Retinal Toxicity after Repeated Intravitreal Carboplatin Injection into Rabbit Eyes

Background. The aim of this study was to assess retinal toxicity in a rabbit model after carboplatin delivered as repeated transcorneal intravitreal injection, in order to determine the highest possible safe dose for use in human retinoblastoma " seeding " tumor chemotherapy. Methods and Results. We used six albino rabbits in an in vivo experiment and injected 0.008 mg of carboplatin intra-vitreally (iv) 4 times at two-week intervals. 0.08 mL saline was injected into the left eye. We recorded electroretinograms (ERGs) before the first carboplatin injection and after the fourth injection. Platinum concentration was measured 1 h after the fifth additional injection. We found reduced dark-adapted b-wave amplitudes and, light-adapted b-wave and a-wave amplitudes. The differences between right and left eyes was significant but we found no histopathologic retinal changes. Conclusions. 0.008 mg of carboplatin is probably the highest possible safe dose for the treatment of retinoblastoma patients. Questionable is direct extrapolation of retinal toxicity from the rabbit eye model to the human eye.


INTRODUCTION
Retinoblastoma is a childhood cancer arising from immature retinal cells in one or both eyes.Chemo therapeutics are used in treatment but vitreous seeding is a serious limiting factor in retinoblastoma therapy.Various modalities to increase intravitreal concentra tions of chemotherapeutics have been tested, for example, coulombcontrolled iontophoresis 1 , peribulbar administra tion 2 , cryotherapy one day before intravenous carboplatin with or without cyclosporine 3 etc.Some are too complex for clinical practice or vitreous concentrations of chemo therapeutic do not reach effective levels and are unstable 1 .Another described treatment is intravitreal transscleral injection of chemotherapeutic agent 4,5 .No serious com plications have been found to date but tumor dissemi nation in human retinoblastoma was demonstrated by subsequent fine needle biopsy or pars plana vitrectomy 6 .Hence, some physicians used a more difficult approach through the limbus, anterior chamber and peripheral iris for fine needle aspiration biopsy to avoid seeding tumor cells into orbital tissues 79 .This approach was also used in this experiment.We assessed possible retinal changes after transcorneal intravitreal carboplatin injection which would preclude its use in clinical practice.We chose the rabbit and we selected a dose of 0.008 mg according to previously published data 10,11 , where a dose of 0.001 mg induced no changes on electroretinograms (ERGs) and a dose of 0.01 mg evoked significant alterations.The dose we chose was near the upper limit in order to achieve the most effective concentration.

Animals and drug delivery
After approval by the Faculty Committee on animal welfare, New Zealand albino male specified pathogenfree (SPF) healthy rabbits (Anlab, Prague, Czech Republic) (n = 6) received a 0.008 mg dose of carboplatin dissolved in 0.08 mL of saline by unilateral transcorneal intravitreal injection into the right eye.The same volume of saline was injected into the left eye of each animal as a sham control.The same procedure was carried out 4 times at twoweek intervals.Rabbits were kept under standard laboratory conditions.Ambient temperature was 20-24 °C, and rela tive air humidity ranged from 55-60%.They were anesthe tized with a mixture of ketamine hydrochloride (50 mg/ kg, Narketan 10 a.u.v.inj, Vétoquinol, Lure Cedex, France) and xylazine hydrochloride (5 mg/kg, Rometar 2% a.u.v.inj, Spofa, Prague, Czech Republic) given intra muscularly (im) before the intervention.Topical oxybu procaine anesthetic eyedrops (0.4%, Benoxi gtt., Unimed Pharma, Bratislava, Slovakia) were instilled into the con junctival sacs and the eyelids were fixed with a sterile speculum.A total of 3 mL of 1% povidoneiodine solution (10% Betadine, EGIS Pharmaceuticals Ltd., Budapest, Hungary) was used for disinfection.Two months after the first injection of carboplatin, all rabbits were euthanatized by exsanguination via the carotid arteries under general anaesthesia and their eyes were processed for histopatho logic examination.
Electroretinograms (ERGs) were recorded before the first and 2 weeks after the fourth injections.Electro retinographic readings consisted of a series of intensities presented under dark and lightadapted conditions ac cording to the ISCEV protocol.Pupillary mydriasis was induced by instillation of one drop of tropicamide 0.5% (Mydrum, Chauvin Ankerpharm GmbH, Germany).After 30 min of dark adaptation, ERGs were recorded simultaneously with a skin electrode and direct corneal ERGjet contact lens electrode 12 .The skin electrode was placed 1 cm behind the lower lid (Fig. 1).A skin electrode on the forehead served as a ground.Stimulation and re cording of the ERGs were performed with the RETIscan system (Roland Consult, Brandenburg, Germany).The rod (scotopic) ERG was recorded with a white flash at an intensity of 0.01 cd.s.m2 and for maximal scotopic answer an intensity of 3.0 cd/m 2 .s.The cone (photopic) ERG was recorded with the same stimuli intensities (3.0 cd/m 2 .s)and background illumination of 30 cd/m 2 .Statistical analysis was done using the software Statistica 9.1 WAN (StatSoft, Tulsa, USA).Data were compared using a ttest.P≤ 0.05 was considered statistically significant.

Carboplatin concentration measurement
To measure the platinum concentration, an additional, i.e. fifth, dose of the carboplatin was injected into the right eyes after the second ERG.Samples of vitreous humour were collected one hour later to verify the concentra tion of carboplatin in the vitreous cavity.Electrothermal atomic absorption spectrometry (ETAAS) was employed to analyze the total platinum concentrations in vitreous humour after the last administration as a suitable method for carboplatin concentration determination.ETAAS de termines total Pt in fluids or tissues, including any forms of the drug subject to hydrolytic action and subsequently inactivated by irreversible binding to protein and thus ren dered inactive and not cytotoxic.The platinum concentra tion was measured by graphitefurnace atomic absorption spectrophotometry with Zeeman background correction (Varian 220 Z, Australia). 100 μL of vitreous humour was diluted 1:14 with a solution containing Triton X100 (0.2 vol %), antifoam A (0.2 vol %), and deionized water.A programmable sample dispenser piped the samples and calibration standard into the furnace.Platinum determina tion was carried out by the standard additions method.The highest possible concentrations of carboplatin imme diately after its administration were calculated according to the globe diameter.

RESULTS
All animals were followed up during the experimental period by a veterinarian.No treatmentinduced weight changes were found as a sign of the general toxicity of carboplatin.One animal perished suddenly during general anesthesia before the fourth carboplatin administration.Its gross dissection showed no other cause of death.We found no structural changes on histopathologic examina tion of the eyes.
Statistically significant reductions in the darkadapted bwave (ra b) amplitudes and in the maximal scotopic answer (msa b) amplitudes and also elevation of cone answer (Fig. 2, Tables 1 and 2) were found in carboplatin treated eyes We also found statistically significant changes in ra b, msa a, msa b and ca b waves in the ERGs records of control eyes (Tables 1 and 3).There were no structural changes on histopathologic examination.The measured average platinum concentration one hour after additional injection was 1349 μg/L.The calculated highest possible average concentration of carboplatin according to the globe diameters was 8422 μg/L immediately after application of 0.008 mg of carboplatin.

DISCUSSION
We found significant changes in the ERG records after repeated injection of 0.008 mg of carboplatin into right eyes.Similar but more serious changes were found after single intraocular injection of higher dose (more than 0.01 mg) of carboplatin 10 .Repeated intraocular injection of 0.05 mg of carboplatin resulted in serious ERG changes and also structural changes -chorioretinal atrophy found by histopathologic examination 11 .Similar ERGs changes (elevation in ca a) were also recorded after lead expo sure 13 .
Due to the effect of carboplatin on right eyes, changes in ERG records were significantly different to changes       All changes are probably reversible and depend on the period of intraocular pressure increase 15 during intravit real injection of carboplatin and saline.Recovery of visual functions was expected as we found no structural changes on histopathologic examinations but an extended period of time for recovery was probably necessary 16 .

CONCLUSION
Intravitreal delivery of 0.008 mg of carboplatin re sulted in damage to retinal function but not structure.Thus, the changes are probably transient.The dose of carboplatin we used is probably the highest possible for the treatment of retinoblastoma patients.Questionable is, of course, the direct extrapolation of retinal toxicity from the rabbit eye model to the human eye.
= rabbit number ra b = rod answer, wave b msa = maximal scotopic answer ca = cone answer right b = right eye before administration right a = right eye after administration left b = left eye before administration left a = left eye after administration

Fig. 3 .
Fig. 3. Mean values of ERG before and after administration of carboplatin.

Table 1 .
ERGs before and after administration of carboplatin (right eyes) and saline (left eyes).

Table 2 .
Right eyes before and after administration of carboplatin.

Table 3 .
Left eyes before and after administration of saline.