Uptake of Tritioted Thymidine in Mitochondria of the Retina

Transcription

1 Investigative Ophthalmology & Visual Science, Vol. 30, No. 12, December 1989 Copyright Association for Research in Vision and Ophthalmology Reports Uptake of Tritioted Thymidine in Mitochondria of the Retina Barbara J. Woodford and Janer C. Blanks Light microscopic autoradiography with 3 H-thymidine was used as a probe for DNA synthesis on the retinas of the guinea pig, rabbit, and monkey. Relatively heavy labeling was found in the ganglion cell and inner nuclear layers as well as in the photoreceptor inner segments in the guinea pig and monkey. Electron microscopic autoradiography demonstrated that in the ganglion cells and photoreceptor inner segments in the monkey, 89% of silver grains, representing thymidine uptake, were on or near the mitochondria. Invest Ophthalmol Vis Sci 30: , 1989 Since most cells of the neural retina of mature vertebrates are postmitotic, it has been thought that retinal cells synthesize little DNA under normal circumstances. ' However, Rapp et al 2 observed light microscopic autoradiographic uptake of tritiated thymidine in retinas of normal rats, particularly in the photoreceptor inner segments. Using biochemical techniques, Kapoor et al 3 confirmed incorporation of radioactive thymidine into total retinal DNA of normal rats, with maximal incorporation 6 hr after injection. Using light microscopic autoradiography in primates, Blocker et al 4 demonstrated intense uptake of thymidine over ganglion cells, the inner nuclear layer, and ellipsoids of inner segments. Since mitochondria contain their own DNA, it was suggested that such thymidine uptake is due to active DNA synthesis in mitochondria of retinal cells, and therefore that DNA synthesis in mature retina is not negligible, as was previously thought. To test whether mitochondria are sites of DNA synthesis in normal adult retina, we used ultrastructural autoradiography to localize thymidine uptake in the monkey. To test the observation of thymidine uptake in the retina in other mammalian species, we performed light-microscopic tritiated-thymidine autoradiography on the rabbit and guinea pig retina, as well as on the retina of the monkey. Materials and Methods. Animals were anesthetized with ketamine and xylazine before injection and before enucleation, and were treated in accordance with the ARVO Resolution on the Use of Animals in Research. Twenty-four hr prior to enucleation and sacrifice, tritiated thymidine (40-60 Ci/mM methyl- 3 H-thymidine, ICN Radiochemicals, Irvine, CA) in sterile saline was injected into the vitreous of each eye. Two hundred yd was injected into each of four monkey eyes, 140 fid into one rabbit eye, and 70 ^Ci into one guinea pig eye. After enucleation under anesthesia, the animals were sacrificed with sodium barbital. Eyes were fixed in 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer, ph 7.4. Nasal regions of the posterior poles of the monkey eyes and superior and inferior regions of the posterior poles of the rabbit and the guinea pig eyes were sampled. The samples were processed and embedded in glycol methacrylate (Polysciences, Inc., Warrington, PA) for light microscopic autoradiography. Two-/*m sections were coated with NTB2 emulsion (Eastman Kodak, Rochester, NY), exposed for 4-6 weeks, developed with D-19 (Eastman Kodak), and post-stained with toluidine blue. The nasal region of the posterior pole from one monkey eye was processed and embedded in epoxy resin (Polybed, Polysciences, Inc.) and examined for electron microscopic autoradiography. Parlodioncoated grids supporting 800 A sections were dipped into Ilford L4 emulsion (Polysciences, Inc.), exposed for 2-3 months, developed in D-19, post-stained with uranyl acetate and lead citrate, and viewed with a Zeiss (Miinchen, FRG) EM-10 electron microscope. To determine what percentage of silver grains had located over mitochondria in preference to other organelles, a random selection of ten electron micrographs of photoreceptor inner segments and ganglion cells, at a final magnification of XI 2,000-20,000, was examined. The number of grains not on or not touching mitochondria was compared to the total number of grains in each micrograph, and this ratio was expressed as the percentage of grains that were nonmitochondrial. Results. Light microscopic autoradiography of the guinea pig and monkey retinas demonstrated silver grains in similar areas (Fig. 1). Regions of comparatively heavy labeling were seen over the ganglion 2528

2 * Fig. 1. Light microscopic autoradiograms of retinas injected intravitreously with 3H-thymidine. (A) Monkey. (B) Rabbit. (C) Tangential section of guinea pig photoreceptor cells. Note the bands of grains over the inner segments (is) and in ganglion cells (arrows), onl, outer nuclear layer; inl, inner nuclear layer; os, outer segment (A, B, X800; Cf X 1,000). OS C

3 2500 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / December 1989 cells, the inner nuclear layer, and the inner segments. In the monkey and guinea pig, the label appeared in the distal portions (toward the outer segments) of the inner segments (Fig. 1A, C). The retina of the rabbit was more lightly labeled than were the other two species (Fig. IB). Labeling over the inner segments was slightly heavier than over the background, but labeling over the ganglion cells was similar to background. It is not clear whether this lack of incorporation in the Vol. 30 rabbit was due to incomplete vitreal injection or to a genuine difference in the uptake of tracer in the rabbit, particularly in the ganglion cells. Heavily labeled nuclei were occasionally observed in the choroid (data not shown) of rabbit and guinea pig. In the monkey retina studied by electron microscopic autoradiography, silver grains corresponding to the labeled thymidine were located over or near mitochondria throughout all layers of the retina, but Fig. 2. Electron microscopic autoradiogram of the monkey retina. (A) Ganglion cell with silver grains in perikaryal cytoplasm (arrows) (XI 3,000). (B) Higher magnification showing silver grains representing 3H-thymidine in mitochondria (m) of ganglion cell perikarya (X90,000). m m B

4 No. 12 Reporrs were most consistently observed over mitochondria of ganglion cells (Fig. 2) and over mitochondria of photoreceptor inner segments (Fig. 3). A semi-quantitative study of electron micrographs of the ganglion cells or photoreceptor inner segments indicated that 89% of the grains photographed were located over mitochondria or were on the cytoplasmic side and touched mitochondnal membranes. Of a total of 84 grains in ten different fields, 9 grains were not on or not touching mitochondria. In five ganglion cell fields, 19% of the grains were nonmitochondrial, and, in five fields of photoreceptor inner segments, 4% were nonmitochondrial. Discussion. In our study mitochondria! DNA synthesis was consistently observed in photoreceptor inner segments of the three species studied and in ganglion cells of the monkey and guinea pig. It is not clear, however, whether these observations reflect especially high rates of synthesis in the retina in general. Gross et al5 reported that, in the rat, mitochondnal DNA has a half-life of 6.7 days in the heart, 9.4 days in the liver, and 31 days in the brain. To our knowledge, there are no reports of the half-life of mitochondrial DNA in the retina. Our findings suggest quantitative analyses, which would compare DNA 2531 synthesis in mitochondria of retinal cells to that of other cells known to have numerous mitochondria and high metabolic activity, such as cardiac muscle and brain. It is also not clear whether the observed mitochondrial DNA synthesis results from synthesis rates that are higher in ganglion cells and photoreceptors than in other retinal cell types, or whether it reflects the high numbers of mitochondria in these cells. Quantitative, ultrastructural autoradiographic analyses of the relationship between mitochondrial labeling and mitochondnal number are currently being performed to compare DNA synthesis rates in diverse cell types of the retina. The retina has the highest respiration rate of any organ, and photoreceptors of rabbit in vitro contribute to 50% of that value.6 Because of this high metabolic rate, the normal functioning of photoreceptors may be particularly dependent on the maintenance and regulation of the mitochondrial life cycle. One might speculate that abnormal mitochondrial synthesis may lead to abnormal photoreceptor function. Several studies have reported that abnormal-appearing mitochondria were among the earliest morphologic indications in some photoreceptor degener- CIS Fig. 3. Electron microscopic autoradiogram of the monkey retina. Note silver grains on both rod (ris) and cone (cis) inner segments (X20,000). Inset: Note that silver grains are located over the mitochondria (X65.OOO). RIS

5 2532 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / December 1989 Vol. 30 ations. 78 In taurine depletion in cats and rats, mitochondria of photoreceptor inner segments were swollen and disrupted before other ultrastructural signs of photoreceptor damage appeared, prior to cell death (G. Hageman, Bethesda Eye Institute, St. Louis, MO; personal communication). Ultrastructural changes in mitochondria of inner segments, such as shortening and a change in matrix density, were reported by Moriya et al 7 in rats as one of the earliest observations after constant light exposure of 80 lux. Collier and Zigman 8 described swollen inner segments in the gray squirrel after near-uv radiation and postulated that mitochondrial damage was an initial event in cell death. Young 9 has reported damage to retinal DNA resulting from UV irradiation. Mitochondrial DNA may be even more susceptible to direct UV-radiation damage than is nuclear DNA because of the lack of associated histones in mitochondria. 10 Alternatively, mitochondrial DNA may be particularly susceptible to adjacent mitochondrial membrane lipid peroxidation, 10 a process thought to be involved in retinal phototoxicity. However, Rapp et al 2 observed that autoradiographic uptake of thymidine by inner segments in retinas of normal and UV-irradiated rats was quantitatively similar. In the latter study, it is noteworthy that uptake in the UV-irradiated rat retinas increased in nuclear layers, but it remains unclear whether this increase is attributable to nuclear or to mitochondrial DNA synthesis. The results of the current study support the hypothesis that mitochondria are sites of DNA synthesis in mature mammalian retina. This report further suggests that there may be a high rate of mitochondrial DNA synthesis in photoreceptors and ganglion cells, and we hypothesize that perturbation of such synthesis may be involved primarily or secondarily in some photoreceptor degenerations. Key words: DNA, retina, thymidine, autoradiography, mitochondria Acknowledgments. The authors thank Randi Goodnight, MS, for expert technical advice on ultrastructural autoradiography, and Diane Gegala for secretarial assistance. From the Departments of Anatomy and Cell Biology and Ophthalmology, University of Southern California School of Medicine, and Doheny Eye Institute, Los Angeles, California. Supported by National Institutes of Health Grant No. EY and Core Grant No. EY Submitted for publication: December 27, 1988; accepted May 22, Reprint requests: Janet C. Blanks, Ph.D., Doheny Eye Institute, 1355 San Pablo St., Los Angeles, California References 1. Young RW: Visual cells and the concept of renewal. Invest Ophthalmol Vis Sci 15:700, Rapp LM, Jose JG, and Pitts DG: DNA repair synthesis in the rat retina following in vivo exposure to 300-nm radiation. Invest Ophthalmol Vis Sci 26:384, Kapoor CL, Waxier M, and O'Brien PJ: In vivo incorporation of radioactive thymidine into rat retina. ARVO Abstracts. Invest Ophthalmol Vis Sci 29(Suppl):97, Blocker Y, Jose JG, Chu LW, Waxier M, and Hutchins V: Incorporation of radioactive thymidine into the monkey retina. ARVO Abstracts. Invest Ophthalmol Vis Sci 27(Suppl):201, Gross NJ, Getz GS, and Rabinowitz M: Apparent turnover of mitochondrial deoxyribonucleic acid and mitochondrial phospholipids in the tissues of the rat. J Biol Chem 244:1552, Moses RA: Metabolism of the retina. In Adler's Physiology of the Eye. St. Louis, C.V. Mosby, 1970, pp Moriya M, Baker BN, and Williams TP: Progression and reversibility of early light-induced alterations in rat retinal rods. Cell Tissue Res 246:607, Collier R and Zigman S: The gray squirrel lens protects the retina from near-uv radiation damage. In Degenerative Retinal Disorders: Clinical and Laboratory Investigations, Hollyfield JG, Anderson RE, and La Vail MM, editors. New York, Alan R. Liss, 1987, pp Young RW: The chemistry of the retina: Function, renewal, rhythms and the nucleus. In Neurochemistry of the Retina, Bazan NG and Lolley RN, editors. Oxford, Pergamon Press, 1980, pp Fleming JE, Miquel J, Cottrell SF, Yengoyan LS, and Economos AC: Is cell aging caused by respiration-dependent injury to the mitochondrial genome? Gerontology 28:44, 1982.