The Absence of Corneal Toxicity With Low-Level Topical

Anesthesia

David M. Maurice, Ph.D., and Tejinder Singh, M.S.

By means of an osmotic pump we infused 0.3%proparacaine solution continuously into the centralstroma of a rabbit cornea. An area of anesthesiaabout 6 mm in diameter was maintained for severaldays. The epithelium remained unaffected andhealed normally where it was scraped away in theanesthetic area. Similarly, the repeated instillationinto the rabbit's eye of 0.05% proparacaine eyedropsat ten-minute intervals throughout the day resultedin continual anesthesia without signs of epithelialdystrophy. It seems that the toxic level of propara­caine is above that required for anesthesia.

THE ATTEMPT to relieve corneal pain by the instilla­tion of anesthetic eyedrops can lead to disastrousulceration of the epithelium. It is not establishedwhether this ulceration results from a direct cytotox­ic effect on the epithelial cells or from interferencewith a trophic action of the nerve. In either case, theinjury need not be related to the excitability of thenerve terminals, and it may be that the threshold ofanesthesia is below that of cytotoxicity.

In our experiments a solution of anesthetic wasinfused constantly into a point of the rabbit's stromaso that concentric circles of diminishing concentra­tion of the drug were reached in the steady state. Inthis way the relationship of anesthesia and toxicity atdifferent drug levels could be observed in a singleanimal. In other experiments solutions of the anes­thetic in the critical range of concentration wererepeatedly given as eyedrops over an extended peri­od to establish a more clinically relevant situation.

Accepted for publication April 15, 1985.From the Division of Ophthalmology, Stanford University

Medical Center, Stanford, California. This study was supportedby grant EY 04121 from the National Institutes of Health (Dr.Maurice).

Reprint requests to David M. Maurice, Ph.D., Division ofOphthalmology, Stanford University Medical Center, Stanford,CA 94305.

Material and Methods

Pure proparacaine was made up at various concen­trations in physiologic saline; 0.01 % fluorescein wasadded when it was to be infused into the cornea. Weused female albino rabbits weighing about 2 kg asthe experimental animals.

Drug delivery-Solutions of pure proparacainewere loaded into minipumps (ALZA series 1701)which delivered 1 "",Vhr for seven days. The output ofthe pump was passed into a polyethylene cannulahot-drawn at its end to an external diameter of about50 urn, The rabbit was placed under general anesthe­sia, the pump was implanted under its scalp, and thetip of the cannula was placed in the stroma at theapex of the cornea according to the technique de­scribed by Eliason and Maurice." In a few separateexperiments the tip was placed subconjunctivallybetween two extraocular muscles and close to thecorneoscleral limbus. The fine tube was sutured tothe sclera at two limbal points with a loop in be­tween.

Because fine air bubbles can block the cannula, weconfirmed that the pump was operating correctly bysubmerging it and the cannula in a tube of sterilesaline overnight before implantation and noting theemergence of fluorescein from the tip.

We tried to maintain a constant level of anestheticin the tear film by bringing out the tip of the cannulainto the conjunctival sac. Our observations on thefluorescein included in the pump, however, showedthat the distribution in the tear film was unequal andsporadic; this we attributed to the low frequency ofblinking in the rabbit. Instead, we instilled eyedropsinto the lower fornix from a Pasteur pipette. This hadthe advantage of being closer to a clinical situation.

Sensitivity testing-We tested corneal sensitivityby stimulators constructed from 40-gauge silverwire, melting its end to form a smooth metal ballabout 0.3 mm in diameter. A length of 1 em of thewire was attached to a wooden stick. Sliding the ballalong the surface was the initial stimulus; if this did

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692 AMERICAN JOURNAL OF OPHTHALMOLOGY June, 1985

not elicit a response, the ball was pressed on the eyealong the direction of the wire. We recorded a nega­tive response if the animal did not make a full orpartial blink at, or within a couple of seconds of, theindentation stimulus, on three successive trials. Thiscorresponded to a force of about 250 mg and wassufficient to indent the cornea noticeably. We record­ed the response as uncertain if the eyelid musclesshowed a slight contraction on one of the three trialsbut no blink developed, or if a blink occurred a fewseconds after the stimulus. All other responses,whether to the light or full stimulus, were noted aspositive. The cornea was routinely tested at its apexat eight positions spaced equally around the periph­ery about 2 mm from the corneosclerallimbus and ateight points roughly equidistant between the centerand peripheral points. When a negative responsewas noted, especially if it was near the tip of thecannula, further trials were made to define the anes­thetic area better.

Wound healing-With the rabbit under generalanesthesia, a 4-mm circle was marked in the epitheli­um with a trephine. Under slit-lamp observation theepithelium was scraped away within the circle with aNo. 11 blade. The course of the regrowth was fol­lowed by staining the stroma with fluorescein.

Electron microscopy-Immediately after the ani­mal was killed we dropped a mixture of 20% glutaral­dehyde and 1% paraformaldehyde on the cornea atfrequent intervals for a total of five minutes. Immedi­ately thereafter, two needles were introduced intothe anterior chamber and a 2% glutaraldehyde solu­tion in 0.67 M cacodylate buffer, pH 7.4, was per­fused through it for the same time. The cornea wasthen rinsed and immersed in 20% glutaraldehyde forfour hours and then in a 0.67 M cacodylate buffer towash out the fixative. We cut a central button 7 mmin diameter and divided it in two. One piece wasdehydrated in acetone and critical point-dried forscanning electron microscopy and the other waspostfixed in osmium tetroxide and sectioned fortransmission electron microscopy by standard tech­niques.

Results

Stromal infusion-While the mirupump deliversfluid to the cornea, there is a small area of edemaabout 1 mm around the cannula tip, and fluoresceindiffuses out to color the surrounding stroma. Other­wise the tissue appears to be normal, and instillationof a drop of 1% fluorescein or sulforhodamine B,which shows up more clearly against the backgroundof fluorescein, discloses no defects in the epithelium.

In the normal cornea, as well as when saline or anineffective anesthetic concentration is being infusedinto the stroma, the entire surface, except on occa­sion some of the points on the outer circle (Fig. 1), isresponsive to touch.

In six of seven eyes the infusion of 0.3% propara­caine quickly led to an extensive area of anesthesiawhich remained until the pumps ceased to operate atthe end of seven days. Although the apparent sensi­tivity of the rabbit cornea fluctuates, separate esti­mates by two observers of the anesthetic area ondifferent occasions were reasonably consistent(Fig. 2). A circular anesthetic area about 6 mm indiameter was found in each of the six eyes.

In none of the eyes did the epithelium break downover the tip of the cannula, and it retained its normalgloss in the slit lamp. The edema around the tip didnot seem to be greater than in the control rabbits andit subsided after the pumps emptied. In three of theeyes, a 4-mm circle of epithelium, centered on the tipof the cannula, was scraped away three or four daysbefore the pump was due to cease operation. Thesewounds healed normally within 48 hours. We madeno attempt to quantitate the area of the defect or todetermine the moment of closure by constant obser­vation, but we noted no gross difference between thetiming and manner of the healing in these eyescompared with untouched eyes.

Ulceration of the epithelium occurred within fourdays with higher concentrations (0.5% and above) ofthe anesthetic. The bare area could be seen under the

Control

Nasal

Fig. 1 (Maurice and Singh). Sensitivity of normal rabbitcornea. The cross, circle, and point correspond respectivelyto positive, negative, and ambiguous responses at thelocation indicated.

Vol. 99, No.6 Anesthetic Toxicity 693

Day 2

D.M.

Day 2

00 0

• + 0 NasalNasal

+ 0

+

Day 1

00 +• + 0

+ + 0 Nasal

+ + 0 Nasal

+ ++ + +

+

T.S. T. S.

Fig. 2 (Maurice and Singh). Anesthetic area defined by two observers on two successive days.

slit lamp but became more evident after the instilla­tion of sulforhodamine B. The ulceration was limitedto an area around the tip of the cannula, and theepithelium appeared to be normal beyond this. Onslit-lamp viewing, the level of fluorescein under thearea of ulceration was noticeably low. Evidently thedye, and presumably the anesthetic, was able toescape into the tear film at this point.

Subconjunctival infusion-The tip of the tube wasplaced subconjunctivally in five eyes. The concentra­tion of proparacaine in the infusion fluid varied from4% to 20% but the results were similar in each eye.We found that 25% to 50% of the area of the cornea

adjacent to the corneoscleral limbus remained anes­thetized throughout the week that the pump wasrunning. In no case was there any ulceration of thecornea or any significant epithelial staining.

In four eyes, a 4-mm circle of epithelium wasscraped away in the anesthetized cornea; of these,two had similar circles scraped away in the samerelative position diametrically across the center ofthe cornea, where the surface retained its normalsensitivity. All defects healed within two days, andthe behavior of the experimental and control circleson the same eye was indistinguishable.

Topical application-In these experiments drops

694 AMERICAN JOURNAL OF OPHTHALMOLOGY June, 1985

of 0.05% to 0.2% proparacaine dissolved in salinewere instilled in the rabbit's eye at various fixedintervals to maintain anesthesia for six to sevenhours. We looked for injury to the epithelium by anincrease in fluorescein staining; if the test for sensi­tivity had been carried out, the continuous pressingof the wire might have been sufficient to cause anincrease in permeability despite the smooth ballformed at the tip. Accordingly, we conducted thetests for anesthesia separately. In preliminary trialswe found that a drop of 0.05% anesthetic kept thecornea insensitive to touch at its center for about 15minutes, 0.1 % for 30 minutes, and 0.2% for 45 min­utes.

In other tests we determined that anesthesia wascontinuous if drops were instilled regularly at theseintervals for six or seven hours. We confirmed that adrop given shortly after the last of the prolongedseries had worn off did not provide a shorter periodof anesthesia than the initial one.

We found in three eyes that 0.2% proparacainecaused a marked disturbance of the epithelium withsmall ulcerated areas after six hours of continuousinstillation at 30-minute intervals. The instillation of0.1 % solution at 30-minute intervals caused a slightstaining over an eight-hour period, and a 0.05%solution in four eyes caused no apparent stainingwhen given at 15-minute intervals over the sameperiod. With the higher concentrations we werecareful to avoid desiccation by manually blinking theeyelids at intervals and by observing the corneaunder the slit lamp to confirm the integrity of the tearfilm.

Scanning electron microscopy of the epithelium offour of the corneas treated with 0.05% proparacaineshowed the greatest part of the surface to be indistin­guishable from untouched eyes. Only in a few smallscattered areas were there signs of disruption of theouter cell membranes of the surface cells. Similarly,transmission electron microscopy of two areas ran­domly taken from each epithelium showed cells at alllevels to be indistinguishable from normal controls.

Discussion

The toxic effects of the prolonged use of anesthet­ics on the human cornea are well documented andgenerally recognized. The rabbit cornea appears tobe more sensitive. Behrendt! noted the developmentof epithelial ulcers and the loss of effectiveness of theanesthetic one or two days after the frequent instilla­tion of 0.2% tetracaine or dibucaine. This may be

associated with desiccation resulting from disrup­tion of the tear film and the infrequent blinking ratein this animal.

Weekerss found a depression of the electrical activ­ity of the epithelium of the isolated rabbit corneawith dilute anesthetics, 0.1 % benoxinate and 0.25%lidocaine, applied. to the medium bathing the frontsurface; the significance of this finding is not clear.Serious damage to the endothelium occurred whenthe anesthetics were applied to the back surface, asevidenced by rapid swelling of the cornea.

In contrast, we found no deleterious effects on theepithelium while infusing 0.3% proparacaine at arate of 1 ul/hr for several days. Although the concen­tration distribution in the tissue was not quite cer­tain, it was sufficient to cause complete insensitivityin the treated region. Similarly, no epithelial distur­bance resulted from the continued instillation of0.05% proparacaine over one day, nor was a lessen­ing of the duration of anesthesia noted. The absenceof any excess edema where the infusion took placeindicated that the endothelium was also undamaged.

The careful investigation of Kuchle" on the effectsof topical anesthetics on the rate of wound healing inrabbits confirmed our own rough observations thatthey had little effect. A comparison of various diluteanesthetics and saline, 22 drops instilled in 50 hours,showed that on the average the treated eyes healed atabout the same rate as the slowest of the controls.Conversely, Marr, Wood, and Storck" found amarked inhibitory effect with 0.5% proparacaine anda slight effect with 0.5% tetracaine on the healing ofpunctate wounds in rats. Lower concentrations suchas we used here were not tested.

Polse, Keener, and [auregui" and Matsumoto andassociates? measured the duration of anesthesia inhuman eyes in relation to the concentration of anes­thetic in the drop. A logarithmic relation between thethreshold of sensitivity and time was found and asimilar relationship for the concentration present atany moment. The level of anesthetic required toalleviate pain probably varies from patient to patient,but it appears from the measurements of Polse,Keener, and Iaurgeui" that doubling the concentra­tion of proparacaine adds only about five minutes ofanesthesia at any level. In a few patients with painfulcorneal injuries, a single drop of 0.05% proparacaineprovided relief for ten to 15 minutes.

In our main series of experiments, the anestheticwas introduced into the stroma so that it reached theepithelial cells from their basal aspect. The questionarises as to what concentration is actually reached atthe cell membranes at all levels of the layer. Becausethe rabbit blinks only a few times each hour, and in

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consequence maintains an undisturbed and stagnanttear film for long periods, the loss of anestheticacross the anterior surface is not likely to be consid­erable. If the anesthetic is lipid-soluble, all layers ofthe epithelium as well as the tear film immediatelyabove become saturated at the same concentration asthat in the stromal fluid. It however, the apical tightjunctions offer a barrier to diffusion, the tear layerwill be free of anesthetic and the outermost cellsurface will remain unexposed to it; at the same time,the drug will presumably saturate the intercellularspaces. There is no reason to believe, however, thatthe outermost surface is more susceptible to toxicagents than the other cell membranes; in many trau­matized corneas, moreover, a topical anesthetic hasdirect access to the inner epithelial layers.

The size and shape of the zone of corneal insensi­tivity after the subconjunctival infusion of the anes­thetic suggest that it was acting on the nerves as theypass through the sclera, rather than spreading intothe cornea through the stroma or the tear film.Although not clinically relevant, it is further evi­dence that the well-being of the epithelium is notdependent on the ability of its nerve supply toconduct axon potentials.

How far any substance constantly infused at apoint in the cornea will spread is determined by therate at which it diffuses along the stroma and that atwhich it is lost across the endothelium into theanterior chamber.' These rates are unknown in thecase of proparacaine, but, if we assume as a firstapproximation that they are similar to those of fluo­rescein, the concentration gradient of the anestheticcan be roughly estimated from that of the dye. In twocases the rabbit was killed before the pump ceased tooperate and the cornea was quickly excised and astrip about 3 mm wide cut from corneoscleral lim­bus to corneoscleral limbus passing near the tip ofthe cannula. This strip was spread out on a transpar­ent scale and the fluorescence of the dye in it mea­sured at I-mm intervals from corneoscleral limbusto corneoscleral limbus with a slit-lamp fluoropho­tometer.

Figure 3 shows the distribution in one case incomparison with the area of anesthesia. Comparisonof the level of fluorescein in the stroma with that inthe infused fluid indicated that the maximum con­centration around the tip would be about 0.15%proparacaine. The threshold stimulus is raised atconcentrations at least five times less. We made noattempt to refine these data because of the uncertain­ty as to the relative behavior of proparacaine andfluorescein. When an ulcer is found under the influ­ence of higher levels of anesthetic, the concentra-

Fluorescein

Concentration

5 0 5

Dilianca from lip (m m)

Fig. 3 (Maurice and Singh). Distribution of fluoresceinaround injection site compared with area of anesthesia.Solid line, definite anesthesia; broken line, ambiguouszone.

tions that result become even more difficult to esti­mate.

Regardless of the actual concentration in the tis­sue, our experiments indicated that a level of localanesthetic can be maintained for several days whichwill make the cornea insensitive but which is appre­ciably less than the level needed to injure the tissue.Possibly pain can be relieved by the constant instilla­tion of the drug below the toxic level without dangerto the patient. This situation is no different frommany others in which the dosage of a potentiallytoxic drug has to be well controlled; in other words,the therapeutic index is low. In this case overdosingis not a risk because the prescribed concentration isbelow the toxic level and systemic absorption isnegligible.

If the anesthetics are dispensed as eyedrops, theywill need to be instilled frequently, perhaps everyten minutes, to maintain their effect. Although this isnot convenient for the patients, they can adjust thefrequency according to need. A system in which thesolution is infused continuously can be imagined"but would be more inconvenient although a lowerconcentration of anesthetic might be effective. As faras we are aware, this form of treatment has not beenproposed before, and we hope it can be given apractical trial in cases in which short-term relief fromsevere pain is needed.

The dilute anesthetic eyedrops should be dis­pensed by the ophthalmologist or pharmacist; ifstandard 0.5% drops are used they should be dilutedto 10% strength in physiologic saline or balanced saltsolution. The preservative in the eyedrops will alsobe diluted, which is desirable in avoiding toxicity.Because the sterility of the solution cannot be guar­anteed and the response to the anesthesia should be

696 AMERICAN JOURNAL OF OPHTHALMOLOGY June, 1985

monitored, only quantities sufficient for two or threedays, about 10 rnl, should be dispensed.

References

1. Eliason, J. A., and Maurice, D. M.: An ocular perfu­sion system. Invest. Ophthalmol. Vis. Sci. 19:102, 1980.

2. Behrendt, T.: Experimental study of corneal lesionsproduced by topical anesthesia. Am. J. Ophthalmol. 41:99,1956.

3. Weekers, J.-F.: Recherches experimentales sur la ge­nese des lesions corneennes dues aux anesthesiques. Arch.Ophtalmol. 34:121, 1974.

4. Kiichle, H. J.: Zur Wirkung der Oberflachenanasthet­ika auf die Regeneration des Hornhautepithels. Klin.Monatsbl. Augenheilkd. 126:313, 1955.

5. Marr, W. G., Wood, R., and Storck, M.: Effect of someagents on regeneration of corneal epithelium. Am. J. Oph­thalmol. 34:609, 1951.

6. Poise, K. A., Keener, R. J., and Jauregui, M. L.: Dose­response effects of corneal anesthetics. Am. J. Optom.Physiol. Opt. 55:8, 1978.

7. Matsumoto, S., Hayashio. K., Tsuchisaka, H., andAraie, M.: Pharmacokinetics of surface anesthetics on re­generation of corneal epithelium. [pn, J. Ophthalmol.25:335, 1981.

8. Dohlman, C. H., Pavan-Langston, D., and Rose, J.: Anew ocular insert device for continuous constant-rate deliv­ery of medication to the eye. Ann. Ophthalmol. 4:823, 1972.