acute inflammation of the eyelid and cornea in staphylococcus

Acute Inflammation of the Eyelid and Cornea inStaphylococcus Keratitis in the Rabbit

Gregory D. Sloop,1 Judy M. Moreau2 Lisa L Conerly,2 Joseph J. Dajcs,2 andRichard J. O'Callaghan23

PURPOSE. The inflammatory response during Staphylococcus keratitis was analyzed biochemicallyand histologically to determine the source of the neutrophils infiltrating the tear film and cornea.

METHODS. Rabbit eyes were swabbed and then examined by slit-lamp microscopy at 0, 5, 10, 15, 20,and 25 hours after intracorneal inoculation with Staphylococcus aureus. Bacterial colony-formingunits were quantified in the cornea, eyelid, and acute inflammatory exudate. Myeloperoxidaseactivity of ocular swabs of acute inflammatory exudate, corneal homogenates, and eyelid homog-enates was determined. Gross and microscopic examinations of corneas and eyelids were per-formed.

RESULTS. The colony-forming units per cornea exceeded 107 after 10 hours, whereas no bacteriawere cultured from the eyelid until 15 hours postinfection. Slit-lamp examination revealed pro-gressive pathology, and the myeloperoxidase activities of ocular swabs, corneas, and eyelidsincreased markedly by 15 hours postinfection. Corneas showed a wave of neutrophils moving fromthe tear film toward bacteria in the central corneal stroma and early neutrophil migration from thelimbus into the stroma. In the eyelid, neutrophils migrated from the stromal vessels to the tear film.

CONCLUSIONS. Staphylococcus keratitis in the rabbit causes acute inflammation in the overlyingeyelid. Neutrophils of the acute inflammatory exudate interact with the infected cornea, whereasneutrophils migrating through the cornea from the limbus remained distant from the site ofinfection. (Invest Ophthalmol Vis Set. 1999;40:385-391)

The infection of the rabbit cornea with Staphylococcusaureus or the application of pure staphylococcala-toxin to the cornea results in a marked inflammatory

response, including severe corneal edema and an influx ofneutrophils into the corneal stroma.1"3 The present study in-vestigates the inflammatory processes inherent in this infec-tion; such processes are possibly predictive of the eventsoccurring in other ocular inflammations.

In vascularized tissue, inflammatory mediators and leuko-cytes arrive at the site of inflammation directly from bloodvessels. However, an alternative mechanism is necessary forthe avascular cornea. Perhaps the most widely held opinion isthat the neutrophils in acute keratitis originate in the limbusand bulbar conjunctiva4 and that they reach the cornea bychemotaxis through the stroma5 or the tear film.67 In contrast,Weissman et al.8 and Majno and Joris9 have emphasized therole of the eyelid in corneal inflammation. The eyelid could bea source of substantial humoral and formed elements in the

immune response because it is relatively large and has a richervascular supply than the bulbar conjunctiva. The central cor-nea is also closer to the eyelid than to the bulbar conjunctiva,especially when the eye is closed.10 Thus, the eyelid is morelikely to respond earlier and more profoundly to a stimulusfrom the central cornea.

Soluble chemotactins of bacterial origin, such as ./V-formyl-methionyl peptides or toxins, mediate inflammation.3" Che-motactins of host origin such as complement and cytokines arealso known mediators of ocular inflammation. " 1 2 These solu-ble mediators could diffuse through the tear film and elicit ahost response in the overlying eyelid and in the limbus. Despitethe attractive features of this hypothesis, histopathologic doc-umentation of the inflammatory response in the eyelid duringStaphylococcus keratitis is lacking. The aim of the presentstudy was to examine the inflammatory response occurring inthe cornea and eyelid during Staphylococcus keratitis. Empha-sis has been given to the role of the overlying eyelid in theinflammatory process.

From the Departments of 'Pathology and of 2Microbiology, Immu-nology, and Parasitology, Louisiana State University Medical Center;and 3the Louisiana State University Eye Center, New Orleans, Louisi-ana.

Supported in part by United States Public Health Services GrantsEY10974 and EY02377 from the National Eye Institute, National Insti-tutes of Health, Bethesda, Maryland.

Submitted for publication June 30, 1998; revised September 18and October 27, 1998; accepted October 19, 1998.

Proprietary interest category: N.Reprint requests: Richard J. O'Callaghan, Department of Microbi-

ology, Immunology, and Parasitology, LSU Medical Center, 1901 Per-dido Street, New Orleans, LA 70112.

METHODS

Bacterial growth and corneal inoculation. S. aureus 8325-4 wasgrown to log phase in tryptic soy broth (TSB; Difco, Detroit,MI), and the culture was diluted to approximately 104 colony-forming units (cfu) per milliliter, as described previously.'3 Analiquot of this bacterial suspension (10 /xl, 100 cfu) was in-jected into the corneal stroma of both eyes of New Zealandwhite rabbits. Previous studies have shown that injection ofmedia does not result in any ocular changes other than the very

Investigative Ophthalmology & Visual Science, February 1999, Vol. 40, No. 2Copyright © Association for Research in Vision and Ophthalmology 385

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386 Sloop et al. IOVS, February 1999, Vol. 40, No. 2

transient changes associated with the needle's insertion andplacement of liquid into the corneal stroma. All rabbits weremaintained according to the guidelines put forth in the ARVOStatement for the Use of Animals in Ophthalmic and VisionResearch.

Quantification of S. aureus per CorneaThe replication of 5. aureus 8325-4 in the rabbit cornea wasmeasured by determining the number of colony-forming unitsper cornea at 0, 5, 10, 15, 20, and 25 hours postinfection.Rabbits intrastromally injected with 8325-4 were killed, andtheir corneas were harvested (four per time point) and assayedfor colony-forming units at the times described above. Quanti-fication of viable S. aureus per cornea has been previouslydescribed.213 Briefly, whole corneas were aseptically re-moved, dissected, and homogenized. Aliquots of corneal ho-mogenates were serially diluted in sterile phosphate-bufferedsaline (PBS, 0.1 M, pH 7), plated onto tryptic soy agar (0.1 ml,Difco), and incubated at 37°C for 24 hours. Colonies werecounted and colony-forming units per cornea expressed as logbase,,, values.

Quantification of 5. aureus in the Tear FilmThe number of 5. aureus colony-forming units in the acuteinflammatory exudate was determined by Ribbing sterile cot-ton swabs across the conjunctiva and cornea at 0, 5, 10, 15, 20,and 25 hours postinfection (swabs of 4 to 12 eyes were cul-tured per time point). The swabs were immersed in 2 ml sterilePBS for 10 minutes. After agitation, the swabs were removed;three 100-/xl aliquots were plated onto tryptic soy agar (Difco)and incubated at 37°C for 24 hours. Colonies were countedand colony-forming units per swab expressed as log base10

values.

Quantification of S. aureus per EyelidThe number of S. aureus colony-forming units per eyelid wasdetermined by harvesting the eyelids at 0, 5, 10, 15, 20, and 25hours postinfection (8 eyelids per time point). Each eyelid wasbisected parallel to the conjunctiva using aseptic technique.The inner half of each eyelid was then homogenized in sterilePBS, and dilutions were cultured on mannitol salt agar (Difco).Each mannitol-positive colony was identified as S. aureus bydetermining its coagulase activity using rabbit plasma (SigmaChemical Company, St. Louis, MO).

Myeloperoxidase Activity of Ocular Swabs,Corneal Homogenates, and Eyelid HomogenatesSterile cotton swabs were rubbed across the conjunctiva andcornea and then immersed in a sterile solution of CTAB (0.5%in water, hexadecyltrimethyl ammonium bromide; SigmaChemical). Swabs submerged in CTAB were frozen at — 20°C.At the time of assay, the immersed swabs were thawed, frozenin liquid nitrogen, and thawed again to lyse neutrophils. Swabswere discarded, and the CTAB solution was assayed for myelo-peroxidase activity as described previously.1415 Corneal ho-mogenates were prepared in sterile PBS (3 ml per cornea) andassayed as described previously.1415 Eyelids were homoge-nized in sterile PBS, and CTAB (0.5%; Sigma) was added. Eye-lids were homogenized again, frozen, thawed, and assayed formyeloperoxidase activity as described previously for cornealhomogenates.l415 There were 72 samples assayed in triplicate:

24 swab samples, 24 cornea samples, and 24 eyelid samples.The myeloperoxidase assay has a sensitivity of 0.01 units,which has been estimated to be equivalent to 103 neutro-phils.14

Slit-Lamp ExaminationOcular pathology was graded by slit-lamp examination (SLE)with a Topcon S1-5D slit-lamp biomicroscope (Koaku KikaiK.K., Tokyo, Japan) using a scoring system that has beenpreviously described.1'216 Seven parameters were scored on ascale of 0 (absent) to 4 (severe), including conjunctival injec-tion, conjunctival chemosis, corneal infiltrate, corneal edema,fibrin in the anterior chamber, hypopyon, and iritis. Twomasked observers performed all SLE scoring.

Histopathologic ExaminationAll rabbits were killed by intravenous injection of sodiumpentobarbital solution (100 mg/ml; The Butler Co, Columbus,MO). Upper and lower eyelids from each eye (eight per timepoint) were harvested at 0, 5, 10, 15, 20, and 25 hours afterinjection and immediately placed in formalin (10%; EK Indus-tries, Joliet, IL). Gross pathologic examination was performedon each eyelid. The left upper eyelid from each rabbit was thenbisected and processed for histopathologic examination usinghematoxylin and eosin staining as previously described.3 Cor-neas were harvested at 15 and 20 hours postinfection forhistologic examination using hematoxylin and eosin staining asdescribed previously.3

Statistical AnalysisThe SEM of Staphylococcus per cornea, SLE scores, and myelo-peroxidase activity was determined using a Statistical AnalysisSystems program.17

RESULTS

Slit-Lamp ExaminationInflammatory changes in the conjunctiva, cornea, aqueous hu-mor, and iris were reflected by the SLE scores, which increasedthroughout the course of infection reaching a maximum of19.75 ± 0.18 by 25 hours postinfection. SLE showed thatcorneal epithelial sloughing began in the central cornea by 10hours postinfection and extended peripherally. Gross exami-nation of the infected rabbit eyes revealed a viscous acuteinflammatory exudate after 10 hours postinfection.

Myeloperoxidase ActivityThe myeloperoxidase activity of ocular swabs, corneal homog-enates, and eyelid homogenates of infected eyes increasedfrom 10 to 25 hours postinfection (Fig. 1). The myeloperoxi-dase activity of ocular swabs was approximately six- to eight-fold greater than that of the corneal homogenates and approx-imately twofold greater than eyelid homogenates. Themyeloperoxidase activity of eyelid homogenates was approxi-mately threefold to fourfold greater than that of the cornealhomogenates.

BacteriologyThe replication of Staphylococcus in the cornea was exponen-tial from 0 to 10 hours postinfection, reaching a maximum of


JOVS, February 1999, Vol. 40, No. 2 Ocular Inflammation in Staphylococcus Keratitis 387

16 n

12

I3 103

VI 6

1

I J

•CorneaDSwabEl Eyelid

10 15 20

Hours Postinfectlon

25

FIGURE 1. Myeloperoxidase activity of corneal homogenates, ocularswabs, and eyelid homogenates of eyes with Staphylococcus aureuskeratitis. Myeloperoxidase activity, expressed in total recovered unitsof enzyme activity, was determined for corneal homogenates, ocularswabs, and eyelid homogenates of eyes with Stapbylococcus keratitis.All values are the means; error bars are the SEM.

approximately 7.0 log basel0 cfu/cornea after 10 hours postin-fection and maintaining 7.0 log base10 cfu/cornea to 25 hourspostinfection. Despite the large number of colony-formingunits in the cornea, the tear film and eyelid contained less than100 cfu at all time points until 25 hours postinfection (Fig. 2).

Corneal HistopathologyHistopathologic examination of corneas at 15 and 20 hourspostinfection revealed corneal ulceration and marked edema, amass of cocci in the central corneal stroma, and migration of

I6

| 3

I• CorneaDSwabH Eyelid

0 5 10 15 20 25

Hours Postinfection

FIGURE 2. Colony-forming units of Stapbylococcus aureus in rabbiteyes with keratitis. The cornea, acute inflammatory exudate on thecorneal surface, and eyelid of eyes with Stapbylococcus keratitis werecultured to determine the colony-forming units at each site. Bacterialcolony-forming units are expressed as log basem values. All values arethe means; error bars are the SEM. In some samples the error bar isimperceptible when compared with the sample value.

-y- ••?-

FIGURE 3. Inflammation in the rabbit cornea at 15 hours after injec-tion of Staphylococcus aureus. Top; a cornea infected with S. aureusfor 15 hours shows a wave of neutrophils (open arrowhead) migratinginto the stroma from the limbus toward the mass of bacteria (arrow)in the central cornea and adhesion of neutrophils to the denudedanterior cornea (closed triangle; magnification, X44). Middle: highermagnification of the central cornea shows the association of neutro-phils in the tear film with the denuded central corneal stroma; alsoseen is the mass of bacteria in the deeper portion of the central cornea(magnification X22O). Bottom: higher magnification of the peripheryshows neutrophils migrating from the limbus into the peripheral cor-neal stroma (magnification, X220). Sections were stained with hema-toxylin and eosin.

neutrophils from the anterior toward the central cornea (Figs.3, 4). At 20 hours postinfection (Fig. 4), the neutrophils in theanterior cornea were approximately 0.3 mm from the cocci.Migration of neutrophils from the limbus to the central corneawas also seen. The advancing edge of this wave of neutrophilswas 1.3 mm from the cocci. Sections of the limbus revealedmarked edema and moderate acute inflammatory exudate (Fig.


388 Sloop et al. IOVS, February 1999, Vol. 40, No. 2

unlike those harvested immediately after bacterial injection (0hours postinfection), revealed extensive edema and erythemaof the mucous membrane. Only mild redness and edema werenoted by gross examination of eyelids harvested at 5 hours.These changes became progressively more pronounced withan increase in time after corneal injection.

Eyelid Histopathology

Microscopic examination of eyelids harvested immediately af-ter injection of bacteria (0 hours postinfection) into the under-lying cornea revealed very rare isolated neutrophils within thestroma. Neither vascular congestion nor adhesion of neutro-phils to vascular endothelium was seen in these eyelids (Fig. 6).By 5 hours after injection of bacteria into the cornea, inflam-matory changes in the eyelids were noted (Fig. 6). Thesechanges progressed with time through 15 and 25 hours postin-fection (Fig. 6). The inflammatory changes included vascularcongestion and acute inflammatory exudation. A gradient inthe number of neutrophils was noted, such that neutrophils inthe eyelid stroma became more numerous toward the epithe-lium of the eyelid. Pavementing of neutrophils on the endo-thelial surface of blood vessels nearest the eyelid epitheliumwas obvious (Fig. 7). In all cases, neutrophil migration througha nonulcerated eyelid epithelium into the tear film was ob-served (Fig. 8). No bacteria or ulceration of the eyelid was seen

FIGURE 4. Inflammation in the rabbit cornea at 20 hours after injec-tion of Staphylococcus aureus. Top: the wave of neutrophUs migratingfrom the peripheral cornea {open arrowhead) has advanced towardthe bacteria in the central cornea {arrow), in comparison to the corneaharvested at 15 hours postinfection (Fig. 3). in addition, neutrophilshave penetrated into the anterior corneal stroma {closed arrowhead;magnification, X22). Middle: higher magnification of the central cor-nea shows neutrophils that have entered the corneal stroma; also seenis the mass of bacteria in the deeper portion of the central cornea(magnification, X110). Bottom: higher magnification of the peripheralcorneal stroma shows neutrophils migrating from the limbus (magni-fication, X110). Sections were stained with hematoxylin and eosin.

5). Migration of neutrophils into the scleral epithelium wasnoted.

Eyelid Gross ExaminationGross examination of eyelids from eyes with infected corneasrevealed general concordance in degree of change betweenthe right and left eyes and between the upper and lower lids.For those eyelid pairs in which mild discordance was evident,pathologic changes were more pronounced in the upper lid.Eyelids harvested at 25 hours after injection of Staphylococcus,

FIGURE 5- Inflammation in the rabbit corneal limbus and sclera 20hours after injection of Staphylococcus aureus. Top: cellular infiltrateis seen throughout the limbus and within the sclera and peripheralcornea (magnification, X44), Bottom: neutrophils are shown infiltrat-ing the corneal stroma at the limbus (magnification, X22O). Sectionswere stained with hematoxylin and eosin.


JOVS, February 1999, Vol. 40, No. 2 Ocular Inflammation in Staphylococcus Keratitis 389

FIOURU 6. l^ogressive inflammation of the eyelid associated with Staphylococcus keratitis. Top, left: no inflammation or edema is seen in an eyelidharvested at 0 hours after injection of Staphylococcus aureus into the adjacent cornea. The remaining three panels show progressive inflammation,edema, and vascular congestion of the eyelid. Infiltration of neutrophils increases with proximity to the eyelid epithelium. Note the absence ofcocci and eyelid ulceration. Bottom, left: 5 hours postinfection; top, right: 15 hours postinfection; bottom, right: 25 hours postinfection. Sectionswere stained with hematoxylin and eosin; magnification, X220.

at any time in histologic sections of the eyelid. In the sclera andthe eyelid, neutrophils could be seen accumulating on thestromal aspect of the epithelial basement membrane (Fig. 9).

FIGURE 7. Pavementing of neutrophils along eyelid vessel walls near-est to the conjunctiva of eyes infected with Staphylococcus aureus.Neutrophils can be seen adhering preferentially to the wall of theeyelid blood vessel (arrow) nearest to the infected cornea of rabbiteyes with Staphylococcus keratitis at 25 hours postinfection. Conges-tion of the vessel with erythrocytes can also be seen. Sections werestained with hematoxylin and eosin; magnification, X430.

DISCUSSION

This study demonstrates that experimental Staphylococcus ker-atitis in the rabbit is associated with acute inflammation of theoverlying eyelid, which contributes to the neutrophil popula-tion of the tear film. The histopathologic data suggest thatneutrophils of the tear film reach the central cornea faster thanthose migrating through the cornea from the limbus. Also, themyeloperoxidase data demonstrate that neutrophils from thetear film are more numerous than those migrating into thecornea from the limbus. An intense neutrophiiic response wasobserved previously in normal eyes injected with purifiedstaphylococcal a-toxin.3

The histopathologic evidence indicates that the inflamma-tion seen in the eyelid is a response to the associated conicalinfection and not a result of an infection of the eyelid itself.Inflammation was noted in the eyelid 10 hours before bacterialcultures of the eyelid became positive. In the eyelid, the lack ofulceration, the adhesion of neutrophils to the capillary endo-thelium nearest the cornea, the concentration gradient of neu-trophils toward the cornea, the migration of neutrophilsthrough a nonulcerated epithelium, and their exudation intothe tear film all suggest that this inflammation was elicited bya stimulus from the cornea, not the eyelid. Furthermore, thebasement membrane is a physical barrier to neutrophil move-ment,9 so accumulation of neutrophils along the stromal aspect


390 Sloop et al. /OV5, February 1999, Vol. 40, No. 2

FIGURE 8. Exudation of neutrophils from the eyelid to the tear film.The movement of neutrophils (arrows) through the eyelid epitheliumcan be seen at 5 (top) and 25 (bottom) hours postinfection. Sectionswere stained with hematoxylin and eosin; magnification, X1063.

of the epithelial basement membrane in both the eyelid and thesclera suggests that neutrophils originating in the stroma ofboth tissues are migrating into the tear film. The contributionof the linibal vasculature to the exudate in the tear film haspreviously been reported.6

The microbiologic data also suggest that the inflammatoryreaction in the eyelid is a response to the keratitis and not to aninfection of the eyelid itself. The low number of bacteriacultured from the eyelid is more consistent with contaminationfrom the heavily infected cornea than infection of the eyeliditself. Bacteria shed from the ulcerated cornea could havepopulated both the tear film and die inner surface of the eyelid,causing the observed result. In contrast to the cornea, in whichabundant bacteria were easily visible, no bacteria were notedon microscopic examination of the eyelid. Overall, these dataindicate that the inflammatory process observed in the eyelid isa response to the associated corneal infection.

Histopathologic examination of the cornea in this modelof Staphylococcus keratitis suggests that neutrophils from thetear film may be more prominent in the central cornea thanneutrophils migrating from the limbus. Neutrophils from thetear film reach the central cornea by 15 hours postinfectionand by 20 hours are seen entering the superficially erodedstroma of the central cornea. Basu and Minta18 also observedthe penetration of neutrophils into a denuded corneal stroma.Like Chusid and Davis,19 we also noted neutrophil migration

from the limbus toward the central cornea. However, neutro-phils traveling across the corneal stroma from the limbus didnot reach the central cornea by 25 hours, suggesting thatmigration from the tear film is a faster route of neutrophilpassage to the site of infection early in the disease process. Thisobservation is supported by the relative myeloperoxidase ac-tivity of the tear film, cornea, and eyelid. The myeloperoxidaseactivity of the eyelid and the tear film, as measured by swab-bing the anterior eye surface, was multi-fold greater than thatof corneal homogenates. Thus, these results confirm that neu-trophils from the tear film are involved in inflammation of thecornea, as has previously been reported.6'7

Taken together, the observations that bacterial infectionof the cornea is associated with inflammation of the overlyingeyelid and that neutrophils from the tear film are present in thecornea during keratitis suggest that the eyelid has an integralrole in corneal inflammation. This is perhaps not surprising

FIGURE 9. Neutrophils adhering to the stromal aspect of the basementmembrane in the sclera and eyelid. Top: neutrophil morphology isclearly seen in neutrophils accumulating on the stromal aspect of theepithelial basement membrane in the scleral limbus (arrowhead).Bottom: neutrophil morphology is clearly seen in neutrophils accumu-lating on the stromal aspect of the basement membrane of the eyelidepithelium (arrowhead), lntraepithelial neutrophils are also seen (ar-row). Sections were stained with hematoxylin and eosin; magnifica-tion, X2126.


IOVS, February 1999, Vol. 40, No. 2 Ocular Inflammation in Staphylococcus Keratitis 391

because there is no obvious reason why soluble inflammatorymediators, which have been implicated in a variety of otherocular inflammatory processes41220 including Pseudomonaskeratitis,21 should not elicit acute inflammation in the eyelid.The elucidation of this source of neutrophils has importantimplications for management of ocular inflammation. -A me-chanical blockade, which limits the passage of neutrophilsfrom the eyelid to the cornea, could be a beneficial adjunct tobactericidal antimicrobials in the management of bacterial ker-atitis.

In summary, this study has shown that experimentalStaphylococcus keratitis is associated with an inflammatoryresponse in the overlying eyelid and that neutrophils from thetear film are more prominent than those migrating from thelimbus in early inflammation of the central cornea. Theseobservations suggest that the eyelid has an important role incorneal inflammation.

Acknowledgments

The authors thank Loan Nguyen, Kiana Nelson, and Chuck Vial fortheir technical services.

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