A C T A O P H T H A L M O L O G I C A VOL. 55 1 9 7 7
From t h e Department of Ophthalmology, Komrnunehospitalet, Copenhagen (Heads: P. Brcendstrup, S. E. Lorentzen, M . S. Norn and K. N s r s k o v )
EFFECTS OF OPHTHALMIC VEHICLES ON THE STABILITY OF THE PRECORNEAL FILM BY
MOGENS S. NORN and ANNA OPAUSZKI
The break up time (B. U . T . ) of the precorneal film has been studied before and after application of 34 different vehicles. T h e material examined comprised 646 eyes. Maximum increase of the B. U. T. was obtained with 2 O / o methyl cellulose (four times) and 10 O / o polyvinyl alcohol (seven times). These vehicles in the usually employed concentrations, fat-free ointment (polyethylene glycol), acetyl cysteine and polysaccharide (dextran) affected a less pronounced prolongation. The B . U . T . was reduced four or five times by fatty, anhydrous ointments and by silicone oil, about two or three times by emulsions and oils, and twice by 0.01 O / o benzalkonium chloride. The clinical significance of the B. U. T. alterations is discussed.
Key words: ophthalmic vehicles - ointment - oil - artificial tears - precorneal tear film - break up time (B. U. T.) - wetting time - wettability - stability.
The stability of the precorneal tear film is a n important factor in the integrity of the epithelium. Some ophthalmic vehicles add to the stability. W e treat an unstable precorneal film (dry eye syndrome) with artificial tears, and we rub corresponding substances (wetting agents) upon hard contact lenses Received September 16, 1976.
23
Mogens S . Norn and Anna Opauszki
in order to protect the cornea. W e add vehicles to eye drops to prolong the period of contact of the active drug in the hope of promoting its absorption. The stated factors (stability of the precorneal film, contact period, and promoted absorption) may vary independently. They are concepts that should definitely be kept apart. Norn in 1969 introduced a clinically useful method for objective measurement of the stability of the precorneal film (wetting time). Lemp et al. in 1970 assessed the same method, calling it break up time (B. U. T.). The time interval is measured from the conclusion of a blink till occurrence of holes in the precorneal film. The B. U. T. is independent of the patient’s age (Norn 1969; Lemp et al. 1973). It tends to be longer in males than in females (Norn 1969, not confirmed by Lemp 1973). In normal eyes the B. U. T. is 10 seconds or longer (Lemp 1973). Lemp et al. (1975a) studied the effect of artificial tears on 12 normal subjects. They tested 13 different commercial preparations, measuring before and repeatedly (at 15 minute intervals) after the instillation. The B. U. T. increased significantly, most often two or three times, as a sign of improved stability of the precorneal film. The effect was most prolonged with those drugs to which “B. P. Polymer” had been added (adapt, adapettes, adsorbotear) and tears naturale (Alcon 0413), about 90 min against 35-60 min for the others (lacril, liquifilm I and 11, isoptotears, contique, ultratears, presert, lytears - though no effect of tearisol). They point out that salt concentration and preservatives may influence the B. U. T. The preservative benzalkonium chloride in a concentration of 0.01 O/O reduces the B . U . T . four times in rabbit and about twice in man (Wilson et al. 1975, 3 1 human eyes). The angle of contact between precorneal film and dry cornea gives us an indirect impression of the stability of the precorneal film, a narrow angle effecting greater extension of fluid and consequently better wettability and film stability. Lemp et al. (197513) developed such a method on enucleated rabbit cornea. However, the results are not directly transferable to human clinical practice. Rocher (1975) and Shively (1975) measure the angle with a view to contact lenses. The contact period and the influence of vehicles on this can be indicated by estimating the reduced outflow to the nose (Linn & Jones 1968), or by measuring the fall of the technetium suspension concentration in the conjunctiva (Patton et al. 1975; Hardberger et al. 1975). The dependence of the absorption on the vehicle can, for instance, be measured by uptake of radioactive 3H thymidine in the epithelial cells of the rabbit 24
Vehicles and Precorneal Film ( B . U.T.) cornea (Swanson e t al. 1968). A s f o r neomycin, the effect of t h e vehicle on pseudomonas keratitis h a s been studied o n rabbit by Bach e t al. 1970. We cannot decide o n the basis of the literature w h e t h e r methyl cellulose (MC) o r polyvinyl alcohol (PVA) is preferable a s an ophthalmic vehicle. T h e results differ, d e p e n d e n t o n the parameters examined, concentration, viscosity, etc. (cf. Krishna et al. 1964, 1965; Linn & Jones 1968; Swanson e t al. 1968; Bach et al. 1970; H a r d b e r g e r et al. 1975; P a t t o n et al. 1975).
Ointment and oil have the opposite effect of MC and PVA. T h e y reduce the B.U.T. and consequently the stability of t h e precorneal film, thus tending to d a m a g e the epithelium ( N o r n 1975). The object of the present study h a s been t o assess o n t h e basis of h u m a n experiments t h e influences of well-known and less familiar vehicles o n t h e stability of the precorneal film. We have concentrated particularly o n investi-
gating the d a m a g i n g effect of ointments o n the precorneal film and whether other h a r m f u l vehicles exist; f u r t h e r , w h e t h e r MC o r PVA is preferable, and which substance and concentration prolongs the B. U. T. maximally.
Method The patient was placed at a Haag-Streit slit lamp in a half-lit room. 10 p l of a fluorescein mixture with a local anaesthetic added was instilled. This anaesthetic had no influence on the B. U. T. (Norn 1969). The mixture was composed as follows: fluorescein, 0.125 O/o, oxibuprocaine (NovesinR), 0.3 Oio, phenylmercuric nitrate, 0.0025 O/o, sodium chloride to isotonicity. The patient was requested to blink two or three times. A stop watch was started immediately after the conclusion of the final voluntary complete blink. The patient then looked straight forward without blinking. The lid was not supported. The smooth fluorescent precorneal film was studied in the slit lamp (ten times magnification). The light was a 1-2 mm broad, obliquely incident, vertical, cobalt-filtered beam of light, which was moved from side to side over the cornea until the first hole was noticed in the precorneal film. The stop watch was stopped, and the B . U . T . read in seconds. The examination was repeated after renewed blinking. The average of the two examinations constituted the initial B. U. T . The ophthalmic vehicle to be studied was then instilled, using the original dropping bottle (about 50 ~ 1 ) . One or two min later the fluorescein mixture was instilled again, and the B . U . T . was read twice.
25
Mogens S. N o r n and A n n a Opauszki Statistics
T he ratio of the initial B . U . T. average to the B . U . T . after the installation of a vehicle was calculated separately for each case. The mean B. U. T.-factor concerning each vehicle was calculated with a standard error of the mean ( S E M ) . T he coefficient of variation of the two initial B. U. T. determinations was calculated by the formula:
where d is the difference between the results of duplicate determinations, n the number of duplicate determinations, and A the mean of the B. U. T.
Material
A total of 336 patients from an ophthalmic out-patient department and an ophthalmic clinic were examined. Their ages ranged from 14 to 90 years. The percentage distribution was as follows, stated for 10-year age groups: 3.3-4.87.1-7.7-15.4-27.4-25.6-8.3-0.3 per cent, i. e. the greatest number in the age groups of 60 and 70. Females predominated (63.7 per cent). They were approximately equally distributed over the different vehicle groups. The investigation comprised 646 eyes. Of these, 20.4 per cent had an abnormal anterior eye section (65 per cent aphakia, 3.3 per cent keratitis, 2.5 per cent exophthalmos, 1.7 per cent iritis, 1.5 per cent glaucoma subjected to operation, etc.). The remaining were eyes subjected to routine examinations, or to examination for refraction anomalies, etc. The initial B. U. T. value was pathological (below 10 seconds) in 25.1 per cent. The pathological cases were fairly equally distributed over the different vehicle groups, and the B. U. T. alterations did not differ from those of the normal material.
Results The results are seen in the tables. Table I shows the vehicles prolonging the B. U. T., i. e. stabilizing the precorneal film, while the vehicles reducing the B. U. T. have been set out in Table 11. Finally, Table 111 comprises the vehicles with no statistically significant effect on the B. U. T. The coefficient of variation for the initial duplicate determination is stated within the individual groups. It was 30.7 per cent for the total material.
26
Vehicles and Precorneal Film (B. U . T.) Table I . Vehicles increasing the stability of the precorneal film.
B. U. T.-
I
0) 1)
2) 3) 4) 5)
Initial
factor increase k SEM
Vehicle
B. U. T. (seconds)
Coefficient of variation (per cent)
Number of eyes
I
Methyl cellulose 0.5 O/ol) Methyl cellulose 1.5 O / o O ) Methyl cellulose 2.0 O / o O )
1.36 f 0.14 4.36 f 0.72 2.48 f 0.45
22.7 28.6 21.3
2.8 41.4 28.4
14 14 12
Polyvinyl alcohol 1.4 O/o2) Polyvinyl alcohol 3 O/oO) Polyvinyl alcohol 10 "00)
1.89 ? 0.26 2.96 f 0.51 7.16 f 2.48
27.5 12.4 21.8
27.8 33.0 15.3
16 11 12
Polyvinyl alcohol 20 O / o O ) Polyvinyl alcohol 40 O/oO) Adapts)
2.82 k 0.57 2.30 k 0.81 5.13 k 1.34
24.4 35.1 17.5
50.9 21.3 38.7
10 11 30
Adapettes4) Adsorbotears) AdsorboNaCl 5 O / o
3.01 k 0.81 2.42 k 0.32 2.55 ? 0.66
11.7 17.0 21.7
36.1 42.6 50.8
20 28 15
Carbowax No. 1500 Dextran 10 O / o Acetyl cysteine 20 "0
2.12 f 0.21 4.06 k 0.59 1.98 k 0.32
16.8 20.5 19.6
27.7 20.8 12.4
21 11 14
no preservative nor other substance added sodium chloridt and phenylmercuric nitrate 0.001 O/o added (oculoguttae viscosae). sodium chloride and chlorbutol 2 O i o added (liquifilm tears). hydroxyethyl cellulose 0.44 "/a, B. P. adsorbobase, thiomerosal 0.002 O / o , EDTA 0.05 O/o. B. P. adsorbobase, thiomerosal 0.001 O i o . polyvinyl pyrrolidine 1.67 Oio, B. P. adsorbobase, hydroxyethyl cellulose C.44 O i o , thiomerosal 0.002 "/a, EDTA 0.05 O / o .
Mucomimetics
Methyl cellulose ( M C ) is usually employed in a 0.50/0 concentration in the treatment of keratoconjunctivitis sicca. At this concentration the B. U. T. value was raised significantly by the factor 1.36. Higher concentrations prolonged the B. U. T., which became four times longer at a concentration of 1.5 O/O. The difference is significant ( P < 0.001).
27
Mogens S. Norn and Anna Opauszki Table 11. Vehicles reducing the stability of the precorneal film.
B. U. T.Vehicle
factor reduct.
Initial
B. U. T. (seconds)
f SEM
Coefficient of variation (per cent)
Number of eyes
3.81 & 0.81 2.17 k 0.33
25.0 11.6
16.3 38.0
12 10
Petrol jelly and liquid 5.44 & 0.78 paraffinl) Petrol. jelly, liquid paraffin 1.79 f 0.21 and emulgator2)
24.2
24.5
20
20.0
33.3
12
Petroleum jelly Stearin
Cetacean ointments) Cosmea moisture cream4) Wool fat w. waters)
3.09 ? 1.04 5.32 zk 1.32 4.19 C 1.07
18.9 18.1 22.8
14.0 54.0 16.0
12 15 10
Glycerol ointments) Arachis oil Olive oil
k 0.17 k 0.1s k 0.59 3.33 k 0.53 2.33 k 0.36 3.72 k 0.63 4.67 C 0.69
15.5 13.7 20.6
38.6 49.0 26.2
26 10 10
24.7 19.2 23.5
43.8 21.2 28.7
10 10 13
22.0 27.9 29.4
11.5 28.3 35.0
10 12 27
15.9 11.0 23.6
28.8 26.7 48.0
18 11 14
Liquid paraffin Ophthasiloxans) Silicone oil 20 O / a V Silicone oil 100 Oio Antifoam A Benzalkonium chloride 0.01 OioS) Cocaine 2 oio Cocaine 4 o/o Albumen film on cornea
1.83 1.50 2.86
7.23 & 1.26 1.90 f 0.26 1.47 1.72 2.19
f 0.10 f 0.15 k 0.27
80 Oio petroleum jelly and 20 O / o liquid paraffin (simple eye Gintment Ph. Nord. 63). 80 o/o petroleum jelly, 20 "/o liquid paraffin with emulgator (simple eye ointment Ph. D. 48). 3) white wax, spermacet, arachis oil and water (cold cream). 4) wool fat, emulgator, monostearin, propylene glycol, water, preservatives. 5 ) glycine and wheat starch. 6) octylphenol polyoxyethylene 0.025 g, distearate of polyethylene glycol 0.05 g, dimethyl polysiloxan g. s. ml 10. 7 ) in simple eye ointment. 8) with O.g0/o sodium chloride added. 9) 25O/o water content. 1)
2)
28
Vehicles and Precorneal Film ( B . U . T.) Table I I I . Vehicles with no significant effect on the stability of the precorneal film.
B. U. T.Vehicle
Tendency
factor
f SEM
Coeff. Number of of eyes (seconds) variation Initial
B. U. T.
I
Sodium chloride 0.9 O / o Sodium chloride 5 O i o Ultracortenol susp.1) Gelatin powderz) Glycerine 100 O / o Glucose 50 Oio Cotton plug in inf. fornix p H buffer 9.24) p H buffer 10.05) Exploration creams)
fall rise rise fall fall fall fall fall fall fall
1.39 ?z 0.20 1.36 ? 0.17 1.07 k 0.11 1.34 k 0.20 1.14 f 0.13 1.74 ? 0.50 1.72 ? 0.29 1.78 ? 0.46 1.83 f 0.49 4.14 ? 1.95
17.0 20.1 10.4 19.4 22.0 28.4 20.8 18.4 24.0 22.7
14.6 9.0 29.4 15.0 30.0 37.6 30.9 61.3 57.0 44.0
14 13
10 10 14 10 14 12 16 21
microcrystalline suspension of prednisolone acetate 0.5 O/o, with sodium chloride, sodium phosphate and benzalkonium chloride added. 2) sieve 0.7 mm. 3 ) glycerol 16.5 "'/o, boric acid, tragacanth mucus. 4) sodium borate buffer with phenetanol 0.5 O / o added, isotonic. 3 sodium borate buffer, adjusted with N a 0 H, slightly hypertonic. 1)
At still higher concentrations (3 O/O, 4 O/O, 5 O/O and 10 O/O) the MC was so thick that it could not pass through a pipette, but had to be applied with a glass rod. It mixed poorly with the precorneal film, in which lumps and air bubbles were seen. At higher MC concentrations than 1.5 O/O the B. U. T. decreased with rising concentrations (the decrease between 1.5 O/O and 2 O/o was significant, P < 0.05). So, the BUT increase is not only a function of the viscocity of the vehicle.
Polyvinyl alcohol (PVA) is generally used in a concentration of 1.4 O/O. This was found to prolong the B. U. T. significantly by the factor 1.89 (Table I). Rising concentrations prolonged the B. U. T. until a concentration of 10 O/O PVA, which gave a seven times increase of the B. U. T. (P < 0.05). An additional rise of the PVA concentration caused a reduction of the B. U. T. 20 O / o PVA easily passed through the pipette, while 40 O / o was of an excessively viscous consistency. 29
Mogens S . Norn and Anna Opauszki
Adapt, Adapettes, and Adsorbotears are artificial tears, which prolonged the B . U . T . , though not more so than optimum concentrations of MC and PVA. (The results of our investigations harmonize with those of Lemp et al. (1975a). The latter having, however, been represented graphically without SEM, no comparison is permissible between the groups. On the other hand, the duration of the effect has not been recorded in the present study). Addition of 5 o/o sodium chloride to Adsorbotears did not seem to alter the effect of this on the B. U. T. Other substances prolonging the B.
U. T.
Dextran in a 10 O/o concentration is a hyperosmotic polysaccharide used as a plasma substitute. It prolongs the B. U. T. to the same extent as optimal concentrations of MC and PVA. Polyethylene glycol (Carbowax 1500, Macrogol) is a viscous fat-free ointment, which prolongs the B. U. T. more than both MC and PVA in the current concentrations. Acetyl cysteine (Mucomyst, 20 o/o) is a mucosolvent, collagenase-inhibiting compound. It prolongs the B. U. T. equally as long as both MC and PVA in the current concentrations. Ointments
Fatty ointments reduce the B. U. T. (Table 11). Holes soon occur in the precorneal film, with resulting exposure and desiccation of the cornea. The most commonly used ointment in Scandinavia (simple eye ointment, Ph. Nord. 63) is a mixture of 80 o/o petroleum jelly and 20 O / o liquid paraffin. This ointment has a significant greater effect than the corresponding emulsion (simple eye ointment with emulgator, Ph. D. 48, P < 0.001). On the other hand, no significant difference was seen for water in oil uersus oil in water-emulsion.
Stearin did not melt at the corneal temperature, but remained in the form of flakes in the precorneal film. Holes soon occurred, sometimes close to a stearin flake and sometimes distant from this. The B. U. T. was reduced in the same manner and to the same extent as by an indifferent albumen film (piece of egg albumen membrane) placed in the precorneal film. Petroleum jelly melted and spread as drops across the precorneal film. It has a greater tendency to reduce the B. U. T. than stearin. The softer simple ointment without emulsifier reduced the B. U. T. as much as petroleum jelly. The fat-free ointment unguentum glyceroli reduced the B. U. T. twice only. 30
Vehicles and Precorneal Film (B. U . T.) Oils
Oil liquefies at room temperature. Instilled oil was seen to spread at a faster rate over conjunctiva and cornea than ointment. Oil had a significantly reducing effect on the B. U. T. (two or three times). Arachis oil caused less damage than olive oil and liquid paraffin (P< 0.01 and P < 0.05, respectively). Arachis oil had the same effect as the mild simple ointment emulsion (petroleum jelly, liquid. paraf. with emulgator, Ph. D. 48). So, the reduction of BUT is not only a question of the melting point. Silicone
Antifoam A is a silicone-containing ointment, which reduces the surface activity (raises the surface tension). The B. U. T. was found to be reduced very considerably, more than with 20 O / O and 100 O/O silicone oil and to the same extent as with non-emulsified simple ointment.
Ophthasiloxan has been specially prepared for the purpose of increasing the evaporation from an intact cornea. The B. U. T. was, however, reduced less by this compound than by Antifoam A.
Other substances reducing the B. U. T.
Cocaine surface anaesthesia reduced the B. U. T., though no more than the simple ointment emulsion. Benzalkonium chloride in the usually employed concentration as a preservative reduced the B. U. T. (Table 11). This bore out Wilson’s result. Benzalkonium chloride in a high - toxic - concentration (0.5 O/O) effected pronounced micropunctate fluorescein staining. This phenomenon rendered B. U. T. reading difficult and may perhaps explain why we found a prolonged B. U. T. (2.09 k 0.40, 11 eyes).
Drugs having no influence on the B. U. T.
The vehicles found to be without any significant influence on the B. U. T. have been set out in Table 111. As might be expected, the B. U. T. was unaffected by physiological saline. Various hyperosmotic vehicles did not alter the B. U. T. ( 5 O/ o sodium chloride, 100 O/O glycerin, 50 O / O glucose). Evidently a cotton plug placed in the inferior fornix did not absorb enough to alter the B. U. T. 31
Mogens S. Norn and Anna Opauszki
A microcrystalline suspension and a gelatin powder floating in the precorneal film did not alter the B. U. T. An alkaline buffer (pH 10) is a mucosolvent, though not sufficiently so to alter the B. U. T.
Vital staining
After introduction of a vehicle and subsequent B. U. T. reading, vital staining was performed with a mixture of 1 O/O fluorescein and 1 O/O rose bengal (Norn 1974). As stated above, a high benzalkonium chloride concentration (0.5 O/n) gave a pronounced widespread micropunctate fluorescein staining, indicating damage and breach of continuity of the epithelium (cf. Tsnjum 1975). No rose bengal staining was seen. Fatty ointments, oils, and silicone gave punctate, irregularly grouped, often lacuna-like rose bengal staining of the cornea and the adjacent exposed part of the bulbar conjunctiva. Only slight fluorescein staining was seen. The vital staining was in some instances so pronounced as to resemble that in keratoconjunctivitis sicca. The reduced B. U. T. caused desiccation of the exposed part of the epithelium. MC and PVA prolonged the B . U . T . They provoked no vital-stainable phenomena, not even in high concentrations.
Discussion Unfortunately, the above procedure (B. U. T.) has a fairly high coefficient of variation. Lemp et al. (1970) on clinical examination overcame this problem by employing the average of five readings in the individual clinical case. Sauter (1976) even undertook ten readings. W e have employed no more than two plus two (before and after application of the vehicle) to avoid tiring the patient too much. On the other hand, B. U. T. reading is the most direct and simple method of examination for the purpose of assessing the precorneal film under practical clinical conditions. Many well-known vehicles influence the B. U. T. so much (about 300-400 per cent) that the changes are demonstrable even though the coefficient of variation is high (about 30 per cent). The stability of the precorneal film is increased by MC and PVA. A maximum increase is obtained by a high concentration of PVA (10 n/o), without the liquid becoming too viscous. The increase effected by raising the MC concentration is
32
Vehicles and Precorneal Film (B. U . T.)
more limited (maximum at 1.5 O / O ) . The B. U. T. can also be prolonged by using fat-free ointment (Macrogol) o r hyperosmotic polysaccharide (Dextran), and others. These factors are of importance in the treatment of pemphigoid and keratoconjunctivitis sicca, in protection of the cornea, and where the period of contact of drugs is concerned. It is worth noting that ordinarily employed ointments and oils reduce the stability of the precorneal film. This harmful effect is demonstrable by rose bengal staining. Such substances are therefore contra-indicated in pemphigoid and keratoconjunctivitis sicca. Ointment is used to protect the cornea from evaporation. However, protection is only yielded in the cases where the patient cannot blink (facial nerve palsy, during sleep, and under a bandage). In all other cases blinking will provoice holes in the ointment layer covering the cornea with resulting desiccation in spots (Norn 1976). In corneal oedema with intact epithelium, the oedema can be reduced by accelerating the evaporation. This can be effected by shortening the B. U. T., e. g. by means of silicone-containing drugs.
Acknowledgment Our thanks a.re due to Mr. S. Trager (Burton, Parson & Co., USA) for valuable discussions.
References Bach F., Riddel G., Miller C., Martin J. A. & Mullins J. D. (1970) T h e influence of vehicles on neomycin sulfate prevention of experimental ocular infections in rabbits. A m e r . /. Ophthnl. GS, 659-662. Hardberger R., Hanna C. & Boyd C. M. (1975) Effects of drug vehicles on ocular contact time. Arch. Ophthal. (Chicago) 93, 42-45. Krishna N. & Brow F. (1964) Polyvinylalcohol as a n ophthalmic vehicle. A m e r . /. Ophthal. 57, 99-106. Krishna N. & Mitchell B. (1965) Polyvinyl alcohol as a n ophthalmic vehicle. A m e r . /. Ophthal. 59, 860-864. Lemp M. A,, Holly F. J., Iwata S. & Dohlman C. H. (1970) The precorneal tear film. Arch. Ophthal. (Chicago) 83, 89-94. Lemp M. A. & Hamill J. R. (1973) Factors affecting tear film break up in normal eyes. Arch. Ophthal. (Chicago) 89, 103-105. Lemp M. A,, Goldberg M. & Roddy M. R. (1975a) T h e effect of tear substitutes on tear film break up time. Invest. Ophthal. 14, 255-258.
33 Acta ophthal. 55, 1
3
Mogens S . N o r n and A n n a Opauszki Lemp M. A. & Szymanski E. S. (197513) Polymer adsorption at the ocular surface. Arch. Ophthal. (Chicago) 93, 134-136. Norn M. S. (1969) Desiccation of the precorneal film. I. Corneal wetting time. Acta ophthal. (Kbh.) 47, 865-880. Norn M. S . (1974) External Eye. Methods of Examination. Scriptor, Copenhagen, pp. 200. Norn M. S. (1975) Correspondance in A m e r J . Ophthal. 79, 335. Norn M. S. (1976) Eye Ointments - Application, Elimination and protective Action. Ophthalmology Digest (to be published). Patton Th. & Robinson J. (1975) Ocular Evaluation of Polyvinyl Alcohol Vehicle in Rabbits. Journ. of Pfiarm. Science 64, 1312. Sauter J. J. M. (1976) Xerophthalmia and measles in Kenya. Drukkerij van Denderen, Groningen, pp. 235. Swanson A. A., Jeter D. J. & Gregor C. R. (1968) The influence of ophthalmic vehicles on 3H thymidine in normal rabbit corneas. Ophthalmologica 156, 425-436. Tsnjum A. M. (1975) Effects of benzalkonium chloride upon the corneal epithelium studied with scanning electron microscopy. A c t a ophthal. (Kbh.) 53, 358-366. Wilson W. S., Duncan A. J. XC Day J. L. (1975) Effect of benzalconium chloride on the stability of the precorneal film in rabbit and man. Brit. J . Ophthal. 59, GGT.
Author’s address: M. S . Norn, M.D., Department of Ophthalmology, Kommunehospitalet, DK-1399 Copenhagen K, Denmark.
34
From t h e Department of Ophthalmology, Komrnunehospitalet, Copenhagen (Heads: P. Brcendstrup, S. E. Lorentzen, M . S. Norn and K. N s r s k o v )
EFFECTS OF OPHTHALMIC VEHICLES ON THE STABILITY OF THE PRECORNEAL FILM BY
MOGENS S. NORN and ANNA OPAUSZKI
The break up time (B. U . T . ) of the precorneal film has been studied before and after application of 34 different vehicles. T h e material examined comprised 646 eyes. Maximum increase of the B. U. T. was obtained with 2 O / o methyl cellulose (four times) and 10 O / o polyvinyl alcohol (seven times). These vehicles in the usually employed concentrations, fat-free ointment (polyethylene glycol), acetyl cysteine and polysaccharide (dextran) affected a less pronounced prolongation. The B . U . T . was reduced four or five times by fatty, anhydrous ointments and by silicone oil, about two or three times by emulsions and oils, and twice by 0.01 O / o benzalkonium chloride. The clinical significance of the B. U. T. alterations is discussed.
Key words: ophthalmic vehicles - ointment - oil - artificial tears - precorneal tear film - break up time (B. U. T.) - wetting time - wettability - stability.
The stability of the precorneal tear film is a n important factor in the integrity of the epithelium. Some ophthalmic vehicles add to the stability. W e treat an unstable precorneal film (dry eye syndrome) with artificial tears, and we rub corresponding substances (wetting agents) upon hard contact lenses Received September 16, 1976.
23
Mogens S . Norn and Anna Opauszki
in order to protect the cornea. W e add vehicles to eye drops to prolong the period of contact of the active drug in the hope of promoting its absorption. The stated factors (stability of the precorneal film, contact period, and promoted absorption) may vary independently. They are concepts that should definitely be kept apart. Norn in 1969 introduced a clinically useful method for objective measurement of the stability of the precorneal film (wetting time). Lemp et al. in 1970 assessed the same method, calling it break up time (B. U. T.). The time interval is measured from the conclusion of a blink till occurrence of holes in the precorneal film. The B. U. T. is independent of the patient’s age (Norn 1969; Lemp et al. 1973). It tends to be longer in males than in females (Norn 1969, not confirmed by Lemp 1973). In normal eyes the B. U. T. is 10 seconds or longer (Lemp 1973). Lemp et al. (1975a) studied the effect of artificial tears on 12 normal subjects. They tested 13 different commercial preparations, measuring before and repeatedly (at 15 minute intervals) after the instillation. The B. U. T. increased significantly, most often two or three times, as a sign of improved stability of the precorneal film. The effect was most prolonged with those drugs to which “B. P. Polymer” had been added (adapt, adapettes, adsorbotear) and tears naturale (Alcon 0413), about 90 min against 35-60 min for the others (lacril, liquifilm I and 11, isoptotears, contique, ultratears, presert, lytears - though no effect of tearisol). They point out that salt concentration and preservatives may influence the B. U. T. The preservative benzalkonium chloride in a concentration of 0.01 O/O reduces the B . U . T . four times in rabbit and about twice in man (Wilson et al. 1975, 3 1 human eyes). The angle of contact between precorneal film and dry cornea gives us an indirect impression of the stability of the precorneal film, a narrow angle effecting greater extension of fluid and consequently better wettability and film stability. Lemp et al. (197513) developed such a method on enucleated rabbit cornea. However, the results are not directly transferable to human clinical practice. Rocher (1975) and Shively (1975) measure the angle with a view to contact lenses. The contact period and the influence of vehicles on this can be indicated by estimating the reduced outflow to the nose (Linn & Jones 1968), or by measuring the fall of the technetium suspension concentration in the conjunctiva (Patton et al. 1975; Hardberger et al. 1975). The dependence of the absorption on the vehicle can, for instance, be measured by uptake of radioactive 3H thymidine in the epithelial cells of the rabbit 24
Vehicles and Precorneal Film ( B . U.T.) cornea (Swanson e t al. 1968). A s f o r neomycin, the effect of t h e vehicle on pseudomonas keratitis h a s been studied o n rabbit by Bach e t al. 1970. We cannot decide o n the basis of the literature w h e t h e r methyl cellulose (MC) o r polyvinyl alcohol (PVA) is preferable a s an ophthalmic vehicle. T h e results differ, d e p e n d e n t o n the parameters examined, concentration, viscosity, etc. (cf. Krishna et al. 1964, 1965; Linn & Jones 1968; Swanson e t al. 1968; Bach et al. 1970; H a r d b e r g e r et al. 1975; P a t t o n et al. 1975).
Ointment and oil have the opposite effect of MC and PVA. T h e y reduce the B.U.T. and consequently the stability of t h e precorneal film, thus tending to d a m a g e the epithelium ( N o r n 1975). The object of the present study h a s been t o assess o n t h e basis of h u m a n experiments t h e influences of well-known and less familiar vehicles o n t h e stability of the precorneal film. We have concentrated particularly o n investi-
gating the d a m a g i n g effect of ointments o n the precorneal film and whether other h a r m f u l vehicles exist; f u r t h e r , w h e t h e r MC o r PVA is preferable, and which substance and concentration prolongs the B. U. T. maximally.
Method The patient was placed at a Haag-Streit slit lamp in a half-lit room. 10 p l of a fluorescein mixture with a local anaesthetic added was instilled. This anaesthetic had no influence on the B. U. T. (Norn 1969). The mixture was composed as follows: fluorescein, 0.125 O/o, oxibuprocaine (NovesinR), 0.3 Oio, phenylmercuric nitrate, 0.0025 O/o, sodium chloride to isotonicity. The patient was requested to blink two or three times. A stop watch was started immediately after the conclusion of the final voluntary complete blink. The patient then looked straight forward without blinking. The lid was not supported. The smooth fluorescent precorneal film was studied in the slit lamp (ten times magnification). The light was a 1-2 mm broad, obliquely incident, vertical, cobalt-filtered beam of light, which was moved from side to side over the cornea until the first hole was noticed in the precorneal film. The stop watch was stopped, and the B . U . T . read in seconds. The examination was repeated after renewed blinking. The average of the two examinations constituted the initial B. U. T . The ophthalmic vehicle to be studied was then instilled, using the original dropping bottle (about 50 ~ 1 ) . One or two min later the fluorescein mixture was instilled again, and the B . U . T . was read twice.
25
Mogens S. N o r n and A n n a Opauszki Statistics
T he ratio of the initial B . U . T. average to the B . U . T . after the installation of a vehicle was calculated separately for each case. The mean B. U. T.-factor concerning each vehicle was calculated with a standard error of the mean ( S E M ) . T he coefficient of variation of the two initial B. U. T. determinations was calculated by the formula:
where d is the difference between the results of duplicate determinations, n the number of duplicate determinations, and A the mean of the B. U. T.
Material
A total of 336 patients from an ophthalmic out-patient department and an ophthalmic clinic were examined. Their ages ranged from 14 to 90 years. The percentage distribution was as follows, stated for 10-year age groups: 3.3-4.87.1-7.7-15.4-27.4-25.6-8.3-0.3 per cent, i. e. the greatest number in the age groups of 60 and 70. Females predominated (63.7 per cent). They were approximately equally distributed over the different vehicle groups. The investigation comprised 646 eyes. Of these, 20.4 per cent had an abnormal anterior eye section (65 per cent aphakia, 3.3 per cent keratitis, 2.5 per cent exophthalmos, 1.7 per cent iritis, 1.5 per cent glaucoma subjected to operation, etc.). The remaining were eyes subjected to routine examinations, or to examination for refraction anomalies, etc. The initial B. U. T. value was pathological (below 10 seconds) in 25.1 per cent. The pathological cases were fairly equally distributed over the different vehicle groups, and the B. U. T. alterations did not differ from those of the normal material.
Results The results are seen in the tables. Table I shows the vehicles prolonging the B. U. T., i. e. stabilizing the precorneal film, while the vehicles reducing the B. U. T. have been set out in Table 11. Finally, Table 111 comprises the vehicles with no statistically significant effect on the B. U. T. The coefficient of variation for the initial duplicate determination is stated within the individual groups. It was 30.7 per cent for the total material.
26
Vehicles and Precorneal Film (B. U . T.) Table I . Vehicles increasing the stability of the precorneal film.
B. U. T.-
I
0) 1)
2) 3) 4) 5)
Initial
factor increase k SEM
Vehicle
B. U. T. (seconds)
Coefficient of variation (per cent)
Number of eyes
I
Methyl cellulose 0.5 O/ol) Methyl cellulose 1.5 O / o O ) Methyl cellulose 2.0 O / o O )
1.36 f 0.14 4.36 f 0.72 2.48 f 0.45
22.7 28.6 21.3
2.8 41.4 28.4
14 14 12
Polyvinyl alcohol 1.4 O/o2) Polyvinyl alcohol 3 O/oO) Polyvinyl alcohol 10 "00)
1.89 ? 0.26 2.96 f 0.51 7.16 f 2.48
27.5 12.4 21.8
27.8 33.0 15.3
16 11 12
Polyvinyl alcohol 20 O / o O ) Polyvinyl alcohol 40 O/oO) Adapts)
2.82 k 0.57 2.30 k 0.81 5.13 k 1.34
24.4 35.1 17.5
50.9 21.3 38.7
10 11 30
Adapettes4) Adsorbotears) AdsorboNaCl 5 O / o
3.01 k 0.81 2.42 k 0.32 2.55 ? 0.66
11.7 17.0 21.7
36.1 42.6 50.8
20 28 15
Carbowax No. 1500 Dextran 10 O / o Acetyl cysteine 20 "0
2.12 f 0.21 4.06 k 0.59 1.98 k 0.32
16.8 20.5 19.6
27.7 20.8 12.4
21 11 14
no preservative nor other substance added sodium chloridt and phenylmercuric nitrate 0.001 O/o added (oculoguttae viscosae). sodium chloride and chlorbutol 2 O i o added (liquifilm tears). hydroxyethyl cellulose 0.44 "/a, B. P. adsorbobase, thiomerosal 0.002 O / o , EDTA 0.05 O/o. B. P. adsorbobase, thiomerosal 0.001 O i o . polyvinyl pyrrolidine 1.67 Oio, B. P. adsorbobase, hydroxyethyl cellulose C.44 O i o , thiomerosal 0.002 "/a, EDTA 0.05 O / o .
Mucomimetics
Methyl cellulose ( M C ) is usually employed in a 0.50/0 concentration in the treatment of keratoconjunctivitis sicca. At this concentration the B. U. T. value was raised significantly by the factor 1.36. Higher concentrations prolonged the B. U. T., which became four times longer at a concentration of 1.5 O/O. The difference is significant ( P < 0.001).
27
Mogens S. Norn and Anna Opauszki Table 11. Vehicles reducing the stability of the precorneal film.
B. U. T.Vehicle
factor reduct.
Initial
B. U. T. (seconds)
f SEM
Coefficient of variation (per cent)
Number of eyes
3.81 & 0.81 2.17 k 0.33
25.0 11.6
16.3 38.0
12 10
Petrol jelly and liquid 5.44 & 0.78 paraffinl) Petrol. jelly, liquid paraffin 1.79 f 0.21 and emulgator2)
24.2
24.5
20
20.0
33.3
12
Petroleum jelly Stearin
Cetacean ointments) Cosmea moisture cream4) Wool fat w. waters)
3.09 ? 1.04 5.32 zk 1.32 4.19 C 1.07
18.9 18.1 22.8
14.0 54.0 16.0
12 15 10
Glycerol ointments) Arachis oil Olive oil
k 0.17 k 0.1s k 0.59 3.33 k 0.53 2.33 k 0.36 3.72 k 0.63 4.67 C 0.69
15.5 13.7 20.6
38.6 49.0 26.2
26 10 10
24.7 19.2 23.5
43.8 21.2 28.7
10 10 13
22.0 27.9 29.4
11.5 28.3 35.0
10 12 27
15.9 11.0 23.6
28.8 26.7 48.0
18 11 14
Liquid paraffin Ophthasiloxans) Silicone oil 20 O / a V Silicone oil 100 Oio Antifoam A Benzalkonium chloride 0.01 OioS) Cocaine 2 oio Cocaine 4 o/o Albumen film on cornea
1.83 1.50 2.86
7.23 & 1.26 1.90 f 0.26 1.47 1.72 2.19
f 0.10 f 0.15 k 0.27
80 Oio petroleum jelly and 20 O / o liquid paraffin (simple eye Gintment Ph. Nord. 63). 80 o/o petroleum jelly, 20 "/o liquid paraffin with emulgator (simple eye ointment Ph. D. 48). 3) white wax, spermacet, arachis oil and water (cold cream). 4) wool fat, emulgator, monostearin, propylene glycol, water, preservatives. 5 ) glycine and wheat starch. 6) octylphenol polyoxyethylene 0.025 g, distearate of polyethylene glycol 0.05 g, dimethyl polysiloxan g. s. ml 10. 7 ) in simple eye ointment. 8) with O.g0/o sodium chloride added. 9) 25O/o water content. 1)
2)
28
Vehicles and Precorneal Film ( B . U . T.) Table I I I . Vehicles with no significant effect on the stability of the precorneal film.
B. U. T.Vehicle
Tendency
factor
f SEM
Coeff. Number of of eyes (seconds) variation Initial
B. U. T.
I
Sodium chloride 0.9 O / o Sodium chloride 5 O i o Ultracortenol susp.1) Gelatin powderz) Glycerine 100 O / o Glucose 50 Oio Cotton plug in inf. fornix p H buffer 9.24) p H buffer 10.05) Exploration creams)
fall rise rise fall fall fall fall fall fall fall
1.39 ?z 0.20 1.36 ? 0.17 1.07 k 0.11 1.34 k 0.20 1.14 f 0.13 1.74 ? 0.50 1.72 ? 0.29 1.78 ? 0.46 1.83 f 0.49 4.14 ? 1.95
17.0 20.1 10.4 19.4 22.0 28.4 20.8 18.4 24.0 22.7
14.6 9.0 29.4 15.0 30.0 37.6 30.9 61.3 57.0 44.0
14 13
10 10 14 10 14 12 16 21
microcrystalline suspension of prednisolone acetate 0.5 O/o, with sodium chloride, sodium phosphate and benzalkonium chloride added. 2) sieve 0.7 mm. 3 ) glycerol 16.5 "'/o, boric acid, tragacanth mucus. 4) sodium borate buffer with phenetanol 0.5 O / o added, isotonic. 3 sodium borate buffer, adjusted with N a 0 H, slightly hypertonic. 1)
At still higher concentrations (3 O/O, 4 O/O, 5 O/O and 10 O/O) the MC was so thick that it could not pass through a pipette, but had to be applied with a glass rod. It mixed poorly with the precorneal film, in which lumps and air bubbles were seen. At higher MC concentrations than 1.5 O/O the B. U. T. decreased with rising concentrations (the decrease between 1.5 O/O and 2 O/o was significant, P < 0.05). So, the BUT increase is not only a function of the viscocity of the vehicle.
Polyvinyl alcohol (PVA) is generally used in a concentration of 1.4 O/O. This was found to prolong the B. U. T. significantly by the factor 1.89 (Table I). Rising concentrations prolonged the B. U. T. until a concentration of 10 O/O PVA, which gave a seven times increase of the B. U. T. (P < 0.05). An additional rise of the PVA concentration caused a reduction of the B. U. T. 20 O / o PVA easily passed through the pipette, while 40 O / o was of an excessively viscous consistency. 29
Mogens S . Norn and Anna Opauszki
Adapt, Adapettes, and Adsorbotears are artificial tears, which prolonged the B . U . T . , though not more so than optimum concentrations of MC and PVA. (The results of our investigations harmonize with those of Lemp et al. (1975a). The latter having, however, been represented graphically without SEM, no comparison is permissible between the groups. On the other hand, the duration of the effect has not been recorded in the present study). Addition of 5 o/o sodium chloride to Adsorbotears did not seem to alter the effect of this on the B. U. T. Other substances prolonging the B.
U. T.
Dextran in a 10 O/o concentration is a hyperosmotic polysaccharide used as a plasma substitute. It prolongs the B. U. T. to the same extent as optimal concentrations of MC and PVA. Polyethylene glycol (Carbowax 1500, Macrogol) is a viscous fat-free ointment, which prolongs the B. U. T. more than both MC and PVA in the current concentrations. Acetyl cysteine (Mucomyst, 20 o/o) is a mucosolvent, collagenase-inhibiting compound. It prolongs the B. U. T. equally as long as both MC and PVA in the current concentrations. Ointments
Fatty ointments reduce the B. U. T. (Table 11). Holes soon occur in the precorneal film, with resulting exposure and desiccation of the cornea. The most commonly used ointment in Scandinavia (simple eye ointment, Ph. Nord. 63) is a mixture of 80 o/o petroleum jelly and 20 O / o liquid paraffin. This ointment has a significant greater effect than the corresponding emulsion (simple eye ointment with emulgator, Ph. D. 48, P < 0.001). On the other hand, no significant difference was seen for water in oil uersus oil in water-emulsion.
Stearin did not melt at the corneal temperature, but remained in the form of flakes in the precorneal film. Holes soon occurred, sometimes close to a stearin flake and sometimes distant from this. The B. U. T. was reduced in the same manner and to the same extent as by an indifferent albumen film (piece of egg albumen membrane) placed in the precorneal film. Petroleum jelly melted and spread as drops across the precorneal film. It has a greater tendency to reduce the B. U. T. than stearin. The softer simple ointment without emulsifier reduced the B. U. T. as much as petroleum jelly. The fat-free ointment unguentum glyceroli reduced the B. U. T. twice only. 30
Vehicles and Precorneal Film (B. U . T.) Oils
Oil liquefies at room temperature. Instilled oil was seen to spread at a faster rate over conjunctiva and cornea than ointment. Oil had a significantly reducing effect on the B. U. T. (two or three times). Arachis oil caused less damage than olive oil and liquid paraffin (P< 0.01 and P < 0.05, respectively). Arachis oil had the same effect as the mild simple ointment emulsion (petroleum jelly, liquid. paraf. with emulgator, Ph. D. 48). So, the reduction of BUT is not only a question of the melting point. Silicone
Antifoam A is a silicone-containing ointment, which reduces the surface activity (raises the surface tension). The B. U. T. was found to be reduced very considerably, more than with 20 O / O and 100 O/O silicone oil and to the same extent as with non-emulsified simple ointment.
Ophthasiloxan has been specially prepared for the purpose of increasing the evaporation from an intact cornea. The B. U. T. was, however, reduced less by this compound than by Antifoam A.
Other substances reducing the B. U. T.
Cocaine surface anaesthesia reduced the B. U. T., though no more than the simple ointment emulsion. Benzalkonium chloride in the usually employed concentration as a preservative reduced the B. U. T. (Table 11). This bore out Wilson’s result. Benzalkonium chloride in a high - toxic - concentration (0.5 O/O) effected pronounced micropunctate fluorescein staining. This phenomenon rendered B. U. T. reading difficult and may perhaps explain why we found a prolonged B. U. T. (2.09 k 0.40, 11 eyes).
Drugs having no influence on the B. U. T.
The vehicles found to be without any significant influence on the B. U. T. have been set out in Table 111. As might be expected, the B. U. T. was unaffected by physiological saline. Various hyperosmotic vehicles did not alter the B. U. T. ( 5 O/ o sodium chloride, 100 O/O glycerin, 50 O / O glucose). Evidently a cotton plug placed in the inferior fornix did not absorb enough to alter the B. U. T. 31
Mogens S. Norn and Anna Opauszki
A microcrystalline suspension and a gelatin powder floating in the precorneal film did not alter the B. U. T. An alkaline buffer (pH 10) is a mucosolvent, though not sufficiently so to alter the B. U. T.
Vital staining
After introduction of a vehicle and subsequent B. U. T. reading, vital staining was performed with a mixture of 1 O/O fluorescein and 1 O/O rose bengal (Norn 1974). As stated above, a high benzalkonium chloride concentration (0.5 O/n) gave a pronounced widespread micropunctate fluorescein staining, indicating damage and breach of continuity of the epithelium (cf. Tsnjum 1975). No rose bengal staining was seen. Fatty ointments, oils, and silicone gave punctate, irregularly grouped, often lacuna-like rose bengal staining of the cornea and the adjacent exposed part of the bulbar conjunctiva. Only slight fluorescein staining was seen. The vital staining was in some instances so pronounced as to resemble that in keratoconjunctivitis sicca. The reduced B. U. T. caused desiccation of the exposed part of the epithelium. MC and PVA prolonged the B . U . T . They provoked no vital-stainable phenomena, not even in high concentrations.
Discussion Unfortunately, the above procedure (B. U. T.) has a fairly high coefficient of variation. Lemp et al. (1970) on clinical examination overcame this problem by employing the average of five readings in the individual clinical case. Sauter (1976) even undertook ten readings. W e have employed no more than two plus two (before and after application of the vehicle) to avoid tiring the patient too much. On the other hand, B. U. T. reading is the most direct and simple method of examination for the purpose of assessing the precorneal film under practical clinical conditions. Many well-known vehicles influence the B. U. T. so much (about 300-400 per cent) that the changes are demonstrable even though the coefficient of variation is high (about 30 per cent). The stability of the precorneal film is increased by MC and PVA. A maximum increase is obtained by a high concentration of PVA (10 n/o), without the liquid becoming too viscous. The increase effected by raising the MC concentration is
32
Vehicles and Precorneal Film (B. U . T.)
more limited (maximum at 1.5 O / O ) . The B. U. T. can also be prolonged by using fat-free ointment (Macrogol) o r hyperosmotic polysaccharide (Dextran), and others. These factors are of importance in the treatment of pemphigoid and keratoconjunctivitis sicca, in protection of the cornea, and where the period of contact of drugs is concerned. It is worth noting that ordinarily employed ointments and oils reduce the stability of the precorneal film. This harmful effect is demonstrable by rose bengal staining. Such substances are therefore contra-indicated in pemphigoid and keratoconjunctivitis sicca. Ointment is used to protect the cornea from evaporation. However, protection is only yielded in the cases where the patient cannot blink (facial nerve palsy, during sleep, and under a bandage). In all other cases blinking will provoice holes in the ointment layer covering the cornea with resulting desiccation in spots (Norn 1976). In corneal oedema with intact epithelium, the oedema can be reduced by accelerating the evaporation. This can be effected by shortening the B. U. T., e. g. by means of silicone-containing drugs.
Acknowledgment Our thanks a.re due to Mr. S. Trager (Burton, Parson & Co., USA) for valuable discussions.
References Bach F., Riddel G., Miller C., Martin J. A. & Mullins J. D. (1970) T h e influence of vehicles on neomycin sulfate prevention of experimental ocular infections in rabbits. A m e r . /. Ophthnl. GS, 659-662. Hardberger R., Hanna C. & Boyd C. M. (1975) Effects of drug vehicles on ocular contact time. Arch. Ophthal. (Chicago) 93, 42-45. Krishna N. & Brow F. (1964) Polyvinylalcohol as a n ophthalmic vehicle. A m e r . /. Ophthal. 57, 99-106. Krishna N. & Mitchell B. (1965) Polyvinyl alcohol as a n ophthalmic vehicle. A m e r . /. Ophthal. 59, 860-864. Lemp M. A,, Holly F. J., Iwata S. & Dohlman C. H. (1970) The precorneal tear film. Arch. Ophthal. (Chicago) 83, 89-94. Lemp M. A. & Hamill J. R. (1973) Factors affecting tear film break up in normal eyes. Arch. Ophthal. (Chicago) 89, 103-105. Lemp M. A,, Goldberg M. & Roddy M. R. (1975a) T h e effect of tear substitutes on tear film break up time. Invest. Ophthal. 14, 255-258.
33 Acta ophthal. 55, 1
3
Mogens S . N o r n and A n n a Opauszki Lemp M. A. & Szymanski E. S. (197513) Polymer adsorption at the ocular surface. Arch. Ophthal. (Chicago) 93, 134-136. Norn M. S. (1969) Desiccation of the precorneal film. I. Corneal wetting time. Acta ophthal. (Kbh.) 47, 865-880. Norn M. S . (1974) External Eye. Methods of Examination. Scriptor, Copenhagen, pp. 200. Norn M. S. (1975) Correspondance in A m e r J . Ophthal. 79, 335. Norn M. S. (1976) Eye Ointments - Application, Elimination and protective Action. Ophthalmology Digest (to be published). Patton Th. & Robinson J. (1975) Ocular Evaluation of Polyvinyl Alcohol Vehicle in Rabbits. Journ. of Pfiarm. Science 64, 1312. Sauter J. J. M. (1976) Xerophthalmia and measles in Kenya. Drukkerij van Denderen, Groningen, pp. 235. Swanson A. A., Jeter D. J. & Gregor C. R. (1968) The influence of ophthalmic vehicles on 3H thymidine in normal rabbit corneas. Ophthalmologica 156, 425-436. Tsnjum A. M. (1975) Effects of benzalkonium chloride upon the corneal epithelium studied with scanning electron microscopy. A c t a ophthal. (Kbh.) 53, 358-366. Wilson W. S., Duncan A. J. XC Day J. L. (1975) Effect of benzalconium chloride on the stability of the precorneal film in rabbit and man. Brit. J . Ophthal. 59, GGT.
Author’s address: M. S . Norn, M.D., Department of Ophthalmology, Kommunehospitalet, DK-1399 Copenhagen K, Denmark.
34
