Maurice Langham

Factors affecting the hydration of the cornea in the excised eye and the living animal.

RECENTLY, the influence of metabolism on the water content of the excised cornea has been studied by several investigators. Schwartz, Danes, and Leinfelder (1954) immersed pieces of excised corneae in a nutrient medium and modified the metabolism by cold, lack of substrates, and the use of metabolic inhibitors. From their studies, they concluded that the mechanism regulating corneal hydration was dependent on energy derived from the aerobic and anaerobic metabolism of glucose. Davson (1954, 1955) studied the changes in weight and water content of corneae in excised eyes with a view to establishing whether in the living eye there is an active transport mechanism that normally prevents the uptake of aqueous humour, tears, and limbal capillary filtrate. It was observed that the corneae of eyes left for 24 hrs in a cold moist chamber increased in water content and decreased again on warming to 31°C., and that anoxia caused a marked increase in hydration. He concluded that his experimental results were consistent with the existence of an active transport mechanism. in the cornea. Similar conclusions to those reached by these workers were reported by Langham (1955a, b) from studies of the effect of aerobic and anaerobic metabolism on corneal thickness and of the movement of sodium and water into the cornea of the excised eye immediately after enucleation. Philpot (1955) injected solutions of acidic and basic dyes, atropine, and inhibitors of cholinesterase, glycolysis, and respiration into the anterior chamber of excised rabbit eyes and observed their action on corneal hydration. Certain of the dyes and inhibitors led to an increased hydration but inhibitors of cholinesterase were without effect. The observation that iodoacetic acid increased corneal hydration when glycolysis was inhibited by over half led Philpot to conclude that glycolysis was necessary to the maintenance of corneal hydration. No attempt, however, was made in Philpots study to ascertain if the action of these compounds on corneal hydration was due to an inhibition of the metabolic processes regulating corneal hydration or to a non-specific action on the permeability of the cornea to water and salt. This consideration is especially important in view of the observations of Herrmann and Hickman (1948) that iodoacetic acid in low concentrations reduced the cohesion of the epithelium to the underlying stroma.

ACTION OF DIAMOX AND AMMONIUM CHLORIDE ON FORMATION OF AQUEOUS HUMOUR

THE investigation was undertaken to establish the cause of the decrease in intra-ocular pressure induced in normal rabbits by 2-acetylamino-1, 3, 4 thiadiazole 5-sulphonamidet, an inhibitor of the enzyme carbonic anhydrase. At the same time, it was hoped that a comparison of these results with the known ability ofDiamox to reduce the intra-ocular pressure in glaucomatous patients for extensive periods would help in assessing whether the intraocular dynamics of normal and pathological eyes are affected in a similar manner and to a comparable extent. The lowering of the intra-ocular pressure by Diamox has led to the suggestion that the enzyme carbonic anhydrase is essential to the formation of the aqueous humour, and this idea has found expression in the hypothesis that the reduction of the intra-ocular pressure by Diamox is due to an inhibition of secretion across the blood-aqueous barrier (Becker, 1955a; Friedenwald, 1955a, b). This view had its origin in the studies of Friedenwald and Stiehler (1938) on the transfer of acidic and basic dyes across the epithelial layers of the ciliary processes. Friedenwald (1949) suggested that hydroxyl ions were secreted into the aqueous humour, and that an excess of bicarbonate was produced from the reaction of the hydroxyl ions with carbonic acid. This view was supported by Kinsey (1950), who observed in rabbits that the concentrations of bydroxyl and bicarbonate ions in the aqueous humour exceeded those in the plasma. The participation of carbonic anhydrase in this secretory activity was supported by the reports of the presence of the enzyme in extracts of the iris and the ciliary processes (Wistrand, 1951; Kauth and Sommer, 1953; Green, Capper, Bocher, and Leopold, 1954; Gloster and Perkins, 1955a). Experiments designed to test either this hypothesis or alternative explanations of the action of Diamox in reducing the intra-ocular pressure have been reported by several investigators (Becker, 1955a, b, 1956; Becker and Constant, 1955; Green, Bocher, and Leopold, 1955; Langham and Lee, 1955a, b, c). There is general agreement that the reduction in the intraocular pressure is accompanied by a decreased rate of formation of aqueous humour, but the means by which this is brought about is still unresolved.

Permeability of the Cornea and the Blood-Aqueous Barrier to Oxygen

INTEREST in the permeability of the cornea to oxygen arose out of recent investigations on corneal metabolism and the influence of metabolism on the hydration of the tissue. Langham (1952) reported that the concentration of lactic acid in the cornea varied inversely with the oxygen tension in the tear film and concluded that oxygen obtained directly from the atmosphere was utilized by the cornea, and that the oxygen entering from the bloodstream and the aqueous humour was unlikely to meet the requirements of the whole cornea. In studies of a rather different nature, Smelser and Ozanics (1953) and Langham and Taylor (1956) observed that the denial of atmospheric oxygen to the anterior surface of the cornea of the living animal led to an increased hydration and thickness of the cornea. Consequently, to confirm whether or not the respiratory requirements of the tissue could be met by the diffusion of oxygen from the aqueous humour, direct measures of the oxygen tension in the aqueous humour and the rate of diffusion of oxygen across the cornea have been made. At the same time, the opportunity has been taken of assessing by an independent means the conclusion of Fischer (1930) that the cornea shows a unidirectional permeability to oxygen in the inward direction. Previous studies relating to this subject include those of de Haan (1922), Friedenwald and Pierce (1937), and Fischer (1930). The oxygen tension in the aqueous humour of rabbits was measured by de Haan (1922), using Kroghs microtonometric technique. He found the tension to be 20-30 mm. Hg, but recognized that some loss of oxygen from the aqueous humour took place during the analysis through autoxidation. Correcting for this loss, he concluded that the oxygen tension was probably nearer 40-45 mm. Hg. Friedenwald and Pierce (1937) introduced a bubble of nitrogen into the anterior chamber of dogs and, after allowing time for equilibration, analysed its oxygen content; from a series of analyses made after different time intervals they reported the mean oxygen tension in the aqueous humour to be 45 mm. Hg. Their experimental technique is, however, subject to the criticism that paracentesis is known to cause a general increase in the permeability of the blood-aqueous barrier and to disturb the intra-ocular circulation. Fischer (1930) measured the change in oxygen content within a glass chamber sealed to the anterior segment of the eye. He observed a loss of oxygen

Observations on the Growth of Blood Vessels into the Cornea. Application of a New Experimental Technique

Ophthalmological Research Unit of the Medical Research Council, Institute of Ophthalmology, London. FEW subjects in opthalmology are more important from the clinical point of view than opacification and vascularization of the cornea; moreover, the problems included have a wide physiological and pathological interest. The unsatisfactory state of our present knowledge of the fundamental processes involved has been pointed out in the foregoing article by Ashton and Cook (1953), who also give a review of the literature. The present paper describes experimental investigations undertaken to elucidate these matters further. A new method of inducing a swelling and subsequent growth of vessels into the cornea has presented a unique opportunity for studying the underlying causes of these pathological phenomena. It has been shown (Langham, 1951) that a neutralized solution. of the organic compound alloxan, when brought into contact with the cornea of the rabbit, can be used to initiate either a completely reversible swelling of the cornea with little or no vascularization, or a swelling followed by a dense ingrowth of vessels around the whole periphery of the cornea. This method has been applied to the present problem in three ways:

Factors affecting the penetration of antibiotics into the aqueous humour.

ESSENTIALLY the blood-aqueous barrier comprises the layers of cells which separate the blood of the capillary vessels within the globe of the eye from the intra-ocular fluids. Elucidation of its nature by dynamic studies has thrown considerable light on how substances circulating in the blood plasma pass into the aqueous humour. Water readily crosses all the cellular barriers bounding the anterior and posterior chambers of the eye at a much greater speed than substances dissolved in the plasma. The latter enter the eye at widely varying rates, but in general it has been found that substances readily soluble in fat solvents pass through the bloodaqueous barrier faster than those readily soluble in water; thus ethyl alcohol travels approximately five times as rapidly as the fastest water-soluble compound studied. Consideration of the solubility characteristics of the chemotherapeutic substances recently introduced into clinical practice reveals that in general they are soluble in both water and lipids. The sulphonamides undergo ionization in the blood stream, and consequently exist as charged ions strongly soluble in water, and as uncharged molecules sparingly soluble in water but markedly soluble in ether and fats. The relative distribution of the two forms is such that the sulphonamides exist mainly in the uncharged form, soluble in fats. In similar manner, penicillin undergoes ionization in the blood stream and exists as water-soluble ions and as uncharged, undissociated fat-soluble molecules. Ionization of penicillin under these conditions is nearly complete, there being approximately one uncharged particle for every thousand ions. Thus sulphonamides in the blood stream exist predominantly as particles soluble in fats, whilst penicillin exists almost completely as ions insoluble in fats. In the present work an attempt has been made to find a correlation between the rate of entry of the antibiotics into the aqueous humour and their physico-chemical character, and to investigate the premise that lipoid solubility is the main factor influencing speed of entry.

Effect of Cortisone on Vascularization and Opacification of the Cornea Induced by Alloxan

THERE are few reports in the literature of experimental work dealing with the effect of cortisone upon corneal vascularization, and the evidence so fair advanced is partly conflicting (Duke-Elder and Ashton, 1951), but there is already some indication from clinical observers that cortisone exerts a considerable inhibitory influence on the formation of ne\v vessels in the diseased cornea. The experimental vwork reported in this paper investigates this subject further by the use ot a recently evolved technique which has particular advantages for this study. Before proceeding to investigate the effect of cortisone upon corneal vascularization it is necessary to consider the possible stimuli which might initiate new vessel formation; although the causes are still quite unknown, many theories have been advanced, some of which have an especial bearing upon the experimental conditions. Thus assuming for the moment that new vessel formation arises in response to a chemical substance liberated in traumatized tissue, as suggested by Michaelson and Campbell (1949). any effect of cortisone may be due to a neutralizing mechanism, and the extent of its action Would then depend upon the, size of the lesion and the quantity of cortisone administered. Similarly, the severity of the lesion would be of importance were vascularization to result from alterations in the normal tissue tension, as suggested by Cogan (1949). In attempting, therefore, to assess the effect of a given dose of cortisone upon corneal vascularization, it is essential that the experimental lesion should be, as far as variation in individual animals will allow, both standardized, in order to permit comparison, and the minimal required to produce vascularization, to ensure adequate sensitivity. Although the work already published on this subject is of great importance in assessing the probable therapeutic value of cortisone, it cannot be held that caustic (Jones and Meyer, 1950; Leopold and others, 1951), or thermal burns (Lister and Greaves, 1951), are entirely satisfactory methods for investigating this problem, for the

Fluorophotometric Apparatus for the Objective Determination of Fluorescence in the Anterior Chamber of the Living Eye

FOR many years considerable attention has been focused on the problem of the origin and circulation ofthe aqueous humour under normal and pathological conditions. One of the methods applied to this study is the detection within the eye of dyes which enter the anterior chamber across the bloodaqueous baffler-a method first suggested by Ehrlich (1882) when he observed that fluorescein injected intravenously passes into the aqueous humour. Ehrlich anticipated that the use of this dye would promote a deeper understanding of the problem, and many ways have since been devised of determining the presence of fluorescein in the eye of the living animal. In the present paper an instrument is described which permits an objective determination of the degree of fluorescence under experimental and clinical conditions.

Corneal thickness in interstitial keratitis.

THE importance of the role played by changes in the thickness of the cornea in the induction of corneal vascularization has recently become increasingly recognized (Cogan, 1948; Ashton, Cook, and Langham, 1951; Langham, 1952, 1953; Ashton and Cook, 1953). However the relationship between the swelling and increase in opacity of the cornea to the rate and progress of vascularization of the cornea in humans has not hitherto been investigated. In the present study, an attempt has been made to bridge this gap in our knowledge by correlating alterations in corneal thickness with the clinical progress of a series of ten cases of specific and non-specific keratitis receiving cortisone therapy. Changes in the general macrosopical and slit-lamp appearances and in the degree of corneal opacity were noted in addition to the measurements of corneal thickness.

COMMUNICATIONS REACTION OF ANIMAL EYES TO SANGUINARINE AND ARGEMONE OIL

OUR interest in the reaction of the eye to the oil extracted from the seeds of Argemone mexicana and to the alkaloid sanguinarine (Fig. 1), a constituent of argemone oil, arose from the recent studies of several groups of investigators. Hakim (1954) observed that subconjunctival injections of either argemone oil or sanguinarine to rabbits and monkeys produced a marked rise in intra-ocular pressure, and he concluded from these and other studies that sanguinarine induced a glaucomatous condition in animals similar to open-angle glaucoma. Lieb and Scherf (1956) studied the effect of intravenous injections of sanguinarine and related alkaloids on the intra-ocular pressure of rabbits, and observed that a single injection of sanguinarine caused an ocular hypertension lasting 30 to 45 minutes. The authors suggested that the alkaloid acted on a pressure regulatory centre located in the central nervous system, and this view found experimental support in the communication of Hakim (1957) that a chronic ocular hypertension could be induced in conscious cats by periodic injections of very small quantities of sanguinarine into the lateral ventricles. Unfortunately, no detailed report on this latter study has yet been published. Finally, Leach and Lloyd (1956) reported that a chronic ocular hypertension in monkeys could be induced by prolonged feeding with sanguinarine, and Shevalev (1957) observed a glaucomatous-like condition in cats injected with argemone oil daily.