INFECrION AND ImmuNrry, Feb. 1971, p. 209-216Copyright © 1971 American Society for Microbiology

Vol. 3, No. 2Printed in U.S.A.

Experimental Pseudomonas aeruginosa Infection ofthe Mouse Cornea

JOHN R. GERKE AND MICHAEL V. MAGLIOCCOCollege of Optometry, Pacific University, Forest Grove, Oregon 97116

Received for publication 14 August 1970

Pseudomonas aeruginosa infection of human cornea is rare but serious. The workof previous investigators using experimental infection primarily of rabbit cornearesulted in successful therapy for 10 to 50% of clinical cases. The advantage ofusing the mouse is demonstrated. The methods we adapted for characterizing theuntreated experimental infection included: incising the cornea to enable establishingthe infection; corneal examination with a steroscopic microscope; grading cornealpathology; qualitative and quantitative monitoring of the infecting bacteria by cul-turing and staining sectioned and dissected tissues. The characteristics of the tissuepathology, host response, and infection were similar to those reported for otheranimals and man. Corneal pathology was frequently nearly maximal 1 day after in-fection; host response involved a progression of events of long duration; pathologypersisted well beyond the period of bacterial infection. The infection was essentiallynoncommunicable, and invasiveness was limited to the tissues of the incised eye. Theresults show the possibility of tests for invasiveness of clinical isolates and for screen-ing for therapeutic and prophylactic measures.

Pseudomonas aeruginosa infections of thehuman cornea, although not common, usuallyresult in the loss of vision of the infected eye.Such infections originate as a result of cornealwounds from improper use of contact lenses (7),puncture, and irritation by foreign objects. Therapid course of corneal destruction (12 to 48 hr)and the resistance of P. aeruginosa to antimicro-bial therapy make the infection of extreme con-cern. P. aeruginosa infections of tissues otherthan the cornea are becoming of increasingimportance in clinical medicine. The threat tolife is greatest in immunologically weak patients.Because the cornea (having no direct bloodsupply) is an immunologically weak tissue, webelieve that the results of research on the cornealinfection may also apply to these life-threateninginfections.

Other investigators have been concerned withP. aeruginosa infections of the cornea in humanpatients. Experimental infection, primarily ofrabbit corneas, contributed basic informationto the understanding of the pathology anddeveloping therapeutic regimens successful in10 to 50%0 of the cases. These studies demon-strated also that wounding precedes the initiationof infection. The current status was excellentlyreviewed by Hessburg (3) and Smith (5). Al-though some progress has been made in treatingand understanding the causes of corneal damage

resulting from P. aeruginosa infection, there isstill much to be done.The basis of our study of P. aeruginosa infec-

tion of corneal wounds is the advantage of themouse for investigating infection. In comparisonto rabbits, mice require less space, are easier tohandle and maintain, are less expensive, andhistological preparations are easier to make andexamine. Another reason for using the mouseis that many of the recent immunological tech-niques employ this animal. Thus, more animalsmay be used to give more information than ob-tained previously from larger animals and itwill become practical to test many Pseudomonasstrains for invasiveness, different antimicrobials,and diverse means of developing effective im-munity.

This report describes the experimental in-fection and demonstrates its utility as a model ofhuman disease. The descriptions include themethods for initiating the infection and observingthe course of host tissue responses and the bac-terial infection. The results illustrate the com-municability and invasiveness of typical P.aeruginosa strains and the resulting pathologyand infection. Results and limitations of at-tempts to determine the specific tissue locationsof infecting bacteria are described. The signifi-cance of the potential application of the methodsand results are discussed, i.e., testing for in-

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vasiveness of P. aeruginosa clinical isolates, ex-ploring and evaluating the treatment and pre-vention of P. aeruginosa human infections.

MATERIALS AND METHODS

Mice. Young, adult white Swiss from our own

colony were used for all experiments. The colonyoriginated from Simonson's, Gilroy, Calif.

P. aeruginosa cultures. For this report, the followingcultures were selected to illustrate a spectrum of in-vasiveness in the mouse eye. Strain 31 (Platou), iso-lated from human infection, was supplied by E.Fisher, Jr., Portland State University. Strains 38, 40,44, 49, and 51 were obtained from R. J. O'Callaghan,University of Mississippi.

Media. The strains were maintained on nutrientagar (BBL). Trypticase Soy Agar (BBL) was used forgrowing inoculum for infecting corneas. For de-tecting bacteria from ocular tissues, we used TYE agar[1.5% Trypticase (BBL), 0.5% yeast extract (BBL),0.5% sodium chloride, 1.5% agar] and distilled water.The pH of TYE was adjusted with sodium hydroxideto 7.3 before autoclaving.

Infecting with Pseudomonas. Mice were anesthetizedwith sodium pentobarbital (0.06 mg/g intraperi-toneally) or ether. Routinely, only the right eye wasinfected. The cornea was incised with a 26-gaugeneedle. The needle, held like a pencil, was forcefullydrawn over the surface of the cornea in the uppertemporal quadrant, making three incisions about 0.5mm apart. Care was taken not to penetrate into theanterior chamber. The cornea was infected by placingon it one drop of a 37 C, 16-hr slant growth suspendedin 1 ml of water, approximately 101' per ml.

In one experiment, the suspension was injected intothe corneal stroma. Borosilicate glass tubing wasdrawn to the diameter of a 32-gauge needle and brokento form a jagged blunt tip. With a twisting-pushingmotion of the needle, the cornea was penetrated. Astereoscopic microscope was used to observe that thetip was not in the anterior chamber. After injecting0.01 ml, the needle was withdrawn and the cornea wasobserved for elevation of the surface, indicating thatsome of the suspension was retained (much less than0.01 ml).

Culture of cornea. The mouse's head was placed onits side with the eye to be streaked facing upwards.The cheek and lateral temple were pressed to causethe eye to protrude. A sterile inoculating loop wasgently drawn across the cornea. The loop contentswere streaked on TYE agar and incubated for 48 hrat 37 C. Diverse types of other bacteria were detectedin two-thirds of the mice; therefore, P. aeruginosawas differentiated by colony morphology, Gram stain,and Taxo N Discs (BBL). In a study of 33 infectedeyes, culture of cornea detected only 90% of thecorneal infections. The advantage of the culture ofcornea was that it did not damage the eye; conse-

quently it could be used repeatedly and routinelyto qualitatively monitor the bacterial flora.

Visual observation of corneas of living mice. Pre-vious investigators studying this infection in animalsand man relied extensively on detailed descriptions

and simple grading of observations of the cornea. Thedetailed descriptions were based primarily on directobservation aided by low-power optical devices. Formicroscopic observation of mouse eyes, we found thestereoscopic microscope at 30 and 60X to be in-dispensable.

In summarizing the results of several animals, agrading procedure such as the 5-grade method ofWiggins (6) was used initially. We frequently ob-served appreciable changes in pathology that were notscorable as different grades by a five-grade method;consequently, the grading key shown in Table 1 wasdeveloped. Although the pathology for no singlemouse has been observed to progress systematicallythrough grades 1 through 10 during infection andgrades 10 through 1 during healing, all the adjacentstages have been observed successively in both infec-tion and healing. Grades 9 and 10 may represent pri-marily complications of physical trauma, because aninfected cornea can become so weak that it is easilyruptured by a scratch.

Stereoscopic microscope examination of eyes ofliving animals was not limited to the gross cornealobservations described above. The following tissueswere examined: corneal epithelium (including itsneovascularization), corneal stroma, anterior cham-ber, limbus (exterior), iris, and conjunctiva; however,the stroma, anterior chamber, and iris were obscuredby corneal opacity. Typical signs of involvment wereneovascularization, edema, infiltration, swelling, andopacity. We routinely recorded observations on thesetissues; however, there have been no distinct differ-

TABLE 1. Corneal pathology key

Grade Description

0 No pathology.1 Injected iris, no other pathology.2 Hazy opacity in wound or central area

only. "Hazy" represents the entirespan from clear to sufficiently opaqueto obscure the iris from view.

3 Hazy opacity of entire cornea.4 Dense opacity in central area with much

of the remainder of the cornea hazy.5 Dense opacity in central area with much

of the remainder of cornea hazy andwith prominent distention of thecornea.

6 Dense opacity of entire cornea.7 Dense central opacity covering all or

most of cornea and ulcer.8 Dense opacity covering all or part of

cornea and perforated ulcer.9 Dense opacity over all or most of cornea

and a distinct central amorphousknob.

10 Dense opacity over all or part of corneaand irregular surface. Also used withadditional description, for all extremepathologies not covered by 7, 8, or 9.

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ences from observations of Pseudomonas corneal in-fection reported for other animals. Furthermore, noneof these, other than those used in the corneal pathol-ogy key, has shown outstanding value in evaluatingprogression of pathology.

Gross examination by stereoscopic microscope. Theeye of the anesthetized mouse was caused to protrudeand was examined at 30 power. A hemostat was at-tached to the lid margin to fold the lid back for ex-amination of the conjunctiva.

Enucleation. For enucleation, the mice were sacri-ficed; as for corneal streak, the mouse was held tomake the eye protrude. The tips of bent tip forcepswere gently worked behind the globe so that the globewas cradled in the bend. The forceps were held to-gether firmly, and a quick, vertical jerk removed theglobe. A largeamount of conjunctiva usually remainedattached to the enucleated globe. The force exerted onthe retina via the stressing of the optic nerve damagedthe retina; however, its integrity was not important toour studies.

Sectioning and staining. Using either fresh orForma-lin-fixed eyes, we cut 20- and 30-,um sections at minus20 C. Ten-micrometer sections could be cut, but manysuffered severe damage during cutting. The commonminor cutting damage to the 20- and 30-,um sectionsincluded: frequent loss of the center of the lens; ageneral fracturing of the lens capsule; warping of theiris; compression of the vitreous; and, in infected eyes,occasional tearing of the ciliary body and iris. Thesections were placed on Tissue Tact (Dade)-treatedslides and divided into four series. One series wasstained with hematoxylin and eosin (H&E), one serieswith periodic acid-Schiff (PAS) stain, one series withorcein-hematoxylin stain (4), and the fourth series withGram stain.

Dissection and microscopic examination. Aftersacrificing the mouse, the radius bisecting the majorarea of corneal pathology was located with 30Xmagnification and marked with a needle dipped ineosin Y powder. The marked eye was enucleated andtransferred to a sterile slide, covered with saline, andexamined at 30 and 60X. A piece of filter paper (2 by4 cm) was inserted between the eye and the slide. Thepaper was flooded with saline, and the slide, paper,and eye were frozen at -1O C. Two thin slices ofsclera, parallel to the plane described by the mark andcenter of the cornea, were cut off and discarded. Athird cut along the plane of the mark and center ofthe cornea separated the globe into hemispheres.Saline was added to the one to be stained with tetra-zolium, and 10% buffered neutral Formalin was addedto the one to be stained with crystal violet-eosin. Bothwere examined at 30 and 60X while being dissectedwith the aid of a blunt pointed needle for holdingand a flattened sharp-edged needle for separating thetissue. Synechiae, if present, were detected duringremoval of the lens. The lens and scleral sections werediscarded. The retina, ciliary body-iris, and cornea-limbus sections were positioned separately on a slideflooded with one of the stains described in the fol-lowing paragraphs, covered with a no. 0 slip, andsealed.

Crystal violet-eosin stain. To prepare crystal violet-

eosin stain, 2 ml of Gram crystal violet working solu-tion and 40 mg of eosin Y were added to 80 ml of30% glycerin. P. aeruginosa appeared as dark, blue-black bipolar cells against an orange-pink tissue back-ground.

Tetrazolium stain. Tetrazolium stain was preparedas follows. A 5.0-ml amount of sterile minimal medium[KNO3, 1 g; KH2PO4, 3 g; K2HPO4, 7 g; Na3 citrate-2H20, 1 g; (NH4)2SO4, 1 g; MgSO4, 1 g; and water,1 liter] was added to 0.5 ml of a 2% solution of 2,3,5-triphenyl-2H tetrazolium chloride, made in sterilewater, plus 5.0 ml of sterile 30% glycerol. Examinationof this stain was most useful about 0.5 hr after prepara-tion. By 2 hr, autolysis of the tissue was sufficient tomake examination meaningless.

Globe mince. The outside of the enucleated eye wasrinsed with sterile water. The eye was placed on asterile slide and minced with two blades drawn backand forth over each other in a scissorlike action. Thetissue on the slide was washed into a sterile tube with5 ml of filter-sterilized 0.1% Pancreatin. A magneticstirring rod was inserted, and the tissue was homog-enized by agitation with a magnetic stirrer in a 37 Cincubator for 15 min. One milliliter of homogenateand dilutions were plated in TYE medium and incu-bated at 37 C for 48 hr.

RESULTSCommunicability and invasiveness of P. aerugi-

nosa in the mouse cornea. The right eyes of 200mice were incised and active infections wereestablished with seven strains of P. aeruginosa.There were no visible signs of infection of theleft eyes. One hundred forty-two of these sameleft eyes plus those of 22 controls (housed in thesame cages as the infected mice) were streaked(culture of cornea) repeatedly and subjected toterminal globe mince. With the exception of twoeyes showing two colonies at one observationperiod, Pseudomonas was never isolated. Intwo experiments, a total of 10 control mice wereincised but not inoculated. They were also housedin the same cages with the infected mice and noPseudomonas was demonstrated by either tech-nique. These observations indicate that thiscorneal infection is essentially noncommunicable.Although invasiveness was not the subject of

intensive study, five mice infected with strain 31were observed either to maintain their weight orto gain weight during a 5-day experiment. Wenever saw any of our experimental mice act sick.Only one mouse died of natural causes duringall of our experiments, and postmortem cultureof the organs revealed no Pseudomonas; but manyother bacteria were present, among which a Pro-teus appeared to predominate. Invasiveness islimited to the infected eye.In different infections, including the corneal

infection of man and experimental animals,the pathogenicity of P. aeruginosa is generally

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believed to be a function of the invasivenessof the specific strain. Our experiments indi-cated that corneal pathology at 1 day after in-fection (for example, see Table 2) was sufficientlyadvanced to be a useful measure of invasiveness.Pathology increased or decreased after the firstday; consequently, the 1-day observation couldnot be the sole criterion for invasiveness.For this reason we summarized results ofcomparative experiments at 1 day and alsoincluded the direction of pathology change,increase or decrease in severity, indicated after1 day. The summary showed that the absoluteseverity of pathology at day 1 for a specific strainvaried from experiment to experiment, suggestinga need for greater standardization of, e.g., theculture method, number of organisms, or main-tenance of invasiveness. For example, the mediangrades at day 1 for successive experiments withstrain 31 were 7, 6, 8, and 7, and for stain 44were 2, 7, 4, 5, and 4. Assuming that relativeinvasiveness is significant in spite of experiment-to-experiment variations, we found that strains31, 44, and 51 were consistently invasive: grade 2or higher at 1 day and increasing or persistingafter 1 day. Stain 40 was consistently nonin-vasive: grade 2 or lower at 1 day and decreasing.Stains 38 and 49 showed variable invasiveness.Although not a definitive study, the resultsillustrate that mouse corneal invasiveness, as

indicated by persisting pathology, is a functionof strain; however, as shown in subsequentsections, strain is not the only determinant.

Effect of the extent of wounding. Early in ourexperimentation, we were concerned that vari-ation in extent of incision might cause variabilityof infection and resulting pathology. We com-pared the results of experiments by employinginfection after our standard incision procedure, a2-mm scratch, a needle puncture, and injection(with a glass microneedle) of the P. aeruginosaculture directly into the corneal stroma. Thetypical course of pathology followed the stand-ard incision procedure and injection. Variablepathology followed the 2-mm scratch and theneedle puncture. Providing the wound wasadequate, the absolute extent or means of makingthe wound was not critical.

Progression of corneal pathology caused byinvasive strains. A major objective was to docu-ment the pathology of the untreated infectionso that the merits of subsequent preventive andtherapeutic experimental regimens could beassessed.The typical progression of pathology, observed

visually after incision and inoculation with in-vasive strains, in nine experiments with a totalof 217 mice is illustrated in Table 2. The data,showing the effect of one strain in two experi-ments on 18 mice,! were selected as being repre-

TABLE 2. Progression of corneal pathology, straini 44

Expt Mouse

19 5-19-49-38-61-21-52-45-76-16-47-38-18-2

22 1-41-51-62-22-4

Grade rangeGrade median

Pathology grades (time after infection)

4 hr

2222222222222

22

Day

1

434344443443344445

3-54

2

44445

5

5

5

4

4

5

4

5

4

4

4

4

5

4-5

4

4444444944545

4-94

5444466945S

49

64

4-94

8

55

10555593

1045

9

3-10lS

12

221022S

2926S

109

2-104

15 17

2 22 210 92 22 25 42 24 42 26 65 54 29 6

2-10 2-94 2

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sentative of the results obtained in experimentsin which pathology was moderately severe.Considering that there is no functional vision atgrade 4 and higher, a moderately severe graderepresents a severe clinical situation. High grades(9 and 10) frequently appear as jumps from lowergrades (for example, mice 5-7 and 8-2), and thejumps may occur after the progression has ap-parently stabilized (9-3 and 6-4). As describedearlier in this report, it is also common forgrades 7 and 8 to occur at day 1. Regression ofhigh grades may be fast (6-4 and 8-1) or slow(5-7 and 8-2). Regression of lower grades isalso variable (6-1, 5-1, and 1-5). The progressionis unpredictable for individual mice and, as shownby the grade range, there is wide variation be-tween replicates at most observation periods.The grade median shows that damage was

evident at 4 hr and for most mice nearly maximalat 1 day, and that significant regression occurredfor most mice by the end of the second week.The time limits are representative of all of ourresults with invasive strains.With less invasive strains (data not shown), the

grades were lower and a decrease to no pathologywas often observed as early as the second day.Even with strains as invasive as strain 44; aregression to no pathology after the third weekoccurs but is not typical. In such regressions thecornea was clear with no scars, pupil response wasnormal, and lens was clear. Visual function wasnot determined. The regenerative processes ofmouse cornea are appreciable.

Progression of ocular infection caused by in-

TABLE 3. Progressiont of ocular inzfection (experi-ments 19, 21, and 22; strain 44)

No. of No. ofmice miceGemti

Time after showing showing Counts (P. Geometricinfecting sosig- signi- aeruginosa per eye) countsnificant ficant cut

count count

4 hr 1 5 35 300 25060 5,000

2501 day 1 6 60 4,000 4,300

100 300,0002,500 300,000

2 days 3 4 30 7,000 1,700100 4,000,000

3 days 2 5 100 5,000 800800

4 days 1 4 50 10,000 1,000100 30,000

5 days 0 1 10,000 10,00012 days 9 019 days 14 0

vasive strains. The progression of the ocularinfection, measured by P. aeruginosa globemince counts, was studied with invasive strains31, 44, and 51. The results for strain 44 (Table3) are typical. All of the eyes included in Table3 had pathology greater than that normal forincised noninfected controls. At 12 and 19 daysand in some animals as early as 4 hr, no P.aeruginosa was detected, indicating that thehost defenses had been effective in eliminatingthe infection. Varying numbers of P. aeruginosacells were detected in most of the eyes duringthe first 5 days of infection, indicating variableonset of efficacy of host defense in limiting theinfection. The progressive relationship in rela-tive numbers of mice showing significant countand in the geometric means of the counts wasirregular, particularly in the initial period.

Correlation of corneal pathology and infection.The general course of pathology is shown inTable 2. Considering the grades for individualmice of Table 3 (data not shown), severity ofgrade did not correlate with Pseudomonascount. Furthermore, 8 mice of the 27 observedin the first 4 days showed no significant count ofPseudomonas at termination. However, previouspresence of Pseudomonas was confirmed by cul-ture of cornea in seven of the eight. These eightdid not differ from the others in progression ofpathology. Twenty-three eyes at 9 days had nosignificant count, yet all were grade 2 or higher.These observations demonstrated that, whereaspathology is a direct consequence of infection,severity of pathology is not related quantita-tively to the number of organisms present.

Observation of ocular pathology in the livingmouse. The following description of pathologyof an infected mouse that was graded 6 at 1, 2,and 4 days is included to illustrate the typicalocular pathology visible in a living mouse byusinga dissectingmicroscope. Cornea: The cornealedema, seen at all observations, appeared to beindependent of the location of the scratch orthe duration of the infection and was mainlyaround the cornea center. Corneal epithelium:No corneal sloughing was seen at the end of 24hr, but appeared at 2 and 4 days in the area ofthe opacity. Corneal stroma: The stroma alwaysappeared swollen far beyond that of the controleye. Dense opacities occurred in the endemicparts, although some areas were translucentbut swollen. External limbus and conjunctiva:The limbus and conjunctiva were highly in-jected at all observation times. The anteriorchamber, iris, and ciliary body were not visible.In summary, the pathology was not localizedin the cornea but involved adjacent tissues.

Observations of ocular pathology aided by

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dissection. Simple dissection of the mouse eye,based on a central anterior-posterior cut, proveduseful for the iris and ciliary body and good forthe cornea, limbus, and retina. Lens dislocations,synechias, dense anterior chamber infiltrates,and retina alterations were observed easily with astereoscopic microscope at 15 to 30 X. Alterationin tissue tenderness was also revealed whileprobing the tissues with dissecting needles.Formalin-treated tissues were tougher and easierto dissect; however, fresh tissue could be dis-sected before the rapid autolysis caused detect-able alterations.

Following is a typical description of an eye 2days after infection. The iris and ciliary body werepoorly defined but distinguishable. The corneain the area of the incision (for infecting) wasswollen to twice the normal thickness and in-filtrated. The swelling did not extend more thanone-fourth of the corneal diameter from the in-cision. The periphery of the anterior chamber wasinfiltrated. The cornea was opaque in the area ofthe incision. The choroid-retina and lens werenormal.

Dissection was a simple and useful procedure,and we soon relied on it for quickly locatingareas of maximal involvement. During dissection,the infection can sometimes cause the retina tobecome a pulpy mush, or merely wrinkled andseparated from the sclera; however, changes inthe retina were rarely seen. We also observedsoftening of the outer layers of the lens and,commonly, free-floating lenses and sloughingof the corneal epithelium. Anterior and posteriorsynechias were common as early as 4 hr afterinfection.

Observations on stained sections. Conven-tional sectioning and staining procedures wereused to characterize the tissues and to attemptto locate the infecting bacteria. Although otherinvestigators had not reported on the specificpresence and distribution of the infecting Pseudo-monas, we believe such information to be valuablein designing and evaluating experimental thera-peutic and prophylactic regimens.

Sections were stained by H&E, PAS, orcein-hematoxylin, and a Gram stain. Parts of thetissue were loosened from the slide by stainingsolutions containing more than 50% water.Substitution of gelatin or untreated slides forTissue Tac-treated slides did not reduce thisproblem. With H&E and orcein-hematoxylin,the loosening was tolerable; however, with PASand Gram stains, the orientation of tissues withinthe eye was usually severely disturbed. Of thefour methods, H&E was the most uniformlyleast destructive.H&E-stained infected eyes (1 to 3 days after

infection) showed extensive thickening and-infiltration of the stroma and ciliary body, fre-quent ulceration of the corneal epithelium andstroma, all degrees of leukocytic infiltration ofthe anterior chamber, and, in some cases, sepa-ration of the choroid from the sclera. Lens dis-location (resulting from sectioning) was socommon in noninfected eyes that no observa-tions of sections for lens position or possiblesynechias were possible. Mounting the sectionin gelatin tinted with eosin demonstrated, bycomparison, that even the best of H&E stainsresulted in the loss of material from the vitreousand some of the infiltrate of infected anteriorchambers. Nevertheless, we concluded that H&Estains of sections would be useful for determininginfection effects at the level of the host cell.No Pseudomonas was seen in sections of in-

fected eyes stained with H&E, PAS, and Gramstains. Even when a dense bacterial suspensionwas placed on top of sections, no Pseudomonaswas detectable after staining. Evidently the cellswere washed away from the sections duringstaining. Washing away Pseudomonas in infectedtissue is reasonable in consideration of sub-sequent observations that tissues infected withPseudomonas were appreciably more tender orsubject to more rapid autolysis, or both. Otherinvestigators, when staining sectioned eyes,have embedded them in collodion or paraffin.None reported seeing or not seeing individualPseudomonas cells. Our failure to see Pseudomonascells may have been due to not embedding;however, it is likely to have been due to thecombined effects of the infection on the tissuesand the washing action of the staining process.

Staining for locating infecting P. aeruginosa intissues. The dissected parts (retina, iris-ciliarybody, and cornea-limbus) were mounted incrystal violet-eosin and observed at 1,250 xunder bright-field and phase-contrast micros-copy. Although the under sides of all tissuesexcept for the iris were obscured by the over-lying tissues, it was usually possible to seestructures in the top half of the tissues. ManyPseudomonas-like objects were seen floatingaround tissue from infected eyes. For example,in one area of a ciliary body, about 1,000 ofthese objects were seen per field, and in parts ofcorneas there were discrete clusters of about 10objects. (Subsequent observations of smallernumbers of similar objects in uninfected eyescaused us to realize that these objects mightalso be mitochondria from ruptured phagocytesand in phagocytes and tissue cells.) Markedlyincreased numbers of irregular cells (15 by 5,um), presumably phagocytes, were found pri-marily in the ciliary body corneal stroma and

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anterior chamber infiltrate of infected eyes.In an attempt to distinguish Pseudomonas

from the Pseudomonas-like bodies, sections werestained with tetrazolium. Whereas no other struc-tures within the eye rapidly reduced the dye tothe colored form, both Pseudomonas and thePseudomonas-like bodies in an uninfected eyerapidly reduced the dye. An additional diffi-culty with the tetrazolium procedure was thatthe dye penetrated intact tissue surfaces verypoorly.

Bacteriological plate counts for locating in-fecting P. aeruginosa in tissues. An attempt todetermine, by plate count of ocular tissues, theintraocular distribution of Pseudomonas wasfrustrated by the inability of preventing mutualcontamination of the tissues during dissection.Thorough washing of tissues from infected eyeswas impractical because they autolyzed so fast,becoming too tender to handle.

Dissection of a frozen eye was considered as ameans of preventing mutual contamination oftissues. Fifteen eyes infected for 1 to 5 days werefrozen and dissected by a single cut along theplane of the junction of the sclera and limbus.For 10 eyes, there were no P. aeruginosa cellsin the posterior section, and 30 to 50,000 (medianwas 4,500) in the anterior section. For the fivehaving P. aeruginosa in the posterior section, thecounts of the anterior sections of four were 60to 3,000-fold higher. The other showed a 300-fold higher count in the posterior section. Controlexperiments with infected eyes and P. aeruginosaadded to noninfected eyes demonstrated thatfreezing killed about 99% of the bacteria. As-suming that freeze-killing is equal in the twosections, it appears that corneal infection involvesprimarily the anterior section; however, the pos-terior section can also become involved in anappreciable percentage.

DISCUSSIONEven the more invasive strains used in our

studies are weak in comparison to other patho-genic bacteria. The infection did not spread tothe unwounded eye or to incised but uninocu-lated eyes of other animals in the same cage,and it did not appear to involve extraoculartissues. The low communicability obviates thecomplexities of isolation of infected animals,such as are required with more communicablemicroorganisms, and offers minimal hazard ofinfection of laboratory personnel. By using micerather than larger animals, experimentation ismore economical, and statistically significantnumbers of animals can be used in each experi-ment.

Providing that the wound was adequate, the

ability to produce significant pathology was uni-form.

Corneal pathology varied appreciably fromexperiment to experiment and from mouse tomouse within an experiment. Such variationdemonstrates, especially if short observationperiods or few mice are used, that grade ofcorneal pathology alone would be a weak cri-terion for assessing the merits of preventive andtherapeutic regimens. It is interesting to notethat a similar, irregular progression of eventsis reported for human corneas undergoing ther-apy for Pseudomonas infection. The 10 gradesof pathology are useful primarily as a conveniencein recording corneal observations; however, thecriterion of persistent pathology of grade 2 orhigher served to demonstrate invasive infection.The relative simplicities of the mouse experi-mental infection justify considering practicalapplications.There is a practical need for methods to rapidly

identify and determine the invasiveness of P.aeruginosa from conjunctival and corneal swabsand scrapings. This need is complicated by thediversity of nonfermentative rod forms likely tobe found in mild conjunctivitis or even in healthyeyes. Our results show that invasiveness indi-cated by the mouse corneal infection could beexplored as a potential test for invasiveness ofclinical isolates. To show such utility, more ex-tensive testing, including fresh isolates fromclinical infections, is required. Invasivenesswould be indicated by the pathology observed ona group of four to six mice 1 day after experi-mental infection and confirmed by comparisonof 1- and 3-day observations. Such a test, ifmouse and human invasiveness were shown tocorrelate, could be useful in establishing theetiology of specific clinical infections, particu-larly when a mixed culture is isolated from theinfection. It is reasonable to expect that the testwould be applicable to infections of tissuesother than cornea.A similar test with strains of established in-

vasiveness, using bacteriological cultures andmicroscopic examination such as we described,could be used to evaluate or screen for thera-peutic and prophylactic measures.The studies of corneal and ocular pathology

showed that the characteristics of P. aeruginosainfection of mouse cornea are similar to thosereported for other animals and man. The pa-thology is not localized but involves other tissues,including iris, ciliary body, and conjunctiva.Mouse tissues respond to the infection in

essentially the same way as the tissues of otheranimals. Severe damage occurs as early as 1 day.As with rabbits but unlike the majority of the

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GERKE AND MAGLIOCCO

reported human corneal infections, the infectionwas generally self-limiting, and without treat-ment regression of pathology or healing began inthe first week or two. Considering that humaninfections are usually complicated by conditionsof immunological limitation or debilitation,this difference between humans and experi-mental animals does not depreciate the value ofthe animal infection; rather, the difference justi-fies extension of experimentation to animalstreated to weaken their immune mechanisms (2).

In untreated mice, the progression of repairresembled that reported for effectively treatedhuman eyes, except that the mouse repair ratewas much faster. This resemblance indicates thatthe experimental infection might be of value fortesting therapeutic regimens for potential ap-plication to infection-damaged human eyes.The study of the correlation of corneal pathol-

ogy and infection demonstrated that, althoughpathology is a direct consequence of infection,it is not quantitatively related to the number oforganisms present. The globe mince procedurewas used to study the dynamics of the Pseu-domonas population subsequent to infection.Although the bacterial count and duration ofthe infection was variable, near maximal countswere obtained, sometimes within 24 hr afterinfection. Apparently the host defenses wereeffective from the beginning in limiting the in-fection and also effective between the 4th and12th day in eliminating Pseudomonas from theglobe and conjunctiva. The corneal pathologypersisted beyond the period of infection. Whereasno similar experiments have been reported pre-viously, the results are consistent with the pre-vious observations that corneal pathology iscaused by the extracellular enzymes (3, 5) andthat such enzymes trigger a long-lasting pro-gression of inflammatory-repair events (1). Agreater understanding of the nature of the de-fense and repair is important to the design offuture experiments on therapy and prophylaxis.A prime objective of our tissue studies was to

detect the presence and to understand the distri-bution and population dynamics of Pseudomonasafter corneal infection. Methods were developedto enable visual detection of individual bacteriain tissues; however, we were unable to distinguishwith certainty the bacteria from P. aeruginosa-like bodies found in uninfected tissue. Platecounts of the anterior and posterior hemispheres.demonstrate that the infection is primarily ofthe anterior hemisphere; however, it is commonfor the posterior hemisphere to become involved.

Experiments based on infection of the mousecornea should enable extension of previous studies.and lead to the discovery of basic informationpertinent to therapy of human corneal infectionsand the life-threatening infections of other tissues.

ACKNOWLEDGMENTSWe are indebted to W. Montagna and N. A. Roman of the

Oregon Regional Primate Center, Beaverton, Ore., for theirassistance and use of their facilities for sectioning and staining.We are grateful to Earl Fisher, Jr., of the Biology Department,Portland State University, for demonstrating his technique forinfecting rat cornea. We thank C. R. Matti, S. Mosher, PamelaAronson, and R. Buffington for technical assistance and RuthShapland and Rose Gerke for clerical assistance.

This investigation was supported by Public Health Serviceresearch grant EY00496 from the National Eye Institute.

LITERATURE CITED

1. Brown, S., C. Weller, and S. Akiya. 1970. Pathogenesis ofulcers of the alkali-burned cornea. Arch. Ophthalmol.83:205-208.

2. Burda, C. D., and E. Fischer, Jr. 1959. The use of cortisonein establishing experimental fungal keratitis in rats: a pre-liminary report. Amer. J. Ophthalmol. 48:330-335.

3. Hessburg, P. C. 1969. Management of Pseudomonas keratitis.Survey Ophthalmol. 14:43-54.

4. Roman, N., S. F. Perkins, E. M. Perkins, and E. H. Dolnick.1967. A. 0. V. stain. Stain Technol. 24:199-202.

5. Smith, D. T. 1968. Pseudomonas aeruginosa and pseudomonia-sis, p. 650-655. In D. T. Smith, N. F. Conant, and H. P.Willett (ed.), Zinsser microbiology. Appleton-Century-Crofts, New York.

6. Wiggins, R. L. 1952. Experimental studies on the eye withpolymyxin B. Amer. J. Ophthalmol. 35:83-100.

7. World Contact Lens Standard Committee Report. 1959.Ophthalmic microbial hazards, antimicrobial agents anduse of contact lens solutions. Contacto, The Contact LensJoumal, p. 258-267.

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