Anesthetic-Induced Corneal Lesions in Developmental^ Sensitive Rats

Ronnie Guiller,* Jeffrey Wyarr.f Raymond D. Boggs,t ond Carol K. Kellogg*

Developmental critical periods for the induction of abnormalities by exposure to exogenous substances need not be confined to the early embryonic stage of organogenesis. The combination of ketamine hydrochloride and xylazine, two commonly used anesthetic agents, resulted in a corneal epithelial calcium deposition in 84% of rat pups whose exposure was limited to a single injection during the third postnatal week only. Concurrent exposure to ketamine hydrochloride, xylazine, and yohimbine, an

alpha2 adrenergic receptor antagonist, resulted in corneal lesions in only 6% of rat pups so exposed. The etiology is presently not understood but may involve interference with neurally directed corneal development. Corneal desiccation may also play a role. Altered drug metabolism, and toxic interac- tions resulting from a changing oxygen or light milieu are less likely etiologic mechanisms. Aspects of corneal development and mechanisms by which drugs can interact with and disturb normal matura- tional sequences can now be approached. Invest Ophthalmol Vis Sci 29:949-954,1988

Developing organisms can be highly sensitive to differential effect of ketamine plus xylazine in juve- drugs, environmental chemicals and viruses during nile vs. adult rats. very limited periods of development. Deleterious effects result from exposure during that precise devel- Materials and Methods opmental period. One classical example of a drug that can induce abnormalities during a limited exposure is Animals the tranquilizing drug thalidomide. Exposure to tha- Rats (Long Evans, Blue Spruce Farms, Altamont, lidomide from 21 to 36 days of gestation can lead to NY) were housed in supervised quarters with con- phocomelia in human offspring.1'2 Most critical pe- stant temperature (72 ± 2°F) and humidity (60 riods for drug-induced structural abnormalities in an ± 5%), in a room having a complete air change every organism relate to the period of organogenesis. We 20 min, and with a 12 hr light/dark cycle (lights on at have identified, however, a much later developmen- 0600). They were housed in plastic cages with Pine- tal critical period for the induction of corneal pathol- DRI bedding and received Purina rat chow (5001) ogy in the rat from combined exposure to ketamine and tap water ad libitum. The day of birth was desig- hydrochloride and xylazine. nated as day 0. Litters were culled to 10-12 pups on Ketamine hydrochloride (2-(o-chlorophenyl)- day 1 and remained with the lactating female until 2-(methylamino)-cyclohexanone hydrochloride) is weaning on day 20-21. an anesthetic agent that is widely used in veterinary and clinical medicine.3 The combination of ketamine Procedures and the sedative analgesic drug xylazine (2(2,6-di- On the day of injection, pups selected from a mini- methylphenylamino)-4OH-5,6-dihydro-1,3-thiazine mum of three different litters were injected with a hydrochloride) is commonly used in laboratory re- 4 solution of ketamine hydrochloride (12.5 mg/kg, i.p.) search and veterinary medicine. Most reports on the and xylazine (15 mg/kg, i.p.) at a final volume of combined use of the drugs are based on studies in 0.1-0.2 cc/100 g. Following recovery (spontaneous adult organisms. This report presents evidence for a locomotion), they were returned to their home cages. Ketamine hydrochloride (Ketalar) was obtained from From the Departments of *Psychology and t Laboratory Animal Parke-Davis through the hospital pharmacy and xy- Medicine, University of Rochester, Rochester, New York. lazine (Rompun) was purchased from Miles Labora- Supported in part by NIMH Grant MH-31850 and NIDA tories through Butler Labs (Rochester, NY). Training Grant T32DA-07232. Submitted for publication: June 4, 1987; accepted December 15, The drugs were administered concurrently to rat 1987. pups on postnatal days 13-14 (n = 16), 16-17 (n Reprint requests: Ronnie Guillet, MD, PhD, Department of Psy- = 11), 20-21 (n = 29), 23-24 (n = 12), 27-28 (n chology, University of Rochester, Rochester, NY 14627. = 12), or 60-90 (n = 24). Of the 16 rats studied at

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examination included gross dissection and histologic examination of the following tissues: eye and adnexal structures, brain (including pituitary), heart, lung, liver, spleen, gut (stomach, duodenum, jejunum, cecum, colon), pancreas, adrenal, kidney, urinary bladder, mammary gland, thyroid, skeletal muscles, and skin. Tissues were fixed (within 1 min of euthanasia for eyes, within 20 min of euthanasia for other tissues) in 10% neutral buffered formalin, embedded in paraffin, sectioned at 5 fitn and stained with he- matoxylin and eosin. Additional stains, including Pe- riodic Acid Schiff, Oil Red O, alizarin red-S, and gold chloride (of whole cornea),5 were used as indicated. Serologic ELISA studies (Microbiological Associates, Inc., Bethesda, MD) for rat coronavirus and sialoda- cryoadenitis virus were also done on five severely af- Fig. 1. Long Evans rat with corneal opacity. fected animals 50 days after exposure. An estimate of the partial pressure of oxygen reaching the corneal epithelial cells was made assum- 13-14 days of age, ten had unopened eyelids, one had ing that the oxygen reaching these cells did so solely eyelids that had opened spontaneously, and five had by diffusion from the limbic blood vessels prior to eye eyelids that were manually opened 3-5 min after ad- opening. PaO in the arterioles approximates 90-100 ministration of ketamine hydrochloride and xylazine 2 mm Hg and thus the PaO2 at the cell must be less and were deemed sufficiently anesthetized. To evalu- ate the role that dessication may play in producing than 90-100 mm Hg. Once the lids opened, however, the lesion, additional rats at 20-21 days of age (n oxygen could diffuse from the atmosphere (PO2 ap- = 10) also received an ocular lubricant ointment proximately 150-170 mm Hg) to the epithelium, (Duratears; Alcon Laboratories, Inc., Fort Worth, providing as much as 170 mm Hg O2. TX) bilaterally throughout the period of combined All procedures conformed to the ARVO Resolu- anesthesia. To assist in defining the etiology of the tion on the Use of Animals in Research. lesion, several pharmacological studies were con- Results ducted, including: ketamine hydrochloride alone (12.5 mg/kg, n = 7; 25 mg/kg, n = 5; or 50 mg/kg, n Administration of the combination of ketamine = 5), xylazine alone (15 mg/kg, n = 6; 30 mg/kg, n hydrochloride (12.5 mg/kg, i.p.) and xylazine (15 = 5; or 60 mg/kg, n = 3), nembutal (25-30 mg/kg, mg/kg, i.p.) to rat pups 13 days of age and older i.p.) (n = 10), or chloropent (3 cc/kg, i.p.; Fort Dodge resulted in rapid onset of anesthesia (within 3-5 min). Laboratories, Inc., Fort Dodge, IA; each cc contains: Absence of response to tail pinch lasted 60-90 min in chloral hydrate 42.5 mg, magnesium sulfate 21.2 mg, 13-14-day-old pups and 30-40 min in pups 21 days pentobarbital 8.86 mg, ethyl alcohol 14.25%, propyl- and older. Spontaneous locomotion resumed within ene glycol 33.8%, and purified water q.s.) (n = 20). 2 hr in the youngest pups and within 30-50 min in Another cohort of rats 17-19 days old (n = 18) re- the older rats. Ketamine hydrochloride given alone at ceived yohimbine, an alpha2 adrenergic receptor an- 12.5 mg/kg resulted in hyperactivity; at 25-50 mg/kg tagonist (2 mg/kg, i.p.), concurrent with or 30 min it induced rigidity and tremors. Xylazine given alone after ketamine hydrochloride (12.5 mg/kg, i.p.) and at 15-30 mg/kg produced sedation; at 60 mg/kg irre- xylazine (15 mg/kg, i.p.). All experimental groups in- versible respiratory depression occurred. The combi- cluded animals from a minimum of three litters and nation of ketamine hydrochloride (12.5 mg/kg), xy- both sexes were equally represented. The eyes of all lazine (15 mg/kg), and yohimbine (2 mg/kg) (whether animals were visualized unmagnified or at X10 mag- yohimbine was given simultaneously with or 30 min nification three to five times weekly from the day of following administration of ketamine and xylazine) injection for up to 7 weeks. produced anesthesia similar to that seen when ketamine hydrochloride and xylazine were given without yohimbine. Both nembutal and chloropent re- Histologic Studies sulted in onset of anesthesia within 10-15 min and Histologic examination was performed at 10-14 or duration of anesthesia was approximately 3-4 hr. 50 days after drug exposure. Necropsies were per- A corneal epithelial and superficial stromal cal- formed in a separate laboratory. A complete necropsy cium deposition (Figs. 1, 2) appeared within 7 days of

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Fig. 2. Photomicrographs of the » i cornea in control and ketamine/xylazine-exposed rats. Posterior endothe- lium (p), corneal stroma (c), and corneal epithelium (a). Bar = 0.05 mm. (A) Normal control cornea devoid of lesions. (B) Mildly affected cornea P. with numerous clusters of large abun- dantly vacuolated cells in the corneal epithelium (arrow). (C) Moderately affected cornea with occasional inflammatory cells in the outer portion of the a stroma. The overlying corneal epithelium is focally mildly eroded with numerous necrotic cells in the affected areas. There is moderate necrotic debris (n) in the outer portion of the corneal stroma. (D) Severely affected cornea with numerous polymorphonuclear leukocytes and abundant necrotic debris (n) in clefts within the stroma, separating the overlying corneal epithelium. The epithelium, most prominently over the central area, is thickened (small arrows). Same animal as shown in Figure 1.

exposure to the combination of ketamine and xyla- red-S staining revealed calcium deposition in the zine in 84% of pups 20-21 days old at the time of areas of necrosis, as is seen in band keratopathy type injection, with or without eye lubrication. The lesion lesions. Periodic Acid Schiff staining was negative, did not occur following exposure to either drug alone, indicating that vacuolization was not due to abnor- when yohimbine was administered either concur- mal glycogen deposition. Gold chloride staining (Fig. rently or 30 min later, nor following either nembutal 3) of whole cornea revealed neovascularization (low or chloropent (Table 1). The severity of the lesion power view, Fig. 3A) in affected rats. High power ranged from mild (involving central cornea with vac- views (Fig. 3B) revealed similar patterns of corneal uole formation in the basal cells only, Fig. 2B) to innervation in control and affected rats. moderate (involving central cornea and extending Develop mentally the rat pup cornea was most sen- throughout most of the cornea and a moderate sitive to effects of the drug combination between days amount of necrosis with inflammatory cells, Fig. 2C) 16 and 21 (Table 2). Lesions were detected either to severe (involving the entire cornea and a severe grossly or histologically in only 1/48 rats 23 days or degree of necrosis with epithelial thickening and the older. No 13-14-day-old rat with unopened eyes had presence of blood vessels, Figs. 1, 2D). The epithe- corneal lesions; whereas 1/1 rat whose lids opened lium was primarily affected with only secondary stro- spontaneously on day 14 and 4/5 rats whose eyelids mal changes in the corneas that were more severely were manually opened on day 14 developed lesions. affected. No histologic changes were seen in any non- These lesions, however, were all characterized as affected rats. In moderate and severe lesions, alizarin "mild." The range of severity of corneal involvement

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Table 1. Corneal lesions in animals injected at 20-21 days

Unilateral Bilateral % Animals Drug regimen (n) Unaffected (n) lesions (n) lesions (n) affected

Ketamine + Xylazine (29) 4 4 21 84 Ketamine + Xylazine + Lubricant (10) 2 2 6 80 Ketamine 12.5 mg/kg (7) 7 0 0 0 25mg/kg(5) 5 0 0 0 50 mg/kg (5) 5 0 0 0 Xylazine 15 mg/kg (6) 6 0 0 0 30 mg/kg (5)* 4 0 0 0 60 mg/kg (3)t — — — — Ketamine + Xylazine + Yohimbine (17-19d)(18) 17 1 0 6 Nembutal(lO) to 0 0 0 Chloropent (20) 20 0 0 0

* One died—irreversible respiratory depression; t all three died—irreversible respiratory depression.

was not age-dependent from 16 to 21 days of age. The resulting in lasting lesions in the visual axis of the lesions that developed in animals 13-14 days of age, juvenile rat. The corneal lesions were induced at however, were all mild. The latency to appearance of doses considerably lower than those generally used as the lesion was shorter in the 16-17-day-old pups (as anesthetic doses in laboratory animals.6 early as 2 days following injection) than in the The production of corneal lesions by drugs is not 20-21-day-old pups (lesions first noted 5-7 days fol- unique to exposure to the combination of ketamine lowing injection). All lesions noted remained stable and xylazine. Corneal opacities in rodents have been (ie, did not change in severity) and did not regress reported to result from exposure to antidepressants,7 during the study period of up to 7 weeks. capsaicin,8 morphine sulfate,9 and 1-a-acetylmetha- No evidence of rat coronavirus or sialodacryaden- dol.10 The described lesions differ histologically to itis virus was obtained. some degree from each other and from the lesion reported here, suggesting that the cornea is a com- Discussion mon target organ with a multiplicity of influences on it. All of the drugs reported to induce corneal lesions, It is evident from these observations that exposure however, act on the central nervous system: ket- to the combination of ketamine hydrochloride and amine, xylazine, and the antidepressants interact xylazine (but to neither alone at dosages up to two to with noradrenergic neurons; ketamine may also act at four times those used in combination) during a par- glutamate receptors11; capsaicin causes damage to ticular developmental period has a high likelihood of primary sensory afferent neurons whose neurotrans-

Fig. 3. Photomicrographs of a moderately affected cornea stained with gold chloride. (A) Low magnification view demonstrating neovascularization (arrows) extending from limbus to lesion (L). (B) High magnification view demonstrating innervation (arrows). There was no qualitative difference in innervation between control and exposed corneas.

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Table 2. Corneal lesions in animals injected with ketamine and xylazine as a function of age at injection

Total studied Unaffected Unilateral Bilateral % Animals Age (days) (n) (n) lesion (n) lesion (n) affected

13-14 (lids closed) 10 10 0 0 0 (lids open) 6* 1 2 3 83 16-17 19 4 7 8 79 20-21 29 4 4 21 84 23-24 12 11 1 0 8 27-28 12 12 0 0 0 60-90 24 24 0 0 0

1 One opened spontaneously, five opened manually.

mitter is believed to be substance P; and morphine logic examination of mildly affected corneas showed sulfate and 1-a-acetylmethadol are narcotic analge- involvement of the epithelial basal cells but not the sics that interact with opiate receptors. In addition, outer layer of squamous cells, as would be expected if although both juvenile and adult animals may be sus- desiccation were a critical factor. Third, neither nem- ceptible to the drug effects, higher and/or multiple butal nor chloropent exposure, which resulted in a doses of drugs were used to produce lesions in adults. longer duration of anesthesia and hence a greater In the only other study8 in which animals of a range likelihood of drying of the cornea, resulted in corneal of ages were used, peak incidence occurred during the lesions. Additionally, when yohimbine was given ei- second and third weeks of life and declined by the ther simultaneously with or 30 min after the combi- eighth week of life. Although no clear etiology of the nation of ketamine and xylazine, corneal lesions did corneal pathology has thus far been elucidated, the not develop. Lesions were prevented despite no ap- greater susceptibility of juvenile animals of a nonpre- parent change in degree or duration of anesthesia. cocial species to centrally acting drugs suggests an Direct toxicity to the corneal epithelial cells by the effect on or via an evolving innervation. Gold chlo- combination of ketamine and xylazine or their me- ride staining of whole cornea seems to suggest, how- tabolites remains a possibility. At the same dosage on ever, that gross innervation patterns are not altered a per weight basis, ketamine plus xylazine was toxic by drug exposure. Therefore, additional studies of (causing irreversible respiratory depression) to a ma- neural function are necessary to elucidate possible jority of rat pups treated at 7 days of age (five of six). mechanisms of action. Duration of anesthesia was prolonged in 13-14- vs. Several possible pathophysiologic mechanisms of 20-21-day-old rats (average 80 min vs. 40 min). If corneal damage were addressed in this study. Evi- simply a slower, less efficient rate of metabolism of dence for a coincident viral infection was sought ini- the drugs were responsible for the toxicity, a more tially due to the presence of inflammatory cells (lym- severe lesion would have been expected in younger phocytes and polymorphonuclear cells) on histologic animals. This was not the case, as 13-14-day-old examination. The negative serologic studies dis- pups developed only mild lesions, whereas 16-17- or counted that possibility. 20-21-day-old pups developed lesions ranging from Although desiccation of the corneal epithelium mild to severe. may be one factor contributing to the production of The limitation of the toxicity to rats 21 days of age corneal lesions, as suggested by Fabian et al9 and by or younger suggests that the cornea itself or its milieu the observation that, in the present study, lesions may be different prior to 23 days. Since the cornea is could be produced in 13-14-day-old pups with an avascular structure, all nutrients, including oxy- opened eyelids and not in pups whose eyelids re- gen, reach the cells by diffusion from the limbic blood mained closed, it is not the sole etiologic factor. Fa- vessels or surrounding environment. The partial bian et al9 could reduce the incidence of corneal opa- pressure of oxygen reaching the corneal epithelial cities by application of warm saline during deep de- cells was estimated to change from a maximum of pression; however, application of an ocular lubricant approximately 100 mm Hg prior to eye opening to during anesthesia in the present study failed to pre- 170 mm Hg following eye opening. Eye opening also vent development of the lesion in 20-21-day-old results in a change in the intensity of light to which pups. Furthermore, despite absence of lubricant in the cornea is exposed. In the presence of ketamine rats 23 days of age or older, no lesions developed. and xylazine (and/or their metabolites), the increased Similarly, Shimizu et al8 demonstrated that tarsorr- light intensity may adversely affect the basal epithe- haphy did not prevent development of corneal opaci- lial cell layer. However, preliminary studies in which ties in animals treated with capsaicin. Second, histo- 18-day-old animals were kept in constant darkness

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from the time of injection until the time of examina- References tion, 6 days later, suggest that this is not the case. All 1. Lenz W: Chemicals and malformations in man. In Congenital of the injected pups developed moderate to severe Malformations. New York, International Medical Congress, bilateral corneal lesions. 1964, pp. 263-276. How the induction of the corneal lesion is related 2. McBride WG: Thalidomide and congenital abnormalities. to mechanisms mediating the anesthetic and analge- Lancet 2:1358, 1961. 3. Lanning CF and Harmel MH: Ketamine anesthesia. Ann Rev sic actions of the drugs remains to be determined. Med 26:137, 1975. Although both ketamine hydrochloride and xylazine 4. VanPelt LF: Ketamine and xylazine for surgical anesthesia in 1213 interact with monoaminergic neurons, they may rats. J Am Vet Med Assoc 171:842, 1977. do so at different sites. It appears that both drugs are 5. 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Roberts E, Wong E, Svenneby G, and Degener P: Sodium-de- The possible importance of monoaminergic neurons pendent binding of GABA to mouse brain particles. Brain Res to the development of corneal function is also un- 152:614, 1978. clear. However, these studies demonstrate that devel- 14. Hedler L, Stamm G, Weitzell R, and Starke K: Functional characterization of central a-adrenoceptors by yohimbine opmental critical periods for the induction of abnor- diastomers. Eur J Pharmacol 70:43, 1981. malities by exposure to exogenous substances need 15. Schmitt H, LeDouarec J-C, and Petillot N: Antinociceptive not be confined to the early embryonic stage of or- effects of some sympathomimetic agents. Neuropharmacology ganogenesis. Determining the mechanisms underly- 13:289, 1974. ing the anesthetic-induced pathology will provide 16. Schmitt H, LeDouarec J-C, and Petillot N: Antagonism of the antinociceptive action of xylazine, a sympathomimetic agent, valuable information about corneal development by adrenoceptor and cholinoceptor blocking agents. Neuro- that can be applied to other species. pharmacology 13:295, 1974. 17. Hatch RC and Ruch T: Experiments on antagonism of ket- Key words: ketamine, xylazine, band keratopathy, neural amine anesthesia in cats given adrenergic, seratonergic, and development, corneal development cholinergic stimulants alone and in combination. Am J Vet Res 35:35, 1974. Acknowledgments 18. Sakiyama T, Kuwayama Y, Ishimoto I, Sasaoka A, Shiodaka S, Tohyama M, Manabe R, and Shiotani Y: Ontogeny of sub- The expert advice of S. Searl and the technical assistance stance P-containing structures in the ocular tissue of the rat: of M. Morgenstern, K. Steinmetz, and L. Johnstone were An immunohistochemical analysis. Dev Brain Res 13:275, greatly appreciated. 1984.

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