脂肪形成

M.ICROS~OPIC STUDIES 03’ THE YENT FORMATION O F FAT I N LIVING ADULT RABRTTS ELIOT R. CLARK AND ELEANOR LINTON GLAKK Dspartment of Anatomy, Univtmity of Pennsylvania, Philadelph ici SIX TEXT FI~UEES AND THREE PLATES (EIQHTEEN FIGURES) INTRODUCTION In the course of microscopic studies on the growth and behavior of blood vessels and lymphatics in the living mam- mal, carried out in transparent chambers permanently installed in the ears of rabbits, the appearance of newly formed intracellular fa t in ccrtain of the chambers mas noted. The fact that small amounts of adipose tissue are normally present in the rabbit’s ear was established by the study of chambers of the “preformed tissue” type, in wliicli the original tissue of the ear is included in the observation area, since in such specimens scattered groups of fat cells in the subcutaneous tissue were frequently seen immediately after t.he insertion of the transparent window. In the case of chambers of the “round table” type (Clark et al., ’30) and in “moat” chambers (Abell and Clark, ’32), in both of which a hole is cut clear through the ear a t the time of installation, leaving a thin observation space of known and controlled thickness, which is subsequently invaded by new capillaries and tissue, it was obvious that the fat which appeared inside cells in this region was all of it newly formed. Thus, it was apparent that in chambers of these two typcs, in which all the cells and tissues of the observation urea are newly formed, an opportunity was afforded for prolonged microscopic observa- tion of the new formation and subsequent. changes in subcu- taneous fat, with a parallel study of surrounding vessels and other tissues, in the living adult mammal. 255 256 RLIOT R. CLA1t.K AND ELEANOR TAINTOX CTARK The present investigation was based chiefly upon the intensive study of thirty-one “round table” chambers all of which were kept under observation for at least 4 months after installation. In about half of the specimens the ohserva- tion period cxtcnded f o r over a year and in one case for as long as 20 months. The method of construction of the “round table” chamber, t.he operation for its installation in the ear, as well as the technique of microscopic observation and of the carc and handling of the rabbits havc been dcscribed (Clark et al., ’30, Clark, Sandison and Hou, ’31). G N N E U L OBSERVATIONS ON THE OCCURRENCE: OF FAT Of the thirty-om chambers used in this study, twenty-one showed no fat formation in thc new tissue which covered the central tablc, while in ten chambers varying amounts of adi- pose tissue developcd. Of the twenty-one “fat free” chambers, six lacked the detached splints ((Ylark and Clark, ’32) which have been in use since 1931, while four others were of the “removable top” variety (Williams, ’34). In chambers of these two types fat formation was not 8een.l Thc conditions present in these ten chambers which caused them to be sub- jected to greater mechanical stimulation and trauma and hcnce to a mild inflammatory reaction are discussed on p. 263. Omitting these ten chambers and comparing the remaining twenty-one stable chambers o€ similar construction, it was found that fat formation occurred in approximately 50% (ten out of twenty-one). Various comparisons were made of the chanibcrs in which fat appeared with those in which it was absent with regard to differences in thickness of the table area, manner of inscrtion and general temperature conditions during the ‘Sincc completing tJ& study a ncw chamber of the modified “removable top” type, recently described by Williams (’39), has been undcr observation. In this modificd chamber, which has a glass bottom, the new tissue and blood vesseh are protected from strain and the coiiditions arc Yimilar to those found in thc moat chamber. In this specimen, ncw fat formation was observed 3 months after installation. FAT FORMATION IN LIVING AXIMAL 257 months of observation. Comparisons were also made between the two groups of chambers with regard to the sex and general condition of the individual animals. The thickness of the space over the central table varied in the diff creril specimens, depending upon the thickness of the celluloid buffers (measured before fastening them to the table) upon which the mica cover slip rests. Chanibers in which the space measured from 7 0 p to 1OOp were more favorable for the ingrowth of nerves, for the development of contractile arteries and for the maintenance of a continuous lumen in regenerated lymphatic capillaries ; chambers with a thinner space, obtained by using buffers of 40 p to 50 p in thickness, were better adapted for the study of finer cytological details of vascular sprouts, endotheliurn, adventitial cells, blood cells and wandering cells, while a space of 20 p or less proved to be too thin to permit the growth of circulating blood vessels (Clark et ul., ’31). The various chambers included in this study were divided into tm7o groups : those measuring 70 p to 100 p and those measuring 40 p to 50 p. Of the twenty-one chambers in which no fat developed, elercii measured 7 0 p or more and ten measured 4 0 ~ to 50p. Of the ten chambers in which fat formation occurred, six measured over 70 p and four mcasured 40 p to 50 p. Hence, differences in the amount of tissue present in the two different groups as well as differences in richness of nerve supply and in the movement of fluid in the lymphatics noted above apparently had 110 ellect in determining the presence or absence of fat in the new tissue. The chambers used in this study also differed as to the side of the ear into which they were inserted. chambers installed on the dorsal side of the car were found to reckive a more abundant supply of nerves and most of the “dorsal” chambers of this group were inserted with the object of studying nerve regeneration. The dorsal chambers were also subjected to more mechanical stimulatior~ during observation periods, since it mas necessary to reverse tlie ear from its natural position in order to place it on the stage of the microscope. On 258 ELIOT H. CLARK AND ELEdNOR LINTON CLARE analyzing the chambers from this standpoint it was found that of the "fat free" chambers, five were inserted 011 the dorsal, and sixteen on the ventral side. Of the chambers in which fat developed, five were dorsal and five ventral. Ap- parently, then, the slight differences in manipulation oc- casioned by the side of the ear used for installation of the chambers had no appreciable effect upon fat formation. Since the various chambers were inserted at different times of the year and were kept under observation for many months, the majority of them were subjected to moderate seasonal variations in temperature. In the series studied thirteen chambers were inserted in t.he fall and winter months and eighteen in the spring and summer. The temperature of the animal house ranged from 60" to 70°F. during the colder months and 80" to 90°F. in the summer months. Of the twenty- one chambers which showed no fat formation, seventeen re- mained under observation during both cool and warm seasons. In the ten in which fat appeared, it was found that fat forma- tion occurred at all seasons of the year and that in individual chambers it frequently increased and later decreased or dis- appeared and again reappeared regardless of the season. For example, in four chambers fat first appeared in cool weather, increased and then decreased during the cool season and again increased and then decreased in warm weather. In three chambers fat first appeared in warm weather, increased dur- ing the succeeding warm months and continued to increase in the cool season. In one chamber fat appeared in warm weather, then decreased and later increased as the weather became cooler. In another chamber fat first appeared in cool weather arid continued to increase throughout the cool season and the succeeding warm season. And, finally, in one chamber fat appeared, increased and decreased in the warm season and subsequently increased and decreased in the cool season. Although the season of the year evidently played no part in the appearance, increase, decrease and disappearance of fat in the chambers, sudden temperature changes, caused either by extreme changes in the weather or by localized FAT FORMATIOX I% LIVING ANIMAL 259 heating of the ear, which affected the circulation in surround- ing Mood vessels, were observed to have a definite effect on fat cells already present in the table area or in certain portions of it. This will be described in connection with observations on the relation of blood vessels and circulatory conditions to fat (p. 262). In considering the formation of fat, in chambers in relation to the sex, age and general condition of thc rabbits used in this series, no essential differences were noted which could explain the appearance of fat in some chambers and its absence in others. More females than males were used in the entire series since the formcr were found as a rule to be quieter and hence better adaptcd to long-time microscopic observa- tion. Of the chambers in whkh no fat appeared, two were in male rabbits and nineteen in females, while of the chambers in which fat formation took placc, four were in males and six in females. The rabbits used in the scries were all healthy young adults bred in the laboratory. The ages at the time of opcration ranged from 5 months to 2+ years, averaging 13 months. All the animals were fed the same diet, employed for many years, consisting of whole oats and alfalfa with the addition of Yurina pellets daily, half a carrot or a few leavcs of lettuce once a week and with u can of water kept in the oages continuously. The rabbits, when not under observation, wcre kept in roomy individual cages, where thcir opportunities for cxercise were necessarily somewhat restricted. The ani- mals used in this series apparently thrived on this regime as they were all free from disease and well nourished, although none showed an excessivc amount of adipose tissue. S s yet no experiments have been carried out in which either over- abundant or restricted rations have been used. I n the case of the two thinnest rabbits of the group, one- a male-showed fat formation in the chambers of both ears, while in the other-a female-fat was present in one chamber during 5 of the 18 months of observation while no fat developed in the chamber in the other ear. Seven animals with chambers 260 ELlOT R. CLAR.K AND ELEANOR TAINTON CLARK in both ears showed no fat formation in either ear. In two rabbits with chambers in both cars fat formation occurred in the observation areas of both ears. In four rabbits, fat appeared in the chamber of one ear and was absent in thc other. The remaining chambers stndiecl-three without and two with fat-were cases in which one car only was used for the experiment. Two chambers inserted in the ears of the same rabbit showed iiitcresting diff ereiices in the time of appearance, increase and decrease and final disappearance of fat cells during thc same period of 11 months in which they were under intensive observation (figs. 7-18, platcs 1 and 2). The chamber in the right ear (11/13/31R) was inserted on the dorsal side and the depth of the space over the table measured 75 p while thc chamber in the left ear (11/16/31L), insertcd 3 days later, on the ventral side, measured 40 p. In the first chamber thc table space was completcly vascularized 19 days after the operation, in the second a t 26 days. The thicker chamber (right ear) slimed more haemorrhages following the operation and the new vascular network was more profuse than in the tllinner chamber. Cells containing fat appeared first in one region in the left ear on December 19, 33 days after the operation and seven days after complete vascularization of thc table area. The number of fat cells increased gradually in this and adjoining regions for almost 2 months (Feb. 5) when a week intervened (E’eb. 6-12) during which the fat again increased, a week in which it was stationarv in amount and two further periods in March in which the fat decreuscd arid again increased. During April the fat diminished slowly and by the cnd of May had disappcared. In the chamber in the other ear, fat cells did not appear until December 29, 46 days after the operation arid 27 days after vascularization of the table space. The fat cells in- crcascd in number for a month then decreased for 10 days and again increased steadily from February 12 to hiarch 22. During April the €at diminished and it disappeared in May, Administrator 高亮 FAT FOR.MATION IN LII'ING ANIMAL 261 a week before it disappeared in the chamber of the opposite car. Although in this rabbit the two chambers showed the first fa t formation and also the final disappearance of fat in the same months (December and May), whcn individual weeks during the period in which fat was prcscnt in both chambers were compared it was found that the tempo of increase arld dccrcase differed in thc two ears. Thus, although fat cells increased in both chambers during January the rate of in- crease was faster in the right ear (the one in which it made its appearance later), while during the first week of February the fat increased in the Ieft ear and diminished in the right (figs. 10 and 16). Later, following a week in which it increased in both ears, therc followed a week (Uarch 11-38) in which fat in9reased in the right ear and diminished iri thc left. B'rom'the studies of these two chambers as well as from the cases already mentioncd (p. 259) in which fat appeared in the chamber of one ear and was absent in that of the other ear of the same rabbit during the same period of study, it is evident that the general metabolic condition of the animal is not the solc factor in regulating thc appcarance, disappear- ance; increase or decrease of localized dcp0sit.s of subcu- taneous fat. Tn none of the chambers studied was therc any sign of fat forniation during the period of invasion of the tahlc! area- a stage characterized by the presence of serum, fibrin and varying amounts of extravascular blood cells in thc center of the tablc and by rapid sprout formation of the advancing capillaries (Clark et al., '31; Clark and Clark, '39). In all of the ten specimens in which fat formation occurred the table area h 'ad been completely vascularizcd, the evtravasated blood had been adsorbed and the observation region had reached a stable diondition frec from inflammation. I n this series of ten chambei-s fat appeared from 1 to 4 months after installation with an average of 29 months. In all of the ten chambers fat formafion was first seen well out on thc table instead of at the pc riphcry. Administrator 高亮 262 ETJOT R. CLAR.R AND ELEANOLl LINTON CLARK RELATION OP FAT PORMATION TO BLOOD VESSELS Frequently the fat cells appeared first in close proximity to a blood vcssel (fig. 19) but this was not invariably the case. In the two chambers in the same rabbit described or1 p. 260 the first fat cells in the right ear (fig. 7) appeared beside a vein, later spreading to the tissue between the blood vessels, while in the left ear (fig. 13) the first fat cclls appeared in a region bctwccn the blood vessels. Frequently the ncw fat was first seen in one or two localized rcgions of the table area and subsequently it gradually spread out from those foci into adjoining regions. In chambers in which the fat vas seen to decrease and later reappear it showed a tendency to rebzm its original site. In general, fat formation occurred in stable chambers in which the circulation was moderatcly slow or sluggish., How- ever, many chambers with sluggish circulation failed to1 show fat formation while in a few cases fat made its first appearance or increased in amount during a period of relativcly qctive circulation accompanied by new formation of capillaries and arterio-venous anastomoses. I n the latter instances, how ever, it was noticeable that fat formation occurred in portiohs of the table area in which the circulation was relatively. less active. It was also noted that decrease in fat already present in a chamber area usually occurred during or soon aftcr pcriods of active circulation in the vessels in its immediate neighborhood. New cartilage and chondroid material, thc format ion of which was observed in some of the chambers, also occurred in stable chambers with sluggish circulation. Chondroid tissue developed in some specimens which showed no fat formation; in other chambers fat appeared and no cartilage, while in still other cases both fat and chondroid tissuc developed in the same chamber. Of twenty-one chambers in which fat was nevcr present, four showed new cartilage in the table area. Of ten in which fat formation took place, five showed c.artilage €ormaiion and five did not. Administrator 高亮 Administrator 高亮 Administrator 高亮 Administrator 高亮 Although fat formation took place in chauil.)c.rs with quiet circulation or in regions of iiidividual chambers in which the circulation was rdatively sluggish, the relative amounts of fat which clercloped apparently bore a o definite relationship to the charac*tci* of the circiilation, asidc from tho decrease following 2111 inflauimatoq- coiidition noted above. Kot, only did maiij- of the chamber6 with the quietest circulation fail to show fat. formation, hit chambers with relative1 y more active flow occasionally containcd greater amounts of fat than (lid those with quieter circulation in the opposite cars of the same animals. This was the case in the two chambers described on p. 260 in which, although the fat appeared first mid remained longer in the chambet. with quieter Mood flow (left car) the amount of Pat at the peak was gi*catcr in the chamber with tlic more active circulation (right ear). (E’ips. 11 arid 17.) The giwtest amount cf fat formation in any chamber. s tud id is shown in figures 1 arid 2. In this case although tlie circula- tion on the tablo area was moderate or quiet during the period of rapid fat increase it was iioi so sluggish as that observed in a number of other chambcrs of the series in vr-liich either n:, fa t formation or a relatively small amount took place. From a comparison of all thirty-one specimeiis TVC could not cletermine definitely from a study of the circulatory coiidiiions preserib iii the different chambers, the reason for the appcar- unce of fat in certain chambers and its abscnco in others, nor for the greater amount of €at formed in certain chambors. Tn those chambers however, in which fat did appoar it was found to form at, times in which the circulation of tho entire tablc was quiet OK* in localized regions in which the blood flow was less activc than in ilic rest of the observation area. New fat was never seen to form in a region in which the circulation of surrounding blood vcsscls was extremely active, while in sevm of the tcn cliambcrs containing fat, the fat was seen to diminish rapidly following periods of active circulation which persisted for several days. Tn five of these chambers, in which the act,ive circulation in such a period was accompanied Iny emigration of leucocytes, tlie fat decreased rapidly until it Administrator 高亮 Administrator 高亮 Administrator 高亮 Administrator 高亮 Administrator 高亮 Administrator 高亮 Administrator 高亮 264 ELIOT R. CLARK AXTI) ELEANOR L l N T O N CLAHK liad entirely disappeared and did not reappear with the return of a quieter flow. This relationship of the increase and c