The Inflammatory Role of Endotoxin in Rabbit Gram-Negative Bacterial Endophthalmitis

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1 The Inflammatory Role of Endotoxin in Rabbit Gram-Negative Bacterial Endophthalmitis Dean R. Jacobs and Howard B. Cohen The authors used the limulus lysate assays to measure the amount of gram-negative endotoxin produced in two rabbits with experimentally induced gram-negative (Escherichia coif) endophthalmitis. A similar amount of purified enodotoxin was injected into the eyes of 14 rabbits to determine the rate of clearance of endotoxin from the rabbit eyes. Endotoxin was found in clinically inflammatory quantities 2 weeks after injection. Results of pathologic examination showed that endotoxin incites severe inflammatory responses in the eye, affecting the ciliary body, vitreous, choroid, retina, and optic nerve. These results suggest that the limulus lysate assay may be useful for detecting early gram-negative endophthalmitis, and that in such cases, early emergency vitrectomy may be needed to remove the inflammation-inciting endotoxin and preserve useful vision. Invest Ophthalmol Vis Sci 25: , 1984 Bacterial endophthalmitis is a rare but devastating complication of intraocular surgery and trauma. Conventional therapy consisting of topical, intravitreous, and systemic antibiotics is often ineffective in preserving useful vision, even when used properly in cases diagnosed early. 1 " 4 Several authors have described the particularly poor prognosis associated with gram-negative bacterial endophthalmitis. 3 " 7 We have observed several cases of endophthalmitis that show increasing inflammation with media cloudiness despite obtaining negative vitreous cultures 24 hr after initial injection of intravitreous antibiotics. This observation suggests that one or more mediators of inflammation damage the eyes after the organisms are killed. Gram-negative endotoxin may be such a mediator. The purpose of this report is to describe the pathologic effects and clearance of purified endotoxin injected into the vitreous of adult rabbit eyes. Identification of the role of endotoxin in clinical endophthalmitis infections is aided by examining the ocular effects of endotoxin without bacteria present. From Ophthalmology Service, Department of Surgery, Letterman Army Medical Center, Presidio of San Francisco, California. Supported by the Department of Clinical Investigations at Letterman Army Medical Center. Submitted for publication: May 10, The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense. Reprint requests: Howard B. Cohen, MC, Box 13, Letterman Army Medical Center, Presidio of San Francisco, CA Materials and Methods Endotoxin in Clinical Infection We obtained vitreous samples for culture from two cases of human bacterial endophthalmitis that were brought to vitreous tap less than 24 hr after the onset of symptoms. Quantitative plating of these samples yielded bacterial growth of cfu (colony-forming units)/ml. To approximate such a bacterial density in rabbit eyes, the vitreous of the right eye in two large (5 kg) male New Zealand albino rabbits was injected with a 0.1 ml solution containing 5 X 10 4 cfu/ml of Escherichia coli (Federal Drug Administration strain #25922). The solution was injected into the vitreous 3 mm from the limbus through the pars plana, which was identified by transillumination. Direct visualization of the needle tip through the cornea allowed proper placement of the organisms in the central portion of the anterior vitreous. We used 27-gauge needles on 1- cc syringes. The rabbits were anesthetized with ketamine (35 mg/kg) and xylazine (Rompun) (5 mg/kg) injected intramuscularly. After 24 hr we sedated the rabbits again and killed them with intravenous injections of sodium pentobarbital and alcohol (euthanol). We enucleated the eyes and removed cc of vitreous through a pars plana approach with a 20-gauge needle on a 3- cc syringe. The vitreous was plated quantitatively on blood agar for bacterial colony counts. The concentration of endotoxin in the infected eyes was measured by the limulus lysate assay. We used a Mallinckrodt assay system that measured the presence of endotoxin in concentrations as low as pg/ 1074

2 No. 9 OCULAR EFFECTS OF ENDOTOXIN / Jacobs and Cohen 1075 ml. 8 All assays were run in duplicate with appropriate positive and negative controls. When reading the assay, we considered the last positive dilution as the breakpoint. The amount of endotoxin present in the vitreous of each eye was calculated as: assay sensitivity X vitreous volume -r- breakpoint dilution. (Vitreous volume averaged 2.0 ml in our large rabbits). This result represents the total amount of endotoxin present in the rabbit vitreous during an active clinical infection of gram-negative bacterial endophthalmitis. Injection of Purified Endotoxin We injected the right eyes of 14 large (4.2 to 5.2 kg), albino, New Zealand, male rabbits in the manner described above with 50 fig of purified E. coli endotoxin (E. coli serotype O55:B5, Mallinckrodt lot #1JO). Fifty micrograms of endotoxin represents the total endotoxin present in the infected eyes (see Results). For controls we injected fellow eyes in each rabbit with 0.1 cc of pyrogen-free saline. To monitor systemic endotoxin absorption, we recorded each rabbit's rectal temperature before the injections were given and 24 hr later. We compared the intraocular pressure of the endotoxin-injected eye with its fellow, using a Schiotz' tonometer prior to killing. To study the clearance of endotoxin from the vitreous we killed two rabbits at 1,2, 3, 5, 7, 10, and 14 days postinjection. We enucleated the eyes and took samples of the vitreous as described above. A drop of each sample was cultured on blood agar at 37 C, and a smear was made for pathologic examination. After the samples were centrifuged at 3,000 rpm for 15 min, the supernatant was removed and stored at 4 C until assayed. The globes were fixed in 10% formalin. Endotoxin in each vitreous sample was measured by the limulus lysate assay in order to determine the rate of endotoxin clearance from the vitreous. Each sample was diluted serially in twofold dilutions from 1:50 to 1:12,800 and assayed in duplicate for endotoxin. Appropriate positive and negative controls also were assayed. Undiluted assays were performed on vitreous samples from the control eyes injected with saline. Sensitivity of this assay was 25 pg/ml. A clearance curve was plotted, and the rate of clearance calculated. Results Endotoxin in Infected Eyes Twenty-four hours after injection, the eyes infected with E. coli had 4.2 and 4.5 X 10 9 cfu/ml in the vitreous. The eyes had clinical endophthalmitis with Table 1. Comparison of endotoxin dilution breakpoints for duplicate limulus assays of vitreous samples taken from rabbit eyes 24 hr after experimental induction of E. coli infection in the vitreous Duplicate assay results^ Dilution* Rabbit 1 Rabbit 2 : 10,000 :50,000 : 100,000 :500,000 : 1,000,000 :5,000,000 : 10,000,000 * Average breakpoint 1:500,000; tassay sensitivity = 50 pg/ml; +, positive; negative. cloudy corneas, marked adnexal inflammation, and poor direct pupillary response. Results of limulus assays on the infected vitreous samples are listed in Table 1. Duplicate assays of the samples from rabbit 1 had clean breakpoints at a dilution of 5 X 10~ 5. Assays of samples from rabbit 2 had breakpoints at 5 X 10~ 5 and 10~ 6. An average breakpoint of 5 X 10~ 5 was used to calculate the amount of endotoxin present in the vitreous of the infected eyes. The sensitivity of these assays was 50 pg/ml. The amount of endotoxin present in the infected vitreous samples, calculated to be 5 X 10 7 pg/ml or 50 tig/m\, represents an approximation of the amount of endotoxin found in the clinically infected rabbit vitreous. Table 2. Endotoxin clearance data from limulus assays of vitreous samples taken from rabbit eyes 24 hours after injection of 50 ng of E. coli endotoxin into the vitreous Day postinjection Breakpoint dilution : 12,800 :6,400 :6,400 : 1,600 : 1,600 :100 : 1,600 :800 :800 :400 Calculated amount of endotoxin in vitreous* Amount of endotoxin (ng) = sensitivity of assay (25 pg/ml) X volume of vitreous (2 ml) -e- breakpoint dilution. i

1076 INVESTIGATIVE OPHTHALMOLOGY b VISUAL 5CIENCE / September 1984 Vol. 25 ative. All vitreous cultures were negative in both the endotoxin and the control groups, ruling out accidental contamination.

3 1076 INVESTIGATIVE OPHTHALMOLOGY b VISUAL 5CIENCE / September 1984 Vol. 25 ative. All vitreous cultures were negative in both the endotoxin and the control groups, ruling out accidental contamination. Pathologic Effects of Endotoxin Time Idavs) Fig. 1. Graph showing rate of endotoxin clearance in rabbit eye vitreous. Vertical axis represents the amount of endotoxin remaining in the vitreous. Horizontal axis represents the number of days after injection of the vitreous with 50 ng of endotoxin. Endotoxin Clearance Results of the clearance study on 14 rabbit eyes are found in Tables 2 and Figure 1. Table 2 lists endotoxin clearance data. Figure 1 shows the rate of endotoxin clearance in the vitreous. The graph demonstrates that significant quantities of endotoxin still remained in the eyes 2 weeks after injection. Regression analysis was used to fit the curve to the equation y = In x. The coefficient of correlation (R) was 0.92 and is significant at the P < level using Student's t-test with N = 13. The 5-ng value that occurred on day 7 was omitted. All but one of the undiluted vitreous samples from control rabbit eyes were negative for the presence of endotoxin. A single, positive test may have resulted from a break or variation in technique, since repeat tests of diluted samples from the same eyes were neg- The rabbits' average rectal temperatures were 39.3 C before and 39.4 C 24 hr after injection of endotoxin into the eyes. This difference is not significant (P = 0.32 by two-tailed Student's t-test). This finding suggests that endotoxin is absorbed poorly into the blood stream, since only 0.1 ng of intravenous endotoxin (1/ 500,000 of the amount of endotoxin injected) can cause a 0.6 C rise in a rabbit's core temperature. 9 The rabbit eyes were observed closely after injection with endotoxin. Evidence of iris injection became evident within 6 hr after injection. Slower direct pupillary response also was evident after only 6 hr, and absence of pupillary response to a 100-watt light source held 10 cm from the eye was observed within 24 hr. Pupils remained normal in the control eyes. Figure 2 shows the clinical effects of endotoxin on the external eye. Corneal clouding, engorged iris vessels, nonreactive pupils, and peri-limbal conjunctival injection characterized these effects at 24 hr. Most effects diminished during the 14-day period, except for pupillary response, which remained absent in all endotoxin-treated eyes. Measurement of intraocular pressure revealed a mean Shiotz' value of 13.2 mmhg in the endotoxintreated eyes and 17.0 mmhg in the fellow eyes. This decrease was significant (P = 0.01). Histopathologic examination of the eyes revealed a marked cellular inflammatory response to the endotoxin. This response was present after 24 hr and subsided only slightly during the 14-day period. Inflammation over the ciliary body was distinct after 24 hr (Fig. 3). Vitreous inflammation was pronounced with organized vitreous present in most samples. An impressive invasion of the choroid with polymorphonuclear neutrophil leukocytes, lymphocytes, and macrophages was present at 24 hr and persisted for 2 weeks. Focal inflammatory cell invasion of the retina and optic nerve occurred with patchy destruction of retinal architecture (Fig. 4). The retinal and choroidal changes were similar to those found in the rabbit eyes infected with E. coli bacteria (Fig. 5). Discussion Fig. 2. Clinical effects on the external rabbit eye 24 hr after injection of E. coli endotoxin into the vitreous. Note corneal clouding and adnexal swelling. Endotoxin is a lipopolysaccharide-protein complex present in the walls of all gram-negative bacteria. It is released in increased amounts after cell lysis and death, 10 and is a known mediator of inflammation, causing mast cell degranulation, 1 ' polymorphonuclear

.:» ' * No. 9 OCULAR EFFECTS OF ENDOTOXIN / Jacobs and Cohen 1077 Fig. 3. Ciliary body inflammation in the rabbit eye 24 hr after injection of vitreous with 50 /xg of E.

4 .:» ' * No. 9 OCULAR EFFECTS OF ENDOTOXIN / Jacobs and Cohen 1077 Fig. 3. Ciliary body inflammation in the rabbit eye 24 hr after injection of vitreous with 50 /xg of E. colt endotoxin (hematoxylin and eosin, X60). leukocyte margination in vessel walls,' 2 and activation of complement and coagulation pathways. 13 Systemically injected endotoxin increases uveal vascular permeability particularly of the ciliary processes. This increased vascular permeability returned to normal by 24 hr. 14 " 17 When endotoxin was injected intravitreally, a prolonged increase in uveal permeability was found in the absence of clinical or histologic evidence of acute inflammation. 18 " 19 Our purpose in measuring the endotoxin in infected rabbit eyes was to obtain a rough estimate of endotoxin present in clinical infections. Pathologic effects on endotoxin injected into healthy eyes then could have relevance to clinical infections. An infection is a dynamic event that occurs with bacterial concentrations varying over time. For this reason, our estimate of the amount of endotoxin present in an infected rabbit's eye at 24 hr is a crude estimate of endotoxin present in a clinical infection. The actual amount of endotoxin present probably varies a great deal at various times during the infection. In addition, since endotoxin is a bacterial cell wall constituent, its chemical makeup and activity will vary among different species and strains of gram-negative bacteria. 10 Endotoxin has been measured by the limulus lysate assay in experimental rabbit endophthalmitis. 20 " 22 Ease of administration, fast results (obtained in a little over

5 t : I KLg. 5. Rabbit retina and choroid 24 hr after experimental E. coli endophthalmitis was induced (hematoxylin and eosin, X100). In the vitreous, 4.5 X 10* cfu were present.

No. 9 OCULAR EFFECTS OF ENDOTOXIN / Jocobs ond Cohen 1079 an hour), and high sensitivity have made this test a potential tool for early detection of gram-negative endophthalmitis.

6 No. 9 OCULAR EFFECTS OF ENDOTOXIN / Jocobs ond Cohen 1079 an hour), and high sensitivity have made this test a potential tool for early detection of gram-negative endophthalmitis. The results of our rabbit experiments clearly show that gram-negative endotoxin alone causes significant damage to the retina, optic nerve, ciliary body, vitreous and choroid. Clearance of endotoxin in the amounts present in this present was slow with small but detectable amounts present at 2 weeks. The presence of gram-negative rods on a gramstained vitreous smear has been considered an indication for emergency vitrectomy in cases of human bacterial endophthalmitis. 6 The use only of antibiotics in these cases results in bacterial death but has not been shown to reduce the level of endotoxin already present at the time the infection is treated. Bacterial death and cell lysis actually may increase the levels of endotoxin in the vitreous. 10 We suggest that a positive limulus assay test, when properly performed, is an indication for vitrectomy in cases of bacterial endophthalmitis, with or without positive smear results. Key words: Endophthalmitis, endotoxin, limulus lysate, vitreous, Escherichia coli Acknowledgment The authors appreciate the significant technical assistance of Dan Brooks, from the Letterman Department of Clinical Investigations, in handling the animals and performing the lysate assay. References 1. Forster RK: Endophthalmitis: Diagnostic cultures and visual results. Arch Ophthalmol 92:387, Forster RK, Zachary IG, Cottingham AJ, and Norton EWD: Further observations on the diagnosis, cause, and treatment of endophthalmitis. Am J Ophthalmol 81:52, Allen HF and Mangiaracine AB: Bacterial endophthalmitis after cataract extraction: A study of 22 infections in 20,000 operations. Arch Ophthalmol 72:454, Vastine DW, Peyman GA, and Guth SB: Visual prognosis in bacterial endophthalmitis treated with intravitreal antibiotics. Ophthalmic Surg 10:76, McGetric FD and Peyman GA: Visual prognosis in bacterial endophthalmitis treated with intravitreal antibiotics. Ophthalmic Surg 10:87, Diamond JG: Intraocular management of endophthalmitis. Arch Ophthalmol 99:96, Rowsey JJ, Newsome DL, Sexton DJ, and Harms WK: Endophthalmitis, current approaches. Ophthalmology 89:1055, Limulus Amebocyte Lysate Pyrogent, Technical Product Data. Mallinckrodt, Inc. Publication number MK-20298, June Greisman SE and Hornick RB: Comparative pyrogenic reactivity of rabbit and man to bacterial endotoxin. Proc Soc Exp Biol Med 131:1154, Morrison DC and Ulevitch RJ: The effects of bacterial endotoxins on host mediation systems. Am J Pathol 93:525, Gustafson GT and Cronberg D: Effect of endotoxin on mast cells and the extension of the local Schwartzman reaction to the hamster. Acta Pathol Microbiol Scand [B] 59:21, Athens JW, Haab OP, Raab SO, Mauer AM, Ashenbruker H, Cartwright GE, and Wintrobe MM: Leudodinetic studies IV. The total blood, circulating and marginal granulocyte pools and the granulocyte turnover rate in normal subjects. J Clin Invest 40:989, Pearlman DS, Sauers JB, and Talmage DW: The effect of adjuvant amounts of endotoxins on the serum hemolytic complement activity in rabbits. J Immunol 91:748, Howes EL, Aronson SB, and McKay DG: Ocular vascular permeability. Effect of systemic administration of bacterial endotoxin. Arch Ophthalmol 84:360, Howes EL Jr and McKay DG: Reports. The effects of aspirin and indomethacin on the ocular response to circulating bacterial endotoxin in the rabbit. Invest Ophthalmol 15:648, Howes EL Jr, McKay DG, and Margaretten W: The participation of the platelet in the vascular response to endotoxemia in the rabbit eye. Am J Pathol 70:1, 25, Howes EL Jr and McKay DG: Comparison of the ocular effects of circulating endotoxin and immune complexes: role of vasoactive amines. J Immunol 114:734, Gamble CN, Aronson SB, and Brescia FB: Experimental Uveitis. The production of recurrent immunologic (Auer) Uveitis and its relationship to increased uveal vascular permeability. Arch Ophthalmol 8:321, Gamble CN, Aronson SB, and Brescia FB: The pathogenesis of recurrent immunologic (Auer) uveitis. Arch Ophthalmol 84:331, Avallone AN, Parrett C, Smith RE, Meyers R, and Chitzian PA: Rapid detection of experimental E. coli endophthalmitis by the limulus lysate test. Invest Ophthalmol Vis Sci 17:528, Ellison AC: The limulus lysate test. A rapid test for diagnosis of Pseudomonas keratitis or endophthalmitis. Arch Ophthalmol 96:1268, McBeath J, Forster RK, and Rebell G: Diagnostic limulus lysate for endophthalmitis and keratitis. Arch Ophthalmol 96:1265, 1978.

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