT-3Ј BDNF 5Ј-AGCTGAGCGTGTGTGACAGTAT-3Ј 293 55 M61178 5Ј-GTCTATCCTTATGAACCGCCAG-3Ј GFAP 5Ј-ATTCCGCGCCTCTCCCTGTCTC-3Ј 437 55 U03700 5Ј-GCTTCATCCGCCTCCTGTCTGT-3Ј FGF-2 5Ј-CGTCAAACTACAGCTCCAAGCAGA-3Ј 235 55 M22427 5Ј-GGATCCGAGTTTATACTGCCCAGT-3Ј CNTF 5Ј-TGGCTAGCAAGGAAGATTCGT-3Ј 468 56 X17457 5Ј-ACGAAGGTCATGGATGGACCT-3Ј GDNF 5Ј-GACTCTAAGATGAAGTTATGG-3Ј 483 56 L15305 5Ј-TTTGTCGTACATTGTCTCGG-3Ј NGF 5Ј-CTGGACTAAACTTCAGCATTC-3Ј 395 55 M36589 5Ј-TGTTGTTAATGTTCACCTCGC-3Ј interrupted. Sham surgery was identical and continued for the same ONT or injection of NMDA, 1 day before the photic injury, and 5 days duration, except that the optic nerve was not transected. after the completion of the photic injury. Excitotoxic RGC Injury Assessment of Retinal mRNA Levels ␮ Anesthetized rats received a 5- L intravitreal injection of 4 mM NMDA The retinal levels of glyceraldehyde-3-phoshate dehydrogenase ␮ (Tocris Neuramin, Essex, UK) in one eye and a 5- L injection of sterile (GAPDH), rod opsin, FGF-2, glial fibrillary acidic protein (GFAP), isotonic saline in the fellow eye. CNTF, BDNF, nerve growth factor (NGF), GDNF, and Thy-1 mRNA were determined with a semiquantitative reverse transcription-poly- Photic Injury merase chain reaction technique (RT-PCR), as described previously.11 Rats were placed separately in cages and exposed to evenly distrib- Briefly, total RNA was isolated, and first-strand cDNA synthesis per- ␮ uted, bright light. The floor of each cage was illuminated by approxi- formed on 2 g DNase-treated RNA. The individual cDNA species were ␮ ␮ mately 2000 lux. The temperature inside each cage was maintained at amplified in a 10 L reaction, containing the 2 L cDNA aliquot, PCR 24°C, and the animals had free access to food and water. The rats were buffer (10 mM Tris-HCl [pH 8.3] and 50 mM KCl), 4 mM MgCl2, 200 ␮ ␮ placed under these conditions at the same time of day for each M of each dNTP, 4 ng/ L of both the sense and antisense primers and experiment (1700 hours) and returned to their normal housing after 48 Taq polymerase (2.5 U). Reactions were initiated by incubating at 94°C hours of constant exposure to light. for 10 minutes and PCR (94°C, 15 seconds; 52°C, 55°Cor56°C, 30 seconds; and 72°C, 30 seconds; performed for a suitable number of Electroretinography cycles followed by a final extension at 72°C for 3 minutes). Prior experiments had determined the linear phase of amplification for each Maximum amplitude responses from dark-adapted rats were recorded set of primers, and the PCR product from each set of primers was as follows: Rats were dark adapted for at least 2 hours and prepared sequenced to ensure its validity. Interexperimental variations were under dim red illumination. After anesthesia, the pupils were dilated avoided by performing all amplifications in a single run. The oligonu- with cyclopentolate (1%), the eyelids were separated by placing a cleotides primer pairs and their annealing temperatures are shown in cotton thread loosely around the equator of the globe, and rats were Table 1. The PCR products of all primer pairs yielded single bands placed on a stereotaxic frame facing the stimulus at a distance of 50 corresponding to the expected molecular weights, which are also cm.