QUARTERLY JOURNAL OF EXPERIMENTAL PHYSIOLOGY

612.397: 612.89.03 QUARTERLY JOURNAL OF EXPERIMENTAL PHYSIOLOGY THE EFFECT OF SYMPATHECTOMY ON THE FATTY DEPOSIT IN CONNECTIVE TISSUE.1 By A. B. L. BEZNAK and Z. HASCH. From the Institute of Experimental Pathology, University of Budapest. (Received for publication 9th November 1936.) IN this paper we describe experiments dealing with the physiology of the fatty deposit in connective tissue. The experiments prove that for the perirenal connective tissue to serve as an organ of fat storage, it is necessary that its sympathetic nerve-supply should be intact, and it seems likely that the same is true, though to a smaller degree, of any other connective tissue. If the sympathetic nerves are severed, changes take place in the metabolism of the connective tissue in the regions supplied by these nerves, and there occurs an increase in the weight of the fatty deposit on the sympathectomised side as compared with the normal, when the cats are fattened after the operation. When the cats are losing weight more fat remains on the sympathectomised side than on the normal. From these and other results the conclusion is drawn that the metabolism of connective tissue deprived of its sympathetic supply is changed in such a way that both the mobilisation and deposition of fat are slowed down, the former more so than the latter. It is well known that the fat content of muscles increases when they are rendered inactive by cutting their mixed nerve-supply. Mansfeld and Muller [1913] subjected this phenomenon to experimental inquiry and came to the conclusion that the increase of fat in denervated muscle is due to the fact that under normal conditions the muscle receives fat-mobilising influences from two sources, namely, the nervous system and the blood. Severance of the nerve-supply deprives the muscle of one of these influences, hence the increase of the fat content. 1 The results of the experiments described in this paper were presented to the Hungarian Physiological Society [Beznak and Harris, 1934; Beznak and Hasch, 1935] and to the Physiological Society [Beznak, Hasch, and Harris] at its Oxford meeting, 1935. VOL. XXVII., NO. 1.-1937. 1

2 Beznak and Hasch The changes in the chemical composition of muscle after tenotqmy and neurotomy were, however, subjected to detailed experimental study by Andora [1933]. Among other changes the fat content of such muscles was found to increase to 2-3 times the normal value, and since the changes after tenotomy were the same as after neurotomy, it is clear that the increase of the fat content in the muscle is the consequence of the inactivity and not the absence of some trophic influence on the part of the nerve. Increase of the fatty deposition in connective tissue is specially interesting, because this tissue, in contrast to muscle, exhibits apparently no function which is subject to nervous control, as is the case, for example, in muscle contraction. Consequently its denervation does not lead to any apparent "inactivity," and an increase in its fat content would not be expected. While this work was in progress there was no direct experimental evidence that the store of fat in connective tissue was under the influence of the nervous system. After the publication of our results [Beznatk and Harris, 1934], however, a paper by Hausberger [1935] appeared, and his conclusions confirm our own. This paper will be discussed later. METHODS. Our conclusions are based on experiments carried out on 98 cats, 20 rabbits,- and 14 rats. In the course of this work more than 160 cats in all were used, but some died from the operation, some from captivity, and some from intercurrent disease. In the first series of our experiments the cats were fed on kitchen scraps. They lived in groups of five in large cages. The individual daily food consumption was not controlled, but the body-weight was frequently taken and only those were used which showed a gradual loss in weight. The cats described in Tables II., III., IV., V., and VI. were fed in this way. In the second series of our experiments (Tables VIII.-X.) the cats received a synthetic diet, referred to in this paper as Diet II., of the following composition: 33 per cent. finely minced horse-flesh, 33 per cent. lard, 33 per cent. bread, salt. The mixture was thoroughly minced, mixed, cooked, and made into sausages. Known amounts of this food were given in excess to cats living in separate cages, and the unconsumed sausage was reweighed. In some experiments the cats were fed on the same diet but its fat was stained with Sudan III. The fat was weighed, melted, and 1 mg. of Sudan III. per g. fat was slowly dissolved therein. In the experiments in which our aim was to fatten the cats every possible measure was taken to make them eat as much as possible. Thus the diet, besides the above described sausage, contained fresh liver, warm milk with sugar, boiled egg, sardines, etc. A series of experiments was also carried out on hungering cats. They received nothing but water.

The Effect of Sympathectomy on the Fatty Deposit The rabbits were fed on green cabbage and oats. The rats received a mixed diet, namely, 10 g. of Diet II. and 10 g. of the following: casein 55 per cent., butter 25 per cent., lard 15 per cent., cod liver oil 5 per cent., salt mixture, and 5 per cent. brewers' dried yeast. When the rats were given Sudan-stained fat the staining was carried out as described in the case of cats. All operations were performed under ether anaesthesia with aseptic precautions. During extirpation of the superior cervical ganglion care was taken not to damage the jugular ganglion of the vagus. Extirpation of the stellate ganglion in cats was done under artificial respiration, the thorax being opened on the right side between the first and second ribs, the ganglion lifted with a pair of long forceps and excised. When the splanchnic nerves were to be divided in cats, rabbits, or rats, this was done on the left side just below the diaphragm and above the cceliac ganglion. In some cats the sympathetic chain and ganglia were extirpated from the 4th lumbar ganglion above to the last sacral below. The following fatty deposits were weighed to the nearest centigram: perirenal fat, fat in the pericardium, fat in the mesentery around the ureter and in the inguinal region, and the subcutaneous fat in the hind leg and abdomen on either side. The fatty deposits were always dissected out on both sides, great care being taken to carry out the dissection as symmetrically as possible. It is easy to find the boundaries of the perirenal fatty tissue since it is well circumscribed. The pericardial fatty tissue forms a W-shaped mass in the parietal pericardium, the top of the W lying on the base of the heart; the middle of the W was cut out, the two sides divided and weighed separately. The calorimetric estimations of the concentration of Sudan III. in the fatty deposits were carried out in the following way: About 200 mg. of the fatty tissue was weighed on the torsion balance, ground thoroughly with quartz sand, and extracted in the centrifuge with hot alcohol or benzene till the solvent was no longer stained. This required 7-8 extractions. The mixed extracts were concentrated under reduced pressure to 4-5 or 8-9 c.c. according to the intensity of the colour. Filtration through paper moistened with solvent followed. The filtrate was poured into a measuring flask of 6 or 10 c.c. capacity according to the depth of colour. The estimation of the Sudan III. content of the extract was made in a Zeiss "Stufen-photometer," the intensity of colour in the extract being compared with that in a known solution of Sudan III. The results are expressed as y of Sudan III. in 1 g. of fatty tissue. EXPERIMENTAL RESULTS. The first series of experiments deals with the effect of unilateral splanchnicotomy on the perirenal fatty tissue in cats, rabbits, and rats, receiving a normal diet, but slowly losing weight. Table I. shows the weight of the perirenal fatty tissue in the normal animals 3

4 Beznak and Hasch TABLE I.-THE EFFECT OF LEFT SPLANCHNICOTOMY ON THE WEIGHT OF THE PERIRENAL FATTY TISSUE IN CATS, RABBITS, AND RATS. Normal animals. Splanchnicotomy on the left side. Average weight of perirenal Average weight of perirenal Animal. No. cases. of fatty tissue (g.). No. cases. of fatty tissue (g.). Left. Right. Left. Right. Cats. 23 601 5*54 8 2-31 096 Rabbits. 9 3-28 3 39 8 2*64 1-88 Rats.. 14 009 009 14 010 007 and in those in which splanchnicotomy preceded the weighing of the perirenal fatty tissue by 18-55 days. In comparing the differences in weight of the perirenal fatty tissue between the right and left sides in the normal animal with the corresponding values in the splanchnicotomised animal, it is evident in all three species that the perirenal fatty deposit weighs more on the sympathectomised than on the intact side. In cats the difference was from +41 to +300 per cent. without an exception in 8 cases (Table II.). In the case of rabbits the difference was from +21 to +75 per cent. in 8 cases, without exception. In rats, after splanchnicotomy in 11 cases out of 14, the perirenal fat weighed from + 16 to + 158 per cent. more. If the weights of the perirenal fatty tissue on the intact right side were compared in normal and experimental animals it is seen that after the left splanchnic nerves have been divided the weight of the perirenal fat tends to decrease. The decrease is well marked in cats, slight in rabbits, and scarcely present at all in rats (Table I.). It may be that this difference is partly accounted for by the probability of error in selecting the cats. Weights as low as those found in the operated cats are, indeed, met with in the control ones. The fact that cats are so variable in this respect and that the number of experimental animals is only 8 increase the likelihood of this cause. Another cause of this difference is certainly the fact that all the animals except the rats refused to eat for a few days after the operation; mobilisation of fat is in consequence going on. In a few experiments, therefore, simple laparotomy under ether anesthesia was carried out, the splanchnic nerves on the left side being exposed but left uncut (Table III.). After the operation the cats were treated as those in Table II. They slowly lost weight, and when they were killed on the 21st day after operation an even loss in the weight of perirenal fatty

The Effect of Sympathectomy on the Fatty Deposit TABLE II.-THE WEIGHT (IN G.) OF THE PERIRENAL FATTY TissuE IN NORMAL CATS AND IN THOSE IN WHICH THE LEFT SPLANCHNIC NERVES HAD BEEN CUT SOME DAYS BEFORE. 5 Normal. Left splanchnicotomy. -- Left. Side. Right. Differences. g. Per cent. Left. Side. Right. Differences. g- Per cent. D. 0-66 12-20 19-10 3-75 3-12 0.99 3-11 0 53 6-45 10-00 5 40 8-80 7 50 5 40 7*80 7-10 3-45 5 90 5 05 5-20 7-35 3 00 6-40 0-64 10-56 16-84 3.75 2-69 0-95 2-63 0-48 7-10 9*00 5-60 7-20 7-35 5-75 7 50 7 30 4-00 4-20 4-90 4-20 6-00 3-45 540 +0-02 +1-64 + 2-26 + 0 00 +0 43 +0-04 +0-48 + 0 05-0-65 +1-00 -0-20 +1-60 + 0-15 - 0 35 + 0 30-0-20-0.55 +1-70 +0-15 + 1-00 +1-35 - 0-45 + 1-00 I + 3 +15 +12 + 0 +16 + 4 +18 +10-10 +10-4 + 22 + 2-6 + 4-3 - 15 +40 + 3-24 + 22-13 +18 _ 2-94 1-50 3 50 3-86 3-10 2-25 093 040 0-96 0-60 1-45 1-80 0-80 1*60 0*35 0-10 +1-98 +0 90 + 2-05 +2-06 +2-30 +0-65 +0-58 +0 30 + 206 +150 +141 +114 + 288 + 41 +165 + 300 D =days after splanchnicotomy. tissue on both sides was found. It follows, therefore, that splanchthe amount of perirenal fat which nicotomy diminishes the fall in follows laparotomy under ether ansesthesia. In the second series of experiments we investigated the effect of extirpation of the cervical sympathetic and of the stellate ganglion on the pericardial fatty tissue. Cats were used in these experiments. Table IV. shows the results which were obtained in normal cats as controls and in cats in which extirpation of the stellate ganglion, either alone or with the superior cervical ganglion, was carried out 6-24 days before the fatty deposits were weighed. The weight of the fatty deposit in the pericardium of normal cats is fairly constant, showing a variation of ± 10 per cent. On the other hand, in 10 out of 14 cats in which the stellate ganglion was removed the weight of the pericardial fatty tissue was on the average 60 per cent. greater on the right sympathectomised than on the normal side. We also carried 21 21 21 21 18 33 33 33

6 Beznak and Hasch TABLE III.- THE WEIGHT (IN G.) OF THE PERIRENAL FATTY TISSUE IN CATS LAPAROTOMISED UNDER ETHER AN.2STHESIA 21 DAYS BEFORE THE WEIGHING. Left. Right. g- Per cent. 4-72 4-42 11-02 4-82 2-25 3 20 5 20 3 60 4-57 3-86 10-05 4-23 2-60 2-72 5-69 3 50 +0-15 + 0-56 +0-97 +0-59 - 0-35 + 0 48 +0-49 + 0*10 + 32 + 14 5 + 9-6 + 14-0 - 13-0 + 17-0 - 8-6 + 30 TABLE IV.-THE WEIGHT (IN G.) OF THE PERICARDIAL FATTY TISSUE IN NORMAL CATS AND IN THOSE IN WHICH THE RIGHT STELLATE GANGLIA ON THE ONE HAND, AND THE RIGHT STELLATE AND SUPERIOR CERVICAL GANGLIA ON THE OTHER, WERE EXTIRPATED. Normal. Right stellate ganglion extirpated. Left. Side. Right. Differences. g. Per cent. Left. Side. Right. Differences. g. Per cent. A. B. D. 1 13 0-18 0-10 0 20 0 54 0 23 0-11 015 0o15 0 13 0-90 0-15 0-10 0 20 0 54 0 27 0 12 0-16 0-13 0 14 + 0.23 + 0-03 0*00 0*00 0*00-0-04-0-01-0-01 +0.02-0-01 +26 +20 0 0 0-15 - 8-6 +15-7 0.05 0 14 0.80 1 20 0 70 0 60 1-00 1.55 1 62 2 70 0 40 0 42 0 45 0 85 0 70 0 75 0 90 1-05 0 57 0 78 + 0 09 + 040-0-10 + 0-55 + 108 + 0O02 +0-40 + 0*05 + 0-15 +0-21 + + i+ i+ 180 50 14 55 675 88 7 16 37 2700 3650 4300 2950 3750 3850 3050 3350 2400 4150 1550 3750 4800 3050 4000 4000 3400 3400 2650 3300 G. stellatum + g. cervic. sup. extirp. 0-95 1-90 + 0 95 + 100 3550 2150 0 90 1-50 +0 60 + 66 3800 2800 1-25 1-00 - 0-25 - 20 4100 4000 1-55 1-15 -0-40 - 25 3500 3650 23 23 22 23 22 22 22 21 21 6 12 11 24 24 A = the weight of the cat at the time of splanchnicotomy. B the weight of the cat at the end of the experiment. D= days after the operations.

The Effect of Sympathectomy on the Fatty Deposit 7 out sympathectomy in the neck, and found, as Table V. shows, an increase in the pericardial fat on the sympathectomised side similar to that found after extirpation of the stellate ganglion. TABLE V.-THE WEIGHT (IN G.) OF THE PERICARDIAL FATTY TISSUE IN CATS IN WHICH THE RIGHT SUPERIOR CERVICAL GANGLION WAS EXTIRPATED. Side. Differences. ~D Right. Left. g. Per cent. 0-66 050 +0-16 + 32 48 0-90 0-61 + 0-29 +48 17 0-52 0-38 +0-14 + 37 17 0 78 0-57 +0-21 + 37 8 1-05 0-90 +0-15 +16 16 0*45 0-40 + 0-05 +12 52 0-40 0*60-0-20-33 52 D =days after extirpation. In the third series of experiments are cats in which the abdominal sympathetic ganglia were excised on the left side before the fatty deposits in the abdominal connective tissue on the left and right sides were weighed. In 8 cats the 1st, 2nd, and 3rd sacral and the 4th and 5th lumbar ganglia were excised. All the cats survived this operation, but after 3-6 days they began to show signs of intestinal disorder. They lost weight quickly and grew so thin that at autopsy practically no fat was found in some of them either in the subcutaneous or in the peritoneal connective tissue. In those cases which survived a reasonably long period and contained sufficiently fatty deposit, the subcutaneous and peritoneal fat on the left and right sides was dissected out and weighed. The results are given in Table VI. From this it is clear that on the sympathectomised side the fatty deposit was much greater than on the normal side. We conclude, therefore, that in this anatomical region also the normal function of the connective tissue as a store of fat depends on the integrity of its sympathetic nerve-supply. We now wished to discover the nature of the change taking place in the connective tissue after division of its sympathetic supply and which is responsible for the increase in the fatty deposit. In these experiments the effect of splanchnicotomy on the perirenal fat was exclusively used for the following reasons. First, the effect is one which can be demonstrated with absolute certainty. Second, Gage and Fish [1924] proved that among the fatty deposits in the body this is the most readily influenced. For example, when Sudan-stained

8 Beznak and Hasch TABLE VI.-THE WEIGHT (IN G.) OF THE FATTY DEPOSITS IN THE ABDOMEN AND SUBcuTANEOuS TIssuE OF THE CAT AFTER THE LUMBAR AND SACRAL SYM- PATHETIC G(ANGLIA HAD BEEN EXTIRPATED ON THE LEFT SIDE. Abdominal Fat. Subcutaneous Fat. Side. Differences. Side. Differences. D. Right. Left.Per Right. Left. g. Per Right. g. cent. cent. 2-0 6-0 +4 0 +200 8-0 12-0 + 4 0 50 14 6-5 9.4 +2.9 + 45 30 7 45.4 +14 7 48 10 1-8 4-6 +2.8 +155 0-8 2-9 + 2.1 263 18 1-4 3.7 +2.3 +165 0-9 2-6 + 1.7 189 10 D =days after the operation. fat is fed to cats it makes its appearance first and most visibly in this situation. Third, the operation itself is so easy that it can be carried out in 6 minutes; trauma of the experimental animal is thus minimal. The amount of fat in the deposits is the result of an equilibrium, the rate of fat mobilisation balancing that of fat deposition. We wished to discover how this equilibrium was changed in such a way that an increase in the fatty deposit results. Theoretically there are the following possibilities: (a) On the splanchnicotomised side, the rate of mobilisation of fat may be slowed down; (b) the rate of fat deposition may be increased; (c) both may be slowed down, but the latter less than the former; (d) the rate of mobilisation decreases and the rate of deposition increases. Now, if the rate of mobilisation is diminished on the splanchnicotomised side, in starved cats more fat should remain on this than on the intact side. That this is actually the case is shown by the data in Table VII. In each of 9 cats the weight of fatty deposit on the splanchnicotomised side was greater than on the normal side, the average difference being 67 per cent. All the cats lost greatly in weight, and there is no doubt that they used their fatty deposits throughout the body. If, therefore, in one circumscribed part of the body more fat remains than in the other symmetrically placed part, it can safely be concluded that on the side where more fat is found, the rate of mobilisation is diminished. We proved this also in another way. Gage and Fish [1924] have shown that Sudan-stained fat is absorbed and deposited as such. They have thus been able to follow the course of fat after absorption. We therefore fed 10 cats with Diet II., in which the fat was stained with Sudan III., till their fatty deposits were well stained by the addition of the Sudan-stained fat. The degree of this staining may

The Effect of Sympathectomy on the Fatty Deposit 9 TABLE VII.-THE WEIGHT (IN G.) OF THE PERIENAL FATTY TISSUE IN STARVING CATS AFTER LEFT SPLANCHNICOTOMY. Perirenal fat. Side. Differences. A. B. D. Left. Right. g. Per cent. 8-30 4*20 +4-10 + 98 3650 2600 16 17-48 10-90 +6-58 + 60 5100 3900 15 15-65 11 30 +4-35 + 39 4300 3300 15 1-29 1-14 +0-15 + 13 3750 2700 16 1.10 0 68 +0-42 + 61 4150 2500 19 3-10 1-36 +1-74 +127 3050 2500 7 1-80 1-10 +0 70 + 63 3600 2650 21 6-85 3-50 + 3-30 + 94 3550 2850 17 7 60 4 90 +2.70 + 54 2700 2200 17 A = the weight of the cat at the time of splanchnicotomy. B = the weight of the cat at the end of the experiment. D = days after splanchnicotomy. be roughly assessed by the colour of the pad, as this becomes pink if sufficient Sudan - stained fat is deposited in the subcutaneous connective tissue. Cats show great individual variation in the degree of subcutaneous staining. Some become deeply stained in a few days (4-8), others less so. As will be seen in columns 1 and 2 of Table VIII., the concentration of Sudan in cats varies between 16 and 116 y/g. of fat. Although cats show such great individual variation in the degree to which they deposit Sudan-stained fat, there is a difference of ± 15 per cent. only between the right and left sides. When the fatty deposits contained sufficient Sudan - stained fat the abdomen was opened with aseptic precautions under ether anaesthesia, and 200-200 mg. of the perirenal fatty tissue were excised on each side for estimation of their Sudan III. contents. On the left side splanchnicotomy was then carried out. The cats were allowed to starve, and when they died, 8-14 days later, the perirenal fatty tissue on both sides was weighed and its content in Sudan III. estimated. Table VIII. shows the results to be the same as those contained in Table VII.; in every case the weight of the perirenal fatty tissue on the splanchnicotomised side was greater than on the normal, the average being + 53 per cent. The Sudan estimations in experiment 7 could not be used, as in that case on the right side too little fat remained and the estimation of Sudan became therefore unreliable. As will be seen in the table

10 Beznak and Hasch TABLE VIII.-THE WEIGHT (IN G.) AND SUDAN III. CONTENT (IN y/g.) Or PERIRENAL FATTY TISSUE IN CATS. RIGHT SIDE NORMAL; LEFT SIDE TOTAL SPLANCHNICOTOMY. AFTER- WARDS THE CATS STARVED. Concentration of Differences Final con- Differences Of Sudan III. in Sudan III.. in final con- Weight of on the day concentra- of Sudan III. centration fat. of splanch- tion. D. Of Sudan III. nicotomy. cenaincntrat ion p erirenal Differences.. Left. Right. Per Left. Pright Per Left. Right. Per cent. Rih. cent. - cent. 103 85 +18 +21 8 37 76-39 -51 059 0*38 +0*21 + 55 41 33 + 8 +24 8 55 34 +21 + 62 5-71 4.04 +1-57 + 39 116 87 +29 +33 8 94 109-15 -13 1.20 0 89 +0-31 + 35 97 64 + 33 +52 14 68 96-28 - 29 2-26 1*13 +1-13 +100 86 95-9 - 9 14 96 88 + 8 + 9 1.07 0.81 +0-26 + 32 47 45 + 2 + 4 11 41 45-4 -8 0.96 0.64 +0-32 + 50 22 26-4 -15 11........ 126 0.74 +0-52 + 70 16 17-1 - 6 11 10 11-1 -9 1*55 0.96 +059 + 62 68 65 + 3 + 5 11 57 64-7 -11 093 0*56 +0-37 + 66 26 22 + 4 +18 11 24 20 + 4 +20 2*24 1-82 +0-42 + 23 D =days after the splanchnicotomy. the Sudan contents of the fatty deposit on both sides at the beginning of the experiments is equal within ± 15 per cent. At the end of the experiments there was again no difference between the Sudan concentrations on the two sides. Moreover, when the Sudan concentration at the end of the experiment is compared with that at the beginning it is seen to lie within the ± 15 per cent. variation. We conclude from these results that at the beginning of the experiment there was homogeneously stained fatty deposit on each side, and that when the fat was mobilised fat and Sudan were liberated at a constant ratio. If this were not so, the concentration of Sudan could not have remained unchanged. Further, since on the splanchnicotomised side more fat was found than on the normal and the Sudan concentration remained equal on the two sides, it follows that during the starvation less fat was mobilised on the operated than on the normal side. The question of the part played by fat deposition was investigated in the following way. Fourteen rats were splanchnicotomised on the left side. For 9 days after the operation they were fed on Diet II. containing unstained fat. After this they were starved for 48 hours and then given the same diet, with the exception that the fat was stained with Sudan III. The rats were killed at different times after the feeding, as indicated in Table IX., column 2. The Sudan content of the perirenal fat on both sides was observed and compared. It

The Effect of Sympathectomy on the Fatty Deposit 11 TABLE IX.-THE SUDAN III. CONCENTRATION (Y/G.) OF THE PERIRENAL FAT OF RATS IN WHICH THE LEFT SPLANCHNIC NERVES WERE CUT 9 DAYS PREVIOUSLY. Weight of Concentration Dfeecs perirenal Differences. of Sudan III. Differences. fat (mg.). No. H. Left. Right. Left. Right. Mg. tpe Left. cent. 1 1 0 0 427 362 +65 +18 15 7.9-6-4-81 2 2 0 0 3 4 + + 4 5 + + + + Estimations carried out in the mixed perirenal fats of rats 5 6 + + + + 11, 12, 13, 14. 6 7 ++ +++ 7 7 + + + + H =hours after feeding the rats. 8 7 + + + + + + =just perceptible staining. 9 7 + + + + + + + =visible staining. 10 7 + + + + + + + + =strong staining. 11 17 + + ++++ + + + =very strong staining. 12 17 ++ ++++ 13 17 ++ ++++ 14 17 ++ ++++ was found that the perirenal fat began to show signs of Sudan-stained fat deposition after the 4th hour. From this time on the intensity of the colour increased till the 17th hour. From the 7th hour, when the staining was well marked in 7 out of 9 cases, the right side was distinctly redder than the left. In one case the right side was only slightly redder than the left and in one case the two sides were equally stained. No case was found in which the left side was more deeply stained than the right. Unfortunately the small amount of perirenal fat in the rat made it impossible to estimate its Sudan content in each case separately. The perirenal fat from the left side of rats 11, 12, 13, and 14 was therefore mixed, the rats being killed after 17 hours, and the Sudan concentration was estimated. This was found to be 1-5 y Sudan/g. fat. The perirenal fat on the right sides of these rats was also mixed and the Sudan concentration estimated. It was found to be 7i9 y/g. In the perirenal fatty tissue of the normal side, therefore, roughly four times as much Sudan-stained fat was deposited during the same time as in that of the splanchnicotomised side. This conclusion is not invalidated by the fact that on the splanchnicotomised side the fatty deposit was greater, seeing that the difference was merely 20 per cent. The differences found in the Sudan concentration, therefore, cannot be explained by the supposition that equal amounts of Sudan-stained fat are deposited on both sides, but that, the perirenal fat being greater in amount on the splanchnicotomised side, the

12 Beznak and Hasch deposited Sudan-stained fat is diluted more than on the normal side. The only explanation is that the quantity of Sudan-stained fat deposited on the operated side is less than that deposited on the normal side. Similar experiments on cats led to the same conclusion. Four cats in which left splanchnicotomy had been carried out 21 days previously were starved for 48 hours. 150 c.c. of goose fat stained with Sudan III. were then given to each by stomach tube. The cats were killed 18 hours later, and 200-200 mg. of perirenal fat were removed for Sudan estimation. The perirenal fat was not weighed, but distinctly more fat was present on the left side than on the right. Table X. contains TABLE X.-SUDAN CONCENTRATION, Y/G. FAT, IN CATS. LEFT SPLANCHNIC NERVES CUT. THE CATS WERE KILLED 18 Hoims AFTER FEEDING. Side. Differences. Right. Left. Per cent. 94 25-69 - 74 42 14-28 - 66 72 19-53 - 73 113 25-88 -80 the results of the Sudan estimation, and it will be observed that the concentration of Sudan III. on the right side was 4, 3, 4, and 4j times greater than on the left splanchnicotomised side. There is, however, one fact which needs consideration. As is seen in Table VIII., cats which have been fed with Sudan-stained fat for a prolonged period are subjected to left splanchnicotomy and then starved, a difference in the final concentration of Sudan III. in the fatty deposit does not appear, although the weight of the fatty deposit is greater on the splanchnicotomised side. The contradiction of the results of acute experiments is merely apparent. In the acute experiments a certain amount of Sudan-stained fat is added to the uncoloured deposit fat. The intensity of staining after a certain time will depend on the rate of deposition of the stained fat. In prolonged experiments, on the other hand, there is time enough for all fatty deposits, whether slow or quick in their uptake of stained fat, to reach the maximum concentration of Sudan-stained fat. Once this maximum concentration is reached with a surplus of Sudan-stained fat in the diet and blood, no difference can be found between the concentrations of Sudan-stained fat on the normal and splanchnicotomised sides. We are justified in concluding, therefore, that in acute experiments the rate of deposition of fat in the connective tissue is less when the connective tissue is deprived of its sympathetic nerve-supply.

The Effect of Sympathectomy on the Fatty Deposit We have seen that the rate of fat deposition is slowed down in the perirenal tissue after ipsilateral splanchnicotomy. From this it follows that if fat mobilisation is proceeding on both sides at the same rate, the amount of fat deposition on the splanchnicotomised side is less than on the normal. We found, on the contrary, an increase on this side. But it was shown in fasting animals, when the above equilibrium is artificially shifted into the direction of mobilisation, that the fat in the perirenal connective tissue on the denervated side is considerably less mobilised than is that on the normal side. We are forced to the conclusion, therefore, that after splanchnicotomy both the deposition and mobilisation of fat in the denervated area are retarded, but the latter more so than the former. This results in the increase of fat which we have observed in the splanchnicotomised region. With this conclusion the results of experiments in Table XI. are in complete TABLE XI.- THE WEIGHT OF PERIRENAL FATTY TISSUE OF FATTENED CATS IN WHICH LEFT SPLANCHNICS WERE CUT. 13 Side. Differences. Left. Right. g. Per cent. A. B. D. 3.45 3-60 -0-15 - 4 2750 3400 35 3 30 2-70 +0-60 +22 2200 2700 35 3-85 2-85 + 1.00 +39 1750 2350 35 9.00 6 20 ± 2-80 +45 2950 3600 35 A =initial weights of cats. B =final,.. D =days after section of splanchnics. accord. In these experiments we fattened 4 cats after splanchnicotomy. Before the fattening the cats were starved for different lengths of time. At the splanchnicotomy we made certain by inspection that the perirenal connective tissue contained little or no fat. After the splanchnicotomy the cats were highly fed. As is seen in Table XI., in 3 out of 4 cases there was more fat on the splanchnicotomised left side than on the normal right side. The increase is no doubt less than that found after starvation, but it is none the less distinct. This is what might reasonably be expected when the deposition of fat is intentionally increased and when both the mobilisation and deposition are slowed down on the denervated side, the former more so than the latter. DISCUSSION. The question arises as to whether the result obtained in the case of the splanchnic nerve may be put in the general form that the

14 Beznak and Hasch connective tissue as an organ of fat storage is under the control of the sympathetic nervous system. If section of a particular motor nerve throws a particular muscle into inactivity, no one questions that the same sort of nerve will have the same effect in the same sort of muscle. Yet in the case of the fat depot a certain caution is justified. A case, for instance, is described by Strandberg [see Giinther, 1920] in which a piece of the abdominal skin was transplanted into the skin of the back of the hand. When general adiposity supervened, this piece of skin became fat at the same rate as did the abdominal skin. This shows clearly that the different parts of the connective tissue exhibit great individuality with regard to their readiness to store fat, and that they preserve their individuality even after transplantation. Moreover, Gage and Fish's [1924] work proving that the deposition of Sudan III. stained fat takes place first in the perirenal fatty tissue shows that this fat depot is more reactive than many others in the body-another reason for caution before generalisation. In the experiments described in this paper, however, we have found the same effect in three different anatomical regions, though with different intensity. The conclusion that the fat depots are under the control of the nervous system has also been reached by Hausberger [1934]. This author severed, in mice and rabbits, the larger regional branches of the mixed nerves supplying the interscapular fatty body. After the operation he found by histological methods that the denervated fatty deposit contained more fat than did the normal. The same phenomenon was observed in acute starvation and in prolonged underfeeding. These results conform with our own observations. It is certain that while cutting the mixed nerves to the interscapular region Hausberger also cut the sympathetic supply running with these nerves; he observed by a histological method the same phenomenon as that to which we were able to give quantitative expression. The results which we have described in this paper fit in well with the supposition, based on clinical observation, that the fatty deposits are under the control of the sympathetic nervous system. They may, indeed, be regarded as the first direct experimental proof of the validity of this supposition. SUMMARY. 1. Unilateral section of both splanchnic nerves in cats diminishes the degree of fall in the amount of perirenal fat which follows laparotomy under ether ansesthesia. On the splanchnicotomised side the perirenal fat weighs 100-300 per cent. more than on the intact side. 2. The same difference is found if the cats starve after splanchnicotomy, or if cats with very little perirenal fat are fattened after the splanchnicotomy.

The Effect of Sympathectomy on the Fatty Deposit 3. When cats are fed with a diet containing fat stained with Sudan III. the stained fat is deposited in the perirenal fat on both sides in equal concentrations. When they are starved after unilateral splanchnicotomy, the amount of the perirenal fatty tissue on the ipsilateral side is greater than on the normal. The concentration of Sudan III., however, remains on both sides the same as it was previous to the splanchnicotomy and starvation. 4. If cats and rats are kept on normal unstained diets for various periods after unilateral splanchnicotomy, are then fed with a diet containing fat stained with Sudan III. and are killed 18 hours after the feeding, the concentration of Sudan III. on the splanchnicotomised side is about I of that on the normal side. 5. The conclusion is reached that, in consequence of the splanchnicotomy, changes take place in the perirenal connective tissue which slow both the deposition of fat and its mobilisation. The mobilisation is retarded more than the deposition. 6. Unilateral splanchnicotomy in the rabbit and in the rat has the same effect on the perirenal fat as in the cat. 7. The effect of unilateral extirpation of the stellate and the superior cervical ganglia on the amount of the pericardial fat and the effect of unilateral extirpation of the lumbar and sacral ganglia on the abdominal and subcutaneous fat on the denervated side make it likely that the effects of the splanchnic nerves on the perirenal fat may be extended to describe the relation between fat storage in and sympathetic innervation of connective tissue in general. At the beginning of this work we had the collaboration of Mr. Zoltan Harris, and for this and for Professor Saindor Belaik's kind interest and helpful suggestions the authors express their best thanks. REFERENCES. ANDORA, A. (1923). Skand. Arch. Physiol. 44, 1. BEZNAK, A. v., and HARRIS, Z. (1934). Ber. ges. Physiol. 81, 568. BEZNAK, A. v., and HASCH, Z. (1935). Ibid. 88, 336. GAGE, S. H., and FISH, P. A. (1924). Amer. J. Anat. 5, 34. GtNTHER, H. (1920). Die Lipomatosis und ihre klin. Formen, Jena. HAUSBERGER, F. X. (1934). Zschr. mikr. anat. Forsch. 36, 231. HAUSBERGER, F. X. (1935). Klin. Wschr., 14, 77. MANSFELD, G., and MULLER, F. (1913). Pfltgers Arch. 152, 61. 15