INFEcTION

AND IMMUNITY,

Aug. 1975, p. 419-432

Vol. 12, No. 2 Printed in U.S.A.

Copyright O 1975 American Society for Microbiology

Rabbit Corneal Damage Produced by Pseudomonas aeruginosa Infection LARRY D. GRAY AND ARNOLD S. KREGER* Department of Microbiology, Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, North Carolina 27103

Received for publication 12 March 1975

Gross, light microscopic, and electron microscopic examination of the rabbit corneal destruction produced by experimental Pseudomonas aeruginosa infections revealed a combination of acute inflammation and liquefaction necrosis of the cornea. Degeneration of the epithelial cells and the start of polymorphonuclear leukocyte infiltration of the comea occurred initially. These changes were followed by loss of the epithelium, degeneration and loss of the keratocytes and endothelium, loss of the characteristic weblike pattern of the proteoglycan ground substance, dispersal of ultrastructurally normal collagen fibrils, extensive accumulation followed by degeneration of polymorphonuclear leukocytes, and accumulation of plasma proteins and fibrin in the necrotic cornea. Histochemical examination of the cornea suggested a loss of the proteoglycan ground substance but not of collagen. Rabbit corneas injected with Clostridium histolyticum collagenase showed gross and cellular changes similar to those observed during the pseudomonal infections; however, histochemical examination suggested a loss of collagen, and electron microscopy revealed ultrastructurally abnormal collagen fibrils. The results support the idea (i) that a bacterial or host-derived collagenase is not required for extensive corneal damage during a P. aeruginosa corneal infection, and (ii) that a P. aeruginosa corneal infection may severely damage the cornea by producing extensive corneal edema and by causing the loss of the corneal proteoglycan ground substance, thus resulting in dispersal of undamaged collagen fibrils, weakening of the cornea, and subsequent descemetocele formation and comeal perforation by the anterior chamber pressure. Pseudomonas aeruginosa is an opportunistic bacterial pathogen capable of producing a severe comeal infection which is difficult to treat, progresses rapidly, and commonly leads to extensive corneal scarring and corneal perforation (6, 13). Despite the fact that P. aeruginosa is one of the leading causes of central corneal ulcers of bacterial etiology (13), the mechanisms by which it elicits corneal destruction have not been fully elucidated. The results of studies by several investigators (1, 7, 12, 25) suggest that the fulminating corneal destruction characteristic of P. aeruginosa infections may result from the in vivo production of extracellular cornea-damaging pseudomonal proteases. The pseudomonal protease preparations studied by these workers elicited gross corneal damage which appeared similar to that observed during experimental and human corneal infections by P. aeruginosa. However, the corneal destruction produced by the infections and by the protease preparations was not extensively examined and compared by light and electron microscopy.

Our interest in the pathogenesis of P. aeruginosa comeal infections prompted our undertaking the light and electron microscopic characterization of corneal damage produced by experimental P. aeruginosa corneal infections and by purified cornea-damaging pseudomonal protease preparations. The purpose of the study was to determine whether the observed structural alterations suggested or supported a mechanism for corneal destruction by P. aeruginosa. In this report, we describe the sequence of the ultrastructural alterations of the cellular and extracellular components in

damaged by experimental P. aeruginosa infections. The observed ultrastructural damage supports the idea that P. aeruginosa comeal infection may severely injure the comea by producing extensive corneal edema and by causing the loss of the corneal proteoglycan ground substance believed to maintain the order and interfibrillar attachments of the corneal collagen fibrils, thus resulting in dispersal, but not degradation, of the collagen fibrils.

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(The research described in this paper was taken in part from a thesis submitted by L. D. G. to the Bowman Gray School of Medicine of Wake Forest University in partial fulfillment of the requirements for the M.S. degree. It was presented in part at the 1974 meeting of the Ocular Microbiology and Immunology Society in Dallas, Tex., and at the 1975 meeting of the American Society for Microbiology in New York, N.Y.) MATERIALS AND METHODS Bacteria and growth conditions. P. aeruginosa, strains 5-31, IFO 3455, and 158, were kindly supplied by J. Gerke (9), K. Morihara (18), and W. Scharmann (21), respectively. The bacteria were cultivated in tryptone-yeast extract-glucose broth (pH 7.2) containing 0.5% tryptone (Difco), 0.25% yeast extract (BBL), and 0.1% glucose. Two-liter flasks containing 200 ml of broth were inoculated with 0.1 ml of a stationary-phase culture of the bacterium and incubated for 20 to 24 h at 35 C on a gyratory shaker (model G-25, New Brunswick Scientific Co., New Brunswick, N.J.) operating at 200 cycles/min. Production of experimental infections. Samples (30 to 40 gl) of bacterial suspensions, prepared by diluting broth cultures containing 109 to 1010 bacteria/ ml with sterile 0.85% saline, were injected intracorneally (30-gauge, 0.5-inch [about 1.25 cm] needles; Storz Surgical Instruments, St. Louis Mo.) into 3- to 5-lb (about 1.4 to 2.3 kg) New Zealand white rabbits. Each dilution was injected into at least 10 corneas and, in most experiments, the rabbits were sacrificed 1 day postinfection. In two studies, however, the rabbits were sacrificed 2 and 4 days postinfection. Before injection, the rabbits were anesthetized with ether and topical 0.5% tetracaine hydrochloride. The number of bacteria per milliliter in each dilution injected was determined by plate counts. Control preparations injected consisted of sterile tryptone-yeast extract-glucose broth diluted with sterile saline. Production of bacterial collagenase-induced corneal damage. Clostridium histolyticum collagenase (349 units of collagenase activity/mg; Worthington Biochemicals Corp., Freehold, N.J.) was dissolved in 0.05 M tris(hydroxymethyl)aminomethane-hydrochloride (pH 7.5) containing 5 x 10-3 M Ca2+. Samples (30 to 40 ,ul) of solutions containing various amounts of the collagenase preparation were intracorneally injected, as previously described for the bacterial suspensions, into New Zealand white rabbits, and the rabbits were sacrificed 4 h postinjection. Light and electron microscopy. Experimental animals were sacrificed by air embolus, and their corneas were immediately excised without disturbing the central corneal lesions. Half of each cornea was fixed in 10% buffered formalin (pH 7) for paraffin embedding and histochemical studies, and the other half, to be examined by light and electron microscopy, was fixed for 6 h in 4% glutaraldehyde in Sorenson phosphate buffer (0.2 M, pH 7.4). Corneal specimens to be studied by electron microscopy were

INFECT. IMMUN. washed overnight in Sorenson phosphate buffer containing 8% sucrose and were cut into wedge-shaped pieces, each piece containing a portion of the central corneal lesion and of the tissue bordering the lesion. The cut tissues were postfixed for 2 h in 2% osmium tetroxide in Sorenson phosphate buffer containing 7% sucrose, washed in buffer, and dehydrated through ascending concentrations of ethanol. All the above procedures were performed at 4 C. Specimens were further dehydrated at room temperature in propylene oxide, embedded in Epon 812 epoxy resin as described by Luft (14), and polymerized one day each at 35, 45, and 60 C. Embedded specimens were sectioned on a Sorvall Porter-Blum ultramicrotome, model MT2-B, with glass knives. Thick sections (1 to 2 gm), stained with basic fuchsin-alkaline methylene blue (11), methylene blue-azure 11 (20), or toluidine blue-pyronin B (24), were examined by light microscopy (Leitz Ortholux microscope) to evaluate embedment and condition of tissues and to select areas for electron microscopic study. Thin sections (60 to 90 nm) were mounted on uncoated 300-mesh copper grids, stained with phosphotungstic acid, uranyl acetate, and lead citrate (4), and examined in a Zeiss 9S-2 electron microscope with an accelerating voltage of 60 kV. Formalin-fixed tissues to be used for histochemical studies were embedded in paraffin, thick-sectioned (5 to 7 um), and stained by the colloidal iron technique to demonstrate comeal proteoglycan ground substance and by the Van Gieson technique to demonstrate corneal collagen (19).

RESULTS During initial experiments we attempted to produce various stages of infection by injecting each member of a group of rabbits with the same number of bacteria and sacrificing individual animals at various times postinjection. This time-variable approach was not satisfactory because of the unpredictable time required for the development of the infections in the different rabbits of the group. The lack of correlation between postinfection time and severity of infection often resulted in different rabbits demonstrating similar degrees of corneal damage at observation periods many hours apart. In addition, comparison of some rabbits often showed markedly different degrees of corneal damage at similar times postinfection. In an attempt to produce more predictable and reproducible stages of infection in a group of rabbits, the number of bacteria injected into different corneas was varied (approximately 37 to 370,000 in multiples of 10), and most of the animals in a particular experiment were sacrificed 24 h postinfection. At that time, each animal showed a different severity of corneal damage which correlated with the number of bacteria injected. Gross corneal damage elicited by experi-

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CORNEAL DAMAGE BY P. AERUGINOSA

mental P. aeruginosa infections. The intracorneal injection of control preparations produced immediate opacification of the cornea at the site of injection. This opacity disappeared and the cornea retumed to total transparency within 4 h postinjection. No conjunctivitis was observed (Fig. la).

The three different strains of P. aeruginosa produced similar gross damage. Twenty-four hours postinfection, corneas injected with approximately 37 bacteria appeared grossly normal; however, a mild conjunctivitis was observed. Intracorneal injection of approximately 370 bacteria resulted in moderate conjunctivitis

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FIG. 1. Gross corneal damage produced by experimental P. aeruginosa infections and by C. histolyticum collagenase. Results shown are 24 h postinfection or 4 h postinjection of collagenase preparation. (a) Control cornea. Note corneal transparency and the light reflecting from the corneal surface. (b) Small, central corneal ulcer observed postinfection with approximately 370 bacteria. (c) Advanced corneal lesion noted postinfection with approximately 3,700 bacteria. (d and e) Front and side view, respectively, of extensive liquefaction necrosis and purulent exudate observed postinfection with approximately 370,000 bacteria. (f) Liquefaction necrosis produced by collagenase preparation containing 1,000 ,g/ml.

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and central corneal opacification (Fig. lb). A severe conjunctivitis and the presence of a purulent exudate were observed 24 h postinjection of approximately 3,700 bacteria. The cornea was almost completely opaque and had a protruding central corneal lesion (Fig. lc). Extensive liquefaction necrosis was observed postinfection with approximately 370,000 bacteria. The cornea was markedly edematous, was coated with a purulent exudate, and appeared as a protruding, milky-white gel (Fig. ld and e). Light and electron microscopic observations of corneas damaged by experimental P. aeruginosa infections. Light and electron microscopic examination of corneas injected with control preparations revealed no obvious cellular or extracellular alterations 4 to 24 h postinjection. The three different strains of P. aeruginosa produced similar structural alterations. Table 1 describes the development of corneal destruction during experimental P. aeruginosa infections. In summary, degeneration of the epithelial cells and the start of polymorphonuclear leukocyte (PMN) infiltration of the cornea occurred initially. These changes were followed by loss of the epithelium, degeneration and loss of the keratocytes and endothelium, loss of the characteristic weblike pattern and colloidal iron staining of the proteoglycan ground substance, dispersal of ultrastructurally normal collagen fibrils, extensive accumulation followed by degeneration of PMNs, and accumulation of plasma proteins and fibrin in the necrotic cornea. Corneal damage produced by C. histolyticum collagenase. Samples (30 gl) of a collagenase preparation containing 10 gsg/ml (3.5 collagenase units/ml) produced corneal opacification by 4 h postinjection. Samples of solutions containing 100 or 1,000 ,ug/ml elicited extensive liquefaction necrosis, descemetocele formation, and a turbid, watery discharge by 4 h postinjection (Fig. lf). Light and electron microscopic examination

INFECT. IMMUN.

of the damaged corneas showed cellular changes similar to those observed during the pseudomonal infection; however, histochemical studies performed to visualize stromal collagen by the Van Gieson staining technique demonstrated almost complete loss of the red-orange color seen in undamaged stromal areas peripheral to the lesions and in control corneal stromas. Heavy accumulations in the necrotic comeal tissue of yellow-staining fibrin and plasma proteins also were observed. In addition, electron microscopy revealed the presence of ultrastructurally abnormal collagen fibrils. The fibrils were dispersed among fine filamentous material and, in cross-section, varied in shape and diameter (Fig. 4a). In longitudinal section, they appeared reduced and variable in diameter (Fig. 4b) when compared with normal corneal collagen fibrils (Fig. 3g).

DISCUSSION Before examination of the experimentally infected corneas by light and electron microscopy, the severity of the grossly observable damage suggested that the major cause of the corneal destruction was not degeneration and loss of the corneal cells, which are minor structural components of the cornea, but the alteration or loss of a major structural component of the cornea, i.e., the corneal collagen and/or proteoglycan ground substance. Histochemical examination of the damaged corneas revealed normal staining of the collagen but a sharp reduction in staining of the proteoglycan ground substance. In addition, electron microscopy showed dispersed but ultrastructurally normal collagen fibrils and an absence of the characteristic weblike pattern of the proteoglycan ground substance. Confirmation that the electron microscopic techniques used in this investigation were capable of detecting damaged collagen fibrils in vivo was obtained by observing ultrastructurally abnormal collagen fibrils in corneas damaged by C. histolyticum collagenase. Furthermore, the

FIG. 2. Light microscopic observations of corneas damaged by P. aeruginosa infections. Results shown are 24 h postinfection. (a-c) Changes observed postinfection with approximately 370 bacteria. (a and b) Areas bordering the gross lesion. Note accumulations of PMNs under the degenerated epithelium (E) and between the damaged endothelium (arrows) and Descemet's membrane (x370). (c) Area in the center of the gross lesion. Note rounding and detachment of the epithelial cells, PMN infiltration of the detached epithelium, and the presence of bacteria (circled) and PMNs (arrows) in the stroma (x 150). (d-f) Changes observed postinfection with approximately 3,700 bacteria. (d) Area bordering the gross lesion showing complete replacement of the detached epithelium by PMNs (x 150). (e) Severely damaged keratocyte (arrow) (x920). (f) Extensive edema and PMN infiltration of the stroma in the center of the gross lesion (x 150). Sections shown in (a), (b), (d), and (f) are stained with methylene blue-azure II. Sections shown in (c) and (e) are stained with basic fuchsin-alkaline methylene blue. (g) Normal staining of control stroma by colloidal iron technique. (h) Poor stromal staining by colloidal iron technique; postinfection with 3,700 bacteria. Dark staining masses and particles are PMNs (x 150).

CORNEAL DAMAGE BY P. AERUGINOSA

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TABLE 1. Light and electron microscopic characterization of the development of corneal destruction during experimental P. aeruginosa infections No. of bacteria injected

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Light microscopy showed degenerative swelling and disorganization. Electron microscopy showed irregularity of the basement membrane and degeneration of cells. The cells lost their characteristic shapes, appeared irregularly round, and displayed vacuole formation and chromatin clumping. b PMNs and bacteria in the stroma. No alterations noted in keratocytes, collagen, and ground substance. c No structural alterations observed. d Tissue bordering the gross lesion showed PMNs collecting under the degenerated epithelium (Fig. 2a). Tissue in the center of the lesion showed extensive detachment of damaged epithelial cells and PMN infiltration of the detached epithelium and Bowman's layer (Fig. 2c, 3a, 3b). e Light microscopy showed only PMNs and bacteria in the stroma. In the center of the lesion, electron microscopy revealed small regions of irregularly spaced but ultrastructurally normal collagen fibrils embedded in a fine filamentous material. In addition, keratocyte degeneration characterized by extensive intracellular edema, dilated Golgi apparatus, and disrupted outer nuclear membranes was observed. ' Tissue bordering the gross lesion showed PMNs collecting between the damaged endothelium and Descemet's membrane (Fig. 2b). A degenerated endothelial cell is shown in Fig. 3j. Center of the gross lesion showed loss of endothelium. g Complete loss of the epithelium and replacement by PMNs (Fig. 2d). h In the center of the lesion, cell debris, severely a

TABLE 1 con't damaged keratocytes, and large numbers of PMNs and bacteria were observed (Fig. 2e, 2f, 3c). Normal, regular, collagen fibril spacing and the interfibrillar weblike pattern of normal proteoglycan ground substance (Fig. 3d, 3f, 3g) were almost completely lost. Collagen fibrils were often irregularly spaced or were dispersed in electron-lucent areas of edema (Fig. 3e) and among fine filamentous deposits or "lakes" of precipitated plasma proteins (Fig. 3e and 3h). The fibrils appeared ultrastructurally normal and their paucity in electron-lucent areas and in areas containing filamentous material appeared to be due to their dispersion rather than to their degradation. Ultrastructurally abnormal collagen fibrils also were not observed in corneas taken from rabbits 2 and 4 days postinfection. i Histochemical studies showed that stromas stained poorly with colloidal iron (Fig. 2h); however, undamaged areas peripheral to the lesions stained similar to control stromas (Fig. 2g). Damaged stromas stained by the Van Gieson technique were similar to control stromas. i Almost complete loss of endothelium. Some endothelial cell remnants remained attached to Descemet's membrane. The few remaining severely damaged cells showed extensive intracellular edema, swollen mitochondria, and distorted nuclei containing clumped chromatin. k Keratocytes were absent. Extensive accumulations of PMNs, bacteria, plasma proteins, and fibrin were observed in areas of severe stromal edema and stromal disorganization. Many of the stromal PMNs had disintegrated, released their granules, and filled the areas of dispersed collagen fibrils with cell debris (Fig. 3i). Some degenerating PMNs contained phagolysosomes having bacteria in various stages of digestion. Dispersal of ultrastructurally normal collagen fibrils was similar to, but more extensive than, that observed 24 h postinfection with 3,700 bacteria. Ultrastructurally abnormal collagen fibrils also were not observed in corneas taken from rabbits 2 and 4 days postinfection. I Complete loss of endothelium. PMNs and fibrin were observed adhering to the posterior surface of the intact Descemet's membrane.

FIG. 3. Ultrastructural corneal alterations produced by experimental P. aeruginosa infections. Results shown are 24 h postinfection. (a and b) Epithelial cell alterations observed postinfection with approximately 370 bacteria. (a) Migration of PMNs between damaged basal cells and basement membrane. Note PMNs replacing epithelial cells (E), and areas of intact (arrows) and missing (*) basement membrane (x5,760). (b) Degenerated cells showing intercellular edema (IE), intracellular edema (*), a vacuole (V), slightly dilated rough endoplasmic reticulum (horizontal arrows), damaged mitochondria (vertical arrows), and chromatin clumping (N) (x14,720). (c) Keratocyte damage observed postinfection with approximately 3,700 bacteria. Keratocyte remnants containing a swollen mitochondrion (*) and cell debris (CD) are shown adjacent to a less severely damaged keratocyte containing swollen mitochondria (arrows) (x 14,720). (d) Normal ordered arrangement of collagen fibril lamellae (L) in control cornea (x30,400). (e) Appearance of lamellae postinfection with approximately 3,700 bacteria. The fibrils are dispersed in electron-lucent areas of edema (E) and among fine filamentous deposits of precipitated plasma proteins (*) (x30,400). (f and g) Normal ultrastructure and ordered arrangement of collagen fibrils in control cornea (x90.000). (f) Cross-section showing regular fibril spacing and the characteristic weblike pattern of the proteoglycan ground substance (arrows) spanning the interfibrillar spaces. (g) Longitudinal section. (h) Dispersed but ultrastructurally normal fibrils observed postinfection with approximately 3,700 bacteria. Cross-sectioned and longitudinally sectioned fibrils are dispersed among fine filamentous deposits of precipitated plasma proteins (*), and the normal weblike pattern of proteoglycan

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