British Jouirnial of Ophthalmology, 1978, 62, 815-820

Effects of intraocular miotics on culturedbovine corneal endotheliutnJEFFREY L. JAY AND MARGARET MAcDONALDFrom the Tennent Institutte of Ophthalmology, University of Glasgow, Glasgow

SUMMARY Two cases of severe corneal oedema occurred after the use of intraocular pilocarpine.Experimental investigations were conducted with cultured bovine corneal endothelial cells exposedfor 5 minutes to 1 % pilocarpine solutions of varying composition. Cells were destroyed in solutionsnot isotonic with aqueous humour, and calcium-free ionic solutions caused loss of cell adhesionwithout loss of viability. Low pH or the presence of 1 % pilocarpine had no detectable effects; 1 %acetylcholine chloride in 5 % mannitol (Miochol) also caused cell destruction, and this preparationwas found to be considerably hypertonic. The minimum requirements for the formulation ofintraocular miotics are discussed.

Corneal oedema after operation for cataract isusually an iatrogenic disorder caused by mechanicaland chemical injury to the endothelium of the cornea.This investigation was carried out after 2 cases ofsevere corneal oedema had followed the accidentaluse of an inappropriate intraocular miotic. Thepreparation used was a sterile, preservative-freesolution of 1% pilocarpine nitrate in water in asingle-dose container (Minims, Smith & NephewPharmaceuticals Ltd.). This miotic has a pH of 4 5and an osmolality of approximately 78 mOsllitreand is not designed for intraocular use.

Experiments were set up to study the morpho-logical changes induced in bovine corneal endo-thelial cell culutures in vitro after a brief exposureto various miotic preparations. The relative toxiceffects of pH, osmolality, absence of calcium, orpresence of pilocarpine were investigated. Theprobable cause of the corneal oedema noted in the 2clinical cases included in this report was identified,and the minimum requirements for the formulationof a miotic solution for intraocular use are outlined.

Case reports

CASE IAn apparently uncomplicated intracapsular cryo-extraction for presenile nuclear cataract wasperformed on a 51-year-old woman. Preoperativeexamination of the cornea by specular reflectionwith the slit-lamp microscope hiad shown slight

Address for reprints: Dr J. L. Jay, Tennenit Institute ofOphthalmology, 38 Church Street, Glasgow G I1 6NT

Fig. 1 Severe corneal oedema 24 hours after intra-capsular lens extraction; I % pilocarpine nitrate in waterhad been utsed as an intraocular miotic (Case 1)

cornea guttata but an otherwise healthy endothelialmosaic. At the end of the operation the anteriorchamber was reformed with a sterile solution of 1%pilocarpine nitrate in water without preservative(Minims). On the first postoperative day severecorneal oedema was present (Fig. 1) and only a1-mm peripheral band of the inferior part of thecornea remained clear. The eye healed quickly with-out further complication, but after 7 months therehad been no improvement in the bullous keratopathy.

CASE 2A 69-year-old woman had a similar procedureperformed by the same surgeon on the sameoperating list. Again preoperative assessment of the

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Table I Miotic solutions and their effect on bovine corneal endothelial cell cultures

EffectSolution pH Osmolality Number oJf

(mOs/litre) experiments Cytotoxic Rounding

1% pilocarpine in water 4 5 81 5 4 0

1% pilocarpine isotonic in NaCI 4 6 282 5 0 3

1% pilocarpine isotonic in NaCI with CaCI..* 4 6 308 3 0 0

1% pilocarpine isotonic in PBSt 7-2 300 5 0 4

Isotonic PBS 7 9 296 4 0 2

1% acetylcholine in 5% mannitoll 6 0 430 8 4 0

5% mannitol 7 5 292 4 0 0

*CalciU,m chloride 200 mg/I (anhydrous). tPhosphate buffered saline (NaH2PO4. 2H.O/Na2 HPO4. 1 2H,O). Miochol (Cooper Laboratories)

corneal endothelium showed mild guttate change onthe endothelial surface but no gross disturbance ofthe endothelial cell pattern. The anterior chamberwas reformed with an identical solution of pilo-carpine. Severe oedema of the whole cornea wasseen on the first postoperative day, but during thefollowing 3 months the oedema cleared slowly frombelow. Observation of the corneal endotheliumafter the oedema had cleared showed severe guttatechanges with a distorted cell mosaic. The unoperatedeye showed a normal endothelial cell pattern.

Material and methods

Adult bovine corneas were removed from eyesobtained at the local slaughterhouse. Within 4 hoursof death the endothelial cell layer together withDescemet's membrane was separated by dissectionas described by Stocker et al. (1958). The membranewas cut into small pieces with scissors and transferredto plastic tissue culture flasks of 25 cm2 surface area.Hepes buffered TC 199 culture medium was usedwith 10% fetal calf serum and benzyl penicillin100 units/ml. The cultures were maintained at 37°C.Secondary cultures were obtained by trypsinisationof primary explants with 5 % trypsin in calcium- andmagnesium- free phosphate buffered saline(Dulbecco's). Tests were performed on establishedcell cultures which had been maintained for morethan 1 year and also on fresh cultures only a fewweeks old.To imitate the time scale of a single dose of intra-

ocular miotic experiments were designed to studythe endothelial cell changes after a 5-minute exposureto various test solutions and during 24 hours ofrecovery in culture medium. 2 ml of test or controlsolutions (Table 1) were exchanged for the culturemedium in each flask and allowed to remain incontact with the cell monolayer for 5 minutes. The

solution was poured off and the cells washed with2 ml of TC 199. The cells were then allowed torecover in the growth medium described above.Four fields in each flask were marked for pho-tography and a series of phase-contrast photo-micrographs were taken of each field. The timeintervals were: before exposure to the solution, after5 minutes' exposure, after 2 hours' recovery ingrowth medium, and after 24 hours' recovery. Theentire experiment was carried out in a hot room at37°C, and during each experiment several differenttest solutions were compared with controlpreparations.The photomicrographs were printed at x 300

magnification and with the identity of the testsolution masked; changes in the appearance of thecells in each sequence were recorded. When theanalysis of the photographs was complete, thesolutions were identified.

Test solutions were made up as shown in Table Iso that possible toxic effects of osmolality, pH,absence of calcium ions, or presence of pilocarpinecould be differentiated. Osmolality was determinedby depression of freezing point using a model 3D'Advance' osmometer, and pH was measured on ablood gas analyser. For the purposes of comparisonthe commercial preparation of 1% acetylcholinechloride in 5% mannitol (Miochol, Cooper Labora-tories) was also tested, as was an isotonic solutionof 5% mannitol. A pilot experiment included steriledistilled water as one of the test fluids.

Results

MORPHOLOGICAL CHANGES IN CULTUREDENDOTHELIUMTwo distinct changes in cell morphology were notedin the cultures. Some solutions produced celldestruction which was not obvious at the end of the

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Effects of intraocular miotics on cultured bovine corneal endothelium

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2c 2dFig. 2 Morphological changes in subcultured bovine corneal endothelial cells induced by exposure to 1% pilocarpinenitrate in water; pH 45, 81 mOsIl, for S minutes. (a) Before exposure, the polygonal cellforms are typical of activelyspreading endothelium. (b) The same cells after 5 minutes' exposure; the nuclei are much more prominent and aperinuclear halo is seen. (c) After 2 hours' recovery in TC 199 culture medium the cells show some cytoplasmicdisruption. (d) At 24 hours severe cell destruction has occurred (phase contrast, x 100)

5 minutes' exposure but developed during thesubsequent 24 hours in normal growth medium.This is called the cytotoxic effect (Fig. 2). Othertest solutions caused the cells to lose adhesion andseparate from each other and from the substrate.This is described as cell rounding, and it occurred assoon as the solutions were added to the culture flasks.

Residual cells rapidly regained their normal mor-phology during the recovery phase (Fig. 3).These changes were seen both in actively dividing

areas at the edges of cell colonies and in confluentareas at the centre of the colonies, but the activecell forms showed more marked changes of eithertype. Recently established cultures seemed just as

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3c 3dFig. 3 Response of'subcultured bovine endothelial cells to calcium-free ionic solutions. (a) Initial appearance. (b) Thesame area after S minutes' exposure to 1% pilocarpine nitrate isotonic in sodium chloride. Cells lose adhesion to thesubstrate and become rounded. (c) After 2 hours' recovery in TC 199 culture medium the cells have again flattenedand spread out. (d) After 24 hours' recovery (phase contrast, x 100)

susceptible to the test solution as did cultures whichhad been established for over a year.

EFFECTS OF SOLUTIONS UNDERINVESTIGATIONCytotoxic cffect.-After 5 minutes' exposure to 1%pilocarpine nitrate in aqueous solution (pH 4 5,

81 mOs/litre) the cells showed increased prominenceof nuclei with a clear perinuclear halo (Fig. 2b).These cells showed some distruption of cytoplasmin the early recovery period at 2 hours (Fig. 2c), andby 24 hours (Fig. 2d) appreciable cell death hadoccurred. Similar but more severe changes occurredwhen the cells were exposed to distilled water.

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Effects of intraocular miotics on cuiltured bovine corneal endothelium

1 % acetylcholine chloride in 5% mannitol (Miochol;pH 6 0, 430 mOs/litre) also caused appreciable celldestruction after 24 hours' recovery, although therewere, no detectable changes in the ealier photographs.Both these preparations causing cell damage hadstubjected the cells to considerable osmotic shock.Rounding.-Exposure to isotonic calcium-free

ionic solutions (Table 1) caused the cells to separate,lose adhesion to the substrate, and form sphereswhich were visible in the 5-minute micrographs(Fig. 3b). This did not affect the viability of the cells,as rounded cells still in contact with the substraterapidly flattened on to the bottom of the flasksduring the recovery period (Fig. 3c and 3d). Againactively spreading cells showed greater changes thandid confluent cell areas at the centre of colonies.Table 1 shows that the cytotoxic effects occurred

only in non-isotonic solutions while roundingoccurred only in isotonic calcium-free ionic solutions.The experiment did not show any identifiablechanges which could be attributed to the low pHof some solutions or to the presence of pilocarpine.

1% pilocarpine isotonic in sodium chloride with200 mg of calcium chloride per litre, pH 4 5, hadno detectable effect on the cells after 5 minutes'exposure. An isotonic solution of 5% mannitol wasalso without effect.

Discussion

The experimental results suggest that the severecorneal oedema which occurred after the 2 cases ofintracapsular lens extraction was related to theextreme osmotic shock which was caused by theintraocular use of a solution of 1% pilocarpinenitrate in water. The presence of pilocarpine whenisotonic in saline with added calcium had no detect-able effect on bovine endothelial cell cultures after5 minutes' exposure, but after several hours'exposure to pilocarpine a dose-related toxic effectdoes occur (Coles, 1975a).Addition of a phosphate buffer to neutralise the

rather acid pilocarpine solutions showed no detect-able benefit and may have accelerated the roundingeffect by combining with residual calcium in thecells to enhance the effect of a calcium-free solution.Buffered preparations would have the disadvantageof rendering the miotic unstable, as pilocarpine inneutral solution undergoes spontaneous isomerisa-tion to inactive isopilocarpine after a few weeks ofstorage at room temperature (Anderson and Cowle,1968). The theoretical benefit of using a neutralpilocarpine for intraocular injection would have tobe balanced by the risk of cold shock to the cornealendothelium if the buffered pilocarpine requiredstorage at low temperature.

Although it has been shown by Kaye et al. (1968)that calcium is essential to maintain the junctionalcomplexes of endothelial cells, it is unlikely that theomission of this divalent cation would causeappreciable endothelial cell death. On the other handthe cell rounding and loss of adhesion demonstratedin this study might be a factor in causing temporarydisruption of the continuity of the endothelialbarrier in vivo. With more severe loss of adhesionsome cells might separate completely fromDescemet's membrane, so that even after recoverythere would be a reduction in the endothelial cellpopulation. This might be enough to provokedecompensation in a cornea with an already reducedendothelial cell density.The damage produced by 1 % acetylcholine in 5%

mannitol may be related to the hypertonicity of thispreparation, but this has not yet been fully clarified.Mannitol alone in isotonic strength (5%) did notseem to cause changes.

Extracapsular lens extraction, phacoemulsifi-cation, intraocular lens implantation, and the use ofsuction cutting instruments in the anterior segmentmay require prolonged irrigation of the anteriorchamber with an artificial aqueous humour.Perfusion fluids should not be toxic to the cornealendothelium, as the use of an unphysiologicalsolution is likely to potentiate any mechanicaldisruption of the endothelium during the surgicalmanipulation. A suitable artificial aqueous forprolonged perfusion has been shown by Edelhauseret al. (1976) to require a bicarbonated Ringer'ssolution with added glucose, glutathione, andadenosine (GBR). The same authors suggest that forlow volume irrigation of the anterior chamber lessfavourable fluids such as sodium chloride 09% orRinger-lactate solution might be adequate.As miotics for intraocular use are likely to bathe

the corneal endothelium only for a short time, theirformulation need be less complex than that forsolutions used for anterior chamber perfusion. Thereare, however, some important factors to beobserved. The pilocarpine should be prepared in aphysiological salt solution containing calcium tomaintain cell adhesion, and the final osmolality mustbe near that of aqueous humour, approximately300 mOs/litre. No bacteriostatic preservative shouldbe used, as these may cause severe endothelialdamage. Maurice and Perlman (1977) have shownthat benzalkonium chloride in the concentrationused in eye drops causes irreversible destruction ofrabbit corneal endothelial function after a fewseconds of exposure. In addition toxic effects ofintraocular epinephrine have been attributed by Hullet al. (1975) to the bisulphate preservative ratherthan the drug itself, and Coles (1975b) has shown

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that the endothelial damage caused by erythromycinis aggravated by the addition of benzyl alcoholpreservative.

Autoclave sterilisation is suitable for pilocarpineand may be preferable to filter sterilisation, as somemembrane filters are coated with a detergent wettingagent which can damage cell cultures if the filtersare not washed thoroughly before use. Alternatively,grades of filters specifically for tissue culture mediamay be used, as these have the wetting agent omitted(Mulvany, 1972).

References

Anderson, R. A., and Cowle, J. B. (1968). Influence of pHon the effect of pilocarpine on aqueous dynamics. BritishJournal of Ophthalmology, 52, 607-611.

Coles, W. H. (1975a). Pilocarpine toxicity. Archives ofOphthalmology, 93, 36-41.

Coles, W. H. (1975b). Effects of antibiotics on the in vitro

rabbit corneal endothelium. Investigative Ophthalmology,14, 246-250.

Edelhauser, H. F., van Horn, D. L., Schultz, R. O., andHyndiuk, R. A. (1976). Comparative toxicity of intra-ocular irrigating solutions on the corneal endothelium.American Journal of Ophthalmology, 81, 473-481.

Hull, D. S., Chemotti, M. T., Edelhauser, H. F., van Horn,D. L., and Hyndiuk, R. A. (1975). Effect of epinephrineon the corneal endothelium. American Journal of Ophthal-mology, 79, 245-250.

Kaye, G. I., Mishima, S., Cole, J. D., and Kaye, N. W.(1968). Studies on the cornea. VII. Effects of perfusionwith a calcium-free medium on the corneal endothelium.Investigative Ophthalmology, 7, 53-66.

Maurice, D., and Perlman, M. (1977). Permanent destructionof the corneal endothelium in rabbits. InvestigativeOphthalmology, 16, 646-649.

Mulvany, J. G. (1972). Sterile filtration. In Animal TissueCulture: Advances in Technique, p. 27. Edited by G. D.Walsey. Butterworths: London.

Stocker, F. W., Eiring, A., Giorgiade, R., and Giorgiade, N.(1958). A tissue culture technique for growing cornealepithelial, stromal, and endothelial tissues separately.American Journal of Ophthalmology, 46, 294-298.

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