THE EFFECT OF ACTIVITY OF RETICULO- ENDOTHELIAL SYSTEM ON THRESHOLD. OF b-wave OF ERG OF DARK ADAPTED RABBIT EYE

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1 THE EFFECT OF ACTIVITY OF RETICULO- ENDOTHELIAL SYSTEM ON THRESHOLD OF b-wave OF ERG OF DARK ADAPTED RABBIT EYE SHIGECHIKA FUJISHITA* Department of Physiology, Osaka City University Medical School, Osaka It has been known that the night blindness occurs in patients with severe liver disease. In 1925 Fridericia and Holm (1) found the cause of night blindness is the dificiency of vitamin A, and in 1931 Tansley (2) reported that the cause of which is the decrease of visual purple in quantity. In1952Kimura and Hosoya (3) found that the absorption maximum (Ď max.) of the visual purple solution extracted from the retinae of liver extirpated toads or frogs, shifted towards shorter wave mĐ and concluded that an insufficiency of visual purple synthesis could be traced in liver extirpated toads or frogs, and that the visual purple obtained may be supposed to be the same material as the isorhodopsin described by Collins (4). In1950Hwang and Hosoya (5) found that choline and betaine were much stronger than pilocarpine in the augmentative effect on visual purple regeneration, and in1950hwang (6) recognized that methionine augmented the regeneration of the visual purple. So-called lipotropic substances such as choline, betaine and methionine, which prevent the appearance of fatty liver, augmented the regeneration of the visual purple. So the auther thought that the retinal functions should be influenced by the activities of liver. So far no attempt has ever been made to study the effect of the activities of Kupffer cells on the retinal functions. But, the effect of vitamin A which is found in Kupffer cells has been observed. In1941, Popper et al. (7) reported that vitamin A is stored chiefly in Kupffer cells, when high amounts of which are present in the liver. The present study deals with the effect of the activities of reticuloendothelial system on the sensitivity of retina. The description of this work will be divided into two parts, one dealing with the method of testing the granulopectic activity of reticulo-endothelial system, and the other with the way in which the threshold of b-wave of electroretinogram (ERG) was obtained. Received for publication December18,

2 RETICULO-ENDOTHELIAL SYSTEM AND ERG 73 PART1. THE CONGO RED TEST METHODS Experimental animals employed were rabbits weighing2.5-3kg. In order to excite the activities of reticulo-endothelial system, rabbit was injected a dose of20mg. carbon per100g. of body weight intravenously. The carbon particles were suspended in a physiological saline solution, and the suspension was centrifuged at3,000r.p.m. for15minutes in order to get rid of small number of large particles of carbon presented in the original preparations. Congo Red Test: 0.1cc. of1% Congo red solution per100g. of body weight was injected into vena retroauricularis of one ear. Four minutes later about 0.9cc. of blood was withdrawn from vena retroauricularis of the opposite ear by a syringe previously containing0.1cc. of3.8% sodium citrate solution. The blood was placed in a tube containing9cc. of3.8% sodium citrate solution and mixed gently. After one hour, another0.9cc. of blood was removed, and treated with sodium citrate as before. The bloods were centrifuged at3,000 r.p.m. for10minutes and the supernatants were obtained. The samples of supernatant were compared in a colorimeter, using the 4- minutes sample as the standard. The standard was set at40mm. and the reading of the1-hour sample was noted. The percentage of dye remaining in the blood was: and the percentage of dye that disappeared was: 100%-% of dye in blood. If the supernatant was contaminated with carbon particles, the following method was employed in order to get rid of these particles. 8cc. of contaminated supernatant was placed into a test tube containing6cc. of isopropyl alcohol and6cc. of isoamyl alcohol and mixed completely. The mixture was centrifuged at3,000r.p.m. for10minutes and two layers were obtained. The upper layer contained Congo red and lower layer contained carbon particles. Using the4-minutes sample as the standard, the samples of upper layer were compared and the calculation was made as before. RESULTS Granulopectic Activity of Reticulo-Endothelial System: Granulopectic activity of reticulo-endothelial system (RES) was calculated by Congo red test. The results of the test obtained from normal rabbits were about35-40%. The results obtained from rabbits which were injected a dose of 20 mg. carbon per 100g. of body weight, were calculated after2hours of injection, and they were about50%. Therefore it seems that the granulopectic activity of RES cells for dye is stimulated by injection of carbon particles.

3 74 S. FUJISHITA PART2. ELECTRORETINOGRAM METHODS The forehead of a rabbit were depilated with strontiumsulfide. The depilatory was wiped away with cotten wool containing a small quantity of ether. Then the rabbit was anesthetized with a dose of0.1g. urethane per100g. of body weight, and was kept more than one hour in a dark room. Electroretinogram was led through a pair of zinc-zincsulphate non-polarizable electrodes placed one on the upper border of cornea and the other on the depilated skin of the forehead. After one hour in the dark, one of the eyes was exposed to monochromatic light for1/15 sec. The wave, lengths of monochromatic light employed were 400, 430, 500, 520, 550, 580, 610,640and670 mit. In order to get monochromatic light, a lamp of 150W (100V) and interference filters were used. The strength of monochromatic light was controlled by a variable resistance, and measured by thermopile in relative energy intensity. If the eye of a rabbit was exposed to light for1/15sec., a- and b- wave of electroretinogram were observed. The present paper deals with the thresholds of b-wave. RESULTS Thresholds of b-wave: In fig.1where the ordinates are given in terms of logarithm of the relative energy and the circles show the thresholds of b-wave of electroretinogram elicited by monochromatic light, the wave lengths of which were400, 430, 500, 520, 550, 580, 610, 640and670mu. Even five hours later, these thresholds of normal rabbit remained constant. Similar results were obtained from20rabbits. Threshold curve of normal rabbit. Scotopic sensitivity curve. Ordinates: logarithm of reciprocal of relative energy at threshold. Abscissae: wave length.

4 RETICULO-ENDOTHELIAL SYSTEM AND ERG 75 In fig.2 the ordinates are given in terms of logarithm of the reciprocal of the relative energy necessary for a threshold response, and the curve shows the scotopic sensitivity curve. In the curve there are two humps, one is in green region (Ď max.=520mu) and the other in red region (Ď max.=610mu). In fig.3 the scotopic sensitivity curve which is drawn in fig.2, by replotting with its points calculated as a percentage of the maximum at520mu, the curve (continuous line) is obtained. This curve is compared with the visual purple absorption curve (brocken line). After the reading of the thresholds of b-wave, a dose of20mg. carbon particles per100g. of body weight was injected into vena retroauricularis under dim light and the rabbit was dark adapted for1hour. After the second dark adaptation, the thresholds of b-wave are obtained in the course of time. In figs.4, 5or6, the uppermost curve is the threshold curve bofore Scotopic sensitivity curve and visual purple absorption curve. Ordinates: percentage of maximum at520mu. Abscissae: wave length : scotopic sensitivity curve. Broken line: visual purple absorption curve. Threshold curve. Ordinates: logarithm of relative energy. Abscissae: wave length. The uppermost curve: scotopic sensitivity curve before injection. Second curve: 2hours after injection. The bottom curve: 4hours after injection. Threshold curve. Ordinates: logarithm of relative energy. Abscissae: wave length. The uppermost curve: scotopic sensitivity curve before injection. Second curve: 2hours after injection. The bottom curve: 3hours after injection.

5 76 S. FUJISHITA FIG. Threshold curve. The upper curve: before injection. Lower curve: 3 hours after injection. Threshold curve. Ordinates: logarithm of relative energy. Abscissae: wave length. the uppermost curve: scotopic sensitivty curve before injection. Second curve: 2hours after injection. Third curve: 22hours after injection. 4th curve: 32hours after injection. The bottom curve: 4hours after injection. Decrease of threshold intensity. after The height of b-wave. A: before injection. B: 3hours injection. injection and the others are threshold curves obtained after injection. It is obvious that after injection the thresholds of b-wave were decreased. In fig.7 decrease of the threshold intensities are plotted against the length of time. Initially the thresholds remain constant for about two hours. During a period of2-4hours after injection they fall rapidly, after which they flatten out again. On the other hand, the carbon particles were cleard from blood in121-2hours. Whenever the threshold decreases, the height of the b-wave elicited by

6 RETICULO-ENDOTHELIAL SYSTEM AND ERG 77 same monochromatic stimulus (equal in energy intensity and in wave length) may be increased. The fact is shown in figs. 8and9. In fig.8, of the two curves shown, the upper was taken before injection, and the lower was taken3hours after injection. The broken line ( in fig.8) shows equal energy level employed in the following experiments. The height of b-wave elicited by monochromatic stimuli before injection is compared with that elicited after3hours of injection. In fig.9 series A shows the b-waves elicited before injeciton and series B shows those elicited after injection. Comparing A with B, the height of b- wave of the latter is larger than that of the former. Therefore it is obvious Increased sensitivity. that the sensitivity of retina was increased by the injection of a dose of injection. Second curve: 32/1hours 20mg. carbon particles per100g. of body weight. In fig.10the scotopic sensitivity curves obtained before and at various intervals after injection are shown. Upper most curve: 4hours after after injection. Third cuve: 22/1 hours after injection. The bottom curve: DISCUSSION According to Popper's work, there was a preponderance of vitamin A in the Kupffer cells over the liver cells under conditions of large vitamin A supply, so the Kupffer cells can be considered as the active cells, which are of great importance in the metabolism of vitamin A. If the metabolism of vitamin A is promoted experimentally, the visual function of retina may be influenced. Under such presumption, a dose of20 mg. carbon particles per100g. of body weight was injected intravenously in a rabbit to accelerate the activities of reticulo-endothelial system. Although the role of reticulo-endothelial system on the metabolism of vitamin A is not yet clear, the author assumes that the metabolism of vitamin A is accelerated when the reticulo-endothelial system is excited. The granulopectic activity of reticulo-endothelial system was tested by Congo red test. The results of Congo red test upon normal rabbits were35-40%, but the results obtained from the rabbits injected with carbon particles were about50 It is certain that the cells of reticulo-endothelial system or the liver cells are excited when a suspension of carbon particles is injected intravenously in a rabbit.

7 78 S. FUJISHITA On the other hand, when a suspension of carbon particles was injected intravenously in a rabbit, increase of scotopic sensitivity was observed. From the facts above described, it may be concluded that at least there is a parallelism between the increase of activity of reticulo-endothelial system (or liver cells) and the increase of sensitivity of dark adapted rabbit retina. The maximum of hump (in green region) of scotopic sensitivity curve is 520 mƒê, and the maximum of visual purple absorption curve is 500 mƒê. Therefore, the cause of the increase of sensitivity in green region may be assumed to be the augmentation of regeneration of visual purple. But, in short wave lengths the scotopic sensitivity curve is shifted toward the long wave length compared with the curve for visual purple absorption. This difference in the curves may be due to the absorption of short wave length in the, refractive media of the eye. The hump in the red region of scotopic sensitivity curve is probably concerned with unknown visual pigment whose maximum of absorption curve is about610mi. The increase of sensitivity in red region of spectrum by a injection of carbon particles may be due to the augmentation of regeneration of the visual pigment. In present study, the metabolism of vitamin A and the regeneration of visual pigment were not observed, so it is better to leave the question open until further evidence is available. SUMMARY (1) This study, performed by Congo red test and electroretinogram, is concerned with the relation between the activity of reticulo-endothelial system and the threshold of b-wave of electroretinogram under normal and under excitatory state of reticulo-endothelial system. (2) The granulopectic activity of reticulo-endothelial system was tested by Congo red test. (3) The activity of reticulo-endothelial system was excited by the injection of a dose of 20 mg. carbon particles per 100g. of body weight. The results of Congo red test obtained under normal state were 35-40%, and that obtained under excitatory state of reticulo-endothelial system (by means of injection of carbon particles) was about 50%. (4) Scotopic sensitivity was calculated as the reciprocal of the threshold of b-wave, so the lower the threshold intensity, the greater was the scotopic sensitivity, and the greater was the increase of the height of b-wave. (5) After2hours of injection of a suspension of carbon particles, the height of b-wave (elicited by monochromatic stimli of which wave lengths are 400, 430, 500, 520, 550, 580, 610, 640 and 670mƒÊ) was rapidly increased. (6) After the injection of a dose of 20mg. carbon particles per 100g. of body weight the threshold intensities of b-wave are decreased. Initially the threshold remained constant for about two hours. During a period of 2-4 hours after injection they fell rapidly, after which they did decrease no more. (7) Scotopic sensitivity curve has two humps, one is in green region and the other in red. The maximum of the hump in green region is 520mƒÊ, and

8 RETICULO-ENDOTHELIAL SYSTEM AND ERG 79 the maximum of the curve for visual purple absorption Therefore, the cause of the decrease of threshold of b-wave in green region may be assumed to be the augmentation of regeneration of visual purple. But the cause of the decrease of threshold of b-wave in red region may be assumed to be the augmentation of regeneration of unknown visual pigment of which maximum of absorption curve The author wishes to thank Prof. Y. Hosoya for helpful suggestions and criticism during the course of the investigation. The expenses of this work were defrayed by a grant from the Ministry of Education. REFERENCES 1. FRIDERICIA, L. S. AND HOLM, E. Amer. J. Physiol. 73: 63, TANSLEY, K. J. Physiol. 71: 442, KIMURA, E. AND HOSOYA, Y. Jap. J. Physiol. 3: 29, COLLINS, F. D. AND MORTON, R. A. Biochem. J. 47: 18, HWANG, T.-F. AND HOSOYA, Y. Jap. J. Physiol. 1: 64, HWANG, T.-F. Jap. J. Physiol. 1: 165, POPPER, H. AND BRENNER, S. J. Nutrition23: 431, 1942.