key: cord-022219-y7vsc6r7
authors: PEIFFER, ROBERT L.; ARMSTRONG, JOSEPH R.; JOHNSON, PHILIP T.
title: Animals in Ophthalmic Research: Concepts and Methodologies
date: 2013-11-17
journal: Methods of Animal Experimentation
DOI: 10.1016/b978-0-12-278006-6.50008-2
sha: 
doc_id: 22219
cord_uid: y7vsc6r7

nan

The component tissues of the eye are both individually and collectively unique and fascinating to both clinician and researcher. We find in a single organ a variety of complex tissues each with its own particular vascular relationships, from the avascular cornea and lens to the extensively vascularized uvea. Charac teristic biochemical features of the tissues exist as well, including the endothelial pump of the cornea, the anaerobic glycolytic pathway of the lens, and the poorly defined transport systems of aqueous humor production and outflow. Besides the dynamic aqueous, we find the most substantial tissue of the eye, the vitreous humor, to be apparently a relatively stagnant blob of hydrated connective tissue whose physiology is still largely a mystery. The neurologic control mechanisms of such diverse processes as pupillary size, accommodation, and probably aque ous humor inflow and outflow add another dimension of potential inquiry. The eye is devoid of lymphatics, and the nature of its tissues further contributes to its immunologie uniqueness. The retina incorporates all the complexities of neuroperception and transmission. Nowhere else in the body are such diverse structure and function so intimately and intricately related.

An appreciation of these perspectives, the importance of vision as a cognitive sense, and consideration of the plethora of infectious, inflammatory, degenera tive, traumatic, toxic, nutritional, and neoplastic diseases that can affect any one or all of the component tissues of the eye, will elucidate the challenge the authors face in attempting to review pertinent conceptual and logistical approaches to eye research in the animal laboratory. While the majority of investigations have had as their objective ultimate correlation with normal and abnormal function and structure of the human eye, laboratory studies have provided an abundance of comparative information that emphasizes that while there are numerous and amazing similarities in the peripheral visual system among the vertebrate (and even the invertebrate) animals, significant differences exist that are important to both researcher and clinician in selection of a research model and in extrapolation of data obtained from one species to another, and even among different species subdivisions.

Details of comparative anatomy and physiology have been reviewed by several authors (Prince, 1956 (Prince, , 1964a Polyak, 1957; Duke-Elder, 1958; Prince et al., 1960; Walls, 1967) . These are classic works and invaluable references. One is reminded that "research" means "to look for again," and while some concepts presented in these earlier texts are indeed outdated, and some facts since disproven, phylogenetic perspectives acquired from familiarity with this literature arms the scientist with valuable information. Throughout this chapter, we will dwell on species anatomic and physiologic differences when they have been specifically defined or where they are of value in regard to specific laboratory methodologies.

While the subhuman primate is frequently described as the ideal laboratory model for eye research, many of these animals are threatened or endangered, and logistical aspects of procurement, maintenance, and restraint should en courage investigators to explore and define the validity of nonprimate animals.

When deemed essential, primates should be utilized with maximum thrift and humane care in mind.

The eye is a sensitive organ, and in all species adequate anesthesia for man ipulative procedures as well as postoperative analgesia should be considered in in vivo studies; the rabbit, for instance, is extremely sensitive to barbiturate anesthesia and prolonged procedures that require intraocular manipulations pose a challenging problem in terms of subject mortality.

The in vitro culture of ocular tissue cell lines offers increasing potential in research as an alternative to animal models. The lens, retinal pigment epithelium, and corneal epithelium and fibrocytes may be readily maintained and manipu lated. Current work with the corneal and trabecular endothelium is perhaps the most exciting advance in ophthalmic research in recent years. In vitro culture techniques offer a number of logistical advantages over animal models.

A knowledge of spontaneous ocular diseases is essential to avoid interpreting pathology as experimental that may well be unrelated to the investigation. Thorough preoperative ophthalmic examination is a prerequisite of any study, and the laboratory scientist will find it to his benefit to be associated with an interested human or veterinary clinical ophthalmologist to assist in defining and managing spontaneous ocular disease. For instance, electrophysiologic studies of the canine retina will be invalid if the subject has chorioretinal lesions of canine distemper, a common entity in laboratory dogs. Examination beyond the target organ is also recommended to detect abnormalities that may directly or indirectly influence results. Frequently encountered spontaneous infectious diseases are reviewed in a subsequent section.

We have limited our discussions primarily to research methodologies involv ing the globe itself; a paucity of information is available regarding the adnexa, orbit, and extraocular muscles, and described techniques are refinements of general approaches rather than those specifically developed for ocular research. In the same perspective, topics of pure physiology or biochemistry are limited, and we did not embark into the unfamiliar and complex area of central visual mechanisms. Our approach, we hope, if not all inclusive, has been comprehen sive, with adequate references to specific detail and additional information for the interested reader.

Spontaneous infectious diseases that involve the eyes of laboratory animals are significantly prevalent to warrent brief discussion. It is important to consider these entities, especially before using animals derived from poorly defined sources. Propensity of a species toward the development of spontaneous infecti-ous diseases that might not only affect general health but also induce ocular pathology in both experimental and control subjects may affect selection of a model of procurement and isolation procedures utilized. Preinvestigation ocular examinations are necessary to detect preexisting pathology.

Of the three large species used in ophthalmic research (cat, dog, and primate) , the primate appears to have a minimal predisposition for spontaneous infectious ocular disease. This may be a result, however, of a lack of ophthalmic examina tion in disease situations. It is also important to note that the hamster and guinea pig rarely develop eye infections. Table I summarizes those infectious diseases that more commonly involve the eyes of laboratory species.

The use of laboratory animals in the investigation of infectious ocular disease has included rats, hamsters, guinea pigs, rabbits, cats, dogs, and subhuman primates. The disease agents studied have ranged from viruses to protozoa, with the rabbit serving as the most popular model. These studies have been of value in defining pathogenesis and the effects of therapeutic agents.

Those disease agents known to cause conjunctivitis in man do not necessarily produce a similar disease in laboratory species. A particular species may require immunosuppression to allow the induction of a specific infection (Payne et al., 1977; Forster and Rebell, 1975) . In other instances, a model may be specifically susceptible to an organism; for example, the chlamydial inclusion body con junctivitis agent in guinea pigs.

An additional and somewhat unusual use of the guinea pig involves the Sereny test (Formal et al., 1972) . Because their conjunctivae are so susceptible to Shigella organisms, this animal, through the use of a drip apparatus, has been used to identify the pathogenic strains (Mackel et al., 1961) . Listeria monocytogenes produces a severe keratoconjunctivitis in the guinea pig, but in the rabbit and monkey the disease is much milder and more difficult to reproduce (Morris and Julianelle, 1935) . This concept has lent itself to studies concerning epithelial cell phagocytosis in the guinea pig (Zimianski et al., 1974) . Organism installation techniques involve either direct swabbing or syringe inoculation into the conjunctival sac.

The owl monkey (Actus trivirgatus) has proved to be the model of choice for the study of Chlamydia trachomatis conjunctivitis; other primate species includ ing orangutans have proved to be only mildly susceptible (Fraser, 1976) .

A number of models are available for investigators interested in studying keratitis. The rabbit has proved to be the most feasible subject, especially in herpes simplex studies (Kaufman and Maloney, 1961) . Many bacterial and fun gal diseases afflicting man have also been examined in this model, including the effects of antibiotics, steroids, interferon, vitamins, and other agents Nesburn and Ziniti, 1971; Smolin et al., 1979; Pollikoff et al., 1972) .

Herpesvirus studies can be significantly influenced by the particular strain of virus utilized. For example, some are more effective in producing stromal keratidities as compared to the epithelial condition (Metcalf et al., 1976) . Con comitant in importance may be the route of inoculation. In some studies viral suspensions are placed into the conjunctival sac (Nesburn and Ziniti, 1971) , while in others they are dropped onto the cornea . Corneal preparation may be important; for example, some studies have employed chalazion currette (Stern and Stock, 1978) , while others have employed spatula scrap ing or circular trephine defects (Kaufman and Maloney, 1961) . Deep inoculations are required to produce a stromal keratitis (Fig. 1) , while the epithelial disease may be induced through shallow scratches (Polli koff et al., 1972) . The rabbit appears to be the species of choice for bacterial keratitis, although Davis et al. (1978) feel that the guinea pig is of equal value. In one study an inbred animal was used (strain 13) to reduce the inherent variability seen in some of these experiments (Davis and Chandler, 1975) . Whether or not the rabbit model has any specific biologic advantage, the guinea pig would appear to be more feasible because of its smaller size, ease of handling, ease to anesthetize, cost of purchase and maintenance, and, perhaps most significantly, resistance to spontaneous Pasteur ella conjunctivitis, which frequently occurs in laboratory rabbits. Methods of bacterial inoculation usually involve an intracorneal injec tion using a microsyringe and small volumes of quantitated microorganisms.

Fungal keratidities have been studied in rats, rabbits, and owl monkeys. Rats were initially popular (Burda and Fisher, 1959) , but rabbits and monkeys have become the select species. Fusarium solarti keratitis was first studied in the subhuman primate, but, due to expense, Forster and Rebell (1975) utilized the pigmented rabbit. This model, having received germinating conidia (as opposed to spores) interlamellarly, developed a sustained progressive infection, but only after pretreatment with subconjunctival steroids. Candida albicans keratitis has also been induced in the corticosteroid pretreated rabbit . Inoculation techniques for these organisms are identical to those described for bacterial and viral keratidities. Ishibashi (1972) suggests that backward flow from the inoculation wound and volume accuracy can be controlled by using a microsyringe and a 27-gauge flat-faced needle.

Recently, human corneal transplants have been incriminated in the transmis sion of rabies to recipients. A model has been developed in the guinea pig utilizing the slow virus agent of Creutzfeldt-Jacob disease (Manuelidis et al., 1977) . Using a method developed by Greene (1950) , this study involved the heterologous transplantation of infected guinea pig cornea sections into the an terior chamber of six recipients with resultant development of spongiform encephalopathy.

Studies designed to produce anterior uveitis usually employ inoculation into the anterior chamber. This procedure is best performed with a 25-to 30-gauge needle directed obliquely through the limbus into the chamber (Smith and Singer, 1964) . Ocular histoplasmosis was effectively studied in the rabbit using this method, the result being a well-defined granulomatous uveitis. The same proce dure may be applied to produce bacterial or viral anterior uveitis (Kaufman et al., 1970) . It is advisable, however, to withdraw initially an equivalent amount of aqueous fluid to avoid intraocular pressure elevation (Zaitseva et al., 1978) . Aguirre et al. (1975) administered canine adenovirus type I intravenously to young beagles to produce the anterior uveitis characteristic of infectious canine hepatitis. Carmichael et al. (1974) demonstrated that this was a complementmediated antigen-antibody complex disease.

The induction of endophthalmitis may utilize direct transcorneal chamber in oculation as described above; this method was used in rabbits to produce a bacterial infection in order to assess diagnostic anterior chamber paracentesis technique (Tucker and Forster, 1972) . The same procedure was also used in the rat to create a fulminating Fusarium solarti endophthalmitis (O'Day et al., 1979) .

The intravitreal route of inoculation is utilized to induce posterior segment infection. This technique requires toothed forcep stabilization of the eye and needle introduction through the pars plana (May et al., 1974) . Indirect ophthalmoscopy may be employed to ensure accurate deposition. Typically a tuberculin syringe with a 5 /s-inch, 27-gauge needle is used with the subject under general anesthesia or deep tranquilization. The needle is inserted at 12 o'clock and directed toward the posterior pole. Small volumes may be injected without signif icant alteration in the intraocular pressure. Avallone et al. (1978) used a similar technique in rabbits to demonstrate the effectiveness of the Limulus lysate test for rapid detection of E. coli endophthalmitis. After producing a staphylococcol endophthalmitis by this method, Michelson and Nozik (1979) tested the perfor mance of a subcutaneously implanted minipump for antibiotic administration in the rabbit.

Ocular toxocariasis, a human disease caused by larval migration of Toxocara canis, was produced in mice by Olson et al. (1970) . Freshly incubated ova were administered per os by stomach tube. Anterior chamber hemorrhage and actual sightings of larval migration were reported. Hobbs et al. (1978) found ocular Mycobacterium leprae and M. lepraemurium in the nine-banded armadillo and mouse, respectively, following intravenous, intraperitoneal, or foot-pad inoculation of the organisms; the uveal tract was primarily involved, although lesions were observed in other ocular tissues. Lesions were more dramatic in mice immunologically depressed by thymectomy and total body irradiation.

Dur to its susceptibility to Toxoplasma gondii, the rabbit has been developed as a model for ocular toxoplasmosis. Nozik and O'Connor (1968) used the California pigmented rabbit and a variation of a method described by Vogel (1954) to study the associated retinochoroiditis. This technique consists of prop osing the eye of an anesthetized rabbit and passing the needle retrobulbarly through the sclera to the suprachoroidal space (Fig. 2) . The authors point out that while the lesions are less active and heal spontaneously in 3 weeks, features are of comparative value. For detailed information concerning other uses of this model the reader is referred to Tabbara's organism recoverability experiments (Tabbara et al., 1979) .

Histoplasmosis has been studied in mice, rats, rabbits, guinea pigs, dogs, pigeons, chickens, and primates. Smith et al. (1978) point out that because it is essentially a macular disease in man, the ideal model should have similar anatomy. Basically two orders of animals qualify: the avian species due to their dual fovea and the primates. The above authors used the stumptailed macaque, M. arctoides, to produce a focal choroiditis by inoculating yeast-phase Histoplasma capsulatum into the internal carotid artery. They emphasized the desirabil ity to create a model that, in addition to focal choroiditis, also exhibits minimal involvement of the retina, no anterior segment disease, and a late macular lesion.

Experimental ocular cryptococcosis has been induced in rabbits, primates and cats, the latter species appearing to be the ideal choice because of its susceptibil ity and related high incidence of chorioretinal lesions. Blouin and Cello (1980) used an intracarotid injection technique which reproduced lesions identical to those seen in the naturally occurring disease.

A septic choroiditis of bacterial origin was produced by Meyers et al. (1978) by intracarotid injection of staphlococcal and streptococcal organisms. This in turn produced a serous retinal detachment, which was the primary objective of the study.

Animal studies have provided an abundance of information dealing with the pharmacodynamics of the eye and drugs used to treat ocular disease. Because of the accessibility of the ocular structures, there are a number of routes of drug administration which are not available in treating other organ systems. On the other hand, there are barriers to drug penetration in the eye which must be considered, including a hydrophobic corneal epithelial layer over the external ocular structures and an internal blood-ocular barrier similar to the blood-brain barrier. The endothelial cells of the iris and retinal vessels, the nonpigmented epithelium of the ciliary body and the retinal pigment epithelium all contribute to this blood-ocular barrier (Cunha-Vaz, 1979) .

Various agents can compromise these barriers to drug penetration. Inflamma tion can also affect any of the ocular barriers. In some experimental designs it may be desirable to create inflammation and/or minimize the barriers. Tech niques include mechanical removal of the corneal epithelium with a scalpel blade, inducing infectious keratitis or treating the external eye with enzymes (Barza et al., 1973) . A more generalized inflammation can be created by sen sitizing the animal to a specific antigen and then challenging the eye with that antigen (Levine and Aronson, 1970) . Certain pharmacologie agents affect spe cific barriers; anticholinesterase inhibitors, for instance, can break down the blood aqueous barrier (von Sallmann and Dillon, 1947) , while agents with epithelial toxicity, such as benzalkonium chloride, are added to many topical preparations to increase the permeability of the corneal and conjunctival epithelium. All these factors regarding method of administration and barrier permeability must be considered in an experimental design.

Once again the rabbit has been most frequently chosen for pharmacologie experiments on the eye. Although at first glance the anatomy of the rabbit eye would seem close,enough to the human to make this a valid experimental model, at least for drug penetration studies, significant differences do exist, as will be pointed out.

Drugs can usually be applied topically to the eye in concentrations much higher than could be tolerated systemically. Topical application has many limita tions, however. If a topical drug is to be effective intraocularly it must traverse the hydrophobic epithelial layer and the hydrophilic corneal stroma; ideally, then, it should exist in an equilibrium between ionized and unionized forms with biphasic solubility characteristics. Thus, both chemical configuration and pH of the vehicle are important. Other important limitations are the small tear volume and rapid clearance of drugs from the tear film (Janes and Stiles, 1963) . Rabbits tend to blink infrequently; consequently corneal contact time is prolonged com pared to humans. Most nonprimate mammals possess a nictitating membrane which may affect tear dynamics. Drugs may be applied with an ointment vehicle to prolong contact time, but bioavailability may be affected. Finally, the position of the animal may be important. A tenfold difference in aqueous drug levels in rabbits has been reported depending on whether the animal was upright or re cumbent during the experiment (Sieg and Robinson, 1974) .

Injection of a drug under the conjunctiva or Tenon's capsule theoretically allows delivery of greater amounts of drug, prolonged contact time, and bypass of the external epithelial barrier. Subconjunctival injection may be given in two ways; by (1) injecting directly through the bulbar conjunctiva or (2) by inserting the needle through the skin of the lids, leaving the conjunctiva intact. The method of injection may be very important; one study showed that in rabbits leakage of hydrocortisone back through the injection site into the tear film ac counted for most of the intraocular absorption (Wine et al., 1964) . Although numerous articles have been written on the kinetics of drug absorption after subconjunctival injection (Baum, 1977) , the subject is still controversial, and recent evidence presented by Maurice and Ota (1978) indicates that absorption into the anterior chamber by this route may be much lower in rabbits than in man. If so, the rabbit may be an inappropriate model for this type of research.

The potential for serious complications makes intraocular injection of drugs a 4 'last resort" option in clinical practice. In many cases the maximum amount of drug tolerated within the eye is small (Leopold, 1964) . Good results have been reported, however, in the treatment of experimental bacterial endophthalmitis in rabbits with intravitreal injection of antibiotics (von Sallmann et al., 1944; Peyman, 1977) . Usually 0.05-0.2 ml of antibiotic solution is injected into the nucleus of the vitreous through the pars plana.

Intramuscular and intravenous are the preferred routes for systemic administra tion of drugs in most laboratory animals. Drugs may be given orally in food, water, or by stomach tube, or more reliably and precisely with pills or capsules in the case of cats and dogs. Whatever the systemic route, intraocular levels are limited by the body's tolerance for the drug and by the blood-ocular barriers. One method of obtaining high ocular levels is arterial infusion, usually of the ipsilateral carotid artery. The highest levels have been obtained experimentally by retrograde perfusion of the intraorbial artery in dogs; details of the technique are discussed by O'Rourke et al. (1965) .

In general the assessment of penetration into the various intraocular compart ments is carried out using radio-labeled preparations of the drugs under study. The labeled drug is administered, and after a predetermined length of time samples of aqueous are removed by paracentesis or the whole eye is removed and standard size tissue samples taken for scintillation counting. O' Brien and Edelhouser (1977) utilized a direct chamber perfusion technique to study penetra tion of radio-labeled antiviral drugs through the excised cornea. In the case of antimicrobials, drug levels in ocular tissues may also be determined using a microbioassay such as the agar diffusion technique described by Simon and Yin (1970) .

Other methods have been used less commonly. Autoradiography has been used to assess intraocular penetration (McCartney et al., 1965) , and distribution in the orbital compartments has been studied using diatrizoate, a radio-opaque contrast media (Levine and Aronson, 1970) .

Techniques used to assess drug efficacy depend primarily upon the type of drug under study. Most drugs used in the treatment of glaucoma, for instance, work through complicated autonomie mechanisms which alter the inflow and outflow of aqueous humor. Tonography, tonometry, and fluorophotometry are used to study these effects and are discussed elsewhere. Methods used to study antibiotic efficacy, on the other hand, are usually less objective. Results are often based on semiquantitative or subjective impressions of clinical response. Actual bacterial counts on samples of tissue following treatment of experimental infec tions are feasible, but the organism and size of original inoculum must be strictly defined (Kupferman and Leibowitz, 1976) .

Steroid efficacy is even more difficult to assess objectively in experimental models. Leibowitz et al. (1974) have reported a method of quantifying steroid response based on the number of polymorphonuclear leukocytes labeled with tritiated thymidine remaining in the tissue following treatment of inflammatory keratitis.

Successful organ and cell culture has been reported with a variety of ocular tissues, including whole eyes. The culture of lens, cornea, and corneal endothelium have become routine techniques in many ophthalmic laboratories. Paul (1975) provides a good manual of basic tissue culture techniques. The early work in culture of ocular tissue is the subject of an excellent review by Lucas (1965) . A number of significant advances have been made since that time, especially in the areas of retinal pigment epithelial and cornea culture.

Suspended between the aqueous and vitreous humors with no direct vascular supply, the lens is maintained in a type of natural organ culture. It has always been felt, therefore, that the lens should be ideal for in vitro culture, and lens culture experiments have been utilized extensively to study the metabolism of this tissue. A great deal of research into ideal media and culture conditions has been necessary, however. Rabbit, mouse, and rat lenses have been most widely used, but culture of bovine lenses is also feasible despite their large size (Owens and Duncan, 1979) . The lens may be cultured in an open (continuous perfusion) or closed system. The advantages and disadvantages of each type of system are discussed by Schwartz (1960a) . Closed systems patterned after that described by Merriam and Kinsey (1950) are by far the most popular, but for certain types of experiments more elaborate perfusion systems are necessary (Schwartz, 1960b; Sippel, 1962) .

The composition of the media for lens culture has been found to be very important. Kinsey et al. (1955) have provided systematic studies on the optimum concentration of various culture media constituents. Presently most lens culture is being done in a modified TCI99 media which has been used by Kinoshita and others with good success (von Sallmann and Grimes, 1974) . Thoft and Kinoshita (1965) have found that a calcium concentration somewhat higher than that in the aqueous is desirable in the culture media. More recently, Chylack and Kinoshita (1973) have shown that removal of the lens with part of the vitreous still attached to the posterior surface improves certain parameters of lens function in vitro. Presumably, leaving vitreous attached to the lens prevents damage to the pos terior capsule.

In addition to culture of whole lenses, cell cultures of pure lens epithelium have been important in the study of lens metabolism. Most of the early work was done with chick epithelium, but more recently cultures of mouse lens epithelium have been shown to retain greater cellular differentation in culture (Mann, 1948; Russell et al., 1977) .

Three different cell types make up the layers of the cornea: epithelial cells, fibrocytic stromal cells, and a single layer of endothelial (mesothelial) cells. All three corneal cell types, as well as whole corneas, can be maintained in culture. The culture of whole corneas has been investigated as a means of corneal preser vation prior to transplantation. Most of these studies have used human rather than animal tissues.

Endothelial cell culture has become an important technique in the study of this very metabolic ly active cell layer. Stocker et al. (1958) described a technique for separating a corneal button by carefully peeling off Descemet's membrane with endothelium from the posterior surface and peeling the epithelium from the opposite side to yield three relatively pure cell types which can be explanted onto separate cultures (Fig. 3 ). Colosi and Yanoff (1977) have used trypsin to obtain endothelial cells or epithelial cells from whole corneas. Perlman and Baum (1974) have used Stacker's method to isolate endothelial cells from rabbits and have had good success in maintaining large endothelial cell cultures for several months using a modified Eagle's MEM supplemented with calf serum, bicarbo nate, glutamine, and kanamycin sulfate. Most exciting are the reports of using tissue cultured endothelial cells, seeded on to donor corneas with endothelium removed, for transplantation in rabbits (Jumblatt et al., 1978) .

Although the corneal stromal cells are the most thoroughly studied ocular fibrocytic cells, other fibrocytes have been successfully cultured, including goniocytes from the anterior chamber angle (Francois, 1975) and hyalocytes from the vitreous (François et al., 1979) .

Tissue culture of immature neural retina has been a popular subject. Early experiments are summarized by Lucas (1965) . Recently, differentation of em bryonic neural retinal cells from the chick has been described both in cell aggre gate cultures (Sheffield and Moscona, 1970) and monolayer cultures (Combes et al., 1977) .

Organ culture of mature neural retina has proved more difficult. Mature retina tends to deteriorate rapidly in a convential Warburg apparatus (Lucas, 1962) . The organ culture technique of Trowell (1959) has been used to maintain mature and nearly mature retina for several days (Lucas, 1962) . This technique requires mechanical support of the retina on a metal grid with receptor cells uppermost, exposed to the gas phase; in this case air as 95% oxygen was found to be toxic. Ames and Hastings (1956) described a technique for rapid removal of the rabbit retina, together with a stump of optic nerve, for use in short-term culture experi ments including in vitro studies of retinal response to light (Ames and Gurian, 1960) .

Until recently only a few studies were available on the behavior of retinal pigment epithelium (RPE) cells in culture, but recent work on the multipotential nature of the RPE cell has aroused new interest in this area. As with neural retina, chick embryos have been most often used for RPE cultures. Hayashi et al. (1978) used EDTA and trypsin to isolate chick RPE cells for culture on Eagles MEM supplemented with 10% fetal calf serum. A similar technique has been used to culture RPE cells from Syrian hamsters (Albert et al., 1972a) . Mandel-corn et al. (1975) have reported an ingenious experiment in which RPE cells from one eye of an owl monkey were transplanted into the vitreous of the fellow eye through the pars plana where the proliferation and metaplasia of those cells could be studied.

The experiment described above is not the first time that investigators have attempted to take advantage of the eye's transparent media and large avascular spaces to create a type of in vivo tissue culture. Many successes have been reported in transplanting homologous and autologous tissues into the anterior chamber. Markee (1932) transplanted endometrium into the anterior chamber of guinea pigs, rabbits, and monkeys where it continued to undergo cyclic changes. Goodman (1934) also reported ovulatory cycles of ovaries transplanted into the anterior chamber of rats. Woodruff and Woodruff (1959) successfully trans planted homologous thyroid tissue into the anterior chamber of guinea pigs. Eifrig and Prendergast (1968) found that autologous transplants of lymph node tissue in rabbits were well tolerated. A variety of embryonic tissues, with the exception of liver, have been successfully transplanted into the anterior chamber of rabbits (Greene, 1943) .

Heterologous transplants of tissue from one species to another have more often than not been unsuccessful. Greene (1947) reported good results in transplanting malignant tumors into the anterior chamber of different species. He observed that transplants from human to guinea pig and rabbit to mouse worked best. Greene's technique of transplantation involves introducing a small piece of donor tissue in a small canula fitted with a stylet through a limbal incision and firmly implanting the tissue into the anterior chamber angle opposite the incision. Morris et al. (1950) reported disappointing results using this technique for heterologous tumor transplants in guinea pigs, but obtained better results by transplanting the tissue into the lens beneath the lens capsule. The reader is referred to Woodruff (1960) for further discussion of intraocular transplantation experiments.

The eye is unique in that it is devoid of lymphatics and thus has no directly associated lymph nodes. Classic animal studies have been utilized to define ocu lar mechanisms of immune response and have demonstrated that the uveal and limbal tissues play a role similar to that of secondary lymphoid tissue elsewhere in the body. Bursuk (1928) injected typhoid bacilli and staphylococci into the cornea of rabbits and found that agglutinins and opsonins appeared in the corneal tissues earlier and in greater concentration than in the serum, suggesting local respon siveness. Thompson and co-workers (1936) obtained identical results using crys talline egg albumin as the antigen, and further showed that when only one cornea of each animal was injected precipitating antibody could not be detected in the contralateral uninjected cornea. Rabbit experiments in which ovalbumin was injected into the right cornea and human serum albumin was injected into the left cornea demonstrated that the respective cornea contained antibodies only to the antigen they had received and not to the antigen present in the contralateral cornea; specific antigen was detectable within 8 days (Thompson and Olsen, 1950) . Using a'fluorescent antibody technique, Witmer (1955) demonstrated immunoglobulin cells in the uvea of the rabbit, and Wolkowicz et al. (1960) showed that excised, sensitized uveal tissue in short-term culture would actively produce specific antibody. Lymphoid cells migrate to the eye in the presence of antigenic stimulus. Thus, x irradiation of the eyes of a rabbit does not impair the ability of either the limbal tissues or the uvea to form antibody, whereas irradiation of the peripheral lym phoid system does (Thompson and Harrison, 1954; Silverstein, 1964) . When the primary ocular response to an antigenic challenge has subsided, sensitized 4 'memory" lymphocytes may persist in the uvea or limbal tissues, since a later exposure to the same antigen introduced at a distant site or injected directly into the circulation results in renewed antibody production within the eye (Pribnow and Hall, 1970; Silverstein, 1964) .

The dog, cat and monkey have all been used as animal models in cornea research. The rabbit, however, has been exploited to such a degree for this purpose that a few comments on the rabbit cornea are appropriate. The rabbit has a large cornea approximately the same diameter as the human cornea. It is significantly thinner, however, with an average central thickness of about 0.40 mm. The thin cornea and shallow anterior chamber have contributed to technical difficulties in performing intraocular lens implantation and penetrating keratoplasty in rabbits (Mueller, 1964) . The corneal epithelium is also thin in rabbits, and the existence of Bowman's layer in the rabbit has been disputed (Prince, 1964b) .

One of the most important qualities of the rabbit cornea is the capacity of the endothelium for regeneration. Following injury, rabbit endothelial cells are able to divide and cover the defect with minimal effect on the endothelial cell density. The dog is similar in this respect (Befanis and Peiffer, 1980) . Cats and monkeys, on the other hand, have been shown to have very little potential for regeneration of endothelium. In these animals, as in man, remaining endothelial cells grow and spread to cover the defect (Van Horn, 1975) . Thus cats and monkeys are probably better models in studies where response to endothelial injury is impor tant.

The recent surge in interest in the nature of the corneal endothelium has been brought about largely by the demonstration that the endothelium is the site of the "fluid pump" transport mechanism essential for maintaining corneal dehydra tion and thus transparency (Maurice, 1972) . Localization of the fluid pump and many subsequent studies have been made possible by the endothelial specular microscope developed for viewing the endothelium of enucleated eyes and modi fied for use with excised corneas in the form of the perfusion specular micro scope (Maurice, 1972) (Fig. 4) . Later the endothelial microscope was adopted for in vivo examination of humans and animals, including cats, rabbits, and monkeys (Laing et al., 1975) .

There is nothing new about viewing the specular reflection from the corneal endothelium, a long established slit lamp technique. The specular microscope, however, is an instrument designed to optimize this type of illumination to produce a high magnification (200-400 x) image of the endothelium suitable for photomicrography, accurate endothelial cell counts, and detailed observation of individual cells (Fig. 5) . A discussion of the optical principles of specular mi croscopy is provided by Laing et al. (1979) . Commercially available endothelial microscopes are designed primarily for use with humans, but can be easily adapted to any laboratory animal with a sufficiently large cornea (which excludes rats and mice). The smallest eye movements blur the image of the endothelium, and, therefore, general anesthesia is usually required when working with ani mals. Technique and observations in dogs (Stapleton and Peiffer, 1979) and cats (Peiffer et al., 1980a) have been described.

In 1951 Ussing and Zehran described an apparatus consisting of two small Lucite chambers separated by a piece of frog skin which could be used to measure transport of materials potential differences across the frog skin. This same technique was modified for study of the cornea, and dual chamber perfu sion experiments form the basis for much of what we know about corneal physiology (Donn et al., 1959; Mishima and Kudo, 1967) . The perfusion specu lar microscope has added an extra dimension to this type of in vitro experimenta tion by allowing direct observation of endothelial cell shape and integrity during perfusion. Atraumatic removal of the cornea avoiding contact with the en dothelium or excessive bending of the cornea is essential to the success of this type of experiment. The technique of Dikstein and Maurice (1972) for excision of the cornea with a scierai rim has been used very successfully.

For longer term in vitro experiments the cornea can be cultured by convential closed organ culture techniques as described elsewhere in this chapter.

The impetus for the intensive investigation of optimum conditions for corneal storage has resulted from the feasibility of corneal transplantation in humans. Most of the research has been done using rabbits, however, and is especially applicable, therefore, to use and storage of animal corneas for whatever purpose.

The method of corneal storage in most eye banks today is moist-chamber storage, storing the intact globe at 4°C in a glass bottle with saline-soaked cotton in the bottom. The stagnant fluid quickly becomes loaded with waste prod ucts, making this method unsuitable for long-term storage. A number of alterna tive techniques are used in laboratory storage including storage of excised cor neas at 4°C, organ culture at 37°C, and cryopreservation for long-term storage.

Several different media for storage of excised corneas have been evaluated. The most popular at present is McCary-Kaufman (M-K) media, modified tissue culture media containing TC-199, 5% dextran 40, and a mixture of streptomycin and penicillin (McCarey and Kaufman, 1974) . Storage in M-K media probably preserves corneal viability longer than moist chamber storage, endothelial dam age occurring at 2 days in moist chamber stored rabbit corneas compared to 4-6 days for M-K stored corneas (Geeraets et al., 1977) . Stocker et al. (1963) reported good success with excised corneas stored in autologous serum at 4°C. Subsequent studies, however, show conflicting results as to the advantage of serum storage over moist chamber storage (Geeraets et al., 1977; Van Horn and Schultz, 1974) .

Corneal organ culture represents an alternative method of prolonging corneal viability. It is similar to convential storage of excised corneas except for tempera ture, which is maintained at 37°C and may allow for preservation of endothelial viability for up to 3 weeks (Doughman et al., 1974) . The culture media most often used is Eagles MEM supplemented with fetal calf serum.

Cryopreservation is the only method which permits indefinite storage of whole viable corneas. The tissue is passed through a graded series of solutions contain ing dimethyl sulfoxide and frozen at a controlled rate over liquid nitrogen. Studies indicate that some endothelial damage may occur during the freezing process but destruction is incomplete (Basta et al., 1975) . In fact, survival of donor endothelial cells following transplantation of cryopreserved corneas has been demonstrated using sex chromatin markers in monkeys (Bourne, 1974) . Techniques of cryopreservation have been reviewed and evaluated by Ashwood-Smith (1973) .

Methods of estimating corneal viability after various storage regimens are all based on evaluating endothelial integrity. Histochemical stains have been used extensively (Stocker et al., 1966) . Dye exclusion stains, such as 0.25% trypan blue applied to the endothelium for 90 seconds and then rinsed off with saline, can be used without apparent toxicity to the endothelium. The pattern obtained with trypan blue staining is more difficult to see in rabbits than other species. A modification of this technique using a combination of trypan blue and alizarin red on rabbit corneas has recently been reported (Spence and Peyman, 1976) (Fig.  6 ). Electron microscopy, both scanning and transmission, are also used to assess the condition of the endothelium (Fig. 7) .

The most innocuous method of estimating corneal viability is simply mea surement of corneal thickness at body temperature which is an indirect indication of the condition of the endothelium, healthy endothelium being able to deturgese the cornea to near-normal thickness. The measurement of corneal thickness, or pachometry, has traditionally been done using an optical technique developed by von Bahr (1948) . The specular microscope, however, provides for a more direct method of measuring corneal thickness by registering the distance from the point of applanation at the corneal surface to the image of the endothelium.

Viraj and bacterial infections of the cornea have been studied mostly in rab bits, especially in the case of herpes simplex keratitis where attempts to study this disease in other animals have been unsuccessful. Techniques for initiating these infections are discussed in Section III. Noninfectious forms of keratitis may result from tear deficiencies, vitamin A deficiency, allergic phenomenon, and chemical or mechanical damage. Keratoconjunctivitis sicca (KCS) resulting from tear deficiency has been studied in the dog, the disorder being surgically induced by removal of the lacrimai gland and nictitating membrane (Helper et al., 1974a; Gelatt et al., 1975) or chemi cally induced with phenazopyridine (pyridium), 60-90 mg/day orally for 3-6 weeks (Slatter, 1973) . François et al. (1976) have used rabbits to study KCS. They found it necessary to remove all the accessory lacrimai tissue, including Harder's glands and the nictitating membrane as well as the lacrimai gland proper to induce the disease in rabbits.

Interstitial stromal keratitis, thought to be an immune-mediated phenomenon, has been demonstrated in mice after intravenous and intracutaneous injections of bovine γ-globulin or bovine serum albumin (Kopeloff, 1976) . A simple method of inducing an inflammatory stromal keratitis in rabbits is injection of 0.03 ml of clove oil into the corneal stroma (Leibowitz et al., 1974) . Phlyctenular keratitis, another allergic form of keratitis, has been reported in animal models, but Thygeson et al. (1962) have failed to produce true phlyctenular disease in rabbits and questions whether the previously reported models are valid.

Most of the clinically significant lesions, hereditary or acquired, affecting the corneal endothelium result in visually disabling corneal edema. Spontaneous dystrophies leading to corneal edema are described in the veterinary literature and are reviewed by Dice (1980) . Corneal edema can be induced by in vivo freezing with a metal probe to destroy endothelial cells. The edema may be temporary or permanent depending on the area, temperature, and duration of cold exposure (Chi and Kelman, 1966) . We have found that two successive freezes with a contoured 10-mm brass probe, cooled to -140°C with liquid C0 2 , consis tently produces temporary corneal edema in dogs (Befanis and Peiffer, 1980) . A slightly smaller probe is used for rabbits. The probe is applied two times for 15-60 seconds with time for complete thawing allowed in between.

Corneal edema and scarring are often irreversible. Corneal transplantation is one method of restoring a transparent visual axis, but a number of prosthetic devices, usually fashioned out of methyl methacrylate, have been used experi mentally over the last 30 years and are now being used clinically on a limited basis. Stone and Herbert (1953) reported a two-stage procedure for implanting a plastic window in rabbit corneas. Dohlman and Refojo (1968) have reviewed the previous 15 years experience with plastic corneal implants.

Many of these procedures require the use of a tissue adhesive to hold the plastic prosthesis in place. Cyanoacrylate adhesives are used. Methyl-2cyanoacrylate creates the strongest bond, but is too toxic for use on the cornea. Butyl-2-cyanoacrylate can be used, and the longer chain substituted forms are even less irritating but do not form as strong a bond (Havener, 1978) . Gasset and Kaufman (1968) have used octyl-2-cyanoacrylate for gluing contact lenses to rabbit and monkey corneas with the epithelium removed. Glued-on contact lenses have also been used for treatment of experimental alkali burns in rabbits (Zauberman and Refojo, 1973; Kenyon et al., 1979) .

Conventional contact lenses without adhesive can be used in animal experi ments. Most investigators have found that both hard lenses and silicone lenses are well-tolerated in the rabbit (Thoft and Friend, 1975) . Hard lenses should be specifically fitted to the rabbits, however, and a partial tarsorraphy may be used if necessary to prevent rabbits from expelling lenses (Enrich, 1976) .

Systemic connective tissue disorders may be associated with local ocular signs of diffuse, nodular, or necrotizing scleritis that can involve the cornea as well. Hembry et al. (1979) sensitized rabbits by intradermal ovalbumin plus Freund 's adjuvant followed by injection of ovalbumin at the limbus to produce a necrotiz ing corneoscleritis.

Animal studies of the composition of the aqueous humor, its function, and the processes controlling the dynamic state of its constituents and volume have two main objectives: (1) appreciation of the physiology, biochemistry, and hence metabolism of the tissues of the anterior segment and (2) definition of the mechanisms that control rate of aqueous humor production and outflow and hence intraocular pressure. Definitive information is available for only a limited number of mammalian species, and these data suggest that species variation in aqueous humor composition and dynamics exists. In addition, anatomical charac teristics of the outflow pathways differ between species. Excellent reviews by Cole (1974) and Tripathi (1974) detail these differences, which emphasize that making inferences from research results using nonprimate mammals to man requires appreciation of these species differences.

The maintenance of normal intraocular pressure (IOP) is dependent upon a critial balance of dynamic equilibrium between the processes of aqueous humor production and drainage. Concepts of aqueous humor formation are based largely upon laboratory work with the rabbit. Aqueous humor is produced by the ciliary processes as a result of active transport and passive ultrafiltration processes; approximately one-half of the aqueous is produced by active secretion across the two-layered epithelium. The exact mechanisms of transport have not been de-fined and may demonstrate species differences; active transport of sodium in the presence of a sodium-and potassium-activated adenosine triphosphatase located in the cell membrane of the nonpigmented epithelium is one of the main primary events in the formation of the aqueous fluid (Cole, 1974) . The remaining 50% of aqueous humor is formed by passive processes, including diffusion, dialysis, and ultrafiltration. The composition of aqueous humor is essentially that of a plasma ultrafiltrate but varies between species. Gaasterland et al. (1979) have studied the composition of rhesus monkey aqueous. Differences between aqueous and plasma levels of potassium, magnesium, chloride, and bicarbonate have been demonstrated and suggest species differences in transport mechanisms and/or anterior segment metabolism. A saturable active transport system for ascorbate has been documented in the rabbit (Barany and Langham, 1955) . Transport mechanisms for amino acids demonstrated in the rabbit may not be present in rat, cat, monkey, and dog (Reddy, 1967) . Total volume of the aqueous humor will vary among species with the size of the globe and relative proportions of the anterior segment (Cole, 1974) .

The rate of aqueous humor formation in the species studied, with the exception of the uniquely high-valued cat, is approximately 1.0-2.0 μ,Ι/minute (Cole, 1974) and is dependent upon ciliary artery blood pressure, the pressure in the ciliary body stroma (essentially equal to IOP), and the facility of flow through the ciliary capillary and capillary wall. Because of these pressure gradients, passive aqueous humor production is decreased as IOP increases; this pressure-dependent component or ' 'pseudofacuity ' ' has been demonstrated to account for up to 30% of total facility in monkeys (Bill and Barany, 1966; Brubaker and Kupfer, 1966) . The aqueous humor enters the posterior chamber, flows through the pupil into the anterior chamber due to thermal currents, and exits the globe by passing through the flow holes in the trabecular meshwork and reentering the peripheral venous circulation via thin-walled vascular channels.

Methodology utilized to quantitate aqueous humor production and flow rates involve the measurement of the turnover of a substance within the aqueous introduced by active and/or passive processes from the peripheral vasculature or intraocular injection. The ideal technique does not involve introduction of a needle into the globe, as this undoubtedly disrupts normal homeostatic mechanisms. Man, guinea pig, rabbit, and cat have been studied utilizing fluorescein turnover with slit lamp fluorometry, a variety of isotopes, and other substances. Reasonable correlation among investigators utilizing different tech niques within species has been observed (Cole, 1974) .

The comparative anatomy of the outflow pathways has been reviewed by Calkins (1960) and Tripathi (1974) . In man and the primates a well-defined trabecular meshwork spans the scierai sulcus from the termination of Descemet's membrane to the base of the iris to the scierai spur. Deep to the trabecular meshwork within the sulcus a single large channel, Schlemm's canal, drains the aqueous into the episcleral veins (Fig. 8) .

In nonprimate mammals, the scierai spur and sulcus are absent; the trabecular fibers span the ciliary cleft, a division of the ciliary body into inner and outer leaves, deep to the fibers of the pectinate ligament. These fibers consist of uveal tissue and extend from the termination of Descemet's membrane to the iris base. Aqueous drains into small saculated vessels, the aqueous or trabecular veins, which communicate with an extensive scierai venous plexus (Figs. 9 and 10). Van Buskirk (1979) utilized plastic lumenal castings and scanning electron mi croscopy to study the canine vessels of aqueous drainage and to demonstrate that in this species the exiting aqueous mixes with uveal venous blood.

The mechanisms of aqueous humor outflow through the trabecular meshwork are not completely understood. Passive pressure mechanisms certainly play an important role; increases in episcleral venous pressure result in decreased outflow and increased IOP. Active transport mechanisms for both organic and inorganic anions have been demonstrated. Transmission electron microscopic studies of the endothelium of the scierai venous plexus have revealed giant cytoplasmic vac uoles that are suggestive of transcellular transport mechanisms and indicate that this is the main site of resistance to outflow (Tripathi, 1974) .

Some aqueous humor may exit through the ciliary body, entering the suprachoroidal space and into the choroidal circulation and sclera. This pressureindependent uveoscleral outflow accounts for up to 30-65% of bulk flow in subhuman primates and has been demonstrated in cats, rabbits, and man to be quantitatively less than in two species of monkeys (Bill, 1961 (Bill, , 1965 (Bill, , 1966 Bill and Phillips, 1971; Fowlks and Havener, 1969) . Qualitative uveoscleral routes have been suggested in the dog utilizing dextran-fluorescein studies (Gelati et al., 1979b) .

Because of the radius of curvature of the cornea and the presence of an overlying scierai ledge, light rays from the base of the iris, the angle recess, and the trabecular meshwork undergo total internal reflection, preventing direct visualization of the outflow structures without the use of a contact lens to elimi nate the corneal curve. Two general types of gonioscopic contact lenses are available; indirect lenses, which contain mirrors and allow examination of the angle by reflected light, and direct lenses, through which the angle is observed directly. A magnifying illuminated viewing system, ideally the slit-lamp biomicroscope, is essential for critical evaluation.

Gonioscopic examination of the canine iridocomeal angle was first reported by Troncoso and Castroviejo (1936) , although Nicholas had previously depicted the canine angle by drawings in 1924. Troncoso in 1948 compared the gross and gonioscopic appearance of the angles of the dog, cat, pig, rabbit, rhesus monkey, and man.

The clinical application of gonioscopy in comparative ophthalmology is rela tively recent. Lescure and Amalric (1961) described its use in the dog, and subsequently numerous investigators have stressed the value of the technique in the diagnosis of glaucoma in the dog (Vainsi, 1970; Gelatt and Ladds, 1971; Bedford, 1973 Bedford, , 1977 . Martin (1969) has correlated the microscopic structure and gonioscopic appearance of the normal and abnormal canine iridocorneal angle.

The technique is straightforward and involves topical anesthesia and minimal physical restraint in dog, cat, and rabbit and ketamine sedation in primates (Fig.  11 ). The concave surface of the lens is filled with artificial tears and placed on the corneal surface; the Franklin goniolens with a circumferential flange is re tained by the eyelids and enables the examiner to have both hands free. The Troncoso or Koeppe lens may also be utilized; vacuum lenses and the Swan lens are smaller and thus adaptable to younger and smaller animals (Fig. 12) .

The structures observed during gonioscopic examination of the nonprimate mammal, using the dog as an example, include, from posterior to anterior, the following:

1. The anterior surface and base of the iris 2. The pectinate ligament 3. Deep to the pectinate ligament, the ciliary cleft, and the trabecular meshwork 4. The deep or inner pigmented zone representing the anterior extension of the outer leaf of the ciliary body 5. The outer or superficial pigmented zone which is variable in presence and density and represents melanocytes in the limbus 6. The corneal dome (Fig. 13) Species variations in appearance do exist. The sub-human primates present a gonioscopic appearance of the iridocorneal angle identical to man. In the cat, the pectinate fibers are thin and nonbranching, while in the dog they tend to be deeply pigmented, stout and arbiform. In the rabbit the pectinate fibers are short and broad (Fig. 14a-c) . 

These tests have been used in man to detect suspicious or borderline glaucoma patients as well as to investigate the heredity of open angle glaucoma by stressing the homeostatic mechanisms of the globe. Provocation results in an increase in IOP that can be characterized by extent and duration. The water drinking and corticosteroid provocative tests have been most useful in open angle glaucoma, whereas the mydriatic and dark room tests have been valuable in narrow angle glaucoma (Kolker and Hetherington, 1976) .

The water provocative test in man, rabbits, and subhuman primates (Macaca irus) has been studied with Schiotz and applanation tonometry and in combina tion with tonography and constant pressure perfusion (Swanlijung and Biodi, 1956; Galin et al., 1961 Galin et al., , 1963 McDonald et al., 1961; Galin, 1965; Thorpe and Kolker, 1967; Casey, 1974) . The procedure has been used in rabbits to test the effects of different drugs on pressure-regulating mechanisms (McDonald et al., 1961) .

The test in dogs has defined normal values and demonstrated significant dif ferences in American cocker spaniels and beagles with spontaneous glaucoma (Lovekin, 1971; Gelatt et al., 1976a) (Fig. 15 ).

It is generally accepted that the increase in intraocular pressure after the administration of a substantial volume of water is primarily related to a sudden decrease in the osmolarity of the blood; the related influx of water into the eye is presumed to increase intraocular pressure proportional to the volume of water administered (Galin et al., 1961 Galin, 1965) . Hemodilution may not be solely responsible; in man the increase in IOP in 20% of the patients occurs before the fall in serum osmolarity (Spaeth, 1967) . The subsequent rate of decay of IOP assesses the ability of the outflow system to cope with the increase inflow.

Clinical measurement of the facility of outflow is accomplished by raising IOP by placing a tonometer on the eye and determining the subsequent rate of volume loss and pressure decrease; as resistance to outflow of aqueous humor increases, the pressure changes will decrease. The principle of the test may be traced to the massage effect, whereby pressure on the eye leads to a softening of the globe due to increased outflow. Schiotz indentation tonography employs placement of an electronically recording Schiotz tonometer on the eye; the weight of the tonome ter will indent the cornea, reducing ocular volume and increasing IOP. The instrument is left on the cornea for a 4-minute period. Tables are utilized to derive the rate of aqueous humor outflow based upon the observed changes in IOP over the 4-minute period as recorded by the tonograph (Grant, 1950; Kronfeld 1952 Kronfeld , 1961 Garner, 1965; Drews, 1971; Podos and Becker, 1973) .

In 1950, Grant showed that the coefficient of outflow facility (C) is related to the change in ocular volume (Δν) occurring over the time interval (t), as a result of the difference between the average pressure during tonography (P t av) a n d the IOP prior to placement of the tonometer on the globe (P 0 ): C = Av/t(P tav -P 0 ). The coefficient is expressed in microliters of aqeous humor outflow per minute per millimeters of mercury pressure (Grant, 1950) .

Tonography has certain limitations. The accuracy is dependent upon the accu racy of the tonometer calibration, since Δν, Ptav» **nd PQ are derived from this data. In addition, six physiologic assumptions are made upon which accurate quantitative results are dependent (Grant, 1950) .

1. That there is a constant continuous flow of aqueous humor 2. That the process of tonography does not alter this flow 3. That the process does not alter outflow facility 4. That the process does not change the uveal vascular volume 5. That tonography does in fact measure the outflow of aqueous humor through the trabecular meshwork 6. That the eye exists in a steady state in regards to aqueous humor dynamics during the 4-minute period.

Previous discussions of pseudofacility-the decrease in aqueous humor pro duction that occurs with increased IOP-and uveoscleral outflow demonstrate that assumptions 2 and 5 are not valid. Evidence exists that outflow facility is depen dent upon IOP. In addition, uveal blood volume is probably influenced by the placement of the tonometer on the cornea (Podos and Becker, 1973) . However, tonographic C values determined for the human eye correlated well with values obtained by constant pressure perfusion, aqueous humor fluorescein disappear ance time, and aqueous humor turnover of certain substances (Becker and Costant, 1956; François et al., 1956; Bill and Barany, 1966) .

Additional factors warrant consideration when evaluating tonographic proce dures in species other than man; animal globes differ significantly from the human eye in terms of ocular volume, radius of corneal curvature, vascular dynamics, and tissue characteristics upon which the accuracy of Schiotz identation tonometry or tonography is dependent; Schiotz tonometric conversion tables are inaccurate if applied to the canine eye (Peiffer et al., 1977) . The technique cannot be utilized in animals without pharmacologie restraint, and the effect of the drugs utilized on the steady state of IOP must be taken into account. Helper and his associates (1974b) used xylazine and ketamine sedation and found C values from 0.05 to 0.32 in normal dogs, 0.03 to 0.38 in Basset hounds, and 0.03 to 0.10 in a small number of glaucomatous patients. Gelatt and his associates ( 1977a) observed a combination of acetylpromazine and ketamine to have mini mal effect on the steady-state IOP of the normal canine eye, and demonstrated impairment of outflow in beagles with inherited glaucoma. Peiffer et al. (1976) reported mean tonographic values of 0.21 using this anesthetic combination in normal mongrel dogs (Fig. 16) .

Applantation tonography may prove more accurate and versatile than Schiotz indentation tonography in the animal laboratory. A 2-minute tonography period is adequate, and the effect of anatomic variables is minimized ). 

Invasive laboratory techniques that have been described to measure facility of outflow involve direct measurement of intraocular pressure via a cannula inserted into the globe and one of three techniques: (1) injection of a known volume of fluid and observing the rapid increase and subsequent slow decrease in IOP (pressure decay curves), (2) perfusion of the globe with fluid at a constant rate and observing the related pressure changes (constant rate perfusion), and (3) perfusion at a variable rate necessary to maintain a given IOP (constant pressure perfusion) (Barany and Scotchbrook, 1954; Grant and Trotter, 1955; Macri, 1959; Melton and Hayes, 1959; Armaly, 1960; Melton and Deville, 1960; Grant, 1963; Langham and Eisenlohr, 1963; Barany, 1964; Brubaker, 1975; Wickham etal., 1976) (Fig. 17) .

These invasive techniques eliminate the variables of scierai creep and changes in uveal blood volume and species differences in corneal anatomy. All require methods in which intraocular pressure, fluid volume in the eye, and flow reach steady state during the measurement. Cannulation of the anterior chamber will induce qualitative and quantitative aqueous humor changes. Despite mathemati cal equivalence, the methods are quite different in practice because they differ greatly in the time it takes for the eye to go from one steady state to another. The time required to reach steady state during constant pressure perfusion is less than 5 minutes compared to much longer times for the other techniques. All are based on the assumptions of pressure-independent facility and secretion rate, which have been criticized (Langham, 1959) .

Facility of outflow can be determined in constant pressure perfusion by measuring average flow from an external reservoir into the eye; time period is a compromise between the desire to achieve accuracy by using a long period and to achieve high temporal resolution by using a short one. A 4-minute averaging period is utilized arbitrarily to facilitate comparison to tonographic values. Facil ity may be calculated at any given pressure utilizing the formula C = FIP, where F is the rate of perfusion flow in microliters of mercury per minute, P is the intraocular pressure in millimeters of mercury, and C is facility expressed in microliters of fluid per millimeter of mercury per minute. In an in vivo system, however, this equation does not consider aqueous humor production and thus provides values lower than actual facility. Barany showed that facility can be calculated at two levels of pressure, Ρ λ and P 2 , utilizing the formula C = (F 2 -F\)K?2 ~ P\)· This formula necessitates assumption of a constant episcleral venous pressure and aqueous humor production. If one estimates a value of 10 mm Hg for the former and 1 ul/minute for the latter, similar results between the two equations are obtainable by dividing rate of secretion by the episcleral venous pressure and adding the result (0.1 ul/minute/mmHg) to the values obtained by the equation C = F IP .

Character of the perfusate can influence facility; 0.9% unbuffered saline causes a decline in resistance on prolonged infusion of the anterior chamber. Barany (1964) utilized phosphate-buffered saline with calcium and glucose added, and Brubaker and Kupfer (1966) perfused the heparinized mammalian tissue culture medium to minimize this "washout" factor. Gaasterland et al. (1979) used pooled heterologous aqueous humor to perfuse rhesus monkey eyes in vivo. Melton and DeVille (1960) studied enucleated canine eyes using constant pressure perfusion and found an average C value of 0.28 at 28 mm Hg IOP. Peiffer and his associates (1976) found the facility of outflow in normal dogs anesthetized with sodium pentobarbital to have a mean value of 0.13 ± 0.07 SD which increased as IOP increased. Perfusion values for outflow were less than those determined tonographically (0.21 ± 0.14 SD with acetylpromazineketamine hydrocholoride sedation, 0.15 ± 0.09 SD with pentobarbital anes thesia) or in the enucleated globe. Van Buskirk and Brett (1978a,b) perfused enucleated canine eyes and found a pressure-dependent facility of outflow of 0.26 ± 0.02SD at 5 mm Hg IOP which increased to 0.62 at 40 mm Hg IOP. Outflow increased significantly during perfusion with hyaluronidase-containing solution and with time in the non-hyaluronidase-perfused eyes. Peiffer et al. (1980a) perfused beagles with inherited glaucoma in vivo and found impairment of outflow facility compared to normal dogs (Fig. 18 ). The limitations of tonographic and perfusion techniques to quantitate aqueous humor outflow in animal species must be appreciated; neither is ideal. The refinement and development of more accurate methodology remains a challenge to the investigator. Either technique is certain to provide more relevant informa tion if performed in vivo rather than on enucleated globes.

The IOP is a differential fluid pressure that measures the vector sum of the forces generated by the intraocular fluids acting at the interface between the fluid and the fibrous coats of the globe. The accurate determination of the IOP is difficult because all the techniques utilized to measure it in some way necessitate altering the parmeter from its original value. In the laboratory, the anterior chamber can be cannulated and IOP determined directly by the fluid level of an open-air manometer; this situation, of course, is not applicable to clinical situa tions where a noninvasive technique is required. Any noninvasive technique, however, must ultimately be compared to simultaneous readings from the cannu lated globe.

Quantitative determination of IOP is achieved by one of two types of tonometry. Clinical estimations depend on subjecting the cornea to a force that either indents (impresses) or flattens (applanates) it. Tonometers that indent the cornea are referred to as indentation tonometers, and those that flatten it are referred to as applanation tonometers. The cornea is utilized because other areas, such as the anterior sclera, have a nonuniform thickness and the variability of additional tissues including conjunctiva bulbar fascia and the underlying anterior uvea.

Normal IOP in many of the animal species, notably the nonprimate mammals, appears to be higher and more variable than that observed in persons (Bryan, 1965; Heywood, 1971) . A number of physiologic variables may affect the IOP. These include the nature of the subject, the time of day, and the position of the subject. Intraocular pressure is related to blood pressure, and it has been demon strated that animals that are excited will have higher IOP. Accurate values are obtained in animals that have been handled and previously subjected to the technique. In persons, a diurnal variation of IOP has been observed with the lowest IOP occurring early in the morning. This variation is probably related to changes in endogenous corticosteriod levels. Diurnal variation has been demon strated in the New Zealand white rabbit (Katz et al., 1975) and in beagles with inherited glaucoma, but not in normal dogs (Gelatt et al., 1979a) . In persons significant differences in IOP are observed with the patient in a sitting position as compared to the prone position. In addition, the variable of technique may contribute to the wide range of normal intraocular pressures observed in animals. Sedation and anesthesia, because of associated cardiovascular effects, are likely to affect IOP. This is especially true of the barbiturates. Bito and co-workers (1979) found that ketamine hydrochloride had minimal effect on IOP of rhesus monkeys (Mucaca mulatta) and reported a mean IOP of 14.1 ± 2.1 SD with higher values and a greater diurnal variation in young animals.

Schiotz indentation tonometry estimates IOP by applying a carefully stan dardized instrument on the cornea and measuring the depth of indentation of the cornea by a weighter plunger. The Schiotz tonometer has the advantages of simple construction, reasonable cost, portability, and relative simplicity of tech nique.

In Schiotz tonometry, a force from a small curved solid surface with a spheri cal curvature which indents the cornea will be balanced by a fluid pressure separated from the solid surface by a thin flexible membrane, the cornea in this case, when the applied force or weight of the tonometer equals the resultant force from the fluid pressure measured in a direction parallel to the direction of the applied force times the area of distortion to the membrane. The surface tension of the tear film exerts a small force parallel to the corneal surface.

The instrument consists of a footplate that approximates the radius of curvature of the human cornea, a plunger, a holding bracket, a recording scale, and 5.5, 7.5, 10.0 and occasionally 15.0 gm weights (Fig. 19) . The cornea is in dented with a relatively frictionless weighted plunger; the amount of plunger protruding from the plate depends upon the amount of indentation of the cornea. The tonometer scale is adjusted so that 0.05 mm of the plunger equals one scale unit. Calibration tables are used to derive the actual IOP from the observed tonometer reading. Most accurate estimations of IOP are obtained with the lighter weights within the middle scale ranges.

The technique for Schiotz tonometry is relatively simple (Bryan, 1965; Vainisi, 1970 ). The cornea is anesthetized with a drop of topical anesthesia. While allowing a few seconds for the anesthesia to take effect, check the recording arm on the tonometer by placing it on the solid convex metal surface provided. The tonometer should read 0, indicating that no indentation of the plunger is occur ring and that the plunger surface is flush with the footplate.

With a bit of practice, Schiotz tonometry can be performed without assistance in the dog and cat. Rabbit tonometry is facilitated with an assistant holding the animal in lateral recumbancy. The animal should be relaxed, and care should be taken not to compress the jugular veins. The first, second, and/or third fingers of the left hand or the thumb may be used to simultaneously retract the lower eyelid of the right or left eyes, respectively. The tonometer is grasped between the thumb and first finger of the right hand and the tonometer scale rotated such that it is easily observed by the examiner. The fourth finger of the right hand is rested on the frontal bone to provide stability and retract the upper eyelid. Care must be taken in retracting the lids so that pressure is applied only to the bony orbital rim and not the globe itself. Excessive eyelid retraction may also create abnormal forces on the globe and should be avoided. The footplate is placed on the cornea as central as possible and gentle pressure applied until the holding bracket glides freely around the footplate shaft (Fig.  20) . The scale reading is noted, and the tonometer is removed. The procedure is repeated two more times; each scale readings should be within one full unit to another. If the readings are to the low end of the scale, additional weights may be applied to the tonometer and the procedure repeated to obtain midscale readings. One potential source of error is the position of the tonometer in relationship to the perpendicular; deviation from the perpendicular will result in an overestimation of IOP directly proportional to the degree of deviation. The process of tonometry will induce an increase in aqueous humor outflow and each repeated measure ment may be slightly lower than the previous estimate of IOP.

Following use, the tonometer should be disassembled and cleaned carefully with a pipe cleaner. Free movement of the plunger within the casing is essential for proper function, as is smooth working of the lever system and the recording arm.

The process of placing the tonometer on the eye will increase IOP between 5 to 20 mm HG; the Schiotz tonometric conversion tables enable the clinician to correlate the tonometer reading with the IOP prior to the placement of the Fig. 20 . Use of the Schiotz tonometer in a dog. The instrument is applied in a perpendicular fashion to the central cornea. Its use is limited to larger primates, dogs, cats, and rabbits, and calibration tables must be devised for each species for maximally accurate determination of intraocular pressure. instrument on the cornea. Because of species differences in corneal curvature, ocular rigidity, and tissue characteristics, the use of a human conversion table will result in inaccurate estimation of IOP. Conversion tables have been de veloped for rabbit (Best et al., 1970) and canine (Peiffer et al., 1977) globes.

Limitations of the Schiotz indentation technique depend upon the species, the clinician, and the instrument. Use in animals is limited to those species with relatively large corneas and animals that can be adequately restrained and posi tioned. It is most useful in larger primates, dog, cat, and rabbit. Ocular rigidity, or the ability of the cornea and sclera to stretch, will vary with age and from species to species and animal to animal (Best et al., 1970; Peiffer et al., 1978b) . Ocular rigidity also increases as the tonometer is placed closer to the limbus. Increases in ocular rigidity provide Schiotz recordings that are higher than actual IOP; with increased ocular rigidity there is less indentation by the Schiotz tonometer, creating a false impression of increased IOP.

Applanation tonometry is based upon the principles of fluid pressures; pressure equals force divided by the area. A force from a plane solid surface applied to a fluid contained by a thin membranous surface will be balanced when the area of contact times the pressure of the fluid equals the force applied by the plane solid surface. This is known as the Imbert-Fick law. Simply stated, in applanation tonometry one may either measure the force necessary to flatten a constant area of the corneal surface or measure the area of cornea flattened by the constant applied force.

Electronic applanation tonometers, notably the Mackay-Marg, are the most versatile and accurate in a wide variety of species. Probe tips are smaller than indentation tonometers, a minimum of intraocular fluid is displaced, IOP is not significantly increased by the procedure, and the technique is independent of ocular rigidity and corneal curvature. It is applicable to smaller laboratory species, and its use has been reported in the chinchilla (Peiffer and Johnson, 1980 

Experimental animal models of glaucoma have been developed to study the effects of elevated IOP on other ocular tissues, to determine the efficacy of medical and/or surgical treatment in reducing IOP, and to define mechanisms of the glaucomatous process itself. Spontaneous animal models of glaucoma have been utilized for the above purposes in addition to investigation to determine their similarities to the disease in man.

Historically, experimental glaucoma has been produced in rabbits by the injec tion of 1% kaolin into the anterior chamber to obstruct the outflow channels, with IOP reaching 50-70 mm Hg within 14 days (Voronina, 1954) . Skotnicki (1957) enclosed rabbit globes with cotton threads to induce glaucoma with resultant optic disc cupping. Flocks and his associates (1959) utilized a similar technique and rubber bands; while initial increases in IOP reached 70-100 mm Hg, pres sures dropped to 35-50 mm Hg within 48 hours. One-third of the eyes developed panophthalmitis and only 12% showed cupping of the optic disc. Kupfer (1962) threaded polyethylene tubing into rabbit iridocorneal angles; IOP increase was observed within 24 hours and remained elevated for 6 months. Loss of retinal ganglion cells and cupping of the optic disc occurred. Samis (1962) and Kazdan (1964) produced glaucoma in rabbits by the intraocular injection of methylcellulose.

Huggert (1957) blocked the outflow of aqueous humor in rabbits using three different techniques but failed to cause significant increases in IOP. De Carvalho (1962) injected cotton fragments into rabbit anterior chambers, which elevated IOP to 38-40 mm Hg; in those animals in which the glaucoma persisted longer than 30 days, retinal and optic disc pathology was observed. Injection of talcum powder or dental cement (Kalvin et al., 1966b) produced elevated IOP in monkeys.

The injection of the enzyme α-chymotrypsin into the globe will produce a variable increase in IOP that may or may not be prolonged; the enzyme particles dissolve the zonules of the lens, the fragments of which collect in and obstruct the trabecular mesh work. The technique has been utilized in the rhesus and owl monkey to study optic nerve and ocular vascular changes (Kalvin et al., 1966a; Zimmerman et al., 1967; Lambert et al., 1968; Lesseil and Kuwabara, 1969) . The enzyme may have direct toxic effects on the retina which must be considered in studying the morphologic and functional effects of elevated IOP on this tissue.

Cyclocryotherapy will cause an acute elevation of IOP in rhesus monkeys (Minckler and Tso, 1976) , and the technique has been utilized to study axoplasmic transport of the axons of the ganglion cells and optic nerve (Minckler et al., 1976) ; the same parameters have been studied by controlled elevation of IOP by cannulation and perfusion (Minckler et al., 1977) .

The use of repeated circumferential argon laser photocoagulation of the iridocorneal angle as described by Gaasterland and Kupfer (1964) results in a predictable sustained elevation of IOP, marked reduction of outflow facility, and progressive cupping of the optic nerve head. The technique has the advantages of being noninvasive and associated with minimal intraocular inflammation and unrelated pathology.

In chickens raised under continuous light exposure from the day of birth onward, IOP rises related to increased resistance to aqueous humor outflow that is detectable as early as 2 weeks of age (Lauber et al., 1972) . Morphologic studies of the trabecular meshwork of affected animals reveals an increase of intercellular collagen and elastic trabecular tissue with resultant densification of the meshwork. There was an absence of endothelial vacuoles, pores, and microchannels observed by transmission electron microscopy in normal birds (Tripathi and Tripathi, 1974a,b; Rohen, 1978) .

While sporadic spontaneous cases of animal glaucoma have been described in a variety of species, only two models are reliably producable by controlled breedings, inherited glaucoma in the rabbit and beagle. These two models will be discussed briefly emphasizing methodologies utilized to define the disease pro cesses.

Buphthalmia (hydrophthalmus, congenital infantile glaucoma) in rabbits is due to an autosomal recessive gene with incomplete prentrance (Hanna et al., 1962) . Histologie abnormalities of the eye are observed at birth; elevated IOP and buphthalmus may be observed as early as 2 to 3 weeks of age in some animals with progressive clouding and flattening of the cornea; ectasia of the globe, particularly in the sclero-cornea region; deepening of the anterior chamber with detachment and fragmentation of the iris membrane; partial atrophy of the ciliary body; and glaucomatous excavation of the optic disk (Hanna et al., 1962) . The primary defect responsible for the development of glaucoma probably in volves impairment of facility of outflow. It has been postulated that the glaucoma may be part of a primary systemic disorder (Hanna et al., 1962) . The gross ocular enlargement which characterizes buphthalmia is accompanied by fibrosis of the filtering angle (McMaster and Macri, 1967) , a decrease in facility of aqueous humor outflow (McMaster, 1960; Kolker et al., 1963) , and a hyposecretion of aqueous humor (Smith, 1944; Auricchio and Wistrand, 1959; McMaster and Macri, 1967; Greaves and Perkins, 1951) . Anatomic studies have em phasized changes at the angle and have postulated that they constitute at least one site of obstruction to aqueous humor outflow. Hanna et al. (1962) demonstrate an absence of the space of Fontana, the iris pillars, and either total absence or a rudimentary development of the trabecular canals and intrascleral channels. McMaster and Macri (1967) observed that the obstruction to outflow lies be tween the trabeculum and the episcleral veins. The angle according to Hanna et al. (1962) is open in the adult buphthalmia but appears closed in the newborn. The combination of hyposecretion and reduced outflow explains why buphthalmic rabbits may have a normal IOP. Rabbits that are genetically buphthalmic but phenotypically normal appear to have an IOP approximately 5 mm Hg higher than normal, and rabbits with clinical signs of buphthalmia may have IOP as high as 50 mm Hg greater. The IOP tends to increase a few weeks prior to observable distention of the globe. Elevated IOP will subsequently return to normal levels. The cause and effect relationship of the striking inverse correlation of aqueous humor ascorbate concentration with the severity of the buphthalmia is not clear; a marked drop in ascorbate levels is present in early preclinical stages of buphthalmia (Lam et al., 1976; Fox et al., 1977; Lee et al., 1978) . The actual sequence of events, involving alterations in IOP, outflow facility, ascorbate concentration of the aqueous humor, and clinical signs are not clearly defined. The rate of progression has been shown to vary with the genetic background. Sheppard et al. (1967) reported that the corneal endothelial cells in a flat preparation from a buphthalmic rabbit were enlarged and of variable size. He postulated that the cells expand to cover the increased corneal area. Van Horn et al. (1977) utilized scanning electron microscopy to confirm this report, but also indicated that there is a loss of endothelial cells in the disease as well. Gelati (1972) published a report on a familial glaucoma in the beagle; pathologic increases in IOP occurred from 9 months to 4 years of age, and affected dogs demonstrated open iridocorneal angles upon gonioscopy.

Additional observations were summarized in subsequent papers (Gelatt et al., 1976b (Gelatt et al., , 1977c . Controlled breedings suggested an autosomal recessive mode of inheritance. The glaucomatous process was divided into early (6 to 21 months of age), moderate (13 to 30 months of age), and advanced (31 months of age and greater) and was evaluated clinically by tonometry, gonioscopy, and anterior and posterior segment examination. In early glaucoma, the iridocorneal angle was open and without anomalies, and IOP was elevated. With moderate glaucoma, variable optic disc atrophy, elongation of the ciliary processes, and focal disin sertion of the zonules from the lens were seen in addition to elevated IOP and open iridocorneal angles. Advanced glaucoma was characterized by increased IOP, narrow to closed iridocorneal angles, lens dislocation, optic atrophy, and progression to phthisis bulbus. Scanning electron microscopy was performed in a small number of dogs and correlated with the gonioscopy observations. Affected dogs responded positively to water provocation (Gelatt et al., 1976a) and dem onstrated decreased facility of aqueous humor outflow at all stages of the glaucomatous process when compared to normal dogs, both tonographically (Gelatt et al., 1977c) and by constant pressure perfusion (Peiffer et al., 1980b) . Responses to topical autonomie agents Whitley et al., 1980) and carbonic anhydrase inhibitors (Gelatt et al., 1978) have been studied, and histochemical studies of adrenergic and cholinergic receptor sites have been performed (Gwin et al., 1979a) . Peiffer and Gelatt (1980) described gross and light microscopic observations of the iridocorneal angle; data sup ported gonioscopic observations that the disease appeared to be an open angle glaucoma, with secondary pathology of the angle structures noted.

Inflammation of the uveal tract is a common and challenging enigmatic clini cal entity that encompasses the variables of inciting stimulus, host response, and associated alteration of ocular structure. The infections uveidites are discussed elsewhere; this section will review noninfectious uveal inflammation (primarily immune-mediated in nature) in animal models, which have proved useful in defining the etiopathogenesis of disease processes; enhancing of our understand ing of the immune response in general and specifically in regards to the eye, a rather unique organ, immunologically speaking; and investigating pharmaco logie mediation of the disease processes.

Limitations of these models should be noted: inflammation of the uveal tract in the animal model, regardless of species, tends to be an acute, self-limited dis ease. In addition to antigens derived from ocular tissue, complete Freund 's adjuvant must be given to induce experimental allergic uveitis (EAU). Models of chronic uveitis have been particularly difficult to develop and require repeated immunizations with the inciting antigen. Even in such models, the inflammation is usually restricted to the anterior segment, and there are minimal retrograde changes compared to chronic human uveitis.

Several studies have shown that the eye is not an immunological privileged site. Allogeneic tissue implanted into the anterior chamber triggers an im munologie reaction. Franklin and Pendergrast (1972) observed that allogeneic thyroidal implants were rejected by the rabbit eye in a histologie manner and chronologic sequence similar to that for implants to other parts of the body. Kaplan and his associates (1975; Kaplan and Streilein, 1977) have reported that although the rejection of allogeneic implants placed in the anterior chamber of inbred rats is delayed, the immunologie recog nition via the afferent limb of the immune response is intact. Since ocular implant vascularization is coincident with that of implants in other areas of the body, immunologie suppression is probably responsible for the delay in the transplanta tion rejection observed (Raju and Grogan, 1969) . In studies of anterior chamber immunization, Kaplan and Streilein (1978) have demonstrated that the allogeneic antigens present in the anterior chamber are processed immunologically by the spleen via a vascular route, since afferent lymphatic channels do not drain the anterior chamber. They suggest that immune deviation occurs as a result of splenic suppressor factors which delay anterior chamber graft rejection. Work by Vessela and his co-workers (1978) in the rat has supported this theory. Their model suggests that antigen processing and recognition occur, but that effector response is delayed. A possible delay mechanism could be low dose antigen sensitization with tolerance because of the lack of lymphatics, but a more likely explanation would be the presence of suppressor factors including nonspecific serologie blocking factors, specific antigen-antibody blocking complexes, sup pressor macrophages, or suppressor T cells.

The majority of experimental animal investigations have been performed using models of sympathetic ophthalmitis or lens-induced uveitis. The role of immunologie mechanisms in the sympathetic ophthalmitis models has been fairly well characterized. The vast majority of this literature does not distinguish whether the immunologie response is primary or whether it is merely an epiphenomenon resulting from an alteration in the ocular antigens produced by another, nonimmunologic, insult.

The physical location and biochemical characterization of antigens responsible for the induction of sympathetic ophthalmitis models of EAU have been studied extensively. Earlier studies dealt with the identification of uveal antigens; bilat eral uveitis can be produced by homologous immunization (Aronson, 1968) . The inflammation tends to be nongranulomatous; however, occasional reports (Col lins, 1949 (Col lins, , 1953 describe lesions histopathologically similar to the granulomatous process seen in human sympathetic ophthalmitis. Vannas et al. (1960) pro duced experimental uveitis in rabbits by enucleating one eye and implanting it in the peritoneal cavity. Aronson and co-workers (1963a,b,c) demonstrated that uveal preparations from albino guinea pigs are antigenic, suggesting that melanin is not a vital antigenic component of the disease process. A number of workers have demonstrated that antigens from the rod outer segments and retinal pigment epithelium can induce experimental allergic uveitis in primates, guinea pigs, and rabbits (Wong et al., 1975; Meyers and Pettit, 1975; Hempel et al., 1976) . While previous workers had demonstrated that crude extracts of uveal tissue can also produce EAU, Faure et al. (1977) have suggested that the activity observed with these uveal preparations was probably due to contamination by retinal antigens, and most investigators accept their hypothesis that retinal antigens are more important than uveal antigens in EAU. Wacker (1973) and Wacker and his associates (1977) have demonstrated that antigens present in the photoreceptor and retinal pigment epithelial layers can elicit the development of chorioretinitis and have partially characterized the responsible antigens. There is a soluble antigen (S antigen) located throughout the photoreceptor layer. This S antigen is most active in the production of EAU; animals with the disease often develop delayed hypersensitivity responses to it. The S antigen appears biochemically similar to a protein subunit of retinolbinding lipoglycoprotein present throughout the photoreceptor layer. S antigen is apparently tissue-specific; crude bovine S antigen was relatively ineffective in the induction of guinea pig EAU. A more purified preparation, however, resulted in a histopathologic lesion, with cellular infiltration in the anterior uvea and destruction of the photoreceptor layer. Thus a purified extract of zenogeneic antigenic material had characteristics similar to those of the tissue-specific allogeneic extract. The S antigen is probably not rhodopsin, since a number of physical characteristics, including solubility, molecular weight, amino acid se quence, specific location, and the absorption spectrum, are different. The authors also identified a particulate antigen (P antigen), located in the rod outer seg ments, that does not elicit a delayed hypersensitivity response and has a lower EAU induction rate. It does elicit the development of antibodies, even in the absence of disease.

In most models of experimental uveitis, cell-mediated, rather than humoral, immune responses are most important in the pathophysiology of these diseases. Delayed hypersensitivity reactions to inciting antigens have correlated with the onset and course of disease in EAU; passive transfer experiments using lymphoid cells have been conducted; cellular mediators (lymphokines) have produced var iations of EAU; and lymphoid cell depletion experiments have been performed. Friedlander and his associates (1974) defined a predominantly eosinphilic re sponse to delayed hypersensitivity in the guinea pig. Meyers and Pettit (1975) demonstrated in the guinea pig both cutaneous de layed hypersensitivity and macrophage migration inhibition reactions toward rod outer segments and retinal pigment epithelial antigens in EAU. In their study, cellular immunity appeared to correlate with clinical disease; specific antibody to the inciting antigen was absent in a number of animals that developed EAU, and there was no correlation with humoral immunity. In a similar study, Wacker and Lipton (1971) demonstrated that delayed hypersensitivity toward the inciting antigen is usually present when EAU is induced; in a number of animals who developed uveitis, no antibody response was detected.

Experimental uveitis can be passively tranferred to normal animals with lym phoid cells but not with serum from animals with EAU (Arnonson and McMaster, 1971 ). Chandler and his co-workers (1973) demonstrated that an ocular inflammation resembling uveitis can be induced with lymphokines produced by sensitized lymphocytes, and other investigators have demonstrated that experi mental uveitis can be abrogated using anti-lymphocyte serum (Bürde et ai, 1974) .

In a mouse viral model the induction and maintenance of lymphocytic choriomeningitis (LCM) virus-induced uveitis is dependent on the thymusderived lymphocytes . Immune spleen cells adaptively trans ferred to immunosuppressed animals did not receive immune lymphoid cells, no uveitis was produced; if the immune spleen cells were treated with anti-theta serum to eliminate the T cells, the uveitis was also aborted.

While cell-mediated immunologie alteration appears to be responsible for the majority of experimental allergic uveitis models, in experimental lens-induced granulatomatous uveitis (ELGU) humoral immunity is important. Needling of the lens will provoke a uveitis in rabbits provided they also receive an intramus cular injection of Freund's adjuvant (Müller, 1952) . Homologous lens antigens injected into the eye have minimal effect in the absence of adjuvant (Müller, 1952; Goodner, 1964) , and even animals previously sensitized by repeated injec tions of lens material into the footpad fail to mount more than a limited uveitis when challenged with an intravitreal injection of either homologous or heterologous lens antigens (Selzer et al., 1966) . Findings such as these prompted the search for a naturally occurring adjuvant which might account for the spon taneous disease; Halbert et al. (1957a,b) were able to show that streptococcal extracts are able to potentiate lens reactions, although to a lesser degree com pared to Freund's adjuvant, while Burky (1934) had already demonstrated a similar effect in rabbits with staphylococcal extracts. Clinical LGU, however, is not associated with bacterial infection.

As lens-induced uveitis is essentially a feature of old and cataractous lenses containing a high proportion of insoluble material, Behrens and Manski (1973a) focused attention on the possible adjuvant effect of albuminoid lens protein.

They found that a single injection of albuminoid into the vitreous of inbred rats produced after about 5 days a uveitis characterized by an initial neutrophil and macrophage response, whereas a similar injection of crystallins was essentially without effect; this suggested that albuminoid may have an effect similar to Freund's adjuvant. Other experiments in rats previously sensitized with whole lens preparations showed that intraocular challenge with albuminoid evokes a cellular response consisting initially of neutrophils followed by macrophages whereas crystallins, particularly a-crystallin, give rise to round cell exudation (Behrens and Manski, 1973b) . These findings suggest that the soluble antigens are responsible for humoral antibody formation and that insoluble albuminoid accounts for cell-mediated responses. Müller (1963) has drawn attention to the enhancing effect of previous sensitization on the uveal response to lens proteins, having found that injection of homologous lens tissue in the presence of Freund's adjuvant some time before needling the lens gives rise to a uveitis of marked proportions. Marak and his associates (1976) have demonstrated that ELGU can be transferred with serum. Immunofluorescent studies of traumatized lenses of sensitized animals have demonstrated the presence of IgG and complement, components of an immune complex-mediated reaction. Depletion of the third component of complement using cobra venom factor or anti-leukocyte serum to inactivate polymorphonuclear lymphocytes (PMNs) decreased the incidence of ELGU. While it is most likely that ELGU is an immune complex disease, immune complexes in the serum or aqueous of experimental animals have not been demonstrated. Carmichael et al. (1974) reproduced the uveitis that accompanied infectious canine hepatitis adenovirus infection or vaccination demonstrating an immune complex disease resulting form soluble virus-antibody complexes and the asso ciated PMN cell response.

There is significant variance in the incidence of experimental allergic uveitis produced in different species of animals. Genetic influences can markedly alter immune responses; in congenic animals which differ only in the region of the chromosome containing immune response (Ir) genes, marked differences in the incidence of autoimmune disease, malignancy, and infections have been noted, and it is possible that genetically determined differences in immunologie reactiv ity may be important in the development of uveitis in both humans and animals. There is a paucity of data using experimental animal models to determine the importance of immune response genes in the development of uveitis. In guinea pigs, the incidence of uveitis differs markedly between different strains. While it is relatively easy to induce uveitis in the Hartley or NIH strains, it is slightly more difficult to induce it in strain 13 and almost impossible to induce it in strain 2 (McMaster et al., 1976) . Mice that have well-characterized histocompatibility and immune response gene systems are a potential model to further study this phenomenon. (Silverstein, 1964; Hall and O'Connor, 1970) . Is there a specific immunologie reaction by these cells toward uveal antigens that is important in the production of human endogenous uveitis, or is the reactivity observed merely as an epiphenomenon? An equally feasible mechanism for the prolongation of an ocular inflammatory response could be the structural alteration induced by a nonspecific immunologie reaction. In the rabbit the development of systemic immune complex disease, with nonocular antigens, results in a change in ocular vascular permeability (Gamble et al., 1970 Howes and McKay, 1975) . It is conceivable that, once an animal receives this type of insult to its vascular system, the structural alteration of the ocular tissue is such that a chronic uveitis either develops or continues despite the lack of specific immune reactivity toward ocular antigens.

The structure, composition, and physiology of the lens has been studied in a wide variety of animal species. Investigators in specific areas of lens research, however, have tended to concentrate on one type of animal model. Thus, much of what is known about the embryology of the lens is derived from research with chick embryos. Likewise, the characterization of lens proteins is based largely on research with bovine lenses, and the physiology and metabolism of the lens has been studied mainly in rabbits and rats. Some caution must be exercised, there fore, in extrapolating data from one animal model to another since significant differences undoubtedly exist. The chick lens, for instance, has a very different protein composition than that of the mammalian lens (Rabaey, 1962) .

There are a number of important differences between the lens of all the com monly studied laboratory animals and the lens of humans and other higher pri mates as have been pointed out by Van Heyningen (1976) . The rat, rabbit, and bovine lens all demonstrate minimal growth during adulthood, whereas the human lens continues to grow throughout life. Also, the human lens tends to maintain its water content at about 65%, while lower animals have a decrease in water content during old age. The composition of the primate lens is different from all others in that it contains a group of tryptophan derivatives, hydroxykynurinines, which are highly absorbant in the ultraviolet range, and, fi nally, only the higher primates appear to have the ability to change the shape of their lens in accommodation.

In addition to the higher vertebrates, the amphibians also deserve mention for their contribution to lens research. Newts and salamanders have long been recog nized for their ability to regenerate a lens from the pigmented epithelium of the iris (Reyer, 1954) . Toads, on the other hand, demonstrate a similar ability to regenerate a lens from cells derived from the cornea. Some of the techniques involved in this type of study are discussed by Stone (1965) . More recently Eguchi and Okada (1973) have caused renewed interest in the relationship be tween pigmented epithelium and lens by demonstrating lenslike structures in cultures of retinal pigment epithelium from chick embryos.

The dry weight of the lens is comprised almost entirely of proteins, the trans parency of which is the sine qua non of lens function. Understandably, then, a large part of lens research has been aimed at analyzing these proteins. The techniques used include the complete armamentarium of sophisticated tools available to the protein chemist. As previously mentioned, the bovine lens, because of its large size and availability, has been most extensively used, al though the rabbit has also been studied in some depth.

Mammalian lenses consist of three major classes of soluble proteins, known as α-, ß, and γ-crystallins, and an insoluble fraction, predominantly albuminoid, most likely derived from a-crystallin. The separation of lens proteins can be achieved in a variety of ways depending on the objectives of the experiment. Most recent studies, however, begin by separating the lens crystallins into major classes by gel filtration chromatography. Specifically, Sephadex G200, Ultragel AC A 34, and Biogel P300 all yield four major protein peaks corresponding to a-, ßn-, ßh~y and γ-crystallin (Bloemendal, 1977) . Sephacryl S-200 has also been used to separate the /3-crystallins into four subclasses (Mostafapour and Reddy, 1978) . Further separation into subclasses is often done utilizing polycrylamide electrophoresis, and still more refined separations can be achieved using twodimensional electrophoresis and immunochemical techniques.

An important concept of lens composition which has been elicited by the use of immunochemical identification of proteins is that of organ specificity, as opposed to the more usual rule of species specificity. Immunoelectrophoresis can be used to demonstrate cross-reactions between lens proteins of species which are widely separated on the phylogenetic scale. In general, antibodies to the lens of one species will show extensive cross-reactivity to lens proteins of species lower on the evolutionary scale, indicating that these antigens have been carried over as evolution has progressed. This technique has been useful in defining some basic evolutionary relationships. Immunoelectrophoresis and other modern methods for analysis of lens proteins are discussed by Kuck (1970) .

More recently a technique for radioimmunoassay of a-and γ-crystillin which is sensitive to very minute amounts of protein has been described (Russell et al., 1978) .

Systems for in vitro study of the intact lens may be of the open type, where the lens is continually perfused with fresh media, or the closed system in which there is no exchange or only intermittent exchange of culture media. Very elaborate open systems have been designed for accurately measuring such functions as metabolite production and oxygen consumption by the lens (Schwartz, 1960a ,b, Sippel, 1962 . Although the open systems theoretically approximate the physiologic state more closely, closed systems similar to the one described by Merriam and Kinsey (1950) continue to be much more widely used because of simplicity and the potential for maintaining several lenses simultaneously. Since 1950 a long series of articles has been published by Kinsey et al. describing a variety of in vitro studies primarily using closed culture systems. These articles may be traced by referring to a recent addition to the series (Kinsey and Hightower, 1978) . If measuring exchange of materials between lens and culture media is not an important part of the experimental design, a compromise between open and closed system has been described by Bito and Harding (1965) which involves culturing lenses in dialysis bags with intermittent changes of the outer media.

There is an overwhelming amount of literature describing agents capable of inducing experimental cataracts in animals and the subsequent chemical and metabolic effects of those agents. An attempt will be made here to review some of the most important and interesting methods of producing experimental cataracts. For a more complete list of experimental cataractogenic agents the reader is referred to reviews by Van Heyningen (1969) and Kuck (1970 Kuck ( , 1977 . Rabbits and rat have been the most popular animals for this type of research. It is interesting that despite the vast amount of information accumulated on the mechanisms of cataractogenesis, the etiopathogenesis of senile cataracts so com mon in man remains undefined.

A variety of physical insults ranging from the relatively gross technique of sticking a needle through the lens capsule to bombarding the lens with a neutron beam will induce cataracts in animals. By far the most widely studied types of cataract under this category is that produced by ionizing radiation. Radiation cataracts have been studied in many different laboratory animals, most of which make satisfactory models with the exception of the bird which seems to be resistant to radiation cataract (Pirie, 1961) . The lenses of younger animals are generally more susceptible to radiation, and the periphery of the lens which contains the actively dividing cells is much more sensitive than the nucleus (Pirie and Flanders, 1957) (Fig. 21 ). X rays, y rays, and neutrons are all potent cataractogens. Beta radiation in sufficient doses can also be used to cause cataracts (von Sallmann et al., 1953) . Much of the early work on ocular effects of ionizing radiation is reviewed by Duke-Elder (1954) .

Because of the recent boom in microwave technology, cataracts caused by this type of radiant energy have received increased attention. The cataractogenic potential for low doses of microwave radiation is probably minimal, however. Hirsch et al. (1977) and Appleton et al. (1975) found that lethal doses of microwaves were often required to produce cataract in rabbits.

Ultraviolet radiation, although not ordinarily thought of as cataractogenic, can cause cataracts experimentally in animals when a photosensitizing agent is ad ministered simultaneously (see toxic cataracts), which illustrates the important concept that two or more cataractogenic factors may act synergistically to pro duce lens opacities (Hockwin and Koch, 1975) .

Diets consisting of more than 25% galactose will consistently produce cataracts in young, rats. This type of experimental cataract has obvious clinical relevance to galactosemia in humans and the secondary cataract which often develops in this disease. The work of Kinoshita et al. (1962) and others in explaining the mechanism of this cataract have demonstrated that this model is also applicable to the study of diabetic cataracts. Effects of cataractogenic sugars (galactose, xylose, and glucose) have also been studied in vitro using rabbit lenses (Chylack and Kinoshita, 1969) .

Diabetic cataracts may also be studied more directly by surgical or chemical induction of diabetes in laboratory animals. Patterson (1953) has used alloxan, 40 mg/kg in a single intravenous dose, to induce diabetes in rats but other tech niques have also been effective including 95% pancreatectomy and intravenous dehydroascorbate (Patterson, 1956) , and intravenous streptozotocin in some spe cies (White and Çinottim, 1972) .

A large number of drugs and toxins have been demonstrated to cause cataracts in both man and animals. Toxic cataracts are reviewed in depth by Kuck (1977) . Many of these agents have been of particular interest because of their therapeutic use in humans. Metabolic inhibitors, not surprisingly, frequently cause cataracts. One such inhibitor, iodoacetate, has been shown to produce cataracts in rabbit lenses in vivo, in vitro, and, interestingly, by direct injection into the lens (Zeller and Shoch, 1961) . Dinitrophenol, a metabolic inhibitor used for weight loss in humans until a high incidence of cataracts was recognized, will also induce cataracts in the chicken (Buschke, 1947) . Sodium cyanate, used experimentally in sickle cell anemia, has recently been shown to cause cataracts in the beagle (Kern et al., 1977) .

Cytotoxin agents are another group of drugs associated with cataracts. Busulfan and triethylene melamine cataracts have been studied in the rat as has dibromomannitol Grimes, 1966, 1970) . Recently bleomycin has been added to the list of cytotoxins with cataractogenic potential, causing cataracts in 3 to 5-day-old rats injected intraperitoneally with a dosage of 75-100 gm/lOgm (Weill et al., 1979) .

Drugs which cause cataracts through more subtle effects on lens metabolism include corticosteroids, anticholinesterose inhibitors, and a number of drugs which interfere with lipid metabolism. Steroid cataracts, although relatively common in humans, have been difficult to reproduce in animals, but Tarkkanen et al. (1966) and Wood et al. (1967) have reported success in rabbits using subconjunctival and topical steroid preparations. Anticholinesterase cataracts have also proved challenging to reproduce in animals, with the monkey being the only animal model available at present (Kaufman et al., 1977) . Triparanol, which blocks cholesterol synthesis, and chloroquine and chlorphentermine, which also affect lipid metabolism, have all been shown to produce cataracts. A reversible cataract is seen in rats receiving a diet containing 0.05% triparanol; the cataract could not be reproduced in rabbits (Harris and Gruber, 1972 ). An un usual anterior polar cataract is produced with chloraquine and chlorphentermine (Drenckhahn, 1978) .

A group of drugs with the potential to produce cataracts by photosensitizing the lens to ultraviolet light has generated considerable attention. Chlorpromazine and methozyproralen have been used in rats to study this effect (Howard et al., 1969; Jose and Yielding, 1978) .

Of the nontherapeutic agents known to cause cataract, naphthalene has been studied most thoroughly, and lens opacities have been induced in the rabbit, rat, and dog. The mechanism of this type of cataract has been studied extensively by Van Heyningen and Pirie (1967) . Naphthalene can be used to create congential cataracts in rabbits (Kuck, 1970) . Toxic congential cataracts have also been seen in mice secondary to corticosteroids (Ragoyski and Trzcinska-Dabrowska, 1969) .

Congenital cataracts can be due to infections as well as toxic and metabolic insults in vitro. Although no animal model of rubella cataract is available for study, three models of viral-induced cataracts in animals have been reported. The Enders strain of mumps virus, injected into the chick blastoderm prior to dif ferentiation of the lens pläcode, has been reported to cause cataracts in the survivors (Robertson et al., 1965) . Hanna et al. (1968) showed that subviral particles of St. Louis encephalitis virus injected intracerebrally into 4-day-old rats would cause cataracts in survivors. Finally, a poorly categorized infectious agent believed to be a type of slow virus, the suckling mouse cataract agent (SMCA), produces cataracts when injected intracerebrally in mice less than 24-hours old (Olmsted et al., 1966) . SMC A has also been studied in vitro by infecting cultured rabbit lenses (Fabiyi et al., 1971) .

Cataracts due to amino acid deficiencies can cause cataracts in rats (Hall et al., 1948) . The most reproducable form of this type of cataract is that due to tryptophan deficiency which can be demonstrated in guinea pigs by feeding a diet containing less than 0.1% tryptophan (von Sallmann et al., 1959) .

Despite the skyrocketing proliferation of intraocular lenses for use in humans following cataract extraction, very little animal experimentation has been done in *ff:##p1 s *" this area. Animal selection should consider size and shape of the anterior chamber, pupil, and lens as well as species-related reactivity to surgery and lens implantation. In general, nonprimate mammals respond to intraocular manipula tions with increased exudation and inflammation compared to primates and hu mans. Sampson (1956) implanted Ridley-type lenses in nine dogs undergoing cataract extraction. Schillinger et al. (1958) reported disappointing results with crude lenses implanted in rabbit and dog eyes. Eifrig and Doughman (1977) used modern iris fixation lenses in rabbits (Fig. 22) . Rabbits were prone to develop corneal edema after lens extraction with or without lens implantation, but edema was prolonged in eyes receiving lenses. Cats have been used for implantation of lenses with slightly less technical difficulty than rabbits, but their vertical slit pupil is not ideal. Because of their large anterior chamber, cats are not suitable for implantation of standard-sized anterior chamber lenses.

Degeneration of the retina has been a leading cause of visual impairment and partial or complete blindness in both man and animals. Many causal factors have been defined and may be categorized as either genetic, chemical, developmen tal, or environmental. The latter category has received the majority of experi mental attention, being primarily concerned with the induction of retrograde changes by chemicals, radiant energy, physical trauma, nutritional deficiency, and viral infections.

The multiplicity of genetic models will be dealt with briefly because rather than specific techniques they are entities within themselves. The Wag/Rij rat is described by Lai (1977) as the "retinitis pigmentosa model," featuring a slowly progressive photoreceptor degeneration thought to be an autosomal dominant trait. At present the genetic model for human retinal dystrophy is the RCA (tan-hooded) or 'Dystrophie rat" (Herron, 1977) . In this animal rod photorecep tor degeneration occurs because the retinal pigment epithelial (RPE) cells do not phagocytize shed rod outer segment photopigment discs. The (rd) mutant mouse has proved to be helpful to evaluate the effects of photoreceptor degeneration on the bipolar cell terminal synapse (Blanks et al., 1974) . Feline central retinal degeneration involving a diffuse atrophy of retinal cones has been described and is a potential model for human macular degeneration (Bellhorn and Fischer, 1970) (Fig. 23) ; the genetics have not been defined (Fischer, 1974) . A tool for studying retinal degeneration in mice and rats involves the use of chimera pro- 

Inherited canine retinal degenerative disease collectively described as "pro gressive retinal atrophy" has been recognized in a number of breeds (Magnusson, 1911; Hodgmen et al, 1949 , Parry, 1953 Barnett, 1965a) (Fig. 24) (Table  II) . Genetic studies have revealed the majority to be inherited as a simple autosomal recessive trait, with three genotypes in the population: the homozygous normal, the heterozygous normal carrier, and the homozygous affected dog. Clinical, electroretinographic, and transmission electron microscopic studies have been utilized to define distinctly different conditions in various breeds including rod-cone dystrophy in poodles (Aguirre and Rubin, 1972) and central progressive retinal atrophy in other species (Parry, 1954; Barnett, 1965b; Aguirre and Laties, 1976 ).

Evans rats and produced a retinal degeneration that involved both photoreceptor cells and RPE. Fluorescein angiography and electron microscopy were utilized to assess altered retinal vascularity. These findings were likened to retinitis pigmentosa in man.

To evaluate retinal changes caused by phenylalinine, the drug was adminis- tered subcutaneously to newborn rats for 1 week, producing profound damage to the bipolar and ganglion cell layers. The presence of the immature blood-brain barrier was suggested to be significant in the development of the lesions (Colmant, 1977) . Hamsters were used by another investigator to produce retinal pigmentary degeneration with yV-methyl-jz-nitrosourea, this model being suggested for use in screening the retinotoxicity of carcinogenic drugs. It should be pointed out, however, that the fundus was difficult to visualize, and the results were derived from histopathology (Herrold, 1967) . Male Dutch-belted rabbits were given different levels of oxalate compounds subcutaneously to study the "flecked retina" condition seen in oxalate retinopathy. This model is also pro claimed valuable for assessing B vitamin deficiencies, ethylene glycol poisoning, and methoxyfluorane anesthesia toxicity (Caine et al., 1975) . Four-to 5month-old pigmented rabbits were used by Brown in 1974 to study the effect of lead on the RPE. The electroretinogram, electrooculogram, and flat mount and histopathological preparations were utilized. The ERG and EOG recordings were normal, suggesting no major disturbance of the RPE. One of the smaller New World primate species, the squirrel monkey, was used by Mascuilli et al. (1972) to evaluate the effects of various elemental iron salts on the retina. Ocular siderosis with RPE and photoreceptor cell damage was evident as early as 4 hours after intravitreal administration. The ERG was used to study these effects.

Synthetic antimalarials have long been recognized as producing ocular com plications in humans, especially macular pigmentary disturbances. Berson (1970) administered chloroquine to cats to assess acute damage to the retina, utilizing intravitreal and posterior ciliary artery injections (Figure 25 ). Hey wood (1974) mentions the affinity of chloroquine and related drugs for melanin pig ment. Miyata et al. (1978) used the Long Evans pigmented rat to evaluate the effects of intramuscular fenthion on the retina. ERG as well as histopathologic changes were extensive 12 months after administration.

A brief review of retinotoxic drug effects illustrated in color is available in the "Atlas of Veterinary Ophthalmoscopy" (Rubin, 1974) . Included are funduscopic representations of ethambutol effects in the dog, naphthalene in the rabbit, chloroquine and related drugs in rabbits and cats, and a selection of other re tinotoxic agents.

In the study of induced retinal degeneration, especially these conditions with RPE involvement, pigmented animals are more desirable than albinos. The sub- human primate is the only species with a macula comparable to the human, although birds may possess dual foveae. Some mammals have an area centralis, a zone of high cone concentration that, while similar, is neither functionally nor anatomically identical to the macula. Species differences in relative numbers of rod and cone photoreceptors varies with the animals' ecological niche, with nocturnal creatures exhibiting a preponderance of rods and diurnal animals more likely to possess a cone dominant retina. With these exceptions (not including variations in retinal vascular patterns, discussed elsewhere), the structural and functional relationships of the neurosensory retina are remarkably similar thorughout the vertebrates.

A multitude of variations producing this type of degeneration have been stud ied including fluorescent, incandescent, ultraviolet, infrared, and colored light excesses with continuous or interrupted exposure patterns, high and low level intensities, and other variations. Additional factors such as age, sex, body tem perature, species, and nutrition have also been simultaneously evaluated. Lai et al. (1978) used Fisher 344 albino rats exposed to a high-intensity fluorescent light to produce a severe peripheral retinal degeneration. A continuous highintensity fluorescent light exposure regimen for albino and hooded rats produced a more severe photoreceptor cell degeneration in the former (Reuter and Hobbelen, 1977). Low-intensity continuous colored lights disclosed that white and blue bulbs produced the most damage in adult albino rats . It is apparent that continuous light sources are the most damaging to the albino animal, the higher the intensity the more severe the degeneration. Newborn albino species, however, will show initial outer segment growth before damage eventually occurs (Kuwabara and Funahashi, 1976) . In 1973, Berso evaluated the 13-striped ground squirrel, which has an all-cone retina, under high intensity illumination. Essentially 200 times the amount of light required to produce cone degeneration in the albino rat produced "retinitis punctata albescens" in the ground squirrel. Zigman et al. (1975) 

A more specific study was performed by Tso et al. (1972) in rhesus monkeys to assess the effects of a retinally focused, low-intensity light beam under normal and hypothermie conditions. Although no difference was detected due to body temperature, a progressive degeneration that involved the RPE and photoreceptor cells was identified in both study groups.

The recent development of laser application in ocular therapy prompted study in laboratory species. Gibbons et al. (1977) used rhesus monkeys to study visible and non visible lasers at high-and low-power levels and found that a 1-hour exposure of the latter was equivalent in RPE damage to 24 hours of the former. In another study Tso and Fine (1979) exposed the foveolas of rhesus monkeys to an argon laser for 10-20 minutes and found cystoid separations of the RPE and Bruchs membrane at 3-4 years post-insult. Good anesthesia and subject stabiliza tion are obvious requirements for these procedures.

The advent of microwave radiation for everyday use has initiated considerable evaluation of its safety for the human eye. Retinal damage in the form of synaptic neuron degeneration was produced in rabbits by exposure to 3100 MHz of microwave radiation (Paulsson et al., 1979) . Ultrastructural changes were dis covered that had not previously been seen, and these resulted from lower levels of energy than anticipated. Solar retinitis has long been a problem in man but recently has increased due to greater numbers of unprotected people being exposed to snow-reflected sunlight. Ham et al. (1979) produced photochemical (short wavelength) and thermal (long wavelength) lesions in rhesus monkeys by exposure to a 2500 W xenon lamp, stimulating in the former case the solar spectrum at sea level. This model demon strated the short wavelength causal nature of sunlight overexposure, resulting in solar retinitis and eclipse blindness.

Higher energy radiation effects have been evaluated by other means including high-speed particle acceleration. Proton irradiation of discrete areas of the retina in owl monkeys produced full thickness retinal damage (Gragoudas et al., 1979) . Rhesus monkey retinal irradiation by highly accelerated (250 MeV) oxygen nuclei produced extensive retinal vascular damage followed by degrees of degen eration and necrosis of the neural layers (Bonney et al., 1977) .

Traumatic optic nerve injuries in humans are not so unusual as diseases that attack and destroy the optic nerve. In either case, however, retinal degeneration frequently follows. Ascending and descending optic atrophy was produced in squirrel monkeys by Anderson (1973) to study the degenerative process. Total optic nerve neurectomy by razor blade knife via lateral orbitotomy was per formed to assess the descending condition, while xenon arc photocoagulation was performed to create the ascending condition. Electron microscopy demon strated degenerative changes effecting the nerve fiber and ganglion cell layers. Both methods produced morphologically similar retrograde processes; photocoagulation, however, provided evidence of ascending atrophy within 2 weeks.

Commotio retinae or traumatic retinopathy was initially induced in rabbits by Berlin (1873) who used an elastic stick to produce retinopathy, Sipperley et al. (1978) employed a BB gun technique that delivered a standard sized metallic BB which struck the cornea between the limbus and pupillary axis to produce the desired contrecoup injury of the posterior pole. The segments of the outer nuclear photoreceptor layer were specifically effected, the authors using fluorescein an-giographic, histopathologic, and electron microscopic results to demonstrate the disease.

Retinal degenerative conditions caused by nutritionally related problems have been studied in rats, cats, and monkeys. These studies have mostly been con cerned with vitamin deficiencies, and for economic reasons have lent themselves well to the rat as a model. The subhuman primate may be geneologically ideal for certain investigations, but it is equally as impractical for fiscal reasons. The cat has been used because of a specific susceptibility to a deficiency of the amino acid taurine (Fig. 26) .

In 1975 Hayes et al. used young and adult cats to assess the effects of taruine deficiency by feeding a diet of casein, which contains very little cystine, the major precursor for taurine. In 3 months a photoreceptor cell degeneration was produced, the initial signs including a hyperreflective granular zone in the area centralis. The ERG recordings indicated a photoreceptor degenerative process, while morphologically the outer nuclear and plexiform layers were destroyed. Recently, Wen et al. (1979) discovered that taurine deficiency in cats produces in addition to the photoreceptor cell a tapetal cell degeneration, thus suggesting that this amino acid also plays a role in maintaining structural integrity in this tissue. These investigators employed the use of the visual evoked potential (VEP) technique to arrive at their conclusions.

Due to its known unusually high concentration in the retina, numerous species, such as chickens, rabbits, rats, and frogs, have been used to define the biologic function of taurine. Pourcho (1977) utilized intravenous and intravitreal radiolabeled 35 S-taurine. The cat, mouse, rabbit, and monkey exhibited higher concentrations of taurine in Müller cells, whereas, by comparison, the chick and frog showed very little.

Vitamin A deficiency has been studied in various species, the rat having been utilized most extensively because it is susceptible to night blindness as well as being less expensive and readily available. Carter-Dawson et al. (1979) used the offspring of vitamin A-deficient pregnant female rats to accelerate the desired condition of tissue depletion. Under low levels of cyclic illumination (1.5-2 ft-c) both rhodopsin and opsin levels decreased, the latter requiring a longer period of time. In addition it was determined that rod cells degenerated before the cone cells. In another experiment, autoradiographic techniques were used to assess the influence of vitamin A deficiency on the removal of rod outer segments in weanling albino rats (Herron and Riegel, 1974) . The process was distinctly retarded by the absence of the vitamin. In a combination study, Robison et al. (1979) evaluated the effects of relative deficiencies of vitamin A and E in rats. Autofluorescent and histochemical techniques revealed that the vitamin E-free diets produced significant increases in fluorescence and lipofucsin granule stain ing regardless of the vitamin A levels. In conclusion vitamin E was thought to prevent light damage by scavenging oxygen radicals and thereby providing pro tection via lipid peroxidation.

Two different species of subhuman primates (Ce bus and M acaca) were used by Hayes (1974) to compare (separately) vitamin A and E deficiencies. After 2 years on a vitamin E-deficient diet a macular degeneration developed, charac terized by a focal massive disruption of cone outer segments. Vitamin A defi ciency, on the other hand, produced xerophthalmia, keratomalacia, and impaired vision in six Ce bus monkeys, the latter due to cone degeneration in the macula and midperipheral retina. Information describing these techniques is available from a previous publication (Ausman and Hayes, 1974) .

The ocular effects of two slow virus diseases have recently been studied: scrapie agent in hamsters (Buyukmihci et al., 1977) and Borna disease in rabbits (Krey et al., 1979) . The scrapie agent when inoculated intracerebrally in young hamsters produced a diffuse thinning of the retina, the photoreceptor cell layer being most severely affected (Fig. 27) . volvement in each foci. Viral antigen was detected in these lesions as well as in nonaffected areas, thus suggesting an immune component involvement. This condition was likened to ocular pathology seen in subacute sclerosing panencephalitis in humans. Friedman et al. (1973) injected newborn albino rats with simian virus 40 (SV40) and demonstrated viral antigen by immunofluorescense at 48 hours. Adult rats injected at birth developed retinal neovascularization, folds, and gliosis, features similar to retinal dysplasia.

In comparison, an extensive retinopathy was produced by Monjan et al. (1972) by infecting newborn rats with lymphocytic choriomenigitis virus. The outer nuclear layers of the retina were initially effected, followed by the inner nuclear and ganglion cell layers and finally total destruction. Due to a modest inflammatory infiltrate, this condition was also suspected to be immunopathologic in nature.

The critical assessment of retinal degeneration has been done through the implementation of five basic techniques: (1) electroretinography (ERG), (2) electrooculography (EOG), (3) visual evoked response (VER), (4) autoradiography, and (5) light and electron microscopy.

The concept of electroretinogram (ERG) was first demonstrated by Holmgren in 1865 using the enucleated eye of a frog. This technique is in principle applica ble to both animal and man and provides a detailed assessment of the rod-cone (visual cell) nature of the retina as well as its functional status. It is based upon the summation of retinal electrical potential changes which occur in response to light stimulation and are measured via corneal or skin surface electrodes (Fig. 28) . Detailed analyses of the ERG can be obtained from Brown (1968) as well as information concerning the use of microelectrodes. Additional information describing the local electroretinogram (LERG), a more precise but invasive technique, is available from Rodieck (1972) .

The organization of the vertebrate retina and the origin of the ERG potential change has been elucidated by the use of intracellular readings. The mudpuppy, Necturia maculosa, has been a favorite subject because of its large retinal cells. Excellent reviews of comparative retinal organization and function are provided by Dowling (1970) and Witkovsky (1971) .

The electrooculogram (EOG) is a clinically applied test of retinal function that was first measured in the eye of a tench by du Bois-Reymond in 1849. This test assesses the standing or corneofundal potential which exists between the anterior and posterior poles of the eye as a subject is taken from a dark to a light adapted state. For detailed information concerning this technique refer to Krogh (1979) (rabbit) or Arden and Ikeda (1966) (rat) .

A newer technique, the visual evoked potential (VEP), has been adapted for use 400 msec in animals by Wen et al. (1979) . This enables the clinical assessment of visual function in the area centralis as compared to the peripheral retina by measuring potential changes in the visual cortex in response to focal or diffuse retinal stimulation. The techniques described for the cat are also applicable to other species. Autoradiography has been available and successfully used in ophthalmologic research for several years (Cowan et al. y 1972) . Tritiated amino acids are readily available in various concentrations and forms and may be injected into the vitreous or other ocular tissues where they may become imcorporated into cellu lar protein metabolism pathways. Distribution may be studied by light or electron microscopy. Ogden (1974) utilized the concept of axoplasmic transport together with autoradiography to trace the course of peripheral retinal nerve fibers to the optic disc. Others have utilized this technique to study outer segment photopigment metabolism. Rods have been shown to regenerate photopigment discs con tinuously with the RPE participating in the process by phagocytizing the shed products. (Young, 1970; Norton et al., 1974; Mandelcorn et al., 1975) . Cones also have mechanisms for photopigment renewal. Specific information concern ing autoradiographic techniques may be obtained from Cowan et al. (1972) , Rogers (1967) , and Kopriwa and Leblond (1962) .

Light and electron microscopy have expanded the morphologic study of the retina beyond the limitations of light microscopy. In 1970 Laties and Liebman discovered by chance that the chlortriazinyl dye, Procion yellow, was a selective stain for the outer segments of retinal cones in the mud puppy and frog. Further investigation by Laties et al. (1976) demonstrated the value of this dye in distinguishing between the outer segments of rods and cones and its use in monitoring outer segment renewal. These investigators studied the dye in rat, dog, rabbit, and monkey eyes as well and discovered that the rod basal saccules could not be visualized in the rat and dog with the same certainty as the gekko (nocturnal lizard) and mud puppy. Specimen preparation involves the injection of aqueous Procion yellow (0.5 to 4%) into the vitreous using a 3 /e-inch, 26-gauge needle.

Although originally used to described a syndrome consisting of CNS and systemic anomalies in the human infant, retinal dysplasia (RD) now applies specifically to any abnormal differentiation of the retina after the formation of the anläge. The histopathologic features normally constituting RD include: (1) rosette formation, (2) retinal folds, and (3) gliosis (Lahav and Albert, 1973) . Table III lists those agents that have either been associated with or used in the induction of RD. The administration of blue tongue virus vaccine to fetal lambs appears to be the one technique that offers all of the features described for RD. This technique involves the use of a live, attenuated virus vaccine, and it is effective only during the first half of gestation (Silverstein, et al., 1971) . The RD inducing effects of x-ray exposure on the retina of the primate fetus have been described (Rugh, 1973) . The ERG was used in this experiment as well as histopathology to demonstrate the lesions. Shimada et al. (1973) and used suckling rats to evaluate the effects of antitumor and antiviral drugs, respectively, the results in both being the induction of rosette formation. In 1973 Silverstein used the fetal lamb and intrauterine trauma to demonstrate the relationship of the RPE in the organizational histogenesis of the retina (Fig. 29 ).

Experimental retinal detachment was first described by Chodin in 1875. The rabbit, dog, and subhuman primate have since become species of choice; the pigmented rabbit model probably the most practical, it being less expensive, more genetically uniform, and easier to manipulate.

A spontaneous inherited detachment has been reported in the collie dog, ac companied by choroidal hypoplasia and posterior staphylomas (Freeman eTal., 1966) . Experimental methods have utilized specifically designed techniques to induce detachment including: (1) traction detachment by perforating injury (Cleary and Ryan, 1979a,b) , (2) intravitreal hyperosmotic injection (Marmor, (Norton et al., 1974) , (4) detachment by experimental circulatory embolization (Algvere, 1976) , and (5) detachment by blunt needle rotation and suction without the use of hyaluronidase (Johnson and Foulds, 1977) . Traction detachments are dependent on vitreal hemorrhage and scarring affects (Cleary and Ryan, 1979a,b), the rabbit and rhesus monkey being successfully utilized in these studies. Vitreal changes are significant in most of these techniques, producing detachments of varying dimensions and duration (Marmor, 1979) . Embolization of the retinal and choroidal circulations with resultant retinal ischemia produced a long-lasting retinal detachment (Algvere, 1976) ; the technique used plastic (polystyrene) beads injected into the central retinal artery and the supratemporal vortex veins of owl monkeys (Aotus trivirgatus). Another approach which produced septic choroiditis and multifocal serous retinal detachments in dogs involved the intracarotid injection of pathogenic bacteria (Meyers et al., 1978) .

Experimental studies of the optic nerve have explored two aspects of this tissue; the pathogenesis of the degenerative changes resulting from increased IOP, and the development of an experimental model for allergic optic nearitis, similar to that seen in multiple sclerosis. The former topic is discussed in the section on glaucoma. In regards to the latter, several workers have observed optic neuritis as well as retinal vasculitis and uvertis in guinea pigs, rabbits, and monkeys affected with experimental allergic encephalomyelitis. (Raine et al. t 1974; von Sallmann et al., 1967; Bullington and Waksman, 1958) . Rao et al (1977) induced papilledema and demyelination of the optic nerve in strain 13 guinea pigs by sensitization with isogenic spinal cord emulsion in complete Freunds adjuvant.

Unlike many other areas of ocular research where evidence obtained from animal models has been accepted with allowances for differences between the human and animal eye, the striking contrasts in ocular blood supply from one species to the next are impossible to ignore. As a result a variety of animal models have been studied in an attempt to sort out those aspects of the vascular anatomy and physiology which are highly variable from those which seem to be generally similar for a number of different species. The rabbit is sometimes used in this line of research, but more often it is supplanted by the monkey, cat, pig, or rat, all of which have an ocular blood supply which is more analogous to that of man.

There are some general trends in the comparative anatomy of laboratory ani mals with respect to the ocular circulation. All of the commonly studied animals have a dual circulation to the eye consisting of (1) the uveal and (2) the retinal blood vessels. In higher primates, including man, both the uveal and retinal vessels are branches of the ophthalmic artery, itself a branch of the internal carotid; the external carotid system contributes very little to the ocular blood supply. In lower animals, on the other hand, the external carotid system, by way of the external ophthalmic artery, supplies the major portion of blood to the eye. Many animals, including the dog, cat, and rat (but not the rabbit), have a strong anastomosing ramus between the external ophthalmic artery and the circle of Willis, and since a relatively large part of their cerebral blood supply comes from the vertebral arteries, the common carotid artery may often be totally occluded with no apparent ill effects to the eye (Jewell, 1952) .

The retinal circulation in primates is supplied by the central retinal artery which branches off from the ophthalmic artery to enter the optic nerve close to the globe (Fig. 30) . In lower animals the retinal circulation is more often derived from a network of anastomosing branches of the short ciliary arteries usually referred to as the circle of Haller-Zinn. In fact, of the nonprimate mammals, the rat is the only animal with a central retinal artery homologous to that of the primates. The presence of a central retinal artery in dogs and cats is disputed (François and Neetens, 1974) , but even if present it is not the main source of blood to the retina (Fig. 31) .

The extent and configuration of the retinal vessels is even more variable than their source. In most laboratory animals, including primates, dogs, cats, rats and mice, the retina is more or less completely vascularized, or holangiotic. Rabbits, however, have only two wing-shaped horizontal extensions of vessels from the optic nerve which are accompanied by myelinated nerve fibers (the medullary rays), and do not actually extend into the retina at all. Horses have only a few small retinal vessels scattered around the optic disc (paurangiotic), and birds have a retina which is completely devoid from blood vessels (anangiotic).

In summary then the primates have an ocular blood supply which is almost identical to that of man. The rat should also be considered as an animal model in studies of the retinal circulation. The ocular blood supply in cats and dogs is somewhat less analogous to that of the human. The rabbit has some very atypical features, especially of the retinal circulation, which should be considered very carefully before including it in experiments on the ocular circulation. One feature of the orbital vascular anatomy in rabbits which has not been mentioned is a peculiar anastomosis between the two internal ophthalmic arteries which has been implicated in the consensual irritative response in rabbits following an injury to one eye (Forster et al., 1979) .

Because of the complexity and small size of the intraocular blood vessels, dissection, even under the microscope, is usually inadequate to study the anatom ical relationships of these vessels. Corrosion casting techniques, involving infu-sion of the eye with neoprene (Prince, l'964b) or plastic resin followed by digestion of the tissues, usually with concentrated sodium hydroxide, have been used for study of the uveal circulation and less often the retinal circulation. In our laboratory corrosion casting with a special low-viscosity plastic has been used with excellent results to study both the anterior and posterior uveal as well as the retinal circulation in cats and monkeys (Risco and Noapanitaya, 1980) . Castings are studied with the scanning electron microscope (Fig. 32) . The retina, because it is thin and relatively transparent, is amenable to simpler studies of vascular morphology. The classic text on this subject is by Michaelson (1954) , who based his observations primarily on flat mounts of retina perfused with India ink. Other dyes have been utilized in a similar fashion (Prince, 1964b) . Another popular technique introduced byKuwabara and Cogan (1960) involves digestion of the retina in 3% trypsin at 37°C for 1-3 hours until the tissue shows signs of disintegration followed by careful cleaning away of the partially digested tissue from the vascular tree and then staining with periodic acid-Schiff (PAS). Other investigators believe that trypsin digestion may destroy some of the more delicate or immature vessels and advocate PAS staining alone to delineate retinal vessels in immature retina (Engerman, 1976a,b; Henkind, et al., 1975) .

So far we have mentioned only in vitro studies of the ocular circulation. In vivo studies can provide information about the dynamics of blood flow as well as morphology. The most widely used in vivo technique is fluorescein angiography. Fluorescein dye is excited by blue light with a wavelength of 490 mm, emitting a yellow-green light with a wavelength of 520 mm. The blood vessels of the retina and iris are relatively impermeable to fluorescein because of their endothelial tight junctions. Thus fluorescein angiography has been useful not only in the study of normal anatomy and time sequence of blood flow in these vessels as demonstrated by the excellent studies in the monkey by Hayreh (1969; Hayreh and Baines, 1972a,b) but also as a test of integrity of the blood-ocular barrier in certain pathological states, especially neovascular lesions. The diffusion of fluorescein across the blood ocular barrier can be assessed quantitatively by fluorophotometric techniques (Cunha-Vaz, 1979) . There is evidence that, in mammals at least, the blood ocular barriers are similar for different species (Rodriguez-Peralta, 1975) .

The basic equipment required is a fundus camera capable of rapid sequence flash photographs and appropriate filters. Satisfactory fluorescein angiograms can be obtained in most laboratory animals, even mice and rats, although general anesthesia is usually required. Angiograms of dogs, cats, and other carnivora are technically more difficult because the reflection from the tapetum interferes with the observation of retinal blood flow. In these animals color fluorescein angiog raphy may give better results. Many of the technical aspects of fluorescein angiography in animals are discussed by Bellhorn (1972) .

Anterior segment angiography of the iris vessels has the advantage of requiring less expensive equipment; a 35 mm camera with a macro lens and rapid recycling flash are essential (Rumelt, 1974) . The rabbit, the most popular experimental animal for anterior segment angiograms, has an anterior segment circulation similar to humans, with the exception of the absence of a contribution from the anterior ciliary arteries to the anterior uveal blood supply, a situation possibly unique among mammals (Ruskell, 1964) . The technique and interpretation of anterior segment angiography is discussed in detail by Kottow (1978) .

Choroidal angiograms are also possible but present a technical problem in that in pigmented animals the retinal pigment epithelium (RPE) absorbs light strongly in both the excitation and emission wavelengths for fluorescein. The RPE is transparent to wavelengths in the infared range and the use of cyanine dyes for infared absorption and emission angiograms of the choroid have been described (Hochheimer, 1979) as well as a technique for simultaneous angiography of the retinal and choroidal circulations using both fluorescein and indocyanine green (Flower and Hochheimer, 1973) . Tsai and Smith (1975) have used fluorescein for choridal angiograms by injecting dye directly into a vortex vein. Angiograms of the ciliary circulation in rabbits have been obtained using conventional radio-opaque media and dental x-ray film (Rothman et al., 1975) .

The measurement of absolute and relative blood flow, velocity of blood flow, and oxygen content in various ocular tissues has been key to the understanding of nervous control of ocular physiology as well as the ocular effects of many drugs. Unfortunately, these measurements have been fraught with technical difficulties. Many of the techniques utilized have been indirect methods requiring compli cated mathematical analysis often based on tenuous assumptions. As a result large discrepancies have resulted from the use of different techniques or even the same technique in different hands. To complicate matters further different inves tigators have expressed results in different terms which are not easily interconver tible. The results of many of these studies have been reviewed and tabulated for comparison by Henkind et al. (1979) .

Measurements of total or localized ocular blood flow have been made based on the washout of various gases including nitrous oxide, 85 Kr and 133 Xe (O'Rourke, 1976) . A heated thermocouple has been used to measure relative blood flow (Armaly and Araki, 1975) , and Bill (1962a,b) measured choroidal flow directly in rabbits by cannulating a vortex vein and in cats by cannulation of the anterior ciliary vein. The most recent technique is the use of radio-labeled microspheres (O'Day et al., 1971) . The labeled microspheres are injected into the arterial system, and shortly thereafter the animal is sacrificed and samples of ocular tissues are taken for quantitation of radioactivity. The microspheres are presumed to embolize in the small vessels of the various ocular tissues in amounts propor tional to the blood flow in that tissue.

Photographic analysis following injection of fluorescein and other dyes has been used to determine retinal oxygen saturation (Hickam et al., 1963) , mean circulation time (Hickam and Frayser, 1965) , and choroidal blood flow (Trokel, 1964; Ernest and Goldstick, 1979) . Oxygen saturation has also been measured directly in monkeys using a microelectrode inserted through the pars plana (Flower, 1976) . Velocity of blood flow has been estimated using Doppler tech niques (Riva et al., 1972; Stern, 1975) and thermistors (Takats and Leister, 1979) .

While absolute measurements may not agree from one study to another, relative measurements may still be valid. Most studies confirm that the choroid receives about 75% of the total ocular blood flow while the anterior uvea receives from 10 to 35% and the retina about 5% of total ocular flow.

One of the most important problems facing clinical human ophthalmologists is the control and treatment of vasoproliferative diseases in the eye. These neovascular proliferations may be confined to the retina or grow into the over lying vitreous and eventually result in retinal detachment or vitreous hemorrhage. They may also involve the iris (rubeosis iridis) and lead to neovascular glaucoma. Diabetes, retrolental fibroplasia, and retinal vein occlusion are all commonly associated with ocular neovascularization.

All of the above conditions have in common some degree of vasoobliteration followed by a period of retinal ischemia and subsequent vasoproliferative response. The most widely accepted theory is that growth of new vessels is stimulated by elaboration of a vasoproliferative factor from ischemie retina, a diffusable substance similar to the embryologie inducing agents discovered by Spemann (1924) . This concept was supported by the isolation of a diffusable factor from certain tumors which stimulated neovascularity (Folkman et al., 1971) . Ryu and Albert (1979) demonstrated a variable nonspecific neovascular response to viable or nonviable melanoma or retinoblastoma cells in a rabbit corneal model. The response was negligible in immune-deficient animals. More recently experiments by Federman et al. (1980) have indicated that implanta tion of ischemie ocular tissues into the rabbit cornea can stimulate a non inflammatory neovascular response distant to the site of implantation.

In diabetes capillary dropout in the retina is a well-documented phenomenon and is probably the precursor of proliferati ve retinopathy. Reports of eye changes, especially capillary changes, in animals with spontaneous or induced diabetes are common (Table IV) , but many of these reports represent sporadic findings. Engerman (1976a,b) has reviewed the subject and feels that the re tinopathy associated with alloxan-induced diabetes in dogs is the best animal model based on morphology and reproducibility of the lesions. Alloxan diabetes produces a similar retinopathy in monkeys but microaneurysms require about 7 years to develop (R. L. Engerman, unpublished communication, 1980) .

The retinopathic changes seen in these animals consists of microaneurysms and other structural capillary changes. We are not aware of any animal model of proliferative diabetic retinopathy with extraretinal neovascularization. Patz and Maumenee (1962) ; Patz et al. (1965) Sibay and Hausier (1967) Gepts and Toussaint (1967) Bloodworm (1965) Engerman et al. (1972) ;

Engerman and Bloodworth ( 

Retrolental fibroplasia (RLF) is a result of oxygen toxicity to the immature retina. Thus premature babies treated with oxygen are most susceptible, and the most immature part of the retina, usually the temporal periphery, is the most common area of involvement. The disease can be induced in newborn puppies, kittens, rats, and mice by exposure to high oxygen concentrations because these animals have an immature incompletely vascularized retina at birth. The kitten has been the most popular animal model for RLF, but the changes seen are comparable only to the early stages of RLF in humans with severe extraretinal disease complicated by retinal detachment being a rare finding in any of the animal models. There is some evidence that there are subtle differences in the pathophysiology of human RLF and that seen in animal models (Ashton, 1966) .

Isolated retinal branch vein occlusion produced with the argon laser has been shown to cause a neovascular response in monkeys (Hamilton et al., 1975) . Once again, however, the new vessels are intraretinal and do not grow into the vitreous.

The injection of inflammatory or toxic substances, such as blood (Yamashita and Cibis, 1961) , ammonium chloride (Sanders and Peyman, 1974), or immunogenemic substances including insulin (Shabo and Maxwell, 1976) into the vitreous may result in intravitreal vessels and fibrous membranes. These eyes show similarities to the late stages of diabetes or RLF in humans, but the pathophysiology is most likely different.

The difficulty in producing intravitreal neovascular growth in animals may be due in part to an inhibitory effect of vitreous on proliferating blood vessels (Brem et al., 1976; Felton et al., 1979) . Rabbit V-2 carcinoma implanted into rabbit vitreous results in a neovascular response only after the tumor has grown into contact with the retina (Finkelstein et al., 1977) . The same tumor implanted into the corneal stroma stimulates a neovascular ingrowth toward the tumor (Brem et al., 1976) , and, as already mentioned, Federman et al. (1980) have reported that corneal implantation of ischemie retina stimulates a similar response.

Rubeosis, or neovascularization of the iris, may accompany diseases which cause retinal neovascularization and often produces glaucoma which is difficult to treat. There is apparently no reproducible way to induce true rubeosis in animals at present. Attempts to produce the condition experimentally have been reviewed by Gartner and Henkind (1978) .

Occlusive vascular disease of the eye includes not only the relatively spectacu lar central retinal artery and vein occlusions but also a number of disorders with more subtle or insidious signs and symptoms. Ischemie optic neuropathy, geo-graphic choroiditis, some forms of persistent uveitis (Knox, 1965) , and possibly acute posterior multifocal placoid pigment epitheliopathy (Ueno et al., 1977) are all examples of ocular disorders which can result from vasoocclusive events.

Experimental occlusion of the larger ocular vessels can be accomplished by occlusion of the vessel with a ligature or clamp. This includes the long and short posterior ciliary arteries (Hayreh, 1964; Hayreh and Baines, 1972a,b; Anderson and Davis, 1974) and the central retinal artery and vein (Hayreh et al., 1978 (Hayreh et al., , 1979 . Lesseil and Miller (1975) reported effects on the optic nerve and retina in surviving monkeys following complete circulatory arrest for 14 to 30 minutes.

Experimental occlusion of the smaller vessels has been produced by embolization of the arterial system with plastic beads or various other particulate matter. Most of the early embolization experiments were limited by the fact that the particulate material was injected into the carotid artery, thus indiscriminately embolizing small vessels throughout the eye (Reinecke et al., 1962; Hollenhorst et al., 1962; Ashton and Henkind, 1965) . More recent reports describe tech niques for selective embolization of the choroidal and retinal circulations based on injection site (Kloti, 1967) or temporary occlusion of the retinal circulation during injection (Stern and Ernest, 1974) . The most selective occlusion of retinal vessels has been accomplished with photocoagulation using the argon laser (Hamilton et al., 1975) or xenon photocoagulator (Ernest and Archer, 1979) .

The concepts of using the globe as an in vivo tissue culture medium has been discussed and exemplified in a previous section; the technique is applicable to a variety of autologous, homologous, and heterologous tumors of a variety of cell line origins. Specifics are more within the realm of the oncologist rather than the ophthalmologist and as such will not be dwelt on. Several experimental tumor models have been well defined and have been utilized to eludiate spontaneous tumorogenic processes and to study the biological behavior of the induced tumors.

Intraocular injection of viruses will induce tumors in a number of laboratory animals. Injection of human adenovirus type 12, a DNA virus, into newborn rat vitreous produced retinoblastoma-like tumors in 3 of 35 animals between 204 and 300 days postinjection . Albert and his associates (1967) had earlier described similar observations in hamsters injected subcutaneously with tissue-cultured ocular cells lines exposed to the virus. Muaki and Kobayashi (1973) produced extraocular orbital tumors in 12 of 59 newborn hamsters 27 to 106 days following intravitreal injection of the same virus; the tumors resembled a neuroepithelioma, suggesting that they were derived from neurogenic primordia in the retrobulbar space. Further investigations demon strated the retinoblastoma in mice (Muaki et al., 1977) . JC polyoma virus was injected into the eyes of newborn hamsters by Ohashi and his associates (1978); 20% of the animals developed retinoblastoma-or ependymoblastoma-like intraocular tumors 7 Vi-\?> months postinjection. Eleven percent developed extraocular tumors, including schwannomas, fibrosarcomas, lacrimai gland carcinomas, and ependymal tumors.

Feline leukemia virus, an oncornavirus, was injected systemically and intraocularly into fetal and newborn kittens; a tumor of apparent retinal origin developed in 1 of 29 subjects (Albert et al., 1977a) . The same virus injected into the anterior chamber of cats results in the development of iris and ciliary body melanomas or, less commonly, fibrosarcomas, in a high percentage of injected animals (Shadduck et al., 1977; Albert et al., 1977b) .

In Taylor and his associates (1971) utilized intravenous 226 Ra to induce ciliary body melanomas in dogs; the tumors appeared to originate from the pigmented epithelium. Albert et al. (1980) transplanted human choriodal melanoma into the vitreous of the "nude" mouse, a homozygous mutant (nu/nu) with a defect in cellular immunity. Serial passage transplantation was possible. The fact that the animal is immunodeficient limits the value of this model to study biologic behavior, but this in vivo system provides abundant tissue for morphologic, biochemical, immunologie, and therapeutic studies.

Ophthalmic Res. 7, 133. Baum

The Eye

Manual of Tonography

System of Ophthalmology. The Eye in Evolution

The Biology of the Laboratory Rabbit

The Eye

Tonography and the Glaucomas

Immunopathology of Uveitis

Ocular Pharmacology

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Cataract and Abnormalities of the Lens

Becker-Schaffer's Diagnosis and Therapy of the Glaucomas

Anterior Segment Fluorescein Angiography

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Cells and Tissues in Culture

Retinal Circulation in Man and Animals

Nuclear Ophthalmology

Cell and Tissue Culture

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Comparative Anatomy of the Eye

Anatomy and Histology of the Eye and Orbit in Domestic Animals

Textbook of Veterinary Internal Medicine

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Atlas of Veterinary Ophthalmoscopy

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A Treatise on Gonioscopy

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The Vertebrate Eye and its Adaptive Radiation

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