Received: 24 April 2020; Published on-line: 27 August 2020
Ultrastructural changes in the rabbit retina after various one-time doses of intravitreal melphalan
N.F. Bobrova, T.A. Sorochynska, N.I. Molachaniuk, A.Iu. Bratishko
SI "Filatov Institute of Eye Diseases and Tissue Therapy of the National Academy of Medical Sciences of Ukraine"; Odesa (Ukraine)
TO CITE THIS ARTICLE: Bobrova NF, Sorochynska TA, Molachaniuk NI, Bratishko AIu. Ultrastructural changes in the rabbit retina after various one-time doses of intravitreal melphalan. J.ophthalmol.(Ukraine).2020;4:50-55. http://doi.org/10.31288/oftalmolzh202045055
Background: Intravitreal chemotherapy (IVC) has become a common eye-preserving treatment for retinoblastoma (RB). However, there are still unsolved issues with regard to dosage of the cytostatic agent, its ultrastructural effects on healthy retinal cells, and late complications of the therapy.
Purpose: To identify and assess ultrastructural changes in the rabbit retina after various one-time doses of intravitreal melphalan.
Material and Methods: Six eyes of three Chinchilla rabbits (age, 5–6 months; weight, 2.5–3 kg) underwent clinical and electron miscroscopy examination. Both eyes of animals ##1, 2 and 3 received a single dose of intravitreal melphalan of 5, 10 and 20 ?g, respectively. Intravitreal injection was performed in a standard manner. The eyes were enucleated 4 weeks after injection. After enucleation, material for histology was taken in a routine manner and examined in a PEM-100-01 electron microscope.
Results: Intravitreal injection of various doses of melphalan had no general toxic effect on the animals. No retinal hemorrhage, vitreous hemorrhage, retinal tear or detachment was observed at all follow-up points, regardless of how large an intravitreal dose of melphalan was administered. No complications were observed postoperatively. Throughout a follow-up period of 4 weeks, there were no focal fundus changes in the eyes that received a single intravitreal injection of 5 ?g melphalan. In the eyes that received a single intravitreal injection of 10-?g or 20-?g melphalan, we noted a range of focal fundus changes from as mild as a few small isolated sites of slight depigmentation to as severe as pigment redistribution with the emergence of brighter and greater depigmentation regions in the retina. Ultrastructural changes corresponded to ophthalmoscopic changes. Electron microscopy revealed destructive changes in the retinal pigment epithelium (RPE), photoreceptors, and M?ller cells of the rabbit retina.
The minimum structural changes in the RPE and photoreceptor cells were seen after an injection of the smallest dose (5 ?g melphalan). After a 10-?g or 20-?g one-time dose of intravitreal melphalan, we observed severe changes (such as cell swelling, hypertrophy, destruction and disintegration) in retinal cells, disorganized retinal layers with loss of typical ultrastructural features, gliosis and necrosis.
Conclusion: On the basis of our findings, we can conclude that intravitreal melphalan is relatively safe when administered as a one-time 5-?g injection, and minimally toxic when administered as a one-time 10-?g injection. However, when administered as a one-time 20-?g injection, it resulted in severe changes such as gliosis and necrosis in retinal cells.
Keywords: intravitreal chemotherapy, melphalan, electron microscopy, retina, experiment
1.Edelhauser HF, Rowe-Rendleman C, Robinson M, et al. Ophthalmic Drug Delivery Systems for the Treatment of Retinal Diseases: Basic Research to Clinical Applications. Invest Ophthalmol Vis Sci. 2010 Nov;51(11):5403-20.
2.Ericson LA, Rosengren BH. Present therapeutic resources in retinoblastoma. Acta Ophthalmol (Copenh). 1961;39:569-76.
3.Inomata M, Kaneko A. Chemosensitivity profiles of primary and cultured retinoblastoma cells in a human tumor clonogenic assay. Jpn J Cancer Res. 1987;78:858–68.
4.Kaneko A, Suzuki S. Eye-Preservation Treatment of Retinoblastoma with Vitreous Seeding. Jpn J Clin Oncol. 2003 Dec;33(12):601-7.
5.Reуnolds ES. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol 1963 Apr;17(1):208-12. doi: 10.1083/jcb.17.1.208.
6.Ueda M, Tanabe J, Inomata M, et al. [Study on conservative treatment of retinoblastoma - effect of intravitreal injection of melphalan on the rabbit retina]. Nihon Ganka Gakkai Zasshi. 1995 Nov;99(11):1230-5.
7.Shimoda Y, Hamano R, Ishihara K, et al. Effects of intraocular irrigation with melphalan on rabbit retinas during vitrectomy. Graefes Arch Clin Exp Ophthalmol. 2008 Apr;246(4):501-8.
8.Shah N, Pham D, Murray T, et al. Intravitreal and Subconjunctival Melphalan for Retinoblastoma in Transgenic Mice. J Ophthalmol. 2014;2014:829879.
9.Cassoux N, Assayag F, Chouchane-Mlik O, et al. Intraocular treatments of a new orthotopic primary human retinoblastoma xenograft. Invest Ophthalmol Vis Sci. March 2012;53(3):6869.
The authors certify that they have no conflicts of interest in the subject matter or materials discussed in this manuscript.