Clive Graymore

METABOLISM OF THE DEVELOPING RETINA III. RESPIRATION IN THE DEVELOPING NORMAL RAT RETINA AND THE EFFECT OF AN INHERITED DEGENERATION OF THE RETINAL NEURO-EPITHELIUM

PREvIous studies in this laboratory have established that the glycolytic activity of the rat retina is doubled between the 10th and 14th days of life. Elimination of the possibility that light stimulationper se is a causative factor, combined with histological considerations, led to the speculation that this sudden increase might be attributed to the differentiation of the visual cells (Graymore, 1958a; 1959). Considerable support for this hypothesis was gained from the observation that subsequent loss of the visual cells, whether chemically induced or as a result of hereditary degeneration, resulted in a reversion of the glycolytic activity to that of the immature tissue (Graymore and Tansley, 1959a,b; Graymore, Tansley, and Kerly, 1959). Comparative estimations of anaerobic and aerobic lactic acid accumulation prompted the suggestion that respiratory activity might well show a corresponding increase at this time of visual cell differentiation, and the purpose of the present communication is to describe experiments designed to test this hypothesis. Corresponding studies on the rat with retinitis pigmentosa are described, and the implications of these findings are discussed in relation to the role of the visual cell in retinal metabolism.

METABOLISM OF THE DEVELOPING RETINA: I. AEROBIC AND ANAEROBIC GLYCOLYSIS IN THE DEVELOPING RAT RETINA

IN recent years considerable interest has been focused on the role of oxygen in the genesis of retrolental fibroplasia, and careful control of ambient oxygen concentration has led to a considerably reduced incidence of this disease in premature infants. Much of the work which led to the ultimate elucidation of this problem and the demonstration that hyperoxia induced vasoobliteration in the immature retina has been reviewed elsewhere (Ashton, 1957). Although the effect of hyperoxia is now recognized as a practical phenomenon little is yet understood of the mechanism of the resulting vasoobliteration, and why it should be confined to the immature retina. The importance of this distinction has been underlined by a recent report in which it has been demonstrated that intravitreal injection of the kitten with sodium fluoride or sodium iodoacetate produces total vaso-obliteration of the retinal vessels in a manner closely resembling that induced by oxygen exposure and that once again this effect is confined to the immature retina (Ashton, Graymore, and Pedler, 1957). There is evidence to suggest that sodium fluoride inhibition promotes retinal oedema both in vitro (Graymore, 1958a, 1959), and in vivo (Pedler, 1958), and it has been tentatively suggested that external pressure resulting from this swelling may be a causative factor in chemically induced vaso-obliteration (Ashton and others, 1957). Whatever the actual underlying cause may be, the differing reaction of immature and mature retinae both to oxygen and metabolic ihnhibitors suggests some basic distinction between the two retinae, either structurally or metabolically or both. The present report is designed to indicate one aspect of retinal metabolism which exhibits a changing pattern during development.

METABOLISM OF THE DEVELOPING RETINA. 7. LACTIC DEHYDROGENASE ISOENZYME IN THE NORMAL AND DEGENERATING RETINA. A PRELIMINARY COMMUNICATION.

The isoenzymes of retinal lactic dehydrogenase have been separated employing electrophoresis on cellulose acetate paper. The results confirm those of other workers using more elaborate procedures in that there are five fractions each of which is capable of reacting with either NAD or NADP. Greater activity is shown by all fractions with the former. Marked variations have been found in fraction five both during the development of the retina and between the retina of normal and retinitis pigmentosa rats. The significance of these findings is discussed with special reference to tissue differentiation.

IODOACETATE POISONING OF THE RAT RETINA II. GLYCOLYSIS IN THE POISONED RETINA

REPEATED intravenous injections of sodium iodoacetate produce a characteristic visual cell degeneration in the monkey, cat, and rabbit (Schubert and Bornschein, 1951; Noell, 1952), the resulting histological picture being very similar to that presented in human retinitis pigmentosa (Noell, 1953). Recently, it has been shown that a similar condition can be produced in the rat by careful injection of two doses of 30 mg. iodoacetate per kg. body weight, an interval of 4 to 5 hrs being allowed between the two injections (Graymore and Tansley, 1958a). Simultaneous administration of sodium malate was shown to decrease the mortality rate of animals treated in this manner, and yet increase the severity of the visual cell damage. In this way it is possible to obtain experimental animals in which the visual cell population is almost completely destroyed. As early as 1924, Warburg and his associates suggested that all the elements of the retina may not make an equal contribution to the general biochemical picture (Warburg, Posener, and Negelein, 1924) and since then there have been indications that the nerve and receptor components may possess essentially different patterns of metabolism (Noell, 1952; Sjostrand, 1953; Strominger and Lowry, 1955; Lowry, Roberts, and Lewis, 1956). Noell (1952), on the basis of the differing effects of anoxia and iodoacetate on the electroretinogram of rabbits, believed that iodoacetate acted on some mechanism which was not dependent on oxygen, and suggested that the specific action of this substance on the visual cells might be due to the fact that these cells possessed a predominantly glycolytic form of metabolism. It was felt that a study of glycolysis in retinae from rats treated in the manner described above might help to elucidate both the mechanism of action of iodoacetate on the retina as well as certain aspects of the differential metabolism of this tissue. A preliminary report of part of this work has been published elsewhere (Graymore and Tansley, 1958b).

IODOACETATE POISONING OF THE RAT RETINA: I. PRODUCTION OF RETINAL DEGENERATION

IT is now well established that retinal degeneration can be produced in several species of laboratory animals by intravenous injection of sodium iodoacetate (Schubert and Bornschein, 1951; Noell, 1952; Berardinis, 1953; Karli, 1954; Rabinovitch, Mota, and Yoneda, 1954; Babel and Ziv, 1956). Thus, Noell (1952) described a destruction of the visual cells in rabbits, cats, and monkeys after repeated doses, and drew attention to the close similarity between the experimental condition and retinitis pigmentosa in man (Noell, 1953). Most workers have used rabbits for these studies, but Rabinovitch and others (1954) recorded the same result in chickens. So far the rat has been found to be an unsatisfactory animal for such experiments; Rabinovitch and his co-workers (1954) failed to obtain retinal degeneration in this species after repeated sub-lethal doses. Noell (1952) has remarked on the difficulty of finding a single sub-lethal dose which would produce histologically recognizable damage to the retina. Most of his studies in rabbits involved the use of repeated doses of 15 to 20 mg./kg. body weight (12 mg./kg. in the cat). In this laboratory, however, it has been found possible to produce histological damage in the rabbit by giving a single injection of40 mg./kg. In order to achieve this without killing the animal it was necessary to administer the dosein at least 10 ml. saline over a period of about 20 minutes (Tansley, 1955). The present observations arose out of attempts to produce retinal degeneration in the rat for metabolic studies. We also failed to achieve this in the rat with a single sub-lethal dose and therefore tried other procedures.

STUDIES ON DEVELOPING RETINAL VESSELS V. MECHANISM OF VASO-OBLITERATION (A Preliminary Report)

SINCE the discovery of the specific effect of oxygen on the immature retina, which manifests itself in vasoconstriction leading to total obliteration of the ingrowing vascular complexes (Ashton, Ward, and Serpell, 1953, 1954), the work of this department has been concerned with experiments designed to elucidate the mechanism of this effect. The evidence that the phenomenon is entirely confined to vessels actually growing in the retina, and then only when the retina is immature and in its normal anatomical position, has been recently reviewed elsewhere (Ashton, 1957). Broadly speaking there would appear to be three main theoretical explanations for the closure of these vessels:

Lactic acid dehydrogenase (LDH) in the cornea: I. Content, nature and distribution

The total content, nature and distribution of lactic acid dehydrogenase have been investigated in the epithelium plus endothelium, and stroma, of the rabbit cornea. An attempt has been made to interpret the information obtained in relation to the general glycolytic and oxidative metabolism of the tissue. The possibility that the lactate formed by the stroma is utilized by the epithelium is considered.

The metabolism of the retina of the normal and alloxan diabetic rat. The levels of oxidised and reduced pyridine nucleotides and the oxidation of the carbon-1 and carbon-6 of glucose.

Abstract The levels of oxidised and reduced NAD and NADP have been determined in the retinae of normal and alloxan-diabetic rats. In the alloxan-diabetic animal the ratios of NAD/NADH2 and of NADPH2/NADP are both reduced, as a result of an increase of NADH2 and a decrease of NADPH2. The relative rates of oxidation of the carbon-1 and carbon-6 of glucose have been studied using C-14 labelled glucose. A C-1/C-6 ratio of 1.13 was obtained for both normal and alloxan-diabetic animals. The levels of the oxidised and reduced co-enzymes in the retina of the normal rat are similar to those of brain and contrast with those of liver; this would suggest a minimum activity for the pentose phosphate pathway. This idea is supported by the ratio of oxidation of C-1/C-6. The co-enzyme changes in the retina of the alloxan-diabetic rat could be taken to infer an increased glycolytic rate, but a reduced rate of breakdown by the shunt mechanism. This possibility is not reflected in the C-1/C-6 ratios. The factors that complicate these interpretations are discussed.

Levels of soluble NAD-linked α-glycerophosphate dehydrogenase and lactic acid dehydrogenase in rat retina

The retina of the rat has a highly active LDH system but shows very little GPDH activity. There is an increase in both these levels during development, but it appears that GPDH plays no significant part in the reoxidation of NADH 2 produced by GAPDH. This regeneration is probably effected by the pyruvate-lactate system, a situation analagous to that in tumour cells. The mechanisms involved are discussed in relation to enzyme distributions.

Coenzyme dependency of alcohol dehydrogenase in the retina of the rat. II. Histochemistry

Abstract The distribution of ethanol and retinol dehydrogenases is investigated histochemically in the retina of the rat. Negligible activity is demonstrable when ethanol is the substrate. When retinol serves as substrate, however, strong activity is seen in the outer layers of the retina, particularly in the outer limbs of the visual cell. NADP appears to be the coenzyme of choice.