The changes in the pancreatic cell of the cuinea-pig during inanition and refeeding.код для вставкиСкачать
THE CHANGES I N THE PANCREATIC CELL OF THE GUINEA-PIG DURING INANITION AND REFEEDING WEN CHAO MA Department of A n a t o m y , Peking Union Medical College, and the Hull Laboratory of A n a t o m y , University of Chicago FOUR FIQURES INTRODUCTION The experimental modification of mitochondria, involving changes, presumably reversible, in the amount of mitochondrial substance in the cell, or in the number, or size of the individual units, is one of the most promising methods for the study of these interesting cytoplasmic structures. In many respects, under experimental conditions, the mitochondria have proved to be the most reactive, as far as visible changes are concerned, of the cell contents. I n various pathological conditions, in nutritional deficiencies or abnormalities, and as a result of the toxic action of various organic substances, the mitochondria respond by changes in amount, or distribution, or form. These changes afford an opportunity for the study of the phases in the disappearance or reduction of the mitochondria, and of the restoration of them, which we may hope will ultimately throw some light on the important question of their origin and antecedents. With this end in view, I have undertaken, at the suggestion of Prof. E. V. Cowdry, the study of the effect of prolonged fasting on the mitochondria in the pancreas of the guinea-pig and the phases of recovery after refeeding. The work was begun at the Peking Union Medical College and completed at the Hull Laboratory of Anatomy, University of Chicago, 47 THE AN-4TOMICAL RECORD, VOL. 27, NO. 2 48 WEN CHAO M A The changes in the mitochondria in the hepatic cell in various experimental conditions have been classified by Mayer, Rathery, and Schaeffer ('14) under the headings, chondriolysis and chondriomegaly. The former implies a considerable augmentation of volume of the cell, associated with a progressive disappearance of the mitochondria ; the latter, diminution in volume of the cell with increase in number and size of the mitochondria1 granules. The final stage of chondriomegaly they call homogenization, in which, apparently, the mitochondria dissolve in the cytoplasm, and the whole cell takes the mitochondrial stains. To these categories Scott ( '16) has added that of agglutination with lipoid change. Mayer, Rathery and Schaeffer express themselves with some reserve as to the possibility of the recovery of the cell from these changes, but indicate that they consider the final stages of chondriolysis and chondriomegaly irreversible. Berg ( '14, '20), who studied the changes due to moderate and prolonged inanition in the hepatic cells of the salamander, seems to be of the opinion that cells which have completely lost their mitochondria may recover. Berg found that moderate periods of fasting produced an apparent increase in the amount of mitochondria in these cells, but if the period was sufficiently prolonged the mitochondria were greatly reduced in number and lost their filamentous shape. In such animals, after the administration of neutral fats, sodium oleate, or sodium glycocholate, he found large numbers of cells which he called empty cells, totally lacking in mitochondria. These cells, as regards other protoplasmic constituents, showed only a moderate degree of change. I n these preparations, also, he found cells which contained no definite niitochondria, but in which the stains revealed a delicate dust-like cloud of material, which, on close study with high magnifications, resolved itself into an aggregation of granules so minute as to be almost at the limit of visibility. These he regarded as new mitochondria arising in the cytoplasm, though he did not definitely assert that they were derived C H A N G E S I N PANCREATIC CELL O F GUINEA-PIG 49 from non-mitochondria1 cytoplasmic antecedents. Unf ortunately, there is no evidence in Berg’s work to show that the empty cells are at all capable of recovery, nor any important indication as to whether the cells containing the dust-like granules were on the down grade or the reverse. I n his article on the chromophile cells of the nervous system, E. V. Cowdry (’16) describes the partial solution of the mitochondria in the cytoplasm of these cells. Miller (’22) studied the tissues of albino rats for mitoehondrial changes under a variety of nutritional conditions. Some of the animals were kept without food, either with or without water. Others were fed on diets deficient in vitamine, or on simple protein diets such as gelatin and zein. I n general his results confirm those of Berg, since he found a general tendency toward reduction of mitochondria, loss of the filamentous form, and, in some cases, total disappearance of mitochondria. His results do not indicate whether the animals were still capable of recovery or whether they were actually moribund when sacrificed for histological study. Similar changes were also found in rats subjected to slow asphyxiation by being inclosed in a chamber with a limited small quantity of air. There seems to be a general agreement, therefore, among the authors quoted, that inanition produces changes in the mitochondria, expressed in reduction in amount of mitochondrial material, change in shape of the units from rods and filaments to spheres, and reduction in number of these units. It is interesting to note, in connection with the asphyxiation experiments of Miller, that N. H. Cowdry (’20) obtained comparable results in plant seedlings submerged for a time in water or grown in a limited air space. With the exception of Berg’s speculations about the new formation of mitochondria in ‘empty cells’ of the salamander liver, I have been able to discover no discussion of the method of recovery from advanced conditions such as those described. 50 WEN CHAO M A MATERIAL AND METHODS Guinea-pigs were kept for varying lengths of time in special cages provided with perforated bottoms. This precaution was necessary, because guinea-pigs kept without food devoured their own faeces, and when two o r more animals were kept together, the stronger nourished themselves at the expense of the weaker. Water was freely supplied throughout the experiment. Animals so kept survived for varying periods. Some animals succumbed in three days, while others survived for as many as twenty days. Another series of animals, after a period of fasting for four to eight days, were refed for the purpose of studying the process of recovery. The pancreas was prepared for histological study by the well-known mitochondria1 methods, including those of Regaud, Benda, and Bensley. I n the case of Benda-fixed material the crystal-violet alizarin method of Benda was employed for staining. In the other cases Bensley's anilin acid fuchsin-methyl green method was used. For distinguishing between zymogen granules and mitochondria, the sections, fixed, as recommended by Bensley, in acetic osmic bichromate, were stained with neutral gentian, followed by ammonium molybdate, and counterstained by the anilin acid fuchsinmethyl green method. I n every case the pancreas was taken for study only from animals killed for the purpose. Those which succumbed in the course of the experiment were discarded. The mitochondria were also studied in the fresh gland in isotonic salt solution, and after supravital staining with Janus green B. STRUCTURE O F T H E NORMAL ACINUS CELL The pancreatic acinus cell of the guinea-pig differs in no noteworthy particulars from that of other animals as described by Bensley ( 'll), Scott ( '16), and Cowdry ( '17), etc. Since, however, the purpose of this work is to define the C H A N G E S I N PANCREATIC C E L L O F GUINEA-PIG 51 changes in the cell produced by inanition, it will be necessary to describe, in greater detail than Bensley has done, the characteristics of this cell (fig. 1). All figures are drawn from preparations fixed in Bensley 's acetic-osmic-bichromate mixture, and stained by the anilin acid fuchsin-methyl green method. Mitochondria black; f a t and zymogen gray. Zeiss 1.5 mm. apo. objective, compen. oc. 8; tube length 135 mm.; camera-lucida outlines a t 32 em. Fig. 1 An acinus of a normal pancreas of guinea-pig. 52 WEN CHAO M A The acinus cell displays under normal conditions two welldefined zones, an outer transparent zone, and an inner zone containing the zymogen granules. The relative sizes of these two zones vary with the secretory condition of the cell. In animals which have fasted for twenty-four hours, and particularly if the secretion has been checked by the administration of atropin sulphate, the zymogen may occupy as much as three-fourths of the cell, while after prolonged secretin stimulation it may be reduced to zero, though usually, even after prolonged secretion, a narrow zone of zymogen is still retained along the lumen. I n the fresh cell, examined in normal salt solution or in blood serum, the outer transparent zone appears to consist of an optically homogeneous ground substance in which are imbedded mitochondria, sometimes fat droplets, and occasionally clear vacuoles. It contains, as is well known, the substance to which the deep staining of this zone is to be attributed, namely, the chromophile substance, called also prezymogen (Mouret, '95), ergastoplasm (Gamier, '00) , chromidial substance, etc. With acid fixations the basal substance coagulates in the form of filaments; hence the name sometimes employed of basal filaments of Solger ( '94). The mitochondria and fat require especial consideration, since they are particularly involved in the inanition changes to be considered. The mitochondria are found chiefly in the basal clear zone of the cell, but not exclusively so. They extend also into the mass of zymogen granules, though they are more difficult to see in this location, owing to the high refractive power of the granules. The mitochondria of the pancreas are remarkable for the size and length of the individual filaments and rods. These appear to lie parallel for the most part to the long axis of the cell, but a study of tangential sections of the acini reveals the fact that many also lie parallel to the basement membrane, and appear in the section frequently as round granules. Their form is various, and has been extensively studied by N. H. Cowdry ('17) in his comparisorl CHANGES IN P A K C R E A T I C C E L L O F G U I S E A - P I G 53 of animal and plant mitochondria. The filament or rod predominates, but Y-shaped ones and even partial nets may be present. They are usually somewhat sinuous, but may be shortened up into rods or bent around into ring-shaped forms. Sometimes, instead of being continuous, the filaments are apparently broken up partially into segments, and successive segments in the same filament may be irregularly swollen. Bulbous enlargements, which have often been interpreted as young zymogen granules, may be seen in the course of the filaments or at their extremities. Blebs containing a clear substance seem to be less numerous in the mitochondria of the guinea-pig's pancreas than in those of the white mouse described by Scott ( '16). Variations in total mitochondria1 content seem to be considerable, and cells which are side by side in the same acinus may differ from one another both in the number of the mitochondria and in their form. The relation of the mitochondria to the nucleus is wholly secondary. I find in my preparations no indications of the growth of mitochondria out of the nucleus, though, to be sure, the mitochondria1 units are often closely applied t o the surface of the nucleus. Fat is a variable constituent of the normal acinus cell of the pancreas of the guinea-pig. It is particularly apparent in the cells of animals which have been feeding freely or in which the pancreas has been stimulated to prolonged secretory activity by means of secretin. It occurs in the form of small droplets of highly refractive substance in the basal clear zone of the cell. These droplets have a faint yellowish color, and a lower refractive power than the usual intracellular fats. They blacken with osmic acid, stain in Sudan 111, and are extracted by the usual fat solvents. Since these granules of fatty nature are involved in the inanition changes, they will be considered more fully later. I N A N I T I O N CHANGES IN THE PANCREATIC CELL Prolonged withdrawal of food produces with constancy, in the acinus cell of the pancreas, profound changes, affecting 54 WEN CHAO MA chiefly the mitochondria. These become reduced in number and in size and change from rods and filaments t o spherules o r irregular granules. At the same time fat droplets make their appearance in the cell, apparently arising in the substance of especially modified mitochondria. The magnitude of the change is not proportional t o the period of inanition, lout is dependent on the condition of the animal as well. Some animals will survive total withdrawal of food f o r as long as twenty days, while others succumb in less than a week o r even in three days. The comparison of figures 2, 3, and 4, which show portions of the cells of three acini taken from an animal which had fasted for a period of twenty days, with figure 1 from a normal animal, will reveal the character and extent of the changes involved. I n figure 2 the mitochondria are much reduced in number and the shape of the individual elements much modified. I n this preparation filaments are rare and the majority of the mitochondria1 units are reduced to the form of irregular granules of various sizes. Some of these granules are arranged in rows, and a close study of them reveals a thin connecting thread of mitochondrial substance. I n other cases continuous filaments may be made out in which the material is irregularly distributed along the narrow connecting thread. Other filaments present clearly the appearance of segmentation. I n most of the cells of this acinus there are present osmic-blackened fat droplets partly or wholly surrounded by a ring of material which stains like mitochondria. Figure 3 is an acinus from the same pancreas in which the mitochondria1 change is less advanced. I n this, however, the tendency of the filaments t o segment is apparent, as well as the irregular swollen aspect of the segments. In two cells of this acinus the mitochondria are reduced to two or three small granules and rods. It is worthy of note that the zymogen content of these cells with much reduced mitochondria is in no wise different from that of the other cells of the acinus. The fat droplets of the inanition pancreas are similar to those found constantly in the pancreas of the well-fed animal, C H A N G E S I N PANCREATIC C E L L O F GUINEA-PIG 55 Fig. 2 An acinus from a pancreas of guinea-pig after twenty days’ total withdrawal of food. Fig. 3 Another acinus from the same pancreas as that from which figure 2 was drawn, showing a less advanced degree of change. Fig. 4 Paratangential section through the edge of a n acinus; twenty days’ inanition; shows arrangement of mitochondria parallel to basement membrane, and f a t granules surrounded by mitochondria1 substance. 56 WEN CHAO MA but are of greater size and are surrounded by a larger amount of stainable substance. The question as to whether this substance is mitochondrial in nature or whether we have to deal here with a special liposome of non-mitochondria1 origin has been difficult to answer. The substance of this envelope stains in dilute Janus green B solutions precisely as do the mitochondrial filaments, except that the fat envelope is more easily stained. This is true also of the fixed material; that is to say, the fat envelope retains the acid fuchsin even when the differentiation of the preparation has been pushed f a r enough t o decolorize all of the filamentous mitochondria. It is also more resistant to fixation, and is preserved in areas of the tissue remote from the surface, where the usual mitochondria have been dissolved. I n the fresh condition, stained with Janus green, the fat globules appear completely snrrounded with a green-stained envelope, which is, however, thicker on one side. I n the fixed preparations this envelope is often in the form of a crescent occupying one side of the fat globule, and often, also, partly broken up into segments which adhere to the surface of the globule. This segmentation is less apparent in the Regaud-fixed material than in that fixed in Bensley’s acetic osmic bichromate mixture. The determination of the mitochondrial nature of this envelope is not easy, because of the difficulty of constructing a continuous series of changes between the normal condition as displayed in figure 1, and the stages of extreme change as seen in figures 2 and 3. The animals develop these changes with such different rates that it becomes a matter of inference only whether a given condition represents an intermediate phase or not. A phenomenon which is common t o the early phases of inanition is the tendency of the mitochondrial filaments to dissociate into smaller units so as t o give the impression of a larger numerical content. These smaller units tend t o aggregate into groups particularly at the base of the cell, but do not, as f a r as I can see, actually agglutinate into a mass. The individual elements as well as the unsegmented filaments tend to swell and assume a club shape, o r CHANGES I N PANCREATIC CELL O F GUINEA-PIG 57 t o be changed in some cases to spheres, in others to filaments with a bulb-like enlargement at one end. The first fat to be seen in the cell is in the form OP small droplets in the interior of mitochondrial spheres. Rarely two or three such droplets may be seen in the course of a filament. Later in the process, however, single fat droplets are the rule, and usually not more than one or two in each acinus cell. This property of forming fat in inanition and in prolonged secretion is peculiarly the property of the acinus cell. Miller ('22) makes no mention of a similar process in the stomach and duodenum, and it is difficult to decide whether the 'Hohlkorper' of Berg ('20) in the liver cell belong to this category or not. At all events, there is no proportional deposit of fat in cells either of the ducts or of the islets of Langerhans. The extraordinary fact that only one or two of the mitochondrial units participate in this fat-forming process is difficult to understand. It is possible, of course, that liposomes are not mitochondrial in origin or that certain of the mitochondria are specialized in some way for this function. I have been unable, however, to distinguish, in the normal pancreatic cell, either by vital staining or by fixation and staining, these fat-forming mitochondria from the other mitochondria of the cell. Another possibility, also, must be considered, namely, that the relation of the mitochondria to the fat is secondary, by application of the mitochondrial substance to the surface of an independently formed fat globule. The zymogen content of the cells in the inanition pancreas does not seem to be much affected. At all periods there is a substantial content of zymogen granules, irrespective of the degree of reduction of the mitochondria. RECOVERY O F T H E PANCREATIC CELL DURING REFEEDING Animals which have been kept without food for a length of time recover at various rates, depending upon the extent to which they have been reduced by fasting. By some animals food is taken sparingly at first, and such animals may even continue to lose weight during the first twenty-four hours 58 WEN CHAO MA after feeding is resumed. Others begin to gain immediately. As soon as the weight shows increase, changes in the pancreatic cell become apparent, which are the beginning of a process leading to full restoration of the cell to the normal condition. These changes involve disappearance of the fat, increase in mitochondria, and return of the latter to the normal form. The disappearance of the fat may begin as early as six hours after feeding is resumed. It is frequently completed in twenty-four to thirty-six hours, though in some cases the f a t may persist for a longer period. The first change that is apparent is the withdrawal of the fat globules from their mitochondrial envelopes. The latter seem to contract and expel the oil globules so that one finds oil globules and mitochondrial spheres lying side by side or with the globule still partly inclosed. I n other cases the mitochondria1 envelope appears t o break up into a number of fragments from which the oil drop escapes. At this stage the cell contains, in addition t o one or more fat droplets, one o r more large spherical masses which stain intensely with acid fuchsin. These represent the fragments of the envelope of the fat droplets. They may persist f o r as long a period as six days after feeding is resumed, but usually they disappear in the first two or three days. I n one of my cases, that of an animal kept without food for eight days, then fed for six days, the spherules were still abundant, but some of them were evidently degenerating. Instead of being spherical, they were irregular in shape, stained feebly, and contained clear vacuoles. Whether others of the spherules contribute to the restoration of the mitochondria of the cell or disappear, I cannot state with certainty. I n those cases where the envelope breaks up into a large number of small spherical masses they soon become indistinguishable from the other mitochondria of the cell and their fate cannot be followed independently. At the same time the mitochondria show changes. The phases of this process are difficult to analyze on account of the inequality of the changes produced in neighboring cells C H A N G E S I N PANCREATIC C E L L O F GUINEA-PIG 59 by starvation. The end conditions are clear enough. The mitochondria again become abundant and the filamentous or rod shape is restored. The following states may be recognized: the spherule state, the short-rod state, the segmented rod, and the filament. I n view of the readiness with which mitochondria are known to change their state as observed in tissue cultures by M. and W. H. Lewis ( '14), such appearances must be interpreted with caution. This much is sure: there is an increase of mitochondrial substance in the cell, and this increase is related to previously existing mitochondrial units. I n the recovering pancreatic cell, I have seen at no time such small premitochondrial granules as were described by Berg in the liver cell of the salamander; nor is there at any time in this process any evidence of the derivation of the mitochondria from the nucleus as claimed by Saguchi. Although the mitochondria may be closely applied to the surface of the nucleus, they never enter it. Accordingly, I interpret the appearances found in the pancreatic cell of the guinea-pig as follows. The spherical mitochondria of the inanition pancreas on the resumption of feeding assume a rod shape, grow in length, and segment. I n this way the number of mitochondria is increased. Finally the filamentous character of the normal mitochondria is attained by growth in length of the segments. CONCLUSIONS 1. Prolonged inanition produces constant changes in the mitochondria of the acinus cells of the pancreas of the guinea-pig. The mitochondria are reduced in number and changed from rod and filamentous forms to irregular granules and spheres. 2. I n no case is the mitochondrial content entirely lost, although the reduction may be extreme. 3. Certain of the mitochondria become enlarged to spheres in which fat is deposited, so that an inanition pancreas always contains, in the base of the acinns cells, large fat droplets each surrounded by an envelope of mitochondrial substance. 60 WEN C H A O M A 4. The change is reversible, and after two to six days of refeeding the cells resume their normal content and form of mitochondria. 5. The new mitochondria are formed from those remaining after inanition, and there is no evidence of the formation of mitochondrial units in the cell independently of previously existing mitochondria. 6. At no time in this period of active increase is there any continuity of mitochondria with intranuclear structures as claimed by Saguchi (’20). The mitochondria are of cytoplasmic, not nuclear, origin. LITERATURE CITED BERG, W. 1914 Ueber periodische Veranderungen der Salamanderleber mit besonderer Beriicksichtigung der Pigmentzellen. Ztschr. f . Morphol. u. Anthropol., Stuttgart, Bd. 18, S. 579-608. 1920 Ueber funktionelle Leberzellstrukturen. I. Arch. f . mikr. Anat., Bonn, Bd. 94, S. 518-608. COWDRY,E. V. 1916 The structure of chromophile cells of the nervous system. Contributions to Embryology, Carnegie Institution of Washington, Washington, no. 11, pp. 27-43. COWDRY, N. H. 1917 A comparison of mitochondria in plant and animal cells. Biol. Bull., vol. 33, pp. 196-228. 1920 Experimental studies on mitochondria in plants. Biol. Bull., V O ~ . 39, pp. 188-206. GARNIER,C. 1900 Contribution l’btude de la structure et du fonctionnement des cellules glandulaires shreuses. J. de l’anat. et de la physiol., annee 36, pp. 22-98. MAYER,A., RATHERP,F., AND SCHAEFFER,G. 1914 Les granulations ou mitochondries de la cellule hhpatique. J. de physiol. e t de pathol. gbn. Par., V O ~ . 16, pp. 581-622. MILLER;S. P. 1922 Effects of various types of inanition upon the mitochondria i n the gastro-intestinal epithelium and in the pancreas of the albino rat. Anat. Ree., vol. 23, pp. 205-210. MOURET,J. 1895 Contribution B l’btude des cellules glandulaires (pancrbss). J. de l’anat. et de la physiol., annee 31, pp. 211-236. LEWIS, M. R., AND LEWIS, W. H. 1915 Mitochondria (and other cytoplasmic structures) in tissue cultures. Am. Jour. Anat., vol. 17, pp. 3 3 9 4 0 1 . SAWJCHI,S. 1920 Studies on the glandular cells of the frog’s pancreas. Am. Jour. Anat., vol. 26, pp. 347-422. SCOTT, W. J. M. 1916 Experimental mitochondria1 changes in the pancreas in phosphorus poisoning. Am. Jour. Anat., vol. 20, pp. 237-253. SOLGER,B. 1894 Zur Kenntniss der seeernierenden Zellen der Glandula submaxillaris des Menschen. Anat. Anz., Jena, Bd. 9, S. 413-419.