Albrecht v. Graefes Arch. klin. exp. Ophthal. 197, 71--81 (1975) 9 by Springer-Verlag 1975

Side-Effects of Local Anesthetics on the Corneal Epithelium of the Rabbit Eye* J. P. Harnisch, F. H o f f m a n n , a n d L. D u m i t r e s c u Ophthalmic Clinic of the Klinikum Steglitz, Freie Universitgt Berlin (Head: Prof. Dr. I-I. I-Iager) Received May 26, 1975

Summary. The toxic side-effects of 0.4 % Novesine and Chibro-Kerakain were investigated by scanning- and transmission electron microscopy in 15 rabbit eorneae each in intervals of 3, 6, 10, 15 and 60 mins. The lesions following Kerakain were larger and occurred earlier. After 60 mins, the specimens after Novesine application showed an almost complete repair process, whereas after Kerakain the epithelial surface appeared to be functionally impaired. Zusammen/assung. Raster- und transmissionselektronenmikroskopischwerden 15 Kaninehenhornh~ute nach Novesin 0,4 %und 15 naeh Chibro-Keraeain in Abstgnden yon 3, 6, 10, 15 und 60 rain auf toxische Nebenwirkungen untersucht. Die L~sionen nach Keracain waren ausgepr~gter und traten rascher ein. 60 min nach Applikation sind die l~eparationsvorg~nge bei Novesin weitgehendst abgesehlossen. Die Pr~parate nach Keracain zeigen zu diesem Zeitpunkt ein in seiner Funktion noeh beeintri~chtigtes 0berfl~chenepithel. The long-term, u n c o n t r o l l e d application of local anesthetics leads to a delay i n regeneration (Mart, 1957) due to a n i n h i b i t i o n of the mitotic ratio, cell division a n d the cell m i g r a t i o n (Linn a n d Vey, 1955; Kiiehle, 1955). F i n a l l y , severe ulceration m a y occur a n d result i n a f u n c t i o n a l or even complete loss of the eye (Itilsdorf a n d Zenklusen, 1973). I n this i n v e s t i g a t i o n t h e effect of a single application of local anesthetics, as used e.g. i n tonography, was tested. The most c o m m o n l y used local anesthetics belong to the benzoic acid ester group (Killian, 1973). For t h e comparative test we chose 0.4% Novesine a n d Chibro-Kerakain. Both preparations differ i n the radical of t h e amino-benzoic acid. Novesine is the d i e t h y l a m i n o e t h y l e s t e r of t h e p - a m i n o - m - b u t o x y - b e n z o i c acid. I n K e r a k a i n t h e b u t a n o l is replaced b y propanol. This s t u d y was done to e x a m i n e the s h o r t - t e r m effect of one application of the c o m p o u n d to the cornea b y scanninga n d transmission-electron microscopy. I t was performed since this t y p e of application is most c o m m o n l y used and, on the other hand, up to now the influence of a single application to the cornea has n o t been i n v e s t i g a t e d electron microscopically. I n addition, we were concerned with the problem of possible differences between the two local anesthetics applied most frequently. P r e l i m i n a r y experiments let us assume t h a t K e r a k a i n effects more severe alterations onto the cornea t h a n 0.4 % Novesine. Materials and Methods Twenty rabbits were used in this investigation. They were offering no resistance during the application of the drugs and, afterwards, their eyes were not manipulated any more. In 15 eyes * This paper was presented in part at the 3rd annual meeting of the European Club for Ophthalmic Fine Structure in Marburg on March 7 and 8, 1975.

72

J . P . I-Iarniseh

et al.

one drop 0.4% Novesine was applied on the cornea and another 15 eyes received one drop Chibro-Kerakain each. Five eyes of the Novesine-series remained completely untreated, they neither received sodium chloride. For control, five eyes of the Kerakain-series were treated with Benzalkonium, since a bacteriostatic from the Benzalkonium-series is admixed to Kerakain. 3, 6, 10, 15 and 60 mins after t r e a t m e n t for b o t h experimental series 4 animals per time interval were killed b y administering t h e m an overdosage of Nembutal, and glutaraldehyde was dripped immediately onto their corneae. Thus, we received 3 treated and 2 control eyes for each experimental time interval. After enucleation the eyes were fixed for 1 h in 3.5% glutaraldehyde. Subsequently, the corneae were excised b y means of a trepan and fixed for another 2 h in fresh glutaraldehyde solution. The 40 cornae were t h e n bisected for scanning- and transmissionelectron microscopy. For scanning electron microscopy the specimens were dehydrated in alcohol series air-dried, a n d coated in the v a c u u m evaporator at 5 • 10 .5 Tort with a 200-500 A gold layer. Afterwards they were examined with the Cambridge Stereoscan. For transmission electron microscopical studies the eorneae were refixed in 1% osmie acid for 1 h, dehydrated in alcohol series and, then, embedded in micropal. Serial sections of the cornea surface were prepared on the P~eichert OmU s for each time interval on several sites. The preparations were then contrasted with uranyl acetate a n d Reynolds-lead and examined with the Siemens Elmiskop 101.

Scanning-Elecronmicroscopical Findings The scanning-electronmicroscopical picture of the r a b b i t cornea has already been described b y Blfimcke a n d Morgenroth (1967) a n d b y Hoffmann a n d Schweichel (1972). Therefore, only the most characteristic criteria of the corneal surface are described briefly in this paper. A t medium magnification, the light-dark contrast of the cells is most characteristic. The darker cells are predominant. A t higher magnification the regular slim microvilli and microplicae become visible. I t is mainly the medium dark cells which show circular focal depressions which we call "craters". The light cells demonstrate single "pits". Necrosis of single cells is not present a n d cells in desquamating process are infrequently observed. Only in the 10th and 15th rain did the control eyes reveal deviations from the normal surface appearance. These included dark margins, single cell necrosis and a slightly increased desquamation. All the treated corneae disclose alterations in their superficial epithelial layer. The findings are summarized in Table 1. The p a t t e r n of damage ensuing from the application of 0.4 % Novesine or Chibro-Kerakain differs in time and severity. Although basically identical, the changes are less severe a n d more delayed alterations were after Novesine t h a n after Chibro-Kerakain. For example, three rains after Novesine an almost normal epithelium is found, b u t after Chibro-Kerakain extended group necrosis appeared (Figs. 1 and 2). Also at 3 rains after Chibro-Kerakain application, sporadic nodular distensions a n d condensatoins of the microvilli are more frequently observed t h a n is t h e case with Novesine after 10 mins (Figs. 3 a n d 4). The p a t t e r n after 6 mins is also consistent with the more intensive damage due to Kerakain. The slightly increased cell necrosis and desquamation following Novesine contrast with the excessive necrotic cell desquamation following Kerakain (Figs. 5 a n d 6). These distinctive alterations of the surface structure have passed their peak of severity b y the 15th min. The most striking phenomenon a t 15 mins is the increase in the n u m b e r of "pits ~ a n d "'craters" (Figs. 7 a n d 8). A t this stage, the Novesine specimens show more necrotic cells t h a n those in the Kerakain treated group. Sixty mins after Novesine, restitution of the epithelium is complete a p a r t from a n increase in the dark borders, sporadic cell necrosis and an increase in " p i t s " (Fig. 9). I n addition to these findings, t h e Kerakain specimens demonstrate extreme " c r a t e r " formation (Fig. i0). The corneal alterations ensuing from Benzalkonium application correspond to those observed in the control eyes.

Transmission-Electronmicroscopical Findings The most superficial cell layers of the untreated corneal epithelium show a typical squamous epithelium with polymorphous microvilll a n d microplicae as being prominent on the surface. Frequently, membraneous fusions are present in the typical unit membrane of these cell protrusions, so numerous vesicles of a diameter of 0.1 Ezra to 1 ~m are formed. The cyto-

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changes of microvilli

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Table 1. Alterations of the ultramicroscopical surface structure of the corneal epithelium after local anesthetic application. Three eyes are combined for each interval and compared with the normal findings and the control eyes

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74

J . P . Harnisch et al.

Fig. 1 . 3 mins after Novesine slight decrease of the light-dark contrast and negligible increase in pits (P). x 500 Fig. 2 . 3 rains after KerakMn large group necrosis (N). The cell nuclei are shrinked a n d show karyorrhexis. The necrotic ceils show no microvilli. • 500

Side-Effects of Local Anesthetics on the Corneal Epithelium

75

Fig. 3. I-Iigher magnification of a necrotic cell 3 mins after Kerakain. The microvilli (M) appear condensed. The crater (C) is a b o u t to decay. • 10000 Fig. 4. 10 mins after Novesine presence of cell necroses and condensation of the microvilli (M). Wide, dark borders (B). • 5000

76

J . P . Harnisch et al.

Fig. 5.6 rains after Novesine beginning formation of cell necroses (N), increased desquamation (D), reduction o~ the light-dark contrast and increase in pits (P). • Fig. 6. 6 mins after Kerakain heavy desquamation (/~) of necrotic cells (N). •

Side-Effects of Local A n e s t h e t i c s o n Che Corneal E p i t h e l i u m

77

Fig. 7 . 1 5 rains after N o v e s i n e p r e p o n d e r a n c e of t h e d a r k cells w i t h n u m e r o u s pits (P). • 1000 Fig. 8. 15 m i n s a f t e r K e r a k a i n presence of necrobiotie cells a n d d i s t i n c t increase of t h e craters (C). • 1000 6 Albrecht v. Graefes Arch. klin. exp. Ophthal.

78

J . P . Harniseh et aI.

Fig. 9.60 rains after Novesine marked light-dark contrast, largely normal cell picture, x 500 Fig. I0. 60 rains after Kerakain numerous, in their appearance unusually unregular craters (o). x 5 o o

Side-Effects of Local Anesthetics on the Corneal Epithelium

79

The application of local anesthetic produces a quantitative decrease in the eutrophic cells in favour of cells in which there was different cloudy sewlling and necrobiosis. The microvilli are rarefied and distorted and the cell borders of the more severely degenerated epithelial cells protrude towards the surface. The liquefaction of the cytoplasm is associated with a smoother membrane which is frequently interrupted. The "craters" are only found in degenerated ceils. The cross-section reveals a prominent folding of the cell membrane (Fig. 12). The more distinct alterations after Kerakain, which were observed with the Stereoscan, did not appear to have a specific comparative change under the transmission electron microscope.

Discussion In contrast to the publications of Schlegel and Swan (1954) and Witmer (1953) which based upon light-microscopical studies only, this investigation has shown that toxic epithelial lesions of the rabbit cornea are determinable after application of one drop of a local anesthetic. More recently, Leuenberger (1973) described toxic lesions in rat eyes following a 5 to 15 fold local anesthetic application, but he did not observe any basic differences in the type of ultrastructural change; however, a connexion between the frequency of application and the degree of lesion was noted and 1% Novesine seemed to have the most rapid effect on the corneal ultrastructure. The epithelial alterations of the untreated control eyes suggest that the morphological lesions of the cornea after local anesthetic application may be provoked by secondary factors. I t is only the intensity- and time-related severity of the lesions after 0.4 % Novesine and Chibro-Kerakain application which permits correlation of the findings with the toxic effect of the drugs. Kerakain effects a more r~pid and more intensive epithelial lesion over a longer period of time. The reason for thsis m a y be either a greater impairment of the cell membrane or the cell metabolism. As this preparation needs a preservative additive we investigated the latter separately without finding, however, an explanation for the more severe effect of Kerakain. In clinical experience, the better subjective compatibility of this local anesthetic is in contradiction to its eytotoxic effect, but its earlier onset of action might be a related factor. Since similar alterations of the microvillar surface of the rabbit corneal epithelium have been described by Hoffmann and Schweichel (1972) after ultraviolet treatment the general reaction of the superficial epithelial structure of the cornea seems to coincide with a common response pattern of the cornea towards pharmacological and physical insult. Thus, the microvilli and the plicae are considered to represent an active structure and not a remnant of the desmosomal adhesions points after the separation of the surface cell, which is an assumption made previously by Pedler (1962) and Lenenberger (1973). Consequently, the mierovillar border reflects the different metabolic conditions of the cells. Damage to the cell metabolism by a toxic effect of the local anesthetic could cause the alterations in the surface relief of the cornea. This assertion is confirmed by the transmissionelectronmicroscopical findings. The rarefaction of the microvilli observed after local anesthetic application runs parallel with an increased cytoplasmic degeneration. With progressive cellular disorganization the surface outlines becomes flatter with swelling and protrusion of the cells. This process can be correlated with the destruction of the mitochondria and the consequent loss of the active phospha-

F i g . l l . Desqu~mating swollen epithelial cells with completely liquefied unstructured cytoplasm and smooth cell membrane (I). Underneath a necrobiotic cell. Lightening of the central part of the cytoplasm, destruction of the mitoehondria, the rough endoplasmic reticulum and the tonofibrils. In the marginal area the dense granular structure of the cytoplasm is Preserved. The surface structure is still recognizable in purt (II). • 30000 Fig. 12. A crater (C) in a cell with advanced necrobiosis. • 20000

Side-Effects of Local Anesthetics on the Corneal Epithelium

81

tes. Conversely, after a reversible cell lesion, r e g e n e r a t i o n of t h e microvilli m a y e m a n a t e from t h e cell center to t h e p e r i p h e r y a n d t h u s causing t h e f o r m a t i o n of d a r k cell borders. If t h e cell is i r r e v e r s i b l y d a m a g e d , i.e. if it is necrotic, its memb r a n e is s m o o t h a n d d i s a p p e a r a n c e of t h e craters occurs. These circular folds of t h e cell m e m b r a n e are p r e p o n d e r a n t l y found in m e d i u m d a r k cells a n d are, as is seen in t h e cross-section, surface r e a c t i o n s of ceils with catabolic m e t a b o l i c conditions. Therefore, we c a n n o t agree with t h e a s s u m p t i o n of Pfister (1972) who s t a t e d t h a t " c r a t e r s " are sites of an e a r l y local lysis of t h e cell. T h e a l t e r a t i o n s of t h e surface s t r u c t u r e which were found even 60 rains after a p p l i c a t i o n i n d i c a t e t h a t t h e d a m a g i n g influence of t h e local anesthetics on m e t a b o l i s m lasts longer t h a n t h e i r p h a r m a c o l o g i c a l effect. I n clinical practice, b o t h substances are e q u a l l y useful for m i n o r procedures d e s p i t e t h e differences in t h e i r toxic effect, since t h e mechanical lesions caused b y a t o n o m e t e r are prop o r t i o n a l l y larger. I n t h e case of a p r e d a m a g e d epithelium, as e.g. a f t e r k e r a t o p l a s t y , d e g e n e r a t i v e corneal diseases a n d longer lasting d r i p anesthesia, however, C h i b r o - K e r a k a i n should r a t h e r n o t be a p p l i e d because of its d i s t i n c t l y s t r o n g e r d a m a g i n g effect.

References Bliimcke, S., Morgenroth, K. : The stereo ultrastructure of the external and internal surface of the cornea. J. Ultrastruct. Res. 18, 502-518 (1967) Hilsdorl, C., Zenklusen, G. : Novesinschiiden der tIornhaut. Klin. Mbl. Augenheilk. 162, 525527 (1973) Hoffmann, F., Schweichel, J.-U. : The microvilli structure of the corneal epithelium of the rabbit in relation to cell function. Ophthalmic Res. 4, 175-184 (1972) Killian, It. : Lokalani~sthesie und Lokalaniisthetika zu operativen, diagnostischen und therapeutischen Zwecken. Stuttgart: Thieme 1973 Kfiehle, It. F. : Zur Wirkung der Obefflgchenan/isthetika anf die Regeneration des Hornhautepithels. Klin. Mbl. Augenheilk. 126, 313-320 (1955) Leuenberger, P. M. : Ultrastruktnrelle Ver~nderungen an Itornhautepithel nach Obeffl~chenangsthesie. Albrecht v. Graefes Arch. klin. exp. Ophthal. 186, 73-90 (1973) Linn, J. G., Vey, E. K. : Topical anesthesia in ophthalmology. Amer. J. Ophthal. 40, 697-704 (1955) Mart, W. G., Wood, R., Senterfit, L., Sigelman, S. : Effect of topical anesthetics on regeneration of corneal epithelium. Amer. J. Ophthal. 43, 606-610 (1957) Pedler, Ch. : The fine structure of the corneal epithelium. Exp. Eye Res. 1, 286-289 (1962) Pfister, R. R. : The normal surface of corneal epithelium: A scanning electron microscopic study. Invest. Ophthal. 12, 654-668 (1973) Schlegel, tI. E., Swan, K. C. : Benoxinate for rapid corneal anesthesia. Arch. Ophthal. gl, 663-670 (1954) Witmer, R. : Uber die Verwendung eines neuen Oberfl~chenan/~sthetikums in der Augenheilkunde. Schweiz. reed. Wschr. 6, 22-23 (1953) Dr. J.P. Harniseh Klinikum Steglitz - - Augenklinik D-1000 Berlin 45 I-Iindcnburgdamm 30