Investigative Ophthalmology & Visual Science, Vol. 31, No. 9, September 1990 Copyright © Association for Research in Vision and Ophthalmology Effects of Fluorouracil and Fluorouridine on Protein Synthesis in Rabbit Retina John A. Leon, James M. Dritt, Richard H. Hopp, Richard P. Mills, and Ann H. Milam 5-Fluorouracil (5-FU) and its active metabolite 5-fluorouridine (FUR) are currently being evaluated for the treatment of proliferative vitreoretinopathy and the control of scarring after glaucoma filtering procedures. To test for retinal toxicity, the authors examined the effect of intravitreal injections of 5-FU and FUR on protein synthesis in rabbit retinal photoreceptors and ganglion cells. In addition, the toxic effect of subconjunctival 5-FU injections, after a trephine filtering procedure, on ganglion cell protein synthesis was examined. Albino rabbit eyes were given either unilateral intravitreal injections of 1 mg of 5-FU, 2.5 mg of 5-FU, or 0.1 mg of FUR, or subconjunctival injections of 3 mg of 5-FU twice daily after a trephine procedure. Quantitative autoradiography was used to study ganglion cells and photoreceptor outer segment renewal, and scintillation counting was used to quantify newly synthesized protein transported axonally from ganglion cell bodies to the superior colliculus (SC). Marked reduction of labeled protein reaching the SC was noted after either intravitreal 0.1 mg of FUR (41% inhibition after a single injection and 53% after two injections) or 2.5 mg of 5-FU (41% after one injection and 26% after two injections). This reduction was still present after 8 days in eyes receiving 0.1 mg of FUR (32%) and 2.5 mg of 5-FU (22%). Quantitative autoradiography of retinal photorecep- tors and ganglion cells corroborated these data, demonstrating inhibition of outer segment renewal after one or two injections of either 0.1 mg of FUR or 2.5 mg of 5-FU. This inhibitory effect was statistically significant using the paired t-test for both drugs. No mean inhibition was observed after intravitreal 1 mg of 5-FU injections or after subconjunctival injections of 5-FU. Invest Ophthalmol Vis Sci 31:1709-1716, 1990 The drug 5-fluorouracil (5-FU) is potent antime- coma filtration procedures in animal models1' and in tabolite that inhibits intraocular fibroblast and retinal initial clinical studies.12"15 It is currently being evalu- pigment epithelium proliferation and contraction in ated in a multicenter clinical trial. animal models1"5 and in vitro.6"9 Currently 5-FU is The 5-FU alters DNA synthesis through inhibition under evaluation for treatment of proliferative vit- of the enzyme thymidylate synthetase and decreases reoretinopathy (PVR), and it was found to signifi- RNA function secondary to incorporation of 5-FU cantly reduce the incidence of traction retinal de- metabolites into newly synthesized RNA.16 The anti- tachment (TRD) produced by injection of cultured proliferative and anticontractile effects of 5-FU are fibroblasts into the vitreous chamber of animal eyes. probably due to production of altered RNA.17 Fluo- A dose of 1 mg of 5-FU injected intravitreally re- rouridine (FUR) is the key 5-FU metabolite that af- duced TRD in nonvitrectomized rabbit eyes.1'2'5 fects RNA synthesis and is up to 100 times more Multiple intravitreal injections of 0.5 mg of 5-FU effective than 5-FU in inhibiting fibroblast prolifera- into vitrectomized rabbit eyes have also been effica- tion in vitro.18 cious against PVR.3 A pilot study on patients with Toxicity of 5-FU and FUR has been assessed by PVR demonstrated a moderate success rate of 65% use of light and electron microscopy and by electro- for retinal reattachment after intravitreal 5-FU.10 physiology. We evaluated retinal toxicity of intravit- When applied topically or injected subconjunctivally, real and subconjunctival injection of 5-FU and FUR 5-FU also inhibits postoperative scarring after glau- in rabbits. Since ganglion cells and photoreceptors have high rates of protein synthesis, we measured the From the Department of Ophthalmology, University of Wash- effects of these drugs on protein synthesis and axonal ington School of Medicine, Seattle, Washington. transport in retinal ganglion cells and on outer seg- Supported in part by NIH Grants EY01730 and EY01311; the Lewis Berkowitz Family, National Retinitis Pigmentosa and ment (OS) renewal in photoreceptors. Chatlos Foundations; and in part by an award from Research to Prevent Blindness, Inc. (R.P.B.). A.H.M. is a Senior Scholar of Materials and Methods R.P.B. Reprint requests to Richard P. Mills, MD, Department of Oph- Fifty-three albino rabbits (2-3 kg) were used for thalmology RJ-10, University of Washington, Seattle, WA 98195. this study. This study was conducted in accordance 1709 Downloaded from iovs.arvojournals.org on 09/28/2021 1710 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / September 1990 Vol. 31 with the ARVO Resolution on the Use of Animals in poral to the optic disc. Inhibition of protein synthesis Research. The eyes of 24 rabbits were anesthetized in ganglion cells was determined by comparing topically with proparacaine 0.5%, and the right eyes counts from a drug-treated eye with the companion (OD) received two intravitreal injections 24 hrs apart control eye injected with saline.21 of 1.0 mg of 5-FU, 2.5 mg of 5-FU, or 0.1 mg of FUR, diluted in 0.1 ml of 0.9% saline, doses previously Outer Segment Renewal in Photoreceptors thought to be nontoxic to rabbit or primate ret- 101819 inas. The needle tip was positioned just anterior Quantitative autoradiography was also used to as- 20 to the optic disc in the manner of Orcutt et al. A sess effects of 5-FU and FUR on protein synthesis in second group of seven rabbits underwent a bilateral retinal photoreceptors. Rod outer segment (ROS) re- Elliot trephine filtering procedure after sedation with newal occurs continuously, reflecting a high level of intramuscular ketamine and Rompun. A 1.0-mm protein synthesis in the rod inner segment and inser- trephine was used, straddling the limbus, followed by tion of newly synthesized protein as a band in the conjunctival coverage with a fornix-based flap. These ROS.22 Fifteen rabbits from the first group and 22 rabbits received subconjunctival injections OD of 3 rabbits from the third group were anesthetized with mg of 5-FU in 0.3 ml of 0.9% saline 180° from the sodium thiopental and perfused intravascularly with surgery wound. These injections were repeated twice 4% paraformaldehyde and 0.5% glutaraldehyde in daily for 7 days, according to the method of Gressel et 0.13 M phosphate buffer 72 hrs after intravitreal in- al," using intramuscular ketamine and Rompun for jection of 3H-leucine. The eyes were immediately anesthesia. Left eyes served as controls in all rabbits enucleated and immersed in the fixative for 24 hr. and received equivalent volumes of saline injected The anterior third of each eye was removed, and the intravitreally or subconjunctivally. Twenty-four retina was sectioned and processed as described hours after the last drug or saline injection, all eyes above for autoradiography. Autoradiographic grains 3 were injected intravitreally with 150 ^Ci H-leucine were counted per unit area of radioactive ROS band (New England Nuclear). A third group of 22 rabbits in a standardized segment of retina, just temporal to received only one injection of 2.5 mg of 5-FU or 0.1 the optic disc, to determine if photoreceptor protein mg of FUR into the midvitreous cavity OD under synthesis, as evidenced by OS renewal, had been af- ophthalmoscopic control. Again, the left eye served fected by prior drug treatment. as a control and received an equivalent volume of saline injected intravitreally. After drug exposure times of 1 day for 10 rabbits and 8 days for 12 rabbits, Protein Synthesis In all eyes were injected intravitreally with 3H-leucine. All rabbits showing evidence of leakage from the in- Ganglion Cells 30 jection site were excluded from the study. 21.8 i Protein Synthesis in Ganglion Cells 20-: 17.5 1 Quantitative light microscopic autoradiography 10- was used to determine the effects of 5-FU and FUR 21 1 on protein synthesis in ganglion cells of the retina. ^ 3 Four hours after intravitreal injection of H-leucine, C nine rabbits from the first group were killed with an -10- overdose of sodium thiopental; the eyes were imme- C <D 1 -14.8 diately removed and fixed in 4% paraformaldehyde 2 -20 i in 0.13 M phosphate buffer for 72 hr. Samples of a> retina with underlying choroid and sclera were em- -30- bedded in methacrylate (Sorvall). Sections 2.5 nm in thickness were processed for autoradiography, in- -40 cluding standardized dipping in photographic emul- 1.0mg. 5-FU 2.5mg. 5-FU 0.1 mg. FUR sion, exposure of all slides for 24 hr, and simulta- Drug Tested neous photographic processing. The slides were then dried and stained with Richardson's methylene blue- Fig. 1. Inhibitory effects of 5-FU and FUR on protein synthesis azure II mixture. Autoradiographic grains were in ganglion cells of the rabbit retina. Mild inhibition was found after two intravitreal injections of 2.5 mg 5-FU and 0.1 mg FUR. counted per unit cytoplasm of ganglion cells from a No reduction was found after intravitreal injection of 1.0 mg 5-FU. standardized segment of the retina immediately tem- The vertical bars represent means ± SEM. Downloaded from iovs.arvojournals.org on 09/28/2021 No. 9 5-FU AND FUR IN RABBIT RETINA / Leon er ol 1711 Fig. 2. Photomicrographs of rabbit retinas demon- strating incorporation of 3H-leucine into ganglion cell protein (originals X800).