Received: 19.10.2022; Accepted: 04.11.2022; Published on-line: 21.12.2022

Clinical and ultrastructural changes in the rabbit retina at various doses and numbers of intravitreal melphalan injections

N. F. Bobrova, T. A. Sorochynska, N. I. Molchaniuk, O. Iu. Bratishko

SI "The Filatov Institute of Eye Diseases and Tissue Therapy of the NAMS of Ukraine";

Odesа (Ukraine)

TO CITE THIS ARTICLE: Bobrova NF, Sorochynska TA, Molchaniuk NI, Bratishko OIu. Clinical and ultrastuctural changes in the rabbit retina at various doses and numbers of intravitreal melphalan injections. J.ophthalmol.(Ukraine).2022;6:39-43.    http://doi.org/10.31288/oftalmolzh202263943


Purpose: To assess clinical and ultrastructural changes in the rabbit retina and choroid with an increase in the dose and number of intravitreal (IVit) melphalan injections.

Material and Methods: Thirteen eyes of seven Chinchilla rabbits (age, 5–6 months; weight, 2.5–3 kg) underwent a clinical electron microscopy (EM) examination. Rabbit eyes were divided into five groups based on the dose and number of IVit melphalan injections given. Group 1 (4 eyes) received three 5-μg IVit melphalan  injections; group 2 (2 eyes), two 10-μg injections; group 3 (2 eyes), two 20-μg injections; group 4 (3 eyes), two 30-μg injections; and group 5 (4 eyes), two 40-μg injections. The interval between IVit melphalan  injections was one month. Enucleation was performed at week 4 after the last injection.

Results: In group 1, ophthalmoscopic changes in the retina (small isolated areas of mild depigmentation) stayed the same until the end of the observation period. On EM, some retinal pigment epithelium (RPE) cells showed signs of destruction, whereas others showed signs of compensatory-and-restorative processes. In addition, discs of the outer segments (OS) of photoreceptors showed pathological changes varying in severity. Other photoreceptor layers and retinal neural cells showed no ultrastructural changes. In group 2, fundus changes were more severe than in group 1, and somewhat increased in severity with each injection. There was EM evidence of significant pathology in RPE cells, extracellular edema in the photoreceptor layer, damage to the photoreceptor inner segments and OS, and focal necrosis of the neural tissue. In group 3, there was retinal pigment redistribution after the first injection, with an increase in the degree and area of depigmentation after subsequent injections. In addition, EM found RPE cells showing various pathological changes (e.g., as severe as destruction), pigment granules scattered throughout the retina, and retinal gliosis due to growth of hypertrophic Müller cell processes. In groups 4 and 5, there were large regions of marked depigmentation which increased in area with each injection, becoming almost confluent, which resulted in a complete discoloration of the retina. In addition, EM found necrosis of RPE cells and photoreceptors and RPE cell debris in the inner retinal layers, and the outer and inner retinal layers were lost and replaced by glia. Ganglion cells exhibited signs of degenerative changes. The choroid showed almost complete loss of choriocapillaries with signs of destruction of choroidal vascular walls.

Conclusion: We demonstrated experimentally that repeated IVit melphalan  injections at various doses did not affect media transparency and the structure of the anterior segment of the eye, and produced no general toxic effects on experimental animals. The clinical and ultrastructural changes in the retina depended on the dose of melphalan, with repeated 5-μg and 10-μg IVit melphalan  injections being relatively safe and causing mild changes only in the outer retinal layers. Care should be taken when using IVit melphalan  injections at hig (20-μg – 30-μg) doses, taking into account that toxic changes with various degrees of degeneration (e.g., as severe as gliosis of the retina and destruction of some choroidal capillaries) can develop.




1.Bobrova NF, Sorochinskaya TA. [Combined (intravitreal and intravenous) polychemotherapy in organ-preserving treatment for retinoblastoma]. Oftalmol Zh. 2011;2:38-44. Russian.


2.Bobrova NF, Sorochinskaya TA. [Intravitreal chemotherapy for retinoblastoma: our five-year experience]. Oftalmol Zh. 2015;3:59-68. Russian.


3.Bobrova NF, editor. [A monograph on retinoblastoma]. Odesa: Izdatelskii tsentr; 2020. Russian.

4.Bobrova NF, Sorochinskaya TA. Local retinoblastoma chemotherapy by intravitreal melphalan injections (the preliminary report). South-East European Journal of Ophthalmology. 2009; 2 (3-4): 28-34.

5.Francis JH, Brodie SE, Marr B, Zabor EC, Monde-sire-Crump I, Abramson DH. Efficacy and Toxicity of Intra- vitreous Chemotherapy for Retinoblastoma: Four-Year Experience. Ophthalmology. 2017; Apr; 124(4): 488-495.

Crossref  PubMed 

6.Kaneko A, Suzuki S. Eye-Preservation Treatment of Retinoblastoma with Vitreous Seeding. Jpn J Clin Oncol. 2003 Dec;33(12):601-7.

Crossref  PubMed  

7.Munier FL, Gaillard MC, Soliman S, et al.Intravitreal chemotherapy for vitreous disease in retinoblastoma revisited: from prohibition to conditional indications. Br J Ophthalmol. 2012; 96(8): 1078–83.

Crossref  PubMed 

8.Shah N, Pham D, Murray T, et al. Intravitreal and Subconjunctival Melphalan for Retinoblastoma in Transgenic Mice.  J Ophthalmol. 2014;2014:829879.

Crossref  PubMed 

9.Shields CL, Lally SE, Leahey AM, Jabbour PM, Caywood EH, Schwendeman R, et al. Targeted retinoblastoma management: When to use intravenous, intra-arterial, periocular, and intravitreal chemotherapy. Curr Opin Ophthalmol. 2014 Sep;25:374–85.

Crossref  PubMed 

10.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(495):50-5.


11.Reynolds ES. The use of lead citrate at high pH an electron-opaque stain in electron microscopy. J Cell Biol. 1963;17:208–12.

Crossref  PubMed 

12.Ghassemi F, Fahimeh Asadi Amoli F. Pathological findings in enucleated eyes after intravitreal melphalan injection. Int Ophthalmol. 2014 Jun;34(3):533-40.

Crossref  PubMed 

13.Ghassemi F, Shields CL. Intravitreal melphalan for refractory or recurrent vitreous seeding from retinoblastoma. Arch Ophthalmol. 2012; 130: 1268-1271.

Crossref  PubMed 

14.Shields CL, Douglass AM, Beggache M, Say EA, Shields JA. Intravitreous chemotherapy for active vitreous seeding from retinoblastoma: Outcomes After 192 Consecutive Injections. The 2015 Howard Naquin Lecture. Retina. 2016 Jun; 36(6):1184-90.

Crossref  PubMed 

15.Inomata M, Kaneko A. Chemosensitivity profiles of primary and cultured retinoblastoma cells in a human tumor clonogenic assay. Jpn J Cancer Res. 1987;78(8):858–68.

16.Ueda M, Tanabe J, Inomata M. et al. [Study on conservative treatment of retinoblastoma - effect of intravitreal injection of melphalan on the rabbit retina].  J Jpn Ophthalmol Soc. 1995;99:1230–35. Japanese.

17.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; 246(4):501-8.

Crossref  PubMed

18.Bar-Sela SM, Zayit-Soudry S, Massarweh A, et al. Retinal toxicity of intravitreal melphalan in albino rabbits. J Clin Exp Ophthalmol. 2018;9(1):707. DOI: 10.4172/2155-9570.1000707.




Author contribution: All authors participated in the conception of the article and in writing the manuscript. The final version of the manuscript has been approved by all authors, who in turn are solely responsible for submitting the final version for publication.

Conflict of interest statement: All authors have no real or potential conflict of interest (financial, personal, professional, or other interests) that could influence the subject matter or material described and discussed in this manuscript.

Sources of support. There are no external sources of funding.