Use of an Animal Model in Studies of Bacterial Corneal Infection

Brigitte A. Cowell, Ph.D., Postdoctoral Fellow, Christine Wu, B.S., Research Assistant, and Suzanne M. J. Fleiszig, O.D., Ph.D., Assistant Professor, are in the School of Optometry, University of California-Berkeley, Berkeley, California. Brigitte A. Cowell, Ph.D., Postdoctoral Fellow, Christine Wu, B.S., Research Assistant, and Suzanne M. J. Fleiszig, O.D., Ph.D., Assistant Professor, are in the School of Optometry, University of California-Berkeley, Berkeley, California. Brigitte A. Cowell, Ph.D., Postdoctoral Fellow, Christine Wu, B.S., Research Assistant, and Suzanne M. J. Fleiszig, O.D., Ph.D., Assistant Professor, are in the School of Optometry, University of California-Berkeley, Berkeley, California. Bacterial corneal infection (bacterial keratitis) is the most common cause of suppurative corneal ulceration ( Wilhelmus 1996 ). Despite continual improvement of available medical therapies, permanent corneal opacity frequently results. Occasionally the infection will be so destructive that a transplant is required. Approximately 0.5 to 1.0% of US cases of corneal infection require surgery. More seriously, bacterial keratitis is a major cause of vision loss in developing countries (trachoma and xerophthalmia being the most common causes). The corneal surface is continually exposed to microorganisms from the air and skin yet is infrequently colonized by bacteria. The eye is normally an immunologically privileged organ, and unique features such as eyelid function and bulk flow of tears are thought to be significant factors in protection against corneal infection ( Foster 1987 ). A breakdown or subversion of normal host defense mechanisms is required for infection of the cornea by bacteria to occur. These defenses commence with eyelid function and lashes, which provide some protection. This protection is augmented by the rinsing action of tear flow and antimicrobial constituents of tears ( Smolin 1987 ). Lysozyme is an antimicrobial tear protein that cleaves bacterial cell wall peptidoglycan of susceptible bacteria, which are mainly Gram positive. Lactoferrin has a direct bactericidal effect as well as iron-binding properties that inhibit bacterial growth. Immunoglobulins (especially secretory immunoglobulin A) and complement components add to the array of host defenses encountered in the tear film ( Franklin 1989 ). Furthermore, corneal epithelial cells are capable of phagocytosis ( Fleiszig and others 1995 ). Potentially, this would be another defense mechanism to use against bacterial contamination in which frequent sloughing of the outermost layers of epithelial cells would remove ingested bacteria. Ocular mucin plays a role in defense against infection because of its ability to bind bacteria and by constant exchange during blinking ( Fleiszig and others 1994 ). Although the cornea is normally avascular, immune cells such as polymorphonuclear leukocytes are rapidly recruited from iris and limbal vessels in response to the presence of infecting bacteria. Clearly, impairment of normal corneal defenses increases the risk of bacterial infection. The anterior route of access accounts for most cases of suppurative keratitis. One of the most common predisposing factors for corneal infection is contact lens wear, which may stagnate the tear film and predispose to infection by decreasing clearance of bacteria from the corneal surface ( Fleiszig and others 1992 ). Lens wear may also directly compromise the integrity of corneal epithelium. The risk of infection is significantly enhanced by overnight wear of lenses compared with a daily wear schedule ( Schein and others 1989 ). Indeed, approximately 40% of corneal infections recorded over a 3-yr period in New York were contact lens related ( Levine and Serdarevic 1998 ). A national study in the Netherlands estimated the infection risk for contact lens wearers to be 1.1 per 10,000 for rigid gaspermeable lens wear, 3.3 per 10,000 for daily soft lens wear, and 22.9 per 10,000 for extended wear ( Cheng and others 1998 ). In 1992, the number of Americans wearing contact lenses was reported to be approximately 24 million ( Buehler and others 1992 ). Although the risk of infection for any particular lens wearer is low, these estimates nevertheless translate into a significant number of infections that are potentially sight threatening. Other factors that may increase the risk of corneal infection include dry eye syndrome and blepharitis, which are reported to predispose to infection, and other ocular diseases, which alter the normal tear film ( Lemp 1990 ). Trauma and surgery disrupt the protective epithelial barrier and allow microorganisms direct access to the stroma ( Kreger 1983 ). Typically the corneal stroma is avascular; therefore, significant bacterial proliferation may occur before immune cell arrival. Occasionally bacterial infection of the cornea may occur via limbal blood vessels during sepsis from infectious scleritis, or through the endothelium during intraocular infection ( Wilhelmus 1996 ). Pathogens have evolved strategies to avoid killing by the host defense system. Although the presence of antibacterial proteins such as lactoferrin and lysozyme in the tear film prevents colonization by some bacteria, ocular pathogens such as Pseudomonas aeruginosa have been demonstrated to be resistant to their effect ( Cowell and others 1997 ). The production of an exopolysaccharide capsule or slime layer has been reported to allow pathogens such as Streptococcus pneumoniae and Staphylococcus epidermidis to resist phagocytosis ( Bowman and McCulley 1994 ). By masking complement binding, the O-side chains of bacterial lipopolysaccharide can allow P. aeruginosa to avoid phagocytosis ( Cryz and others 1984 ). P. aeruginosa is one of the most commonly isolated pathogens from contact lens-associated ulcerative incidents ( Levine and Serdarevic 1998 ). Patients infected with P. aeruginosa develop severe ulcers and often require more extensive treatments than patients with infections caused by other pathogens ( Cheng and others 1998 ). Corneal destruction during P. aeruginosa infection is rapid, and perforation and/or loss of vision is possible within 24 hr ( Hazlett 1995 ). P. aeruginosa is an opportunistic Gram-negative pathogen that is ubiquitous in the environment and commonly found contaminating lens cases and solutions ( Holland and others 1993 ). Once adhered to the lens or trapped under the lens proximal to the cornea, bacteria are more difficult to remove by the normal mechanical action of the eyelid and may additionally resist the host defenses of the tear film. P. aeruginosahas recently been demonstrated to cause damage to the intact cornea in vitro after only hours of contact ( Fleiszig and others 1998 ). This damage indicates that stagnation of tear fluid under the lens is potentially involved in the development of some corneal infections. Both bacterial and host factors are considered to be involved in corneal damage resulting from P. aeruginosa infection ( Holland and others 1993 ). P. aeruginosa produces many toxins and proteases that cause tissue damage, while host proteases released by neutrophils (especially elastase) are also destructive. Exotoxin A, exoenzyme S, and phospholipase C are some of the identified virulence factors of P. aeruginosa reported to be important in disease progression ( Holland and others 1993 ). ExoU has recently been demonstrated to be essential for acute cytotoxicity of some strains of P. aeruginosa ( Finck-Barbanc, on and others 1997 ). Protease production (such as alkaline protease and elastase) is thought to be responsible for corneal damage during infection ( Hobden and others 1993 ), and pili and lipopolysaccharide have also been reported to be involved ( Gupta and others 1994 ). The host immune system (which includes antibodies, complement, and phagocytic cells) has a significant effect on disease progression ( Berk and others 1991 ). The inflammatory cell response, in particular, is critical to development of disease ( Hazlett and others 1991 ). The primary immune cells recruited in the initial stages of corneal infection are neutrophils. Lysosomal products released during activation and phagocytosis include cationic proteins, acid proteases, and neutral proteases ( Bowman and McCulley 1994 ). Cationic proteins increase vascular permeability and are chemotactic for mononuclear phagocytes. Acid proteases degrade basement membrane, and neutral proteases are active against fibrin, elastin, and collagen. Neutrophils, when exposed to bacteria, release abundant reactive oxygen species that damage ocular tissue in addition to the targeted bacteria ( Henson and Johnston 1987 ). Upregulation of expression of numerous cytokines and chemokines has been detected in response to bacterial corneal infection, possibly contributing to corneal damage by augmenting or prolonging the inflammatory response ( Kernacki and others 1998 ). We need to better understand the pathogenesis of corneal infections if we are to develop new strategies for prevention or therapy. P. aeruginosa infections do not always respond well to antibiotics to which they are susceptible in vitro. Furthermore, bacteria such as P. aeruginosa are rapidly becoming resistant to current antibiotic agents ( Ciofu and others 1994 ). Contact lens wearers who develop bacterial keratitis generally do not report recent trauma ( Wilson 1996 ). Symptoms usually commence with a foreign body sensation or irritation that increases in intensity to persistent pain. This development is accompanied by redness, photophobia, and blurred vision. Focal stromal suppuration is characteristic of early bacterial keratitis, which consists of a dense yellow-white or gray-white stromal infiltrate with an overlying epithelial defect and adherent mucopurulent exudate ( Wilhelmus 1996 ). multiple infiltrates are sometimes seen in contact lens wearers. Stromal ulceration and liquefactive necrosis may develop if the disease progresses without treatment. Stromal edema and striae usually surround the area of ulceration. A ring-shaped infiltrate may develop, indicating an inflammatory reaction to endotoxin produced by Gram-negative bacteria ( Wilhelmus 1996 ). Active migration of inflammatory cells in the adjacent stroma can blur the edges of the focal infiltrate. Intraocular inflammation can range from a few aqueous cells and flare to hypopyon formation (accumulation and settling of white blood cells in the anterior chamber) correlating with vascular congestion of the iris. Corneal infection induced by P. aeruginosa typically presents as a rapidly progressing suppurative stromal infiltrate with marked mucopurulent exudate ( Wilhelmus 1996 ). Yellowish coagulative necrosis surrounded by epithelial edema resulting from inflammation is characteristic of infection caused by this pathogen. Rapidly escalating ulcer size and degree of suppuration can result in substantial stromal loss. A hypopyon is usually present, and descemetocele formation (protrusion of Descemet's membrane due to thinning of overlying tissue) or perforation may occur. Like most tissues of the human body, the cornea is complex in nature. The corneal epithelium is a highly organized and regular, stratified squamous epithelial cell layer ( Figure 1 ; also Gipson and Sugrue 1994 ). Basal cells of the epithelium adhere to a basal lamina called Bowman’s layer. The underlying stromal layer is composed of collagen fibrils tightly packed in parallel arrays and fibroblasts in a proteoglycan containing matrix ( Olsen and McCarthy 1994 ). The single layer of endothelial cells that complete the corneal complex secretes a thick basement membrane (Descemet’s membrane) that separates the endothelium from the stroma. A mucin containing glycocalyx is secreted by corneal and conjunctival cells that stabilize the overlying tear film. An intact tear film is necessary for corneal cell health, for maintenance of clear vision, and for protection of the corneal surface from foreign materials, including bacteria ( Dartt 1994 ). Blinking augments the spreading of tears and promotes corneal epithelial cell sloughing. Although the cornea is normally avascular, immune cells are readily recruited during infection and play a critical role both in the development of inflammation and in the resolution of bacterial infection. The complexities of the cornea, the immune system, and their interaction with invading bacteria make the use of animal models of corneal infection critical to developing an understanding of pathogenesis of corneal infection and subsequently devising effective therapies for this potentially blinding disease. Structure of the cornea. The use of animals in models of bacterial infection must be fully justifiable, and the ethics of animal experimentation have been vigorously debated for a number of years ( Goodwin and Gordis 1991 ; Naquet 1993 ; Parker 1994 ). Whenever possible, scientists are obliged to reduce the numbers of animals used, refine models to reduce stress to the animals, and finally seek alternative methods ( Russell and Burch 1959 ). Relative replacement, a strategy widely practiced in biomedical research ( Balls 1994 ), involves the humane killing of animals to provide cells, tissues, and organs for in vitro studies. Although animals are still required for the initial source of tissue, development of an cell significantly the for animals in many of of organs in vitro infecting a animal not reduce the number of animals required to complete a One to pathogenesis of infection of the cornea in vitro that has been by is the of cell by cell such as corneal epithelial This of the bacteria and the particular cell cells are may be and that are seen in with stratified tissue such as the cornea. These can result in bacterial in the system. bacteria in numbers with epithelial cells than with cells on cornea ( Fleiszig and others 1995 ). In the the epithelial layer is composed of epithelial cells that in and function as they the is one cell characteristic that of cells to bacterial ( Fleiszig and others ). to cell can also the of epithelial Other cell in corneal tissue from epithelial cells (such as and endothelial cells) may be involved in the infectious directly or via on epithelial cell Bacterial with epithelial cells also may be human corneal cells from by ocular ( Fleiszig and others 1992 ; and Wilson ). The of this are that to study human cells and to who have of infection. Although this may provide initial after contact of the bacteria with corneal epithelial cells, cells in this are to cells in that they and of the tissue from which they as as they are from the Furthermore, only a number of cells can be from of tissue of bacterial infection of tissue in which cells have in and in which important such as and are of to the of the host immune system in the of which is when the direct of bacteria on host tissues are into the pathogenesis of P. infection of the cornea have been an corneal epithelial cell and of ( Fleiszig and others 1995 1996 1998 ). of P. has also been human corneal epithelial cells by corneal ( Fleiszig and others 1992 ). these bacterial virulence factors to be important for infection of cells in vitro have been demonstrated to with virulence in in the ( Fleiszig and others 1994 1996 ). the infectious can be in vitro in must also study to fully understand the of the infectious The study of pathogenesis of infectious disease in the cornea has because of the development of animal models for in studies. and have been the most commonly animals for the study of corneal infections ( and ; Hazlett and others ; Hobden and others ), models and have also been ( and others ; and others 1996 ). of the features of the human corneal disease can be in the models ( Berk 1993 ). 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