Low-dose-rate ionizing irradiation for inhibition of secondary cataract formation

PII S0360-3016(00)01512-1

BIOLOGY CONTRIBUTION

LOW-DOSE-RATE IONIZING IRRADIATION FOR INHIBITION OFSECONDARY CATARACT FORMATION

ANTONIA M. JOUSSEN, M.D.,* BERTHOLD HUPPERTZ, M.D.,† HANS-REINHARD KOCH, M.D.,‡

NORBERT KERNERT,§ KEVIN CAMPHAUSEN, M.D.,\ KLAUS SCHLOSSER, PH.D.,§

ANDREAS M. H. FOERSTER, M.D.,* FRIEDRICH E. KRUSE, M.D.,¶ ALEXANDRA LAPPAS, M.D.,* AND

BERND KIRCHHOF, M.D.*

Departments of *Ophthalmology and†Anatomy, University of Technology (RWTH) Aachen, Aachen, Germany;‡Dardenne Klinik, BadGodesberg, Germany;§Forschungszentrum Karlsruhe, Cyclotron Laboratory, Karlsruhe, Germany;\Department of Radiation Oncology,

Harvard Medical School, Boston, MA;¶Department of Ophthalmology, University of Heidelberg, Heidelberg, Germany

Introduction: Secondary cataract formation limits visual function after cataract surgery. Various experimentalmethods utilizing the pharmacologic inhibition of lens epithelial cell proliferation have been proposed. However,diffusion into the anterior chamber may lead to damage of corneal endothelial cells. This study evaluated theinhibition of lens epithelial cell proliferation with a capsular bag ring, labeled with a b-emitting radioisotope.Methods and Materials: In vitro studies using rabbit lens epithelial cells were performed to investigate thedose-dependent effect of irradiation. Based on these results, P-32–labeled PMMA rings were implanted into thecapsular bag of NZW rabbits in vivo after phacoemulsification. Animals were examined for development ofposterior capsule opacification over a period of 12 weeks following surgery. Radiation damage to the surroundingocular tissue was subsequently analyzed in histologic sections using TUNEL assay and proliferation marker.Results: Irradiation of lens epithelial cells in vitro with >5 Gy resulted in a dose-dependent decrease in thenumber of cells. BrdU testing demonstrated a near complete inhibition of cell proliferation.In vivo, implantationof P-32–labeled PMMA rings led to inhibition of epithelial cell proliferation and secondary cataract formationbut was not able to fully inhibit aberrant differentiation of some remaining cells. Histologic examination showedno evidence of radiation damage of the ciliary body or the corneal endothelium.Conclusions: Low-dose beta irradiation exhibits the potential for inhibition of lens epithelial cell proliferationboth in vitro and in vivo. Further investigation of various nuclides and their radiation profiles is needed tooptimize the prevention of posterior capsule opacification due to epithelial cell proliferation. © 2001 ElsevierScience Inc.

Posterior capsule opacification, Beta irradiation, Capsular tension ring, Lens epithelial cells, Inhibition ofproliferation.

INTRODUCTION

With present technologies, cataract extraction and intraoc-ular lens implantation result in improvement of visual acuityin up to 95% of cases. However, postoperative complica-tions that cause deterioration of the visual acuity, such asposterior capsule opacification (PCO), occur in approxi-mately 50% of these patients (1). A regenerative secondarycataract can be due to the proliferation and migration of the

remaining lens epithelial cells from within the capsular bag.Although low-trauma surgery, vigorous hydrodissection,and mechanical polishing of the posterior capsule duringcataract surgery lowers the incidence of PCO considerably,the current surgical techniques do not allow removal of alllens epithelial cells from the equatorial zone. Thus, it is notpossible to completely prevent ingrowth of the lens epithe-lial cells into the visual axis. Besides remaining lens epi-

Reprint requests to: Antonia M. Joussen, M.D., Ph.D., SurgicalResearch, Enders 1025, Children’s Hospital, Harvard MedicalSchool, 300 Longwood Ave., Boston, MA 02115, USA. E-mail:[email protected]

This work was funded by Start Programm 1/2000 RWTHAachen and DFG Jo 324/3-1 (A.M.J.)

Part of the work was presented at the 97th and 98th meeting ofthe German Ophthalmological Society in Berlin, 1999 and 2000and at the 2nd Joint International Meeting ABS/GEC-ESTRO/GLAC, Washington, D.C., May 2000.Acknowledgments—Dr. Schmidt Intraocular Lenses, St. Augustin,Germany generously provided silicon lenses for implantation and

designed the intraocular capsular bag ring for subsequent activa-tion. All measurements of elasticity of the material after labelingwith P-32 were also kindly performed by Dr. Schmidt IntraocularLenses. Geuder GmbH, Heidelberg, Germany provided the surgi-cal equipment for phacoemulsification. The authors thank G.Kolling, M.D., and K. Rohrschneider, M.D., both Department ofOphthalmology, University of Heidelberg, for helpful discussions.Robert Cormack, Ph.D., Department of Radiation Oncology,Brigham Women’s Hospital, Harvard Medical School, Boston,MA, USA calculated the isodose curves.

Accepted for publication 4 October 2000.

Int. J. Radiation Oncology Biol. Phys., Vol. 49, No. 3, pp. 817–825, 2001Copyright © 2001 Elsevier Science Inc.Printed in the USA. All rights reserved

0360-3016/01/$–see front matter

817

thelial cells, the opacity can also be due to cells of nonlen-ticular origin, such as nonpigmented uveal epithelial cells(2).

The most frequent clinical treatment for PCO is theNd:YAG laser capsulotomy, which causes a photodisrup-tion and leads to dehiscence of the central posterior capsule.This allows for visual rehabilitation in most cases, but maycause serious complications, including damage to or sub-luxation of the intraocular lens (IOL). Laser capsulotomymay also affect the retina by causing cystoid macular edema(3, 4) or retinal breaks and detachment (5–7). A selectiveremoval of the migrating epithelial cells while maintainingposterior capsule integrity cannot be achieved. The disrup-tion of the posterior capsule may also prevent the use of theintact capsular bag to hold accommodative fluid lens fiberreplacements in the future.

Therefore, there has been a wide search for methods toselectively inhibit and remove the proliferating lens epithe-lial cells without damaging the surrounding ocular tissues.Both pharmacologic and enzymatic devices have been re-ported (8–14), most of which have not entered clinical trialsdue to their side effects on the corneal endothelium or theciliary body. The aim of this study was to investigate theutilization of a localized radiation source within the capsularbag. Since there are no data available regarding inhibition oflens epithelial cell proliferation following low-doseb-irra-diation, in vitro studies were performed to determine thedose response for lens epithelial cells. Finally, the efficacyof a radionuclide-impregnated capsular tension ring to pre-vent proliferative secondary cataract due to epithelial cellproliferation was investigated.

METHODS AND MATERIALS

AnimalsFemale New Zealand white (NZW) rabbits weighing

2.5–3 kg were purchased from Charles River (France).Animals were used in observance of German state laws andin accordance to the resolution on the use of animals inresearch as published by the Association for Research andVision in Ophthalmology. All surgical procedures such asphacoemulsification and implantation of the capsular ring,as well as the biomicroscopical observation of the posteriorcapsule opacification, were performed under general anes-thesia (xylazine hydrochloride [5 mg/kg] and ketamine hy-drochloride [35 mg/kg]). Animals were held in a group of15 animals with hay as bedding and with free access towater and food.

Radioactive labeling of the capsular tension ringP-32 was selected as the radioactive isotope, as it can be

safely enclosed inside the polymethylmethacrylate(PMMA) of the capsular tension ring. Radioactive labelingof the PMMA ring was achieved by ion implantation withan energy of 60 keV, resulting in a low washoff rate of P-32out of the PMMA material of the capsular tension ring.Physical parameters: T1/2 (half-life) 5 14.3 days,b-emitter,

Emax 5 1700 keV, Eaverage; 700 keV,b-range: 8 mm formax energy and;2.7 mm for average energy.

Pretreatment of PMMA rings before ion implantation.The rings were washed in 0.9% NaCl in an ultrasonic bathfor 10 min at 40°C and rinsed with sterile saline in ultra-sonic bath in the same conditions. The handling was per-formed inside laminar flow boxes to maintain microbiologicand particulate cleanliness of the product.

Ion implantation process.The implantation was per-formed at the ECRIS ion implanter at ForschungszentrumKarlsruhe/HZY. The P-32 was ionized in the ECR sourceand accelerated to 60 kV to achieve an implantation depthof ;110 mm (straggle;30 mm) in PMMA (15). The ionbeam passed a mass analyzing magnet to separate all ionswith massesÞ 32, mainly to separate P-31. The capsulartension rings were positioned on a special device in thevacuum irradiation chamber. The rings were rotated aroundthe center axes to achieve a homogeneous circumferentialactivity distribution.

Quality control of activated PMMA rings.The qualitycontrol tests included the measurement of the total activity,the circumferential activity distribution, and the washoffrate to determine the retention of the P-32 within thePMMA. The activity was measured with a silicon surfacebarrier detector. The P-32 activity was in the range of563–677 kBq (15.2–18.2mCi) at the end of ion implanta-tion, which achieved an activity of;12 mCi at the time ofclinical application. The homogeneity of the circumferentialactivity distribution was620% of the average activity;however, the measurement accuracy was not better than10%. For the determination of the washoff rate, the PMMArings were washed in an ultrasonic bath, first in 0.9% NaClsolution and second in injection water (10 min at 40°C,same wash procedure as for the pretreatment). The activityof the washoff solutions was determined with a liquidscintillation detector. The average washoff rate was 0.7% inNaCl and 0.05% in H2O. The much higher washoff rate in0.9% NaCl results from the fact that the P-32 located on orclose to the surface was removed during the first washprocess.

Implanted rings turned slightly brown in color withoutopacification. However, as verified by the manufacturer, theelasticity of the PMMA remained unchanged, and therewere no defects in the material after impregnation with thenuclide. Both control rings and irradiated rings were steril-ized at low temperature using a plasma sterilization device.Upon implantation into the rabbits’ eyes, the activity of eachcapsular ring was 12mCi. The cumulative radiation dosewith this activity approximates 15 Gy after 5 half-lives.

Implantation of the P-32 labeled capsular tension ringsand biomicroscopic examination

After pretreatment with gentamicin and ibuprofen eyedrops, the pupils were dilated with tropicamide (1%) andphenylephrine hydrochloride (5%). In addition to the gen-eral anesthesia, topical analgesia was achieved with lido-caine (2%). Epinephrine (1:1000) and gentamicin (80 mg/l)

818 I. J. Radiation Oncology● Biology ● Physics Volume 49, Number 3, 2001

were routinely added to the irrigating solution. Heparin(5U/ml) was added to lower fibrin exudation during surgery.After a temporal limbus near clear cornea incision, centralcapsulorrhexis was performed. The preferred size of theanterior capsular opening was about 6.0 mm. Due to the softnuclei, irrigation and aspiration were predominantly used,and phacoemulsification using ultrasound was added whenrequired. All procedures were performed by the same sur-geon (H.R.K.). Special care was taken to mechanicallyclean the posterior capsule and the periphery by low vac-uum. Hydroxymethylpropylcellulose (HPMC, Adathocel)was used as a visco-elastic. After mechanical cleaning of thecapsular bag, the PMMA ring was implanted. Eyes of thetreatment group received P-32 activated rings; control eyeswere implanted with PMMA rings without activation. Fold-able human silicone intraocular lenses were then inserted.The corneal wound was closed using 10-0 nylon. Postoper-atively gentamycin sulfate ointment was instilled into thefornix for 4 days. Slitlamp examinations were performeddaily for the first 4 postoperative days. After stabilization ofthe eyes after surgery, the observations included weeklyclinical slitlamp examination and photography of each eye.

Determination of proliferation, apoptosis, and histologicexamination

Animals were sacrificed 12 weeks after surgery. Eyeswere enucleated and fixed in 4% neutrally buffered forma-lin. Hematoxyline-eosin staining and periodic acid shiftwere routinely performed. To detect apoptotic cell death inthe ocular tissues after irradiation, DNA single-strandbreaks were visualized usingin situ terminal deoxynucleo-tidyl transferase-mediated dUTP nick-end labeling (TUNELassay, Calbiochem, Germany). TUNEL-positivity was al-ways correlated to the morphology of the respective nu-cleus. Only if the typical features of late apoptosis such asannular chromatin condensation or generation of apoptoticbodies were found TUNEL positivity was taken as specific(16). Proliferation was analyzed immunohistochemicallyusing monoclonal antibodies against PCNA (1:500, Sigma,Germany) and Ki-67 (Mib-1; 1:20, Dako, Germany). Im-munohistochemical reactions were performed using a stan-dardized sequence based on the streptavidin-biotin tech-nique for detection of a biotinylated link antibody asdescribed previously (17).

In vitro examination of irradiation on lens epithelialproliferation

Rabbit lens epithelial cells were isolated from freshlyharvested eyes from NZW rabbits according to Knorret al.(12). In short, the lens was removed from the eyes understerile conditions, and the capsular membrane was carefullypeeled off. After detaching cells from the capsular mem-brane by trypsin / dispase digestion (Seromed, BiochromKD, Berlin, Germany), cells were cultivated in SHEM/Ham’s F12 (1:2) (Bio-Whittaker/Seromed). The culture me-dium was supplemented with 10% FCS, 1% penicilline/streptomycine (20000 U/ 20000mg/l), and 1%

amphothericine B. Seeding density was 13 105 cells per60-mm well. These cells exhibit a specific epithelial appear-ance and can be passaged up to 12 times. Preconfluentcultures from passages 3 to 5 in 60-mm petri dishes wereused for these experiments. All experiments were performedat a minimum in triplicate.

Cells were irradiated after settling in the culture disheswith a photon beam (6 MV) with a buildup depth dose rateof 2 Gy/min. The radiation was applied by a linear accel-erator (Siemens Mevatron kD2), which is also used inclinical routine irradiation. Three dishes were each irradi-ated with a single fraction with a dose ranging from 0.25 to30 Gy. After exposure, the cells were examined with aphase contrast microscope. For each dish, 10 standardizedvideoprints of randomly chosen areas were taken with a400-fold magnification. Attached living cells were countedon these pictures.

To determine the effect of the irradiation on cellularproliferation, we used a Bromodeoxyuridine test (BrdU Kit,Boehringer Mannheim, Germany) on Day 6 after irradia-tion. BrdU is incorporated into DNA during S-phase. Cellswere incubated with 10mmol/l BrdU for 6 hours underculture conditions using the above culture mediums. There-after, cells were washed with fresh medium and fixed inmethanol at220°C. A primary antibody to BrdU was addedto the culture dish at a concentration of 4 U/ml. Afterwashing, a color-labeled secondary was used to selectivelyvisualize cells that had incorporated BrdU during S-phase.Such cells could be identified by brown nuclear staining.The BrdU labeling index was calculated as the percentageof stained cells from the total number of cells.

Statistical analysisAll results are expressed as means6 SD. The data were

compared by analysis of variance (ANOVA). Differenceswere considered statistically significant whenp values wereless than 0.05.

RESULTS

Inhibition of lens epithelial proliferationin vitroPreconfluent cultures of rabbit lens epithelial cells were

irradiated. Photon beam was used, due to similar biologicproperties tob-radiation. As shown in Fig. 1, there was adose-dependent effect on the cell counts after irradiation.Control cultures increased in cell counts from 466 9/fieldon Day 3 to 1606 20/field on Day 12. In contrast, irradi-ation with 5 Gy resulted in a reduction of cell counts to38 6 8/field on Day 3 and to 546 12 on Day 12 (p #0.02).Less than 3 Gy did not result in significant reductionof the cell counts over the observation period.

When compared to cultures treated with a dose of 15 Gy(34 6 9/field on Day 3, 436 9/field on Day 12), there is nosignificant difference between irradiation doses of 5 and 15Gy on Day 3. On Day 12, 20 Gy reduces cell numberssignificantly greater than irradiation with 5 Gy (p # 0.05).Cultures irradiated with 30 Gy showed no vital cells after

819Irradiation for prevention of secondary cataract● A. M. JOUSSENet al.

Day 9. Morphologically, irradiated cells showed a morefibroblast-like appearance, with an inhomogenous outerborder and elongated cytoplasmic processes on Day 16 afterirradiation. Control cultures, in contrast, were completelyconfluent, with a round cytoplasm and an inconspicuousnucleus-cytoplasm ratio (data not shown).

To quantitate the antiproliferative effect of irradiation,BrdU labeling for proliferative activity of cultured cells wasperformed (Fig. 2). After Day 6, the irradiated cell numbersdid not significantly change within the irradiated cultures,although there was a dose-dependent effect on proliferativeactivity (Fig. 3). BrdU labeling index was 0.846 0.16 in

control cultures compared to 0.326 0.05 after irradiationwith 5 Gy and 0.026 0 after irradiation with 20 Gy.

P-32 embedded capsular bag rings (emittingb-irradiation) inhibit lens epithelial proliferationin vivo

Cataract surgery was performed in each of 10 eyes. Noeye exhibited severe fibrin exudation or signs of inflamma-tion extending 24 h after surgery. On slitlamp examination,sufficient wound closure was detected in all 10 eyes. In oneof the control eyes, an anteluxation of the implanted IOLoccurred over 2/5 of the circumference 8 weeks after sur-gery. Proliferating lens epithelial cells from the anteriorcapsule grew onto the anterior surface of the IOL in oneother control eye 4 weeks after surgery. Due to furtherproliferative and fibrotic changes, this eye exhibited anteriorsynechiae between the anterior lens capsule and the iris 8weeks after cataract surgery without showing any signs forintraocular inflammation (data not shown). In the controleyes, no visible fibrosis of the anterior capsule or prolifer-ation of lens epithelial cells onto the anterior surface of thelens was apparent. Figure 4 shows two clinical slitlampphotographs of a control eye 12 weeks after implantation(A) in comparison to an eye with P-32 embedded capsularbag ring (B). In the control eye, proliferative PCO is grow-ing into the optical axis, whereas only distinct fibroticchanges of the posterior capsule are seen in the irradiatedeye.

Histologic examination was able to confirm the clinicaldifference between control and treated eyes. Despite metic-ulous cortical cleaning, there were remaining epithelial cellsin all eyes. These cells created adherence between theanterior and the posterior leaflet of the capsule in the pe-riphery of the capsular bag due to proliferating lens epithe-lial cells in 3 out of 5 control eyes, in contrast to 1 out of 5

Fig. 1. Cell counts of vital rabbit lens epithelial cells after irradi-ation. As an example, irradiation with 5 and 20 Gy is shown out ofall doses. The decrease of cells per counted area correlates to theirradiation dose and the time gap between irradiation and obser-vation.

Fig. 2. BrdU test for proliferation in rabbit lens epithelial cells 6days after irradiation. Whereas controls show multiple dark-ap-pearing BrdU positive cells (A), total cell density is reduced incultures irradiated with 15 Gy (B) and 20 Gy (C), in which also theBrdU test is negative (original magnification3600).

Fig. 3. BrdU index (percentage of BrdU positive cells) 6 days afterirradiation shows dose dependently a significant reduction of pro-liferation.


in the P-32 treated group (data not shown). Fig. 5a showsthe histopathologic appearance of the central posterior cap-sule 12 weeks after implantation of the P-32 impregnatedcapsular bag ring. In control eyes, the central area of the thinposterior capsule is covered by accreting cells, which areTUNEL negative, but exhibit some proliferative activity asshown by Ki-67 (Mib-1) and PCNA immunohistochemis-try. Mainly in the periphery, the posterior capsule is wrin-kled due to fibrosis and shrinking induced by the lensepithelial cells. In two eyes these changes were also seen inthe central posterior capsule. In comparison, in treated eyesthe central posterior capsule is free of cells. In a fewsamples there is some distinct cellular material. The equatorzone of the capsular bag is filled with cells and massiveregenerative cortical material in control eyes, whereas pe-ripheral zones of the capsular bag were cell-free in alltreated eyes. Representative sections are shown in Fig. 5b.Macroscopically and microscopically, the zonules appearedsimilar in both control and treated eyes. The edges of thethick anterior capsule at the side of the previous capsulor-rhexis are coiled in control eyes, which also show a signif-icant aberrant differentiation of anterior epithelial cells, aspreviously referred to as pseudofibrous metaplasia.

To determine damage to the surrounding tissue, sectionswere reviewed for signs of increased apoptosis or inhibitionof proliferative activity in the ciliary body and the cornealendothelium. No difference in proliferative activity of cili-ary epithelium or the endothelial cells of the iris capillariesbetween control and treatment group was apparent (Fig. 6a–f). There was no increase in the number of TUNELpositive cells. However, in one animal, the ciliary processesextending toward the capsular bag showed few TUNELpositive cells (Fig. 6 B).

DISCUSSION

Secondary cataract formation from the proliferation ofepithelial cells lining the capsular bag or by seeding of uvealelements (2) is a significant problem following primarycataract surgery. We demonstrate here that the use of a P-32impregnated capsular ring inhibits secondary cataract for-mation with minimal side effects in a 12-week follow-uptime in our rabbit model.

The use of irradiation near the eye at first appears con-tradictory, as the cataractogenic potential of ionizing radi-ation is well known (18, 19). In animals, irradiation can leadto the formation of abnormal fibers and the formation of asubcapsular cataract (20). Induction of a cataract with irra-diation has been shown with doses lower than 10 Gy (21).Interestingly, total body irradiation with a cumulative esti-mated dose of 10 Gy results in a 54% incidence of cataractformation within 5 years, whereas only 10% of the patientswho underwent cataract surgery after the total body irradi-ation exhibited PCO formation (22, 23). This diminution ofsecondary cataract formation following irradiation led to theconcept of using radiation for PCO prevention.

Local irradiation is best performed by utilizing an im-plantable source, in this case a P-32–impregnated capsularring. P-32 was chosen for its relatively short half-life (14.3days), its short path-length in tissue (6 mm maximum inwater), and its relative low cost of production compared tootherb-emitting nuclides. Recent studies in cardiovascularsurgery have demonstrated safety and efficacy using P-32impregnated vascular stents (24, 25). It was therefore pro-posed that a P-32 impregnated ring could be utilized todeposit a fixed radiation dose to a defined anatomical space.This would prevent ingrowth of proliferating lens epithelialcells from the equator to the optical axisin vivo. Due to theshort half-life and the lack of apparent damage to thePMMA material, the activated capsular bag ring could bemaintainedin situ after losing its activity, as its peripheralsite prevents visual disturbance. In this study, the siliconelens material showed no changes in flexibility or clearnessdue to the P-32 irradiation. However, as different lensmaterials might react differently to irradiation, the compat-ibility between the activated capsular bag ring and theimplant lens material should be the subject of investigationsbefore intraocular implantation.

The method of ion implantation of P-32 is useful toenclose ab-emitting radioactive source into the capsular

Fig. 4. Slit-lamp photographs of control eye 12 weeks after im-plantation (A) in comparison to eyes with radioactive capsular bagring (B). Whereas in the control proliferative PCO is growing intothe opical axis, there are only distinct fibrotic changes of theposterior capsule in the treated eye. The figures each show one eyeout of a series of 5 eyes.


bag ring. The P-32 activity applies a radiation dose within arange of 6 mm from the ring surface. The measurement ofthe washoff rate shows a good retention of the P-32 insidethe material. In thein vitro studies, a photon beam was usedto define the inhibition of the proliferation of lens epithelialcells. Although the photon beam is not the ideal surrogatefor the P-32 exposure, it is reasonable for approximation ofthe required dose for inhibition of lens epithelial cell pro-liferation. Irradiation with more than 5 Gy showed a markedantiproliferative effect on cultured lens epithelial cells. Thein vitro doses were applied in a single fraction, in contrastto the in vivo irradiation with P-32, which emits at a con-tinuous rate, accumulating a total dose of 15 Gy after fivehalf-lives. The utilization of low-dose-rate interstitial im-plants is well known. Using this lower dose rate may lead toincreased cell killing by allowing the cells to progress to themore radiosensitive G2 cell cycle phase (26). There they aremore likely to be damaged by ionizing radiation and un-dergo a terminal event. This was the effect seen in theinvivo portion of this study. There was an apparent antipro-

liferative effect on the remaining epithelial cells that pre-vented PCO.

The major concern when using radioisotopic sources isthe dose to normal structures. The first aspect of this issource placement and migration. The capsular bag acts tohold the tension ring in place and prevent its migration. Inthis regard the location is constant. The second aspect is thecalculation of the dose to the surrounding normal structures.As no previous data exist concerning the dosimetry of thisarea, calculations were made for the dose to the ciliarybody, the artificial lens, the corneal endothelium and epi-thelium, and the retina. A computer dosimetry model uti-lizing 100 P-32 point sources was constructed. From thismodel an estimate of the total dose to critical structure wasmade. A reference distance of the radius of the ring plus theaverage energy distance of P-32 was set as the 15-Gyisodose line (Fig. 7). With this calculation, the posteriorcapsule received 15 Gy. The ciliary processes, which wereclosest to the ring, received 7.7 Gy, while the peripheralciliary body received 0 Gy. The dose to the iris was 4.1 Gy

Fig. 5. (a) Histopathology 12 weeks after implantation of the capsular bag ring. In control eyes, the central area of thethin posterior capsule is covered by extracellular material and intermitting lens epithelial cells, which are TUNELnegative, but show some proliferative activity as shown by Mib-1 and PCNA immunohistochemistry. In some areas, theposterior capsule is wrinkled due to fibrotic changes of the lens epithelial cells. In comparison, the central posteriorcapsule is free of cells in treated eyes (arrow, posterior capsule; arrowhead, anterior vitreous membrane). The fewremaining cells are negative for proliferation marker; however, some cells exhibit positive TUNEL staining (originalmagnification3400). (b) Equator zone of the capsular bag in treated eyes is cell free, in contrast to the control eyes,which show massive regenerative cortical material in the periphery. According to the findings in the central posteriorcapsule, only control eyes show proliferative activity, but no TUNEL staining. Control eyes show a significantpseudofibrous metaplasia of anterior epithelial cells (original magnification3400).


and to the cornea 0.9 Gy. The calculated dose to the retinabehind the ora for both humans and rabbits was 0 Gy, as wasthe dose for the macula, using an average sagittal diameterof the adult globe of 23 mm (calculations for the rabbit eyeused an average anterior-posterior diameter of 16 mm).

Immunohistochemistry revealed no difference betweenPCNA staining or Mib-1 in treated eyes compared to con-trols. Neither the corneal endothelium nor the epithelialcells of the ciliary body showed considerably increasedapoptosis rate as analyzed by TUNEL staining. However,especially within the capsular bag, quantitation of the radi-ation effect remains difficult. Radiation as well as UVexposure induces breaks within the DNA (23, 27). In theP-32 labeled capsular tension ring, the effect of the irradi-ation (decrease of proliferation, increase of apoptosis) ishighest during the first weeks after implantation, due toshort half-life. In contrast, all histologic studies were per-formed 12 weeks after implantation. Therefore, the effect onproliferation and apoptosis was likely past and might only

be detected earlier in the course. Most TUNEL-positivecells are found 24 h after a single UV exposure, and there-after this number decreases due to a phagocytosis of thedead cells from the epithelium (27). This recovery process,which has also been described after wounding of lens epi-thelial cell layers (22), might also be active in PCO devel-opment. The TUNEL staining showed almost no positivecells within the capsular bag of control eyes. In treated eyes,the total number of cells was small, which prevents quan-tification of TUNEL-positive cells.

Histology was able to reconfirm the clinical impressionthat no lens epithelial proliferation was seen in the treat-ment group. However, there were fibrotic changes in bothcontrols and treated eyes, although to a different extent.Immunohistochemistry shows lens epithelial cells, whichstain positive for proliferation markers in the controls butnot in eyes with a P-32 activated capsular tension ring.This indicates that the proliferative component of PCOcan be prevented by the P-32 labeled ring; the aberrant

Fig. 5. (Cont’d)


differentiation, however, is reduced in this setting but notfully prohibited.

Based on these first experiments with intraocular low-dose irradiation, P-32 labeled capsular tension rings appear

to inhibit epithelial migration and proliferation and there-fore the formation of secondary PCO. The use of P-32embedded implants could be combined with other mechan-ical devices for inhibition of secondary cataract formation,such as sharpened edges of the tension ring or convexdesign of the implanted IOL. However, in comparison to theslow release systems of pharmacologic substances (28–30),which still maintain the risk of diffusion and damage of thecorneal endothelial cells, the use of a P-32–embedded ten-sion capsular ring is superior in preventing secondary cat-aract formation with fewer side effects. We believe that theuse of appropriate radioactive sources with adjusted half-life times and energies could even prevent a compensatoryhyperplasia, which was observed following short-term irra-diation (31–32).

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Fig. 6. Histopathology of the ciliary body (A–D) and Iris (a–f) ineyes implanted with the P-32 labeled ring compared to eyes witha nonradioactive control ring. HE staining of an eye with P-32labeled capsular bag ring shows a regular histology (A). There wasno increase in the number of TUNEL-positive cells in the treat-ment group. However, in one animal, the ciliary processes extend-ing toward the capsular bag showed few TUNEL-positive epithe-lial cells in one distinct area (B). In both control eyes and treatedeyes, ciliary epithelium showed PCNA-positive cells (shown fortreated eyes in C) and few Mib-1 positive cells (shown for treatedeyes in D). Histology and immunohistochemistry showed no dif-ference between the two groups (control: (a), (b), (c); with P-32labeled ring: (d), (e), (f)). HE staining in a, d. Iris stroma showsnegative TUNEL staining in both control (b) and radiated group(e). PCNA staining is focally positive in endothelial cells of the irisvessels (control (c), after radiation (f)) (original magnification,3400).

Fig. 7. Isodose curves of a P-32 labeled capsular tension ringwithin the capsular bag of a human eye. Activity of 12mCi uponimplantation of the ring (accumulating to 15 Gy after 5 half-lives)was used for reference (bold inner ring). The surrounding isodosecurves from the center toward the periphery stand for activities of7.5 Gy, 3.75 Gy, 1.5 Gy, and 0.75 Gy. Calculations of tissue-specific activities were made using standard anatomical and geo-metric measurements (33). Approximate data can be drawn fromthe schematic figure.


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Low-dose-rate ionizing irradiation for inhibition of secondary cataract formation