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Investigative Ophthalmology March 1976 216 Reports munologic stimuli such as corneal allografts1 as well as prolong graft survival.1"4 However, in considering the adoption of this technique to human corneal transplantation, several considerations make a homologous antibody preparation more desirable than a heterologous preparation. There are now available techniques for measuring the number and quantity of various antibodies against human transplantation antigens. Thus, it would be possible to repeatedly make a given antibody preparation for use in corneal transplantation. A homologous preparation is less cytotoxic and therefore carries less potential for damage to the graft. Likewise, it is not necessary to chemically modify the homologous preparation to avoid this problem. The results of the present experiments indicate that homologous antibody against transplantation antigens is successful in prolonging rabbit corneal allograft survival. Based on these studies, it would appear that similar pretreatment of human corneal allografts might be successful in prolonging graft survival and even, perhaps, in reducing the incidence of allograft rejection. The author acknowledges the technical assistance of Ms. Patricia Skahen and Ms. Karen Vedder. From the Department of Ophthalmology University of Washington School of Medicine, Seattle, Wash. 98195. Supported by Grant EY-01282 from the National Eye Institute. Submitted for publication Sept. 17, 1975. Reprint requests: Dr. J. Chandler. REFERENCES 1. Burde, R. M., Waltman, S. R., and Berrios, J. H.: Homogaft rejection delayed by treatment of donor tissue in vitro with antilymphocyte serum, Science 173: 921, 1971. 2. Chandler, J. W., Gebhardt, B. M., and Kaufman, H. E.: Immunologic protection of corneal allografts: preparation and in vitro testing of heterologous "blocking" antibody, INVEST. OPHTHALMOL. 12: 646, 1973. 3. Chandler, J. W., Gebhardt, B. M., Sugar, J., et al.: Immunologic protection of rabbit corneal allografts: survival of corneas pretreated with succynilated anti-lymphocyte globulin, Transplantation 17: 146, 1974. 4. Binder, P. S., Gebhardt, B. M., Chandler, J. W., et al.: Immunologic protection of rabbit corneal allografts with heterologous blocking antibody, Am. J. Ophthalmol. 79: 949, 1975. 5. Kaliss, N.: Micromethod for assaying immune cytolysis by the release of 51Cr, Transplantation 8: 526, 1969. 6. Khodadoust, A. A.: Penetrating keratoplasty in the rabbit, Am. J. Ophthalmol. 66: 899, 1968. 7. Khodadoust, A. A., and Silverstin, A. M.: Transplantation and rejection of individual cell layers of the cornea, INVEST. OPHTHALMOL. 8: 180, 1969. 8. Guttman, R. D., Carpenter, C. B., Lindquist, R. R., et al.: Renal transplantation in the inbred rat. III. A study of heterologous antithymocyte sera, J. Exp. Med. 126: 1099, 1967. 9. Wilson, W. E. C , Kirkpatrick, C. H., and Talmage, D. W.: Immunologic studies in human organ transplantation. III. The relationship of delayed cutaneous hypersensitivity to the onset of attempted kidney allograft rejection, J. Clin. Invest. 43: 1881, 1964. 10. Sell, S.: Antilymphocytic antibody: effects in experimental animals and problems in human use, Ann. Intern. Med. 71: 177, 1969. Dissecting ocular tissue for intraocular drug studies. ROBERT ABEL, JR.* AND GERARD L. BOYLE. This report describes a convenient reproducible ocular dissection technique which has important applications for ocular antimicrobial penetration studies. Different ocular tissues can be sectioned while frozen and then plated directly on culture medium containing the test organism; after the zones of bacterial inhibition are measured at 18 hours following incubation, the tissue specimens are weighed providing more reliable evidence regarding drug concentrations. In such a fashion, a drug can be administered topically, subconjunctivally, or systemically, and assayed, from the cornea to the optic nerve at various time intervals. Analysis of antibiotic in the vitreous body, which has important application in the therapy of endophthalmitis, can be routinely performed in the experimental model. The judicious use of antimicrobial therapy is based on controlled ocular penetration studies. Traditionally, these evaluations are limited to analyzing drug concentrations in the aqueous humor and serum. Recently, certain authors1-3 have incorporated tissue dissection, in addition to performing anterior chamber paracentesis in order to study drug uptake in the eye. The purpose of this report is to present a convenient method of ocular dissection which facilitates antibiotic analysis of separate tissues with minimal contamination. For this reason, cefazolin sodium was selected as a sample antibiotic to illustrate the information that can be provided by ocular dissection. Methods. In a previous report,4 albino rabbits received subconjunctival (one eye only), intramuscular, and intravenous cefazolin sodium at various time intervals before sacrifice. The discussion will be confined to the group of 46 rabbits (two to four animals for each time period) L Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933066/ on 05/14/2017 Volume 15 Number 3 which received unilateral subconjunctival antibiotic injection. Painless eardiorespiratory arrest was accomplished by the administration of sufficient intravenous sodium pentobarbital. Samples of primary aqueous humor were immediately obtained from each eye by anterior chamber paracentesis under sterile conditions using a 25-gauge needle mounted on a disposable syringe. Simultaneously, 10 ml. of blood was also collected by intracardiac aspiration. The sterile containers of aqueous humor and serum samples were frozen at -70° C. until the time of the bioassay. The eyes of each rabbit were enucleated removing as much episcleral and leptomeningeal tissue as possible leaving at least 5 mm. of optic nerve. Each eye was positioned, cornea-side up, in a sterile disposable capped tube, which was immediately frozen by immersion in a mixture containing solid carbon dioxide and acetone. The frozen eye samples were then stored in a deep freeze at -70° C. until the time of dissection. The tube dilution technique"' was employed in the determination of the aqueous humor and serum levels of ccfaznlin sodium with tin; lest organism, Staphyhcoccus aureus (ATCC 12(300). The cefazolin sodium activty of the other ocular tissues and fluids was determined by the agar diffusion assay technique employing Mueller-Hinton agar and the same test organism. The test organism was grown and standardized, after which the sensitivity plates were seeded with a 10-a dilution of this culture. The seeded plates were then dried at 37° C. for 15 minutes. A standard curve for determining antibiotic concentration in ocular tissue and vitreous humorsamples was prepared from data obtained when sterile filter paper discs (6.35 mm. in diameter) were immersed in known cefazolin concentration in trypticasc soy broth and then placed in triplicate on the inoculated plates containing the test organism. After overnight incubation at 37° C, the zones of bacterial inhibition were measured and results averaged in order to plot the standard curve for the known concentrations of cefazolin sodium. In such a way, concentrations of antibiotic in ocular tissue could be expressed in micrograms per 100 mg. of tissue, whereas the vitreous humor antibiotic concentration was determined in mierograms per milliliter of fluid. The frozen eyes were removed for dissection using sterile technique. Each globe was bisected just through the back of the lens in order to provide an anterior and posterior vitreous compartment, by using a No. 11 Bard-Parker blade and scalpel in a sawing movement (Fig. 1). This step was facilitated by the use of an adjustable wire clamp which limits excursion of the globe during the initial dissection. The anterior and posterior vitreous samples were rapidly eviscerated Reports 217 Fig. 1. Bisected eye showing the frozen lens in the anterior half of the globe. Fig. 2. Dissection of the corneoseleral cap, iris, and ciliary bod)' away from the lens and anterior vitreous. Frozen aqueous can be seen lying on the sterile Petri dish between the solera and iris. (Fig. 2) and placed in separate sterile tubes; it is essential to complete this step before thawing occurs. Lens, iris, cornea, sclcra, and optic nerve tissue samples were rinsed in sterile saline (to eliminate surface contaminants), drained, and placed on the previously dried agar plates inoculated with the test organisms. At a later time they were weighed and measured. Sterile filter paper discs were absorbed with anterior and posterior vitreous samples and then placed on the Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933066/ on 05/14/2017 Investigative Ophthalmology March 1976 218 Reports Table I. Comparison of in vitro susceptibilities of various organisms to cefazolin sodium with the aqueous and vitreous humor levels attained after subconjunctival injection (25 mg.) Average antibiotic concentration (ng/ml.) Vitreous level Pathogen Citrobacter sp. Herellea sp. Ps. aeruginosa Serratia sp. Prot. sp. (ind. pos.) Enterobacter sp. Enterococcus D. pneumoniae Pr. mirabilis Shigella sp. Salmonella sp. Bacteroides H. influenza Esch. coli Kleb. pneumoniae Coryn. diphtheriae Str. viridans S. aureus N. gonorrhoeae Str. pyogenes Cl. perfringens Cl. tetani M.I.C. (ng/ml.) Cefazolin (5) Aqueous level 0.25 hr. 1.0 hr. Anterior 0.25 hr. Posterior 1.0 hr. 0.25 hr. 1.0 hr. >128 > 128 > 128 >128 32->128 8-M28 32.0 0.12-25 8.0 8.0 4.0 2-4.0 2-4.0 1-4.0 2.0 2.0 0.5-1 0.25-5 0.25 0.12 0.06- .12 0.06 30.8 surface of the inoculated plates. Following overnight incubation at 37° C. for 18 hours, zones of bacterial inhibition (i.e., a clear zone surrounding the various ocular samples) were measured. Controls included similar ocular tissue samples obtained from uninjected rabbits. Results. Table I indicates the aqueous and vitreous humor antibiotic concentrations at 0.25 and 1 hour following 25 mg. subconjunctival injection of cefazolin sodium juxtaposed on a list of the in vitro susceptibilities of many potential pathogens to this agent. Fig. 3 illustrates the cefazolin concentrations achieved at varying time intervals in aqueous humor and anterior and posterior vitreous following subconjunctival administration. The results of bioassay of cornea and sclera samples are shown in Fig. 4; peak concentrations are attained in 30 minutes. Discussion. Investigations of the intraocular penetration of new antimicrobial agents have usually been based on aqueous humor and serum determinations. Employing ocular tissue dissection in such experimental studies can provide meaningful information about antibiotic distribution in other ocular fluids and tissues whatever the route of drug administration. Immediate freezing of the eye permits the later analysis without complication of biodegradation of drug or difficulty of keeping tissues separate. Freezing also allows the investigators to dissect 9.0 18.0 15.3 2.92 1.08 all of the eyes at one time without being disturbed by other procedures. More importantly, the gelatonoid vitreous humor can now be conveniently obtained and analyzed. The concentrations of antibiotic in solid tissue were calculated by comparing the zone of bacterial inhibition from weighed tissue specimens to the zones obtained from filter paper discs immersed in known cefazolin concentrations, in a manner similar to the technique of Barza and co-workers.3 However, these tissue levels of antibiotics must be considered relative rather than absolute because diffusion of drug from tissue samples does not necessarily coincide with diffusion from filter paper. It would be disadvantageous to employ tissue homogenization because the dilutional factor would affect the quantitation of small amounts of antibiotic and many separate blenders would have to be used to eliminate contamination. Significant antibiotic penetration was demonstrated in sclera, cornea, and iris tissue samples after subconjunctival injection (as might be expected), but also following intramuscular and intravenous cefazolin administration. 4 There is some antibiotic absorption into the anterior and posterior vitreous humor (Fig. 3) and even the optic nerve after subconjunctival injection of antimicrobial agents. Perhaps even higher drug levels in the vitreous, retina, and choroid could be at- Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933066/ on 05/14/2017 Volume 15 Number 3 Reports 219 A Aqueous o Anterior Vitreous • Posterior Vitreous A 30 n.a.a. A O A 20 A A A 0 t No antibiotic assayable A A 0 10.0 o 7.5 0 A 5.0 • o -. 2.5 0 o o o • 0 A A A O O O O A A 0 15 m a 30 45 0 2 1 MINUTES 3 HOURS Fig. 3. Comparison of aqueous and vitreous humor drug (cefazolin sodium 25 mg.) levels following subconjunctival injection. A CORNEA 3000 2000 A , • SCLERA A n.a.a. 1000 No antibiotic assayable • *• 200 80 : 60 'A 40 • 20.0 A A A io.o; A A 5.0 REFERENCES • 2.5 • 0 n.a.a. A •• 0 15 30 45 MINUTES 1 3 From the Departments of Ophthalmology, Mount Sinai School of Medicine of the City University of New York, and Temple University Hospital, Philadelphia, Pa. This study was supported in part by Grant No. EY-00340 of the National Eye Institute, National Institutes of Health. Submitted for publication Aug. 13, 1975. Reprint requests: Dr. R. Abel. ^Present address: 1300 Harrison St., Wilmington, Dela. 19806. Key words: ocular tissue dissection technique, antibiotic penetration studies, cefazolin sodium, cryodissection, aqueous humor drug level, vitreous body drug level. A 7.5 hematogenous distribution. Although our method of assaying cefazolin sodium was not sensitive enough to quantitate the aqueous humor drug level in the contralateral eye after subconjunctival injection, another study demonstrated adequate bactericidal concentrations of gentamicin in the aqueous humor of the opposite eye.7 This ocular cryodissection technique is especially useful for analyzing antibiotic activity in anterior and posterior vitreous tumor, in addition to the other ocular tissue samples. In the cefazolin study, significant vitreous absorption was present from five minutes to three hours after subconjunctival inoculation of 25 mg. of antibiotic; also, minimal cefazolin levels were detected in the contralateral eye for 15 to 45 minutes after the injection. The ability to achieve high antibiotic concentrations in the vitreous humor is an extremely important aspect in the prevention and treatment of intraocular infections. Such an ocular tissue dissection method can be applied to various avenues of drug delivery, can include retina and choroid specimens, and can provide meaningful information regarding drug penetration in the inflamed or postoperative eye. 4 5 HOURS Fig. 4. Antibiotic (cefazolin sodium 25 mg.) activity in cornea and sclera following subconjunctival administration. tained after retrobulbar or intracameral administration. Orally administered agents, such as amoxicillin, a new semisynthetic penicillin, have also been found to adequately penetrate ocular tissues with this dissection technique.6 Interestingly, significant antibiotic content can be found in the cornea and sclera of the contralateral eye after subconjunctival administration.4 This can probably be explained on the basis of 1. Salminen, L., Jarvinen, H., and Toiva.nen, P.: Distribution of tritiated penicillin in the rabbit eye, Acta Ophthalmol. 47: 115, 1969. 2. Bloom, M. A., Golden, B., and McKee, A. P.: Antibiotic concentration in ocular tissues, Arch. Ophthalmol. 83: 78, 1970. 3. Barza, M., Baum, J., Birkby, B., et al.: Intraocular penetration of carbenicillin in the rabbit, Am. J. Ophthalmol. 75: 307, 1973. 4. Abel, R., Boyle, G. L., Furman, M., et al.: Intraocular penetration of cefazolin sodium in rabbits, Am. J. Ophthalmol. 78: 779, 1974. 5. Schneierson, S. S., and Torarsky, B.: A method for the determination of Aureomycin in the blood, J. Bacteriol. 57: 483, 1949. 6. Faigenbaum, S. J., Boyle, G. L., Prywes, A. S., et al.: Intraocular penetration of amoxicillin, submitted for publication. 7. Abel, R., Jr., Mascuilli, L., and Boyle, G. L.: Subconjunctival gentamicin prophylaxis against post-operative staphylococcus endophthalmitis in the rabbit, submitted for publication. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933066/ on 05/14/2017