Graefe's Archive

Graefe's Arch Clin Exp Ophtha]mo] (1992)230:488-495

for Clinical and Experimental

Ophthalmology © Springer-Verlag 1992

Toxicity of 1-(fl-o-Arabinofuranosyl)cytosine after intravitreal injection in the rabbit eye Joke J.A.Th. Diets-Ouwehand 1, Rob J.W. de Keizer 1, Gijs F.J.M. Vrensen 1' 2, Sylvia Groen-Jansen 1, and Jaap A. van Best 1 1 Department of Ophthalmology, Leiden University Hospital, Rijnsburgerweg 10, 2333 AA Leiden, The Netherlands z Department of Morphology, The Netherlands Ophthalmic Research Institute, Amsterdam, The Netherlands ReceivedJuly 7, 1991 / AcceptedJuly 20, 1991 Abstract. The retinal toxicity of intravitreally injected 1-(/~-D-arabinofuranosyl)cytosine (cytarabine) was examined in 7 chinchilla rabbits to determine if cytarabine can be used as local therapy for vitreoretinal non-Hodgkin's lymphoma. Fractionated dose of 600 lag, 1500 lag, and 2700 lag cytarabine in stabilized saline were given intravitreally in one eye (2 x 300 lag, 5 x 300 lag, and 3 x 900 lag, respectively, with an interval time of 24 h) and stabilized saline in the other eye as control. Toxic effects were evaluated with biomicroscopy, direct ophthalmoscopy, fluorophotometry, electroretinography, light, and electron microscopy. Toxic effects were found with the 1500 lag and 2700 lag doses only. They consisted of a temporary impairment of the blood retina barrier function for fluorescein as measured by fluorophotometry and an irreversible change of the b-wave in the electroretinograms. No histopathologic changes were seen under the light microscope. Electron microscopic examination showed aberrations in the synaptic pedicles of the photoreceptor cells at a dose of 1500 lag cytarabine. The results suggest that the cytarabine dose that is expected to be therapeutic for vitreoretinal non-Hodgkin's lymphoma (about 90 lag given in three doses of 30 lag) is non-toxic for ocular structures.

Introduction Intraocular reticulum cell sarcoma, also known as primary intraocular large B-cell lymphoma or vitreoretinal non-Hodgkin's lymphoma (NHL), is a tumor that occurs incidentally [21, 35]. About 100 cases have been reported since the first report was published in 1951 by Cooper and Riker [35]. This tumor must be differentiated from systemic N H L with choroidal dissemination and from uveitis [7, 21]. About 70%-80% of the cases are bilateral [7, 8, 35]. Central nervous system (CNS) involvement occurs in 75%-85% of all cases within weeks up to 10 years after Correspondence to: J.A. van Best

diagnosis [7, 8, 25, 30]. In cases of CNS involvement, the mean survival time is 20 months [23, 15] (range 3 months-6 years [25]). In recurring CNS involvement, most patients died within 3 months [25, 37]. The diagnosis of vitreoretinal N H L is difficult to make and requires vitrectomy or vitreous biopsy [7, 15, 30]. N H L responds very well to local radiotherapy [8, 15, 34, 36]. Additional cranial irradiation is recommended in isolated orbital or vitreoretinal NHL, because most patients have subclinical involvement of the CNS at the time they present with vitreoretinal N H L [8]. The permeability of the blood-retina barrier for chemotherapeutic agents is uncertain; therefore, Rouwen et al. [34] recommend a combination of systemic chemotherapy and intrathecal chemotherapy with additional wholebrain and spinal cord irradiation. Others propose this combination only in patients with proven CNS involvement [8, 37]. Another method is the use of high doses of the antimetabolite 1-(/?-I>arabinofuranosyl)cytosine (cytarabine). The effectivity of cytarabine is cell cycle dependent; cells in S phase, the D N A synthesis phase, are most susceptible to its toxic effect [6, 10, 40]. Mitotically quiescent tissues, such as the retina and the lenticular and ciliary epithelium, are less likely to experience a toxic effect [17]. The cancer cell mass can be reduced very effectively when an optimal interval of treatment is used, due to recruitment and synchronisation [10]. Cytarabine has to be metabolized intracellularly to its active metabolite by phosphorylation and can be deactivated by deamination [6, 19]. Tumor cells without the activating enzyme or with high concentrations of the deactivating enzyme will not respond [6, 17, 20]. Cytarabine has proven to be effective against N H L cells in vitro [9, 10, 13, 17] and in vivo [4, 34]. Local application by instillation or subconjunctival injection of any kind of medicine is known to have a very low intravitreal penetration, resulting in an insufficient intravitreal therapeutic dose [3] and local eye complications [4, 33].

489 Table 1. Inhibition concentrations of cytarabine in vitro

Cell type

Reference

Exposition time (h)

ICso (lag/ml) 0.8 0.06 0.008 0.004

Human scleral fibroblast

15

Human retinal pigment epithelial cell Mouse leukemia cell Mouse leukemia cell type 1210 Human B lymphocyte Human T lymphocyte type 60 Cytarabine-resistant human leukemia cell

15

1 24 72 24

19 19

24 12

12 12

24 24

12

24

In vitro studies using several types of cells have reported an ICso for cytarabine ranging from 0.004 ~tg/ml to 100 gg/ml for normal cells and an IC99 of 0.024 lag/ml to 10 ~tg/ml for malignant cells, depending here, too, on the exposition time (Table 1). The large spread in ICso and IC99 is due to the fact that the effectiveness of cytarabine depends on various factors [22]. Intravenously administered cytarabine leads to spinal fluid levels at a dose of 3 g/m 2 body surface [4, 18, 33]. Cytarabine can also be combined with intrathecal methotrexate (MTX); this treatment requires calcium-leucovorin rescue as methotrexate induces severe local and systemic toxic complications [16] and cytarabine, keratitis [38]. In the literature it is not clear how the vitreoretinal N H L is spread to the CNS. The data of the study by S.J. Qualman et al. [30] suggest that it may spread through the optic nerve and that the intraocular disease usually presents itself with visual symptoms years before CNS location occurs. We considered a more local therapy since intravitreal treatment of the primary vitreoretinal N H L with a cytostatic may prevent spreading to the CNS, and so contribute to the improvement of the long-term prognosis. Furthermore, local therapy may preserve the visual function in the local vitreoretinal N H L with less severe side effects than in c o m m o n therapies and may be used as an additional therapy for remaining vitreoretinal N H L after successful treatment of the CNS focus. The half-time (tu2) of intrathecally administered cytarabine is much longer than that of intravenously administered cytarabine due to the low concentration of the deactivating enzyme in the cerebrospinal fluid [20]. Extrapolating this to the vitreous, which is also a structure of the embryonal neural tube with a blood barrier and low concentrations of the deactivating enzyme, we expect that the tl/z after intravitreal injection will also be longer. This favors the assumption that a long exposure to low concentrations is as effective on tumor cells as a short exposure to high concentrations, but with less toxic effect on the normal structures [17, 31]. It should be noted that the clearance of drugs can be faster in the vitrectomized eye than in the intact eye.

IC99 (lag/ml)

10 2.8 2.4 0.024 24

We decided to inject cytarabine intravitreally in rabbits, which is analogous to intravitreal treatment with antibiotics in endophthalmitis [3, 14]. To evaluate the toxicity of this mode of therapy, we used biomicroscopy, ophthalmoscopy, electroretinography, and light microscopy as described previously [2, 26, 28] and, in addition, fluorophotometry to determine the extent of the bloodretina barrier function and electron microscopy for ultrastructural changes.

Materials and methods

Animals and intravitreal injection Seven pigmented rabbits (chinchilla greys), weighing 2.3 3.2 kg, were used in this study. Multiple intravitreal injections of 0.05 ml each of cytarabine dissolved in stabilized saline were given in one eye. The total cytarabine dose in an eye amounted to 600 lag, 1500 gg, or 2700 lag (Table 2). The other eye received a similar volume of saline and was used as a control. Two rabbits were used twice with an interval time of 48 and 122 days. Before the injection the rabbits were sedated with 1 ml Hypnorm, and their pupils dilated with Mydriaticum and phenylephrine HC1 5%. The cornea and conjunctiva were anesthetized by instillation of oxybuprocaine HC1 0.4%. The intravitreal injection was given through the pars plana, 2 mm behind the limbus, just beside the superior rectus muscle in the superonasal quadrant, using a tuberculin syringe with a 30-gauge needle. The bevel of the needle was held to the anterior side of the eye during the injection, and hereafter turned 90° and quietly taken away. During ophthalmoscopy, biomicroscopy, fluorophotometry, and electroretinogram (ERG) study, the animals were sedated with 1 ml Hypnorm, their eyes were maximally dilated with Mydriaticum and phenylephrine HCL 5%, and for ERG the cornea was anesthetized with oxybuprocaine HC1 0.4%. The rabbits were sacrified by an i.v. injection of 5 ml sodium pentobarbital 60 mg/ml, and the eyes were immediately enucleated.

Biomicroscopy and ophthalmoscopy Biomicroscopy and direct ophthalmoscopy were performed before, immediately after, 0.5 h, I h, and 3 h after each injection. The examinations were repeated every 24 h for 1 week, then every 48 h for 1 week, and finally, twice a week until the day of sacrifice (Table 2).

490 Table 2. Cytarabine doses, time, and type of histological evaluation

Vitreousfluorophotometry

Rabbit

Vitreous fluorophotometry was performed with the Fluorotron Master (Coherent Radiation, Palo Alto, Calif.) twice before the injections and on days 2, 5, and 9 after the last injection according to a technique described previously [29]. Thereafter, scans were performed once a week until the end of the experiment. The effect of intravitreally injected cytarabine on the bloodretina barrier permeability was quantified by the ratio between the vitreal fluorescein concentration of the experimental eye and the fellow control eye. Differences between the ratios before and after cytarabine injections were statistically evaluated using Student's t-test.

1

Weight (kg)

2.4

2

2.6

3

2.5

3

2.5

4

2.2

4

2.2

5

2.3

6

2.6

7

3.1

Eye (left/ right)

r

1 1 r 1 r r 1

Total dose (I.tg)

Histology Time a (days)

600 b

saline 600 b saline 600 b saline °'° 1500 e saline

r

600 b

1 1 r r 1 r 1 1 r

saline 2700 d' f saline 1500 c saline 1500 c saline 1500 ° saline

6

90 215 88 112 61 6 65 145

Type

LM LM EM EM EM EM EM EM LM LM LM LM LM LM LM LM EM EM

a Time between last dose to the eye and sacrifice LM, Light microscopy; EM, electron microscopy b 300 pg cytarabine daily for 2 days ° 300 [xg cytarabine daily for 5 days d 900 gg cytarabine daily for 3 days e 122 days after the last dose to the left eye f 48 days after the last dose to the right eye

Electroretinography ERG recordings were performed 2-3 times in all rabbits over a period of 7 days prior to the intravitreal injections to establish an individual standard. Then recordings were made on days 3, 6, and 10 after the last injection and once a week hereafter until the day of sacrifice. The pupils of both eyes were dilated as described above. Both for the adaptation light and the stimulus light, a Ganzfeld was applied. Processing and amplification were done with a physiological amplifier (C 2000, Nicolet Biomedical Instruments, Brussels, Belgium). ERGs were recorded with a hard contact lens electrode with one drop of 2% hydroxypropylmethylcellulosesolution between the lens and cornea. The reference electrode and ground were common Ag/AgC1 skin electrodes applied in a row on two shaven spots at the vertex. The interelectrodal resistance was always less than 25 kOhms. After 10 min dark adaptation, scotopic ERGs were elicited by flashes of 1 J at a frequency of 1 Hz, filtered by a combination of a blue filter and neutral filters of various densities (Dz.6, D2.o, D1.6, Dl.o, Do.6, or Do). Photopic ERGs were elicited by flashes of I J at a frequency of 4 Hz superimposed on a bright blue background. Each tracing was the average of 32 artefact-free responses. The bandpass was 1-100 Hz and the time base for signal analysis was 500 ms under scotopic conditions and 200 ms under photopic conditions. We evaluated both scotopic and photopic ERGs by comparing the experimental eye with the fellow control eye. The ratio of the amplitude and that of the latency time of the b-wave between experimental and control eye were calculated for each measurement.

Histology Acute cytarabine toxicity was studied by light microscopy in 2 eyes (rabbits I and 5 in Table 2). Chronic toxicity was studied in 7 eyes, 3 by light microscopy and 4 by electron microscopy (corresponding to rabbits 4, 6 and 2, 3, an 7 in Table 2, respectively). Three control eyes were used for light microscopy and 2 for electron microscopy (rabbits 1, 5, 6 and 2, 7, respectively). The eyes for light microscopy were fixed in buffered formaldehyde 4.0% immediately after enucleation and thereafter dehydrated and embedded in nitrocellulose 20%. The material was serially cross-sectioned at 17 gm in a vertical-diagonal plane, and every tenth section was stained with hematoxylin-eosin. The eyes for electron microscopy were fxed at 4° C in a phosphate-buffered solution of 1% paraformaldehyde and 1.25% glutaraldehyde supplemented with 3% sucrose (pH 7.4), immediately after enucleation with an opened anterior chamber. A small slice of the central retina near the visual streak and a small slice of the peripheral retina of each eye were taken out. The slices were rinsed with phosphate buffer and postfixed in 2% OsO4 in 0.5 M phosphate buffer. The slices were dehydrated in ethanol and embedded in Epon 812. Ultrathin sections were cut from the Epon blocks and subsequenty stained with lead citrate and uranyl acetate. They were studied under a Philips EM201 electron microscope.

Results

Biomicroscopy and ophthalmoscopy D r o p s o f c y t a r a b i n e were seen in the vitreous directly after each i n j e c t i o n ; they d i s a p p e a r e d in 5-15 min. N o other changes in the vitreous were seen after the first 2 injections. Some swelling a n d redness o f the superior rectus muscle were observed after each injection. N o f u r t h e r changes were observed in the eyes that received a total dose o f 600 gg cytarabine. I n the eyes t h a t received 1500 gg c y t a r a b i n e , a slight v a s c u l a r n a r r o w i n g was observed directly after each o f the last three injections for u p to 3 h later. A persisting v a s c u l a r n a r r o w i n g was also observed in these eyes 1 week after the last injection, together with a pale retinal reflex. T h e eye that received 2700 gg c y t a r a b i n e showed these reactions after the second injection (1800 gg). O n e h o u r after the third injection (2700 gg), i n t r a v i t r e a l bleeding together with exudates were observed. T h e y resorbed w i t h i n a week, b u t the r e t i n a r e m a i n e d pale a n d the vessels contracted.

491

_ D1.6b1D20b1'~ ,

1500 pg cytarabine in the right eye

600 pg cytarabine in the left eye

Reference ERG

..

f2_ ~

R

2700 pg cytarabine in the left eye

"

..2 .

_

R

80 IJV[ 55 ms

A 2.0 >.

~o 1.5 tO 0

t--{

>, 1.0 x 0 "4= 0.5 e5

[ .... i ......

¢r"

i

I

2700 640 1500 2700 0 600 i 1500 Intravitreally injected dose of cytarabine (pg)

Fig. 1. A Scotopic electroretinograms (ERGs) elicited (after 10 min dark adaptation) by flashes of 1 J at a frequency of 1 Hz, filtered by a blue filter and 6 different neutral density filters. Each tracing was the average of 32 artifact-free responses. R, Right eye; L, left eye. B Ratios of the amplitudes and latency times between experimental and control eyes as a function of the cytarabine dose. Closed triangles, Mean ratio of the b-wave amplitudes; open squares, mean ratio of the latency times; bars, standard deviation of the ratios; left panel, scotopic ERG, maximal b-wave amplitude with no a-wave present; right panel, photopic E R G ; 0 gg (before treatment) : Nm = 22, N~ = 7 ; 600 p.g : Nm = 11, Nr = 4; 1500 pg: Nm = 15, Nr = 4 ; 2700 pg ; Nm = 3, N~ = 1 with Nm = number of measurements and Nr = number of rabbits. The time between the last injection and the measurements amounted to 2-14 days

B Electroretinography • Three of the 4 eyes that received 1500 pg cytarabine showed an irrecoverable change in the shape of the scotopic b-wave 2-3 days after the last injection. This change consisted of a widening of the b-wave and a slow return to the standing potential (Fig. 1 A, 3rd column). On average, these eyes showed a permanently decreased ratio of the scotopic and photopic b-wave amplitudes as well as an increased ratio of latency time (Fig. 1 B). None of the eyes that received 600 pg cytarabine showed a significant change in shape, amplitude ratio, or latency time of the scotopic or photopic b-wave (Fig. 1 A, 2nd column, Fig. 1 B). The eye that received 2700 l-tg cytarabine showed the same change of the scotopic b-wave shape as the eyes that received 1500 pg (Fig. 1 A, 4th column). This eye also showed an additional large decrease of the scotopic and photopic b-wave amplitude ratio and an increase of the ratios of the latency times (Fig. 1 B).

Vitreousfluoropho tometry An example of the effect of intravitreally injected cytarabine on the leakage of fluorescein through the bloodretina barrier is presented in Fig. 2. The ratio (R) between vitreous fluorescein concentrations in a treated and control eye measured about 1 h after fluorescein injection is presented in Fig. 3 as a function of the time after treatment with 600 pg and 1500 pg. The ratios of the rabbits that received 1500 pg or 2700 pg cytarabine increased significantly during the first 2 weeks after treatment and decreased to values before treatment in the following 2 weeks (P<0.001; Fig. 3). The ratios of the rabbits that received 600 ~tg cytarabine did not change significantly ( P > 0.3 ; Fig. 3). Four weeks after the last injection, there was no significant difference between the two populations (P > 0.1 ; Fig. 3).

Histology Light microscopy. In all experimental and control eyes, a slight disorganization of the retinal pigment epithelium

492

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R

C

D

A

• 200 I

50 br,

I

20

,,""/l

'; ~,~',",,,'-,

,

r

,' ]

2O 10 Relative distance along the optical axis

3O

Fig. 2. Fluorescein concentration in the eye 1 h after i.v. fluorescein administration, 2 days after the last intravitreal cytarabine injection. Dashed line, 1500 ~tg cytarabine; solid line, control lateral eye; R, retina; A, anterior chamber; C, D, distance interval for leakage evaluation; ng.eq/ml, rig. equivalent fluorescein/ml

¢

3

II> {1>

subretinal space and the outer plexiform and inner nuclear layer, based on the initially increased fluorescein leakage and the persistent change in the b-wave. No obvious differences in ultrastructure of the treated and control pigment epithelium were observed. The junctional complexes, including the tight junctions, were morphologically unaltered, and the cytoplasm did not reveal ultrapathological changes. The subretinal space and the outer segments of rods and cones of treated animals did not differ from those of control animals. Characteristic elements of the border zone between the outer plexiform and inner nuclear layer are the synaptic pedicles of rods and cones (Fig. 4A). These pedicles, either in close proximity to the nucleus of the photoreceptor cell or as separate structures, are characterized by a multitude of small, uniform synaptic vesicles and a synaptic ribbon (SR) contacting invaginating elements of horizontal and bipolar cells. The separated pedicles are fully ensheathed by elements of M/iller cells. In control animals, the synaptic ribbon proved to be lined by regularly arranged synaptic vesicles on both sides. In the eyes treated with 1500 gg (Fig. 4 C), the pedicles exhibited a normal ultrastructure with no obvious abnormalities except for the arrangement of the synaptic vesicles along the synaptic ribbon, which is often largely disorganized. This disorganization is far less pronounced in the animals treated with 600 gg (Fig. 4 B). In all treated animals, the pedicles also contain larger vesicles and clear vacuoles (Fig. 4 B, C).

•

°

Discussion

rr I

2

,

,

I

,

,

,

,I

t

,

,

I

,

5 10 20 50 Time after the last injection (days)

,

,~I

100

Fig. 3. Ratio between vitreous fluorescein in experimental and control eye versus time after the last cytarabine injection. Open triangles, 1500 gg eyes; reversed solid triangles, 600 ~tg eyes; bars, SEM of 3 eyes

and photoreceptor layer was observed. This may possibly be an artifact due to the fixation method. However, it seemed more intense in the eyes that received 1500 gg or 2700 pg than in those that received 600 gg. In all eyes treated with cytarabine, the injection site was marked by a substance that gave a crystalline impression. This could be found throughout all layers (pars plana, choroid, sclera, and conjunctiva). No other pathologic changes were observed in the eyes that received 1500 gg or 600 gg. The eye that received 2700 gg cytarabine had some erythrocytes and leukocytes in the vitreous just above the lower peripheral retina. Some erythrocytes and a few leukocytes could also be found in the photoreceptor layer and underneath the retina. The retinal structure was intact.

Electron microscopy. The analysis concentrated on the ultrastructure of the pigment epithelium and adjacent

In this study, repeated intravitreal injections of cytarabine were given to pigmented rabbits. Our results show that two consecutive intravitreal injections of 300 gg cytarabine were found to be nontoxic. Five consecutive injections of 300 l.tg or three of 900 gg had slight toxic effects. The toxicity of cytarabine was evidenced in the following ways: I. A marked and irreversible change in the shape of the scotopic b-wave, a decrease of the amplitude ratios, an increase of the latency times (ERG), and a slight ultrastructural disorganization of the ribbon synapses of the rod pedicles (EM). The b-wave is generated by the M/iller cells in response to changes in extracellular K ÷ concentration ([K+]o) [11, 24, 27, 32]. These [K+]o changes arise from the reaction of the neurons (photoreceptor, amacrine, bipolar, and horizontal cells) to light [5, 24]. The M/iller cells regulate this [K+]o by means of K ÷ channels and a local difference in K ÷ conductance of their membranes in order to maintain the optimal extracellular conditions for the neurons. It can be assumed that the scotopic b-wave change in the ERG recordings is probably caused by disturbance of the [K+]o, giving rise to alterations in the intracellular K ÷ currents in the M/iller cells [11, 24, 32]. This change of [K+]o cannot be explained by the major mechanism of action of the antimetabolite cytarabine.

493

Fig. 4A-C. Panel of electron micrographs of the border zone between outer nuclear layer and outer plexiform layer in the rabbit retina illustrating the synaptic pedicles (large asterisks) of the photoreceptor cells in controls (A) and in animals treated with cytarabine (B 600 gg, survival time 215 days; C 1500 gg, survival time 88 days). The pedicles are characterized by numerous synaptic vesicles of uniform size, synaptic ribbons (arrowheads), and post-synaptic elements of bipolar and horizontal cell origin (small asterisks). Some of the synaptic pedicles are located in the cytoplasm of the photoreceptor cells adjacent to the nucleus (Nu); others are separate and ensheathed by elements of Miiller cells (M). In control animals, the synaptic ribbons are lined by regularly arranged synaptic vesicles (A). In the animals treated with 1500 gg (C), the vesicle lining of the ribbons is partly absent and distorted. In the animals treated with 600 I.tg (B), the vesicle lining is still present but less regular than in controls

Chabner [6] stated that cytarabine treatment can alter the m e m b r a n e structure, antigenicity, and function at high concentrations by means of inhibition of the synthesis of cell surface glycoproteins by A r a C M P and inhibition by A r a C T P of the synthesis of an essential substrate in the sialylation of glycoproteins. The synaptic ribbon is k n o w n to be involved in the guidance of synaptic vesicles towards the synaptic contact sites with the postsynaptic elements o f the horizontal and the bipolar cells, thus facilitating synaptic transmission. For synapses in the nervous system, it has been determined that the release of neurotransmitters depends on

numerous factors in the m e m b r a n e s of the pre- and postsynaptic elements [39]. The possibility that the effets of cytarabine, as mentioned above, m a y influence the organization o f the synaptic pedicles, has not been ruled out. This m a y lead to an impairment of the transmitter release that would explain the persistent b-wave change. Thus, the disorganization of the ribbon synapse could be a primary effect o f cytarabine, but a secondary effect caused by disturbance of the [K÷]o, either by dysregulation of the Miiller cell or abnormal conductance by the inner nuclear layer neurons (both by means of the abovementioned effects on cell membranes), can also be postulated.

494 2. A t e m p o r a r y leakage o f the blood-retina barrier o n vitreous f l u o r o p h o t o m e t r y . T h e leakage can be attributed to a d y s f u n c t i o n o f the cell m e m b r a n e o f the retinal p i g m e n t epithelial cells and the recovery o f the barrier to an effective repair m e c h a n i s m [29]. 3. Retinal pallor a n d vascular n a r r o w i n g seen with the o p h t h a l m o s c o p e a few weeks after the injections. We c a n n o t explain these features since they could n o t be depicted by L M or E M . O u r results are in disagreement with the toxic effects after intravitreal injection o f 60 gg described previously [28]. To determine whether or n o t this discrepancy was caused by differences in m e t h o d , we repeated the experiments a c c o r d i n g to the p r o c e d u r e described in that study which includes an anterior c h a m b e r aspiration before intravitreal injection and using cytarabine (Cytosar, upj o h n - N e d e r l a n d , Ede, The Netherlands) a n d albino rabbits. N o toxicity could be found. The d a t a presented in this article suggest that the intravitreal injection o f cytarabine can be o f value as a local t h e r a p y for vitreoretinal n o n - H o d g k i n ' s l y m p h o ma. The cytarabine dosage that is expected to be therapeutic for vitreoretinal N H L a m o u n t s to two to three times 30 ~tg [12, 10]. Since the n o n t o x i c dose in our exp e r i m e n t is m o r e t h a n 10 times this dose, the therapeutic dose will p r o b a b l y n o t be toxic to the ocular structures, even w h e n one considers that vitrectomized eyes (vitrect o m y is usually p e r f o r m e d for diagnostic reasons in vitreoretinal N H L ) are m o r e susceptible to toxic substances t h a n intact eyes [1, 2].

Acknowledgements. We wish to thank G. van Lith M.D; Ph.D. for his help with the evaluation of the ERGs. We also wish to thank A. de Wolf for her work concerning the electron microscopic evaluation and D. de Wolff-Rouendaal M.D, Ph.D, and J. van Delft, Ph.D, for their help with the light microscopy evaluation. We are very grateful to L. Colly M.D, Ph.D, W. Swart, M.D, and L. Laterveer, M.D, for their inspiration.

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