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

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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. A reference electrode was placed through the tongue, a grounding shown in Table 1. PCR reaction products were separated on 1.5% electrode was attached to the scruff of the neck, and a platinum agarose gels, with using ethidium bromide used for visualization. The electrode was placed in contact with the central cornea. The cornea relative abundance of each PCR product was determined by digital was intermittently irrigated with a physiologic saline solution (BSS; analysis of gel photographs on computer (Labworks software; Ultra- Alcon, Fort Worth, TX) to maintain the baseline recording and to violet Products, Cambridge, UK). For semiquantitative analysis, the prevent keratopathy from exposure. The dim red illumination was ratio of the densitometric readings between the preconditioned and switched off during recordings, and the rats were kept warm during sham-treated eyes was calculated and compared with an internal stan- and after the procedure. dard mRNA ratio (GAPDH). Ten responses to a flash (10 ␮s, 0.1 Hz) from a photic stimulator (PS33-plus; Grass Telefactor, Inc., West Warwick, RI) set at maximum Histopathology and Immunohistochemistry brightness (setting 16; approximately 17.5 cd-s/m2) were amplified (gain set at 300), filtered (100-Hz low-pass filter, DC high-pass filter, Anesthetized rats were transcardially perfused with 50 mL of 10 mM and 50-Hz filter, notch activated) and averaged (model 1902 Signal phosphate-buffered saline, followed by 4% paraformaldehyde. The Conditioner/1401 Laboratory Interface; CED, Cambridge, UK) The eyes were enucleated and immersion fixed for 1 hour in 4% parafor- b-wave amplitude was measured from the trough of the a-wave to the maldehyde, transferred to 10% neutral-buffered formalin overnight, peak of the b-wave, and the a-wave was measured as the difference in and processed for routine paraffin-embedded sectioning on an auto- amplitude between the recording at onset and the trough of the mated tissue processor (Shandon Pathcentre, Thermo Shandon Inc., negative deflection. Baseline recordings were taken 1 to 4 days before Pittsburgh, PA). Eyes were embedded sagittally and 5-␮m serial sec-

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tions were cut with a rotary microtome (Microm HM 330, McBain Instruments, Chatsworth, CA) and stained with hematoxylin and eosin. For immunohistochemistry, the eyes of saline-perfused rats were enucleated and immersion ﬁxed in 4% paraformaldehyde for 20 minutes, hemisectioned, ﬁxed for a further 20 minutes, and placed in 30% sucrose and 0.1 M sodium phosphate at 4°C overnight. Frozen sections (10 ␮m) were cut on a cryostat (Bright Instruments Ltd., Huntingdon, UK) and suitable hemiglobe sections within approximately 1 mm of the optic nerve head were recovered onto gelatin-coated slides. Air- dried slides were incubated at 4°C overnight with the primary antibody (rat anti-ChAT, 1:10; Roche Diagnostics, East Sussex, UK) and at room temperature for 45 minutes with the secondary antibody conjugated to FITC (anti-rat IgG-FITC conjugate, 1:100; Sigma-Aldrich, St. Louis, MO).

Immunoblot Analysis Proteins were extracted simultaneously with RNA from retina samples by using a standard procedure (Tri Reagent; Sigma-Aldrich). Protein samples were solubilized in buffer (20 mM Tris-HCl [pH 7.4], containing 2 mM EDTA, 0.5 mM EGTA, 1% SDS, 0.1 mM phenylmethylsulfonyl ﬂuoride, 50 ␮g/mL aprotinin, 50 ␮g/mL leupeptin, and 50 ␮g/mL pepstatin A). An equal volume of sample buffer (62.5 mM Tris-HCl [pH 7.4], containing 4% SDS, 10% glycerol, 10% ␤-mercaptoethanol, and 0.005% bromophenol blue) was added, and the samples were boiled for 3 minutes. Electrophoresis of samples was performed using 10% polyacrylamide gels containing 0.1% SDS. Proteins were then blotted onto nitrocellulose. The blots were incubated with anti-actin (1:1000; Chemicon, Chandlers Ford, UK), anti-rhodopsin kinase (1:1000; Cam- bridge Bioscience, Cambridge, UK), anti-FGF-2 (1:200; Santa Cruz Bio- technology, Santa Cruz, CA), or anti-NF-L (1:1000; Chemicon) for 3 hours at room temperature, and appropriate secondary antibodies conjugated to horseradish peroxidase were subsequently used. Nitro- cellulose blots were developed with a 0.016% (wt/vol) solution of 3-amino-9-ethylcarbazole in 50 mM sodium acetate (pH 5.0) containing

0.05% (vol/vol) Tween-20 and 0.03% (vol/vol) H2O2. Experimental Groups The effect of ONT and NMDA-induced excitotoxicity on LIPD was investigated in three experimental groups. The ﬁrst group (ONT-7 day group) was subjected to photic injury 7 days after unilateral ONT, the second group (ONT-21 day group) was subjected to photic injury 21 days after unilateral ONT (fellow eyes received a sham ONT), and the third group received a unilateral intravitreal injection of NMDA (fellow eyes received saline) 7 days before the photic injury (NMDA group).

Statistical Analysis For comparison of independent samples and normalizing for slight day-to-day variation in the ERG, the ratio of the a- and b-wave ampli- FIGURE 1. The effect of ONT on the a- and b-wave amplitudes. (A) The tudes between both eyes of each rat (one eye treated and the fellow a-wave amplitude was significantly reduced 3 and 10 days after ONT sham-treated) were used as the unit of statistical analysis. Similarly, the compared with sham surgery, but had returned to normal by day 21. paired-eye ratio of the RT-PCR and immunoblot densitometric readings (B) The b-wave amplitude was not significantly affected at day 3 but was used as the unit of statistical analysis. A Student’s t-test or one-way was reduced 10 and 21 days after ONT. Sham surgery did not signifi- ANOVA was used to compare means between two or more indepen- cantly affect the amplitudes compared with no treatment. Ratios are dent groups, a Tukey honest significant difference (HSD) test was used shown between (f, n ϭ 8–9) axotomized and untouched fellow eyes, o ϭ Ⅺ ϭ for post hoc comparisons, and a Bonferroni correction applied for ( , n 13) sham-axotomized and untreated eyes; and ( , n 6) untreated right and left eyes. *P Ͻ 0.05, **P Ͻ 0.01. comparisons at multiple time points within an individual experiment. A repeated-measures ANOVA was used to compare serial changes within a group. All statistical determinations were performed on computer (SPSS for Windows, ver.10; SPSS Science, Inc., Chicago, IL), and Ϯ Ͻ in the axotomized eyes showed a small but significant reduc- all data are expressed as the mean SEM, with P 0.05 considered ϭ statistically significant. tion at day 3 and 10 (P 0.018 and 0.01, respectively) but had returned to baseline by 21 days (Fig. 1A). In contrast, the b-wave amplitude in the axotomized eyes was unchanged from RESULTS baseline at day 3, but was significantly reduced at days 10 and 21 (P ϭ 0.009 and 0.006, respectively; Fig. 1B). The Effect of ONT and NMDA on the ERG Six days after the intravitreal injection of NMDA, the mean The changes in the a- and b-wave amplitudes 3, 10, and 21 days b-wave amplitude in the NMDA-injected eyes (as a percentage after ONT are shown in Figure 1. The mean a-wave amplitude of the saline-injected fellow eye) was significantly smaller than

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0.007, n ϭ 8), 5 days after the photic injury, the amplitude was significantly greater than baseline (P Ͻ 0.001, n ϭ 8; Fig. 2B). Hence, ONT provided functional protection against LIPD, and this protection was evident at both 7 and 21 days after the axotomy. In the NMDA group 5 days after the photic injury, both the a- and b-wave amplitudes were significantly greater than baseline (P Ͻ 0.001, n ϭ 8; Fig. 2), indicating that the NMDA- injected eyes also displayed relative functional protection. There was no statistically significant difference between the ONT-21 day and the NMDA groups in the mean a-wave (P ϭ 0.38) or b-wave (P ϭ 0.75) amplitudes after photic injury. However, the mean a-wave amplitudes in both the ONT-21 day and the NMDA groups were significantly greater after the photic injury than the mean a-wave amplitudes in the ONT-7 day group (P ϭ 0.01 and 0.028, respectively). Similarly, the mean b-wave amplitudes in these groups were significantly greater than the mean b-wave amplitude in the ONT-7 day group (P Ͻ 0.001). Representative ERG recordings from the ONT-21 day group and the NMDA group are shown in Figure 3. RT-PCR Data. Figure 4 shows the mean GAPDH, rod opsin, and Thy-1 mRNA levels in all three groups 5 days after the photic injury. In all three experimental groups, the level of GAPDH mRNA (the internal control gene) did not differ significantly (P ϭ 0.87) between eyes, as indicated by the nearly 100% ratios in all three groups (Fig. 4). In the ONT-7 day group, the level of rod opsin mRNA was significantly greater than the level of GAPDH (P ϭ 0.006, n ϭ 8), whereas the Thy-1 mRNA level was significantly smaller than the GAPDH level (P Ͻ 0.001, n ϭ 8), indicating relative protection of the photoreceptor cell bodies (the location of the rod opsin mRNA) in the axotomized eyes, and a significant damage to RGCs. Similarly, in the ONT-21 day group, the rod opsin mRNA level was significantly greater than the GAPDH level (P ϭ 0.002, n ϭ 6), and the Thy-1 mRNA level was significantly smaller (P Ͻ 00.001, n ϭ 6). The NMDA group also displayed a similar pattern. The rod opsin mRNA level was significantly greater FIGURE 2. Relative preservation of the a-wave (A) and b-wave (B) ϭ ϭ against light-induced photoreceptor degeneration 7 and 21 days after than the GAPDH level (P 0.003, n 6), and the Thy-1 mRNA ONT and 7 days after intravitreal injection of NMDA. Recordings after level was significantly smaller (P Ͻ 0.001, n ϭ 6). injury were made 6 or 20 days after ONT and 6 days after NMDA; Immunoblot Analysis. Figure 5 shows the relative pres- recordings after exposure to light were made 5 days after the comple- ervation of rhodopsin kinase (an outer segment protein) 5 days tion of the photic injury. Amplitudes are expressed as the mean ratio after photic insult in both the axotomized eyes (P ϭ 0.035, n ϭ between the eyes of each rat. One eye receiving either an ONT or an intravitreal NMDA injection, and the fellow eye receiving sham treatment. *P Ͻ 0.05, **P Ͻ 0.01, ***P Ͻ 0.001.

the ratio at baseline (P Ͻ 0.001, n ϭ 9; Fig. 2B) but the mean a-wave amplitude was unchanged (Fig. 2A).

Protective Effect of ONT and NMDA-Induced Excitotoxicity Electroretinographic Data. Figure 2 shows the protective effect that ONT and NMDA-induced excitotoxicity had on the a- and b-wave amplitudes of all three groups. Although, 6 days after ONT the mean a-wave amplitude of the axotomized eyes in the ONT-7 day group was significantly smaller than the ratio at baseline (P Ͻ 0.035, n ϭ 12), 5 days after the photic injury, the amplitude was significantly greater than baseline (P Ͻ 0.001; Fig. 2A). Similarly, the mean b-wave amplitude of the axotomized eyes in the ONT-7 day group was significantly greater after the photic injury than the amplitude at baseline (P Ͻ 0.001, n ϭ 12; Fig. 2B). In the ONT-21 day group, the mean a-wave amplitude of the axotomized eyes was also sig- FIGURE 3. Representative ERG recordings. (A) Uninjured control. (B, nificantly greater after the photic injury than the amplitude at C, E, F) Recordings made 5 days after the termination of the photic baseline (P Ͻ 0.001, n ϭ 8; Fig. 2A); and, although, 20 days insult: (B) 21-day ONT eye and (C) its fellow sham-treated eye; (E) after ONT, the mean b-wave amplitude of the axotomized eyes NMDA-injected eye after photic insult and (F) its fellow vehicle-in- was significantly smaller than the amplitude at baseline (P ϭ jected eye. (D) Six days after NMDA injection but before photic insult.

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FIGURE 4. RT-PCR measurements after RGC insults and photic injury. There was relative preservation of rhodopsin mRNA and relative loss of Thy-1 mRNA compared with the housekeeping gene product (GAPDH) after light-induced photoreceptor injury in axotomized eyes and eyes that had previously received an injection of NMDA. Rats underwent a unilateral ONT 7 or 21 days before the photic injury or 7 days before an intravitreal injection of NMDA and were killed 5 days after the photic injury. Mean mRNA levels are expressed as the ratio between the treated eye and the sham-treated eye. **P Ͻ 0.01, ***P Ͻ 0.001.

4) and the NMDA-injected eyes (P ϭ 0.011, n ϭ 4–6). In both the ONT-21 day group and the NMDA group the level of actin (the internal control protein) did not differ significantly (P ϭ 0.87) between eyes, as indicated by the nearly 100% ratios in all three groups (Fig. 5). There was a dramatic loss of neurofilament light (NFL, an axonal marker) by 21 days after ONT (P ϭ 0.004) and a smaller, but significant loss of NFL 7 days after NMDA injection (Fig. 5; P ϭ 0.023). Histopathology and Immunocytochemistry. The axotomized eyes showed a marked loss of RGCs at both 7 and 21 days that was associated with slight thinning of the inner plexiform layer (IPL), but, after the photic injury, the axotomized eyes exhibited better preservation of the outer nuclear layer (ONL; Figs. 6A–D) than did the sham-axotomized eyes. The NMDA-injected eyes also exhibited loss of RGCs and thinning of the IPL, but showed better preservation of the ONL after the photic injury than the saline-injected eyes (Figs. 6E, 6F). A comparison of the cell body counts in the ONL is shown in Table 2.

The Effect of ONT and NMDA on ChAT Immunostaining The characteristic ChAT staining 21 days after ONT was similar to that in the sham-axotomized eyes, indicating that cholinergic amacrine cells, in the inner nuclear layer (INL) and the ganglion cell layer (GCL; displaced amacrine cells) were relatively unaffected by ONT (Figs. 7A, 7B). Similarly, ChAT staining was unaffected in the saline-injected eyes (Fig. 7C), but was abolished in the NMDA-injected eyes (Fig. 7D), indicating some degree of injury to subganglionic retinal layers.

The Effect of ONT and NMDA on Trophic Factor Levels RT-PCR Data. Table 3 shows the increase in mRNA levels in the axotomized and NMDA-injured eyes compared with the sham-treated eyes. Of the factors measured, GFAP and FGF-2 FIGURE 5. (A) Representative immunoblots after RGC insults and pho- displayed the greatest increase after both axotomy and NMDA tic injury and (B) graphic display of the densitometric analysis. There injection. FGF-2 mRNA levels were significantly greater 21 days was relative preservation of RK and relative loss of NFL compared with after axotomy than at 7 days after (P ϭ 0.006). BDNF displayed the housekeeping protein (actin) after light-induced photoreceptor a small but significant reduction 21 days after axotomy com- injury in axotomized eyes and eyes that had received an injection of pared with 7 days after (P ϭ 0.032). CNTF levels were also NMDA. Rats underwent a unilateral ONT 21 days before the photic Ͻ injury or 7 days before an intravitreal injection of NMDA and were elevated after ONT and NMDA-induced injury (P 0.001). killed 5 days after the photic injury. Mean protein levels are expressed NGF levels were only slightly elevated by ONT and unaffected as the percentage ratio between the treated eye and the sham-treated by NMDA injection. Retinal GDNF levels were not significantly eye. *P Ͻ 0.05, **P Ͻ 0.01. Ax, axotomized eye; Sh, sham-axotomized altered by the ganglion cell injuries. eye; RK, rhodopsin kinase; NFL, neurofilament light.

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was also increased 7 days after NMDA injection, compared with the expression of actin (P ϭ 0.018, n ϭ 4).

DISCUSSION Reports of the effect of ONT on the flash ERG are conflicting. ONT has been reported to have no effect on the flash ERG in rats and cats,12,13 and the few recordings of the flash ERG in humans after traumatic or surgical ONT proximal to the entry of the central retinal artery were reported as normal or very mildly affected.14,15 In contrast, Gargini et al.16 have reported that the flash ERG is significantly altered 3 to 4 weeks after ONT in rats. They showed b-wave changes over a wide range of stimulus intensities and more subtle a-wave changes at high intensities. They attributed the changes to FGF-2–induced suppression of retinal function, an explanation supported by the finding that an intravitreal injection of FGF-2 also suppressed the b-wave. Vaegan et al.17 have recently reported that the flash ERG in cats is mildly affected by ONT, and evidence has accrued indicating that at least one optic neuropathy (glaucoma) affects the flash ERG.18–20 A unified interpretation of these observations is complicated by the varied and dynamic effects that ONT has on the retina: ONT not only disconnects the retina from the remainder of the brain, triggering retrograde death of RGCs and causing possible transsynaptic degeneration, but also acts as a form of conditioning, albeit extreme,

FIGURE 6. Retinal sections demonstrating relative preservation of photoreceptors 5 days after photic injury in eyes that had undergone RGC injury. (A) A sham-axotomized eye that underwent photic insult 7 days later and (B) the axotomized fellow eye showing better preservation of the ONL. (C) A sham-axotomized eye that underwent photic insult 21 days later and (D) axotomized fellow eye showing better preservation of the ONL. (E) Saline-injected eye that underwent photic insult 7 days later, and (F) NMDA-injected fellow eye showing better preservation of the ONL.

Immunoblot Data. Figure 8 shows the relative increase in FGF-2 protein after ONT and NMDA-induced injury compared with the internal control protein actin. Both 7 and 21 days after ONT, the expression of FGF-2 was signiﬁcantly increased compared with the expression of actin (P ϭ 0.032 and 0.015, respectively, n ϭ 4). Similarly, the expression of retinal FGF-2

TABLE 2. Effect of Retinal Ganglion Cell Injury Followed by the Photic Insult on the Photoreceptor Cell Bodies

Treatment Control Group Group Group

Untouched control 10–12 NA 7-day ONT 6–74–5 FIGURE 7. The effect of ONT and NMDA injection on ChAT immuno- 21-day ONT 6–73–4 staining. (A) Twenty-one days after sham axotomy and (B) after axo- NMDA 8–93–4 tomy, the characteristic staining pattern is still clearly evident. (C) Saline injection did not affect the staining pattern, but was abolished 7 Data are the number of rows of cells in the ONL. days after NMDA injection (D). Arrows: amacrine cell bodies.

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TABLE 3. The Effect of ONT and NMDA on Total Retinal mRNA Levels of Various Trophic Factors and GFAP

Treatment FGF-2 CNTF BDNF NGF GDNF GFAP

NMDA 141.8 Ϯ 16.8* 79.9 Ϯ 6.9* 2.6 Ϯ 5.8 5.4 Ϯ 20.1 Ϫ1.3 Ϯ 13.4 281.9 Ϯ 60.2* ONT 7 days 67.3 Ϯ 90.0* 48.9 Ϯ 8.7* Ϫ2.39 Ϯ 4.1 24.1 Ϯ 7.9† Ϫ10.7 Ϯ 9.6 92.6 Ϯ 13.2* ONT 21 days 122.8 Ϯ 21.3* 40.2 Ϯ 5.9* Ϫ8.1 Ϯ 3.5† 23.6 Ϯ 7.1‡ 9.9 Ϯ 9.3 76.6 Ϯ 9.8*

Data are expressed as the mean percentage increase in mKNA in the treated eyes (relative to the control eyes). Ϯ SEM. Statistical signiﬁcance treated eye versus control eye by Student’s paired t-test (*P Ͻ 0.001, †P Ͻ 0.05, ‡P Ͻ 0.01), where n ϭ 8–17 (NMDA), n ϭ 12 (ONT 7 days) and n ϭ 17–23 (ONT 21 days).

that protects photoreceptors from light-induced injury. Fur- mediated by Mu¨ller cell activation, indicated by elevated GFAP thermore, all these effects appear to be species dependent. mRNA levels after ONT—findings that are consistent but less ERG changes after ONT must be interpreted cautiously, marked than those reported by Gargini et al.16 because they may reflect inadvertent ischemic changes. The In 1991, Bush and Williams4 found that ONT protects pho- vascular supply to the rat retina is derived from a single vessel toreceptors against light-induced injury. To our knowledge, the (the posterior ciliary artery), a branch of the ophthalmic artery present report is the first confirmation of this intriguing effect. that travels along the ventromedial aspect of the optic nerve Our histologic results were very similar to those previously and divides into retinal and uveal branches as it enters the reported4 and the ERG, RT-PCR, and immunoblot data all posterior globe.21 Ischemic injury can occur if the globe is provided evidence in support of the protective effect of ONT retracted for extended periods, because traction interferes against LIPD. The relatively greater loss of rhodopsin kinase with the retinal blood supply, as evidenced by whitening of the protein compared with rod opsin mRNA may be explained by fundus during this maneuver. In addition, the posterior ciliary the greater light-induced loss of rod outer segments compared artery may be transected during the division of the optic nerve; with the loss of cell bodies in the ONL. It is interesting to note however, if this occurs, complete ocular ischemia occurs, and that this protection of the axotomized eye occurs despite a the ERG is flat. Intracranial transection of the optic nerve may behavioral tendency for the rats to shield the sighted eye exclude the possibility of disruption of the retinal blood sup- during intense light exposure, a phenomenon also reported by 4 ply, but may not affect the retina in the same way as an Bush and Williams. 5 intraorbital axotomy.22 Kostyk et al. showed that FGF-2 immunoreactivity was Given that there is no evidence to suggest that RGCs con- increased in the ONL of the retina 3 to 4 weeks after ONT and tribute to the flash ERG, the serial changes in the ERG observed related the known neurotrophic properties of FGF-2 to the after ONT and NMDA injection could be explained either by a photoreceptor protection. Several insults have been shown to direct or indirect insult to the anatomic substrates that are be protective against light-induced injury and to upregulate 1–3,25 responsible for the ERG in the middle and outer retina. ONT FGF-2. This fact, together with the neuroprotective prop- 1 per se directly affects only the GCL, but inadvertent disruption erties of exogenous FGF-2, has led to the concept that FGF-2 of retinal blood supply during ONT may affect deeper layers. may be an important endogenous survival factor in the rat 26,27 However, immunostaining with ChAT, a marker for cholin- retina, particularly for photoreceptors. We showed a sig- ergic amacrine cells that forms distinct laminae in the IPL,23 nificant upregulation of FGF-2 mRNA and protein after ONT, an was normal after ONT, as previously described in neonatal increase that was greater 21 days after ONT than at 7 days, rats.24 ChAT-containing retinal neurons are very sensitive to corresponding with the greater degree of photoreceptor pres- ischemic injury23; hence, the normal staining after ONT is ervation that we observed at this later time point. There was, further evidence that the ONT did not significantly affect the however, a discrepancy between the level of FGF-2 protein and mRNA expression after ONT and NMDA injection. Other in- blood supply. Therefore, our findings suggest that ONT causes 16 subtle indirect effects to the middle and outer retina, possibly vestigators, using in situ hybridization to measure FGF-2 mRNA, have reported a large discrepancy between the expression of FGF-2 protein and mRNA after ONT, but it is possible that the discrepancy in the present study reflects the use of semiquantitative rather than quantitative techniques. How- ever, in general, the findings support the concept that endogenous FGF-2 is an important photoreceptor protectant. LaVail et al.26 have demonstrated that a variety of sub- stances delivered intravitreally protect mouse photoreceptors against injury, and many of these survival factors are synthe- sized endogenously. Increased CNTF expression by Mu¨ller cells has been reported to occur after ONT.28 However, given that photoreceptors lack receptors for CNTF and BDNF,29,30 but that Mu¨ller cells have receptors for most of the molecules involved in photoreceptor rescue and become activated after retinal injury, it has been suggested that Mu¨ller cells play an integral role in endogenous neuroprotection.6,26,31–34 Harada et al.33,34 have provided evidence for a macroglial–neuronal and microglial–macroglial communication system involved in endogenous neuroprotection after LIPD. Of note, they hav NTR FIGURE 8. Relative upregulation of FGF-2 compared with the house- shown that p75 ligand (probably NGF) reduces FGF-2 pro- 34 keeping protein (actin) 7 and 21 days after ONT and 7 days after duction in Mu¨ller cells, which is consistent with the lack of intravitreal injection of NMDA. *P Ͻ 0.05 upregulation of NGF, as found in the present study.

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Stone et al.27 have hypothesized that the reason FGF-2 leading to long-term photoreceptor survival in hereditary retinal expression is not permanently upregulated is that the struc- degeneration. Invest Ophthalmol Vis Sci. 1999;40:1298–1305. tural protection comes at a functional cost. They suggest that 8. Frasson M, Picaud S, Leveillard T, et al. Glial cell line-derived FGF-2 induces a form of metabolic suppression in the photo- neurotrophic factor induces histologic and functional protection receptors and predicted that the a-wave of the ERG would be of rod photoreceptors in the rd/rd mouse. Invest Ophthalmol Vis suppressed by FGF-2. We observed precisely this effect 3 and Sci. 1999;40:2724–2734. 10 days after ONT, but the response had normalized by day 21. 9. Cayouette M, Behn D, Sendtner M, Lachapelle P, Gravel C. Intraoc- ular gene transfer of ciliary neurotrophic factor prevents death and Therefore, our results support the findings of Stone et al. and increases responsiveness of rod photoreceptors in the retinal de- lend weight to their prediction; however, the recovery of the generation slow mouse. J Neurosci. 1998;18:9282–9293. a-wave, despite the increasing expression of FGF-2, implies the 10. LaVail MM, Unoki K, Yasumura D, Matthes MT, Yancopoulos GD, presence of other mechanisms. Steinberg RH. Multiple growth factors, cytokines, and neurotro- Although NMDA-induced excitotoxicity has been used as a phins rescue photoreceptors from the damaging effects of con- method of inducing RGC death,11 it also injures other cells, stant light. Proc Natl Acad Sci USA. 1992;89:11249–11253. including a subset of cholinergic amacrine cells in the INL 11. Nash MS, Osborne NN. Assessment of Thy-1 mRNA levels as an known to express the NMDA receptor.35,36 The effect on these index of retinal ganglion cell damage. Invest Ophthalmol Vis Sci. cells was evidenced by the loss of the characteristic ChAT 1999;40:1293–1298. immunostaining in the NMDA-injected eyes. To our knowl- 12. Berardi N, Domenici L, Gravina A, Maffei L. Pattern ERG in rats edge, the effect of NMDA on the ERG has not been reported, following section of the optic nerve. Exp Brain Res. 1990;79:539– 546. but the ischemia-like changes (reduction in the b-wave ampli- 13. Mafei L, Fiorentini A. Electroretinographic responses to alternating tude with preservation of the a-wave) can probably be ex- gratings before and after section of the optic nerve. Science. plained by NMDA-induced injury to cells within the INL. 1981;211:953–955. NMDA-induced excitotoxic injury to the inner retina pro- 14. Dawson WW, Maida TM, Rubin ML. Human pattern-evoked retinal tected against subsequent LIPD in a manner similar to ONT. responses are altered by optic atrophy. Invest Ophthalmol Vis Sci. This indicates that the protective effect is generalizable, at least 1982;22:796–803. to some extent, and that inner retinal injury, particularly RGC 15. Harrison JM, O’Connor PS, Young RS, Kincaid M, Bentley R. The death initiates a response from the retina that results in a pattern ERG in man following surgical resection of the optic nerve. delayed protection of the photoreceptors against photic injury. Invest Ophthalmol Vis Sci. 1987;28:492–499. Furthermore, intravitreal NMDA caused an increase in GFAP 16. Gargini C, Belfiore MS, Bisti S, Cervetto L, Valter K, Stone J. The mRNA and an increase in retinal FGF-2 mRNA and protein impact of basic fibroblast growth factor on photoreceptor func- levels, suggesting that macroglial cell activation and the release tion and morphology. Invest Ophthalmol Vis Sci. 1999;40:2088– of FGF-2 may be an essential component of this protective 2099. response. 17. Vaegan, Anderton PJ, Millar TJ. Multifocal, pattern and full field electroretinograms in cats with unilateral optic nerve section. Doc In conclusion, ONT in adult Wistar rats caused small but Ophthalmol. 2000;100:207–229. significant serial changes in both the a- and b-wave of the flash 18. Vaegan, Graham SL, Goldberg I, Buckland L, Hollows FC. Flash and ERG. Intravitreally injected NMDA caused more marked reduc- pattern electroretinogram changes with optic atrophy and glau- tion of the b-wave without affecting the a-wave and clearly coma. Exp Eye Res. 1995;60:697–706. affected cholinergic amacrine cells in the INL. Furthermore, 19. Velten IM, Korth M, Horn FK. The a-wave of the dark adapted ONT and NMDA-induced excitotoxicity produced upregula- electroretinogram in glaucomas: are photoreceptors affected? 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