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The changes in the pancreatic cell of the cuinea-pig during inanition and refeeding.

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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.
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