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Histological studies of the ground squirrel adrenal following hibernation and cold exposure.

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Histological Studies of the Ground Squirrel
Adrenal Following Hibernation
and Cold Exposure'
MARILYN L. ZIMNY
Department of Anatomy, LouisCPna State University School of Medicine,
New Orleans, Louisiana
Hibernating rodents apparently have
adrenals which are free of discrete fat
droplets except in the zona glomerulosa
(Bourne, '51). Since this characteristic
also occurs in ruminants and certain nonhibernating animals, it has not been associated with the ability to hibernate
(Bourne, '51; Nicander, '52). It has been
suggested by Verne and Herbert ('51) that
hibernators and ruminants do not store
hormone precursors in the adrenals.
Among the effects of exposure to low
environmental temperature (4°C) those
upon ground squirrels have shown an interesting effect upon the adrenal glands.
In the adrenal glands of animals exposed
to cold the cortex is more highly developed
and differentiated than in controls, whereas the medulla is not affected (Zalesky and
Wells, '40).
In the present work, the adrenal of the
ground squirrel has been reinvestigated in
control, hibernated and animals exposed
to cold. Histochemical procedures, glandular volume determinations and zonular
measurements have been used to assess
the physiological activity of the adrenal
glands in the experimental groups.
MATERIAL AND METHODS
A total number of 77 adult 13-striped
ground squirrels, Citellus tridecemlineatus, were placed in groups as follows: (1)
controls, 26 animals kept at an environmental temperature of 25-27°C; ( 2 ) coldexposed, 12 animals kept at an environmental temperature of 3 4 ° C for 30-60
days and never hibernated; and ( 3 ) hibernated, 39 animals kept at a temperature
of 3-5°C and hibernated 3-5 days without
interruption.
The adrenal glands of 15 controls, 6
cold-exposed and 25 hibernated animals
were fixed in Bouin's fluid, embedded in
paraffin and sectioned serially at 25 u. The
volumes of cortex and medulla were calculated from planimetric measurements
and a modification of Jackson's paper
weight method. In the ground squirrel
these measurements are quite accurate because it has a well demarcated cortex,
medulla and cortical zonation. The relative widths of the cortical zones were
measured with an ocular micrometer
One adrenal gland was fixed in formalin, from 11 controls, 6 cold-exposed and 14
hiberated animals, washed, and sectioned
on a freezing microtome. Sections 15 M in
thickness were stained for total lipids using Sudan black B in 70% alcohol for 7
minutes; 25-p sections were stained for
cholesterol by the Schultz method (Cowdry, ' 5 2 ) ; and 5-cl sections were stained
for periodic acid-Schiff positive material.
One-half of the other adrenal gland of
these animals was embedded in paraffin
and 7-u sections treated by the periodicacid Schiff reaction and Gomori's chromaffin staining procedure. The remaining
halves were fixed in a 2.5% solution of potassium bichromate, pH 5.6, for 24 hours.
RESULTS
The absolute volume of the adrenal decreases but the volume relative to body
weight is the same in hibernated animals
(table 1 ) . Therefore no specific response
is noted. In the cold-exposed animals the
1 This investigation was supported by research
grant (A-2027) from the National Institute of
Arthritis and Metabolic Diseases, Public Health
Service.
279
280
MARILYN L. ZIMNY
TABLE 1
Control
Vol. (cc) of Cortex
Hibernated
Cold-exposed
5.92
0.439
4.00
0.190
32% dec.
4.44
0.197
25% dec.
0.69
0.042
0.64
0.034
7% dec.
0.82
0.042
20% inc.
Total volume
70Cortex
% Medulla
C / M ratio
6.61
89
4.64
86
14
6.3/1
5.26
84
16
5.4/1
Average wt. ( g m )
96 Experimental wt. loss
23 1
192
20
155
34
Vol. ( c c ) of Cortex Fer gm body wt.
0.0274
0.0031
0.0219
0.0015
20% dec.
0.0290
0.0017
6% inc.
0.0029
0.0035
0.0003
21% inc.
0.0054
0.0005
86% inc.
S.E.
% Change
Vol. (cc) of Medulla
S.E.
% Change
S.E.
% Change
Vol. (cc) of Medulla Fer gm body wt.
S.E.
% Change
absolute volume of the adrenal remains
unchanged but the volume of the medulla
per gram body weight increases greatly.
This illustrates a calorigenic response of
the adrenal in the mediation of the chemical regulation of heat production.
Measurements of the cortical zones are
of doubtful significance due to the great
range of variability within each experimental group. In all groups the zona
reticularis had the greatest width ranging
from an average value of 403 u in the
control to 280 p in both the hibernated and
cold-exposed groups. The zona glomerulosa had the smallest width, ranging from
an average of 74 in the hibernated and
cold-exposed groups, respectively, to an
average of 63 in the controls. The zona
fasciculata ranged from an average width
of 210 u in the cold-exposed group to
185 p in the control and 152
in the
hibernated animals. Both the zona fasciculata and zona reticularis of glands
from all groups showed two strata frequently, the outer stratum consisting of
small, densely arranged cells, the inner
stratum consisting of larger, loosely arranged cells. The outer stratum exhibited
a greater basophilia than the inner stratum. Occasionally the zona fasciculata
could be seen to have merged with the
zona glomerulosa.
11
8.6/1
0.0004
The zona glomerulosa of adrenals from
all three groups of animals showed intense
sudanophilia and a positive Schultz cholesterol reaction (figs. 1 and 3 ) . In the
adrenals of control and cold-exposed animals diffuse areas of lipid material appeared throughout the remainder of the
cortex. However, in the adrenals of hibernated animals both sudan and Schultz reactions were definite for the reticularis
giving a second concentric pattern of lipid
material (figs. 2, 4 and 5).
Medullary response to Gomori's chromaffin stain showed no marked differences
among the groups except for the appearance of a few more deeply stained cells
in the medullae of the hibernated animals.
The bichromate reaction was marked in
the adrenals from hibernating animals
(figs. 6 and 7).
The capsule and other connective tissue
components of glands from all groups gave
a positive mucopolysaccharide reaction.
No evidence of glycogen granules was
seen although small traces of periodic
acid-Schiff positive material could be seen
throughout the cortex and medulla.
DISCUSSION
Zalesky and Wells ('40) found that
cold-exposure produced a more highly developed and differentiated cortex and did
HIBERNATION AND THE ADRENAL
not affect the medulla. In this study, however, the cortical volume in the cold-exposed group decreased with an increase
in medullary volume and there was a
greater decrease in cortical volume in the
hibernated group with only a slight decrease in medullary volume. The adrenal
medullae of the cold-exposed group show
the largest volume per gram body weight
while the cortices show no significant
change. The 34% loss in body weight
which occurs during cold-exposure must
also be considered. The calorigenic action
of adrenaline has been described by many
(Cannon et al., '27; Cori and Cori, '28;
Griffith et al., '47; Soskin and Levine, '52).
Therefore it is possible that in the ground
squirrel exposed to conditions which would
naturally cause a lowering of body temperature, the activity of the adrenal medulla is invoked and an increased discharge
of adrenaline takes place. This would
stimulate skeletal muscle metabolism for
the maintenance of body temperature.
Since adrenaline has been shown to increase lactate output and glucose retention by tissue (Griffith et al., '47) this
increase in adrenal medulla volume in the
cold-exposed group may aid in explaining
the decreased lactate levels of cardiac muscle and liver and the increased lactate
level of skeletal muscle as previously reported (Zimny, '56).
During hibernation there was no enlargement of adrenals or any histo-physical evidence of hyperactivity of the zona
fasciculata in hamsters (Deane and Lyman, '54). Variations in the adrenal cortex in hibernants follow the seasonal cycle
whereas the medulla does not show signs
of cyclic changes but fluctuates throughout the year depending upon adrenaline
(Kayser and Aron, '50; Kayser, '50). Great
variability in the adrenaline content of the
adrenal medulla in winter suggests a possible relationship between the frequency
of awakenings and the lessening of the
hormone in the gland (Kayser, '53).
Although the volumes of both cortex
and medulla decrease during hibernation
the C/M ratio of this experimental group
is nearest that of the control group. This
is also true for adrenal volume per gram
body weight. The volume of cortex per
28 1
gram body weight decreases from the normal while that of the medulla increases
approximately to the corresponding degree.
It is possible that during this state of depressed metabolism, lack of movement and
a 20% loss in body weight the decreases
in absolute volumes are related to body
weight loss while the medulla volume per
gram body weight increase reflects a response to cold which occurred during refrigeration but prior to actual hibernation.
It may be that this increase would lessen
as hibernation is prolonged. During metabolism studies pertaining to hibernation
and starvation (Zimny and Tyrone, '57)
it was found that of the 20% body weight
loss reported to have taken place during
hiberna.tion, 3/4 of this occurs during refrigeration but prior to hibernation and the
remaining 1/4 occurs during the 5-day
period o f uninterrupted hibernation. During this perioid of refrigeration and weight
loss an increased adrenaline response cited
in regards to the cold-exposed group may
have been responsible for an increase in
medulla volume which then decreases during hibernation.
Of particular interest histochemically
were the sudan and Schultz reactions.
Since it has been reported that hibernating
rodents have lipid droplets in only the
zona glomerulosa (Bourne, '51 ) the sudanophilia and positive reaction for cholesterol in the zona glomerulosa of the
control ground squirrel adrenal could be
expected. This also substantiates the vacuolated appearance of this zone upon routine examination following staining with
hematoxylin and eosin.
Cholesterol and its derivatives are considered the possible precursors of the
adrenocortical hormones (Jones, '57) and
Sayers and Sayers ('48) found that the
cholesterol content of the rat adrenal, estimated chemically, corresponded to functional changes. However, Verne and Herbert ('51 ) suggest that hormone precursors
are not stored in the adrenals. Considering the distribution of fat droplets and
choIesterol material in the zona reticularis
in addition to the zona glomerulosa in
glands from hibernated animals, it appears that hormone material is being
stored during hibernation. The depressed
282
MARILYN L. Z I M N Y
metabolism and lack of bodily movement
characteristic of hibernation may account
for this storage phenomenon. In general,
the ground squirrel possesses a lipid-poor
adrenal which in this respect is similar to
the hamster (Meyers and Charipper, '56;
Knigge, '54).
The most marked histochemical response of the adrenal medulla was that
of the bichromate reaction on the glands
from hibernated animals. With Gomori's
chromaffin stain a few more deeply stained
cells appeared in glands from hibernated
animals but not to the extent to establish
major differences among the groups.
These responses may also be indicative of
hormone storage during hibernation.
In studies of the effects of prolonged
hypothermia on the rat Kaufman, Gavin,
and Hill ( ' 5 8 ) found that there was no
evidence of glycogen in the adrenals of
control and hypothermic rats but only
small traces of periodic acid-Schiff positive material in the zona glomerulosa and
medulla. Nicander ('57) demonstrated
glycogen in adrenals, both cortex and medulla, of horses, cattle, sheep, goats, pigs,
dogs, rabbits, guinea pigs and rats. The
capsule in particular contained much glycogen. The capsule and supporting connective tissue elements of the ground
squirrel adrenal were specific for glycogen
but only small traces of periodic acidSchiff material were found scattered
throughout the cellular zones. In general
the adrenal gland of the ground squirrel is
poor in carbohydrate material.
From the measurements and histochemical observations of this study i t appears
( 1) that hormone precursors are stored
in both the cortex and medulla of the
ground squirrel adrenal during hibernation; and ( 2 ) that cold is not a stress
agent.
SUMMARY
Absolute cortical volume of the adrenal
decreased in both hibernating and cold
exposed animals. However, in relationship
to body weight, total glandular volume in
hibernants remained the same. The most
marked increase in volume was shown by
the medulla in the cold-exposed animals
illustrating a calorigenic response.
Lipid material was concentrated in the
zona glomerulosa of adrenals from control and cold-exposed animals and the
medullary chromaffin response was the
same. In addition to the zona glomerulosa
the adrenals of hibernating animals
showed a concentric pattern of lipid in the
zona reticularis. The medullary staining
response was most marked in adrenals
from this group. Periodic acid-Schiff positive material was seen in the connective
tissue components of all glands but was
not specific for any cell area.
The depressed metabolism and lack of
bodily movements, characteristic of hibernation, may account for the storage phenomen of hormone material in the cortex
and medulla. Since the adrenals from the
control and cold-exposed animals showed
no histochemical differences, cold apparently is not a stress agent to the ground
squirrel.
ACKNOWLEDGMENT
The author acknowledges the generous
and able technical assistance of Mrs. Margo Dyess in preparing the slides for this
study.
LITERATURE CITED
Bourne, G. 1951 Cytology and Cell Physiology.
Clarendon Press, Oxford.
Cannon, W. B., A. Querido, S. W. Britton and
E. M. Bright 1927 Studies on the conditions
of activity in endocrine glands. XXI. The role
of adrenal secretion in the chemical control
of body temperature. Am. J. Physiol., 79: 466507.
Cori, C. F., and G. T. Cori 1928 The mechanism of epinephrine action. I. The influence of
epinephrine on the carbohydrate metabolism
of fasting rats, with a note on new formation
of carbohydrates. J. Biol. Chem., 79: 309-319.
Cowdry, E. V. 1952 Laboratory Technique in
Biology and Medicine. Williams and Wilkins,
Baltimore.
Deane, H. W., and C. P. Lyman 1954 Body
temperature, thyroid and adrenal cortex of
hamsters during cold exposure and hibernation, with comparisons to rats. Endocrinology,
55: 300-315.
Griffith, F. R., Jr., A. Omachi, J. E. Lockwood
and T. A. Loomis 1947 The effect of intravenous adrenalin on blood flow, sugar retention, lactate output and respiratory metabolism
of peripheral (leg) tissues in the anesthetized
cat. Am. J. Physiol., 149: 64-76.
Jones, I. C. 1957 The Adrenal Cortex. University Press, Cambridge.
HIBERNATION AND THE AIDRENAL
Kaufman, N., T. L. Gavan and R. W. Hill 1958
The effects of prolonged hypothermia on the
rat. A.M.A. Arch. Path., 66: 96-99.
Kayser, C . 1950 Le sommeil hibernal. Biol.
Rev., 25: 255-282.
1933 L'hibernation des Mammiferes.
Ann&e Biol., 29: 109-150.
Kayser, C., and N. Aron 1950 Le cycle saisonnier des glands endocrines chez les hibernants.
Arch. Anat. (Strasbourg), 33: 21-42.
Knigge, K. M. 1954 The effect of acute starvation on the adrenal cortex of the hamster.
Anat. Rec., 120: 555-582.
Meyers, M. W., and H. A. Charipper 1956 A
histological and cytological study of the adrenal gland of the golden hamster (Cricetus
auratus) in relation to age. Ibid., 124: 1-25.
Nicander, L. 1952 Histological and histochemical studies on the adrenal cortex of domestic
and laboratory animals. Acta Anat., 14: suppl.
16.
1957 A histochemical study of adrenal
glycogen. Acta Anat., (Basel), 31: 388-397.
283
Sayers, G . , and M. A. Sayers 1948 The pituitary-adrenal system. Recent Progr. Hormone
Res., 2 : 81.
Soskin, S., and R. Levine 1952 Carbohydrate
Metabolism. University of Chicago Press,
Chicago.
Verne, J., and S. Herbert 1951 Les types lipidiques de la cortico-surrenale chez les mammiferes; leur classification et leur signification.
Ann. Endocrinol. (Paris), 12: 192-198.
Zalesky, M., and L. J. Wells 1940 Effects of
low environmental temperature on the thyroid
and adrenal glands of the ground squirrel,
Citellzis tridecemlineatus. Physiol. Zool., 13:
268-276.
Zimny, M. L. 1955 A study of lactate and
pyruvate levels during hibernation and cold
exposure. Anat. Rec., 121: 385.
Zimny, M. L., and V. Tyrone 1956 Carbohydrate metabolism during fasting and hibernation in the ground squirrel. Am. J. Physiol.,
189: 1297-300.
HIBERNATION AND THE ADRENAL
Marilyn L. Zimny
PLATE 1
1
Adrenal of a control animal. A 15-p frozen section stained with Sudan black B showing
intense sudanophilia in zona glomerulosa. X 100.
2
Adrenal of a hibernated animal. A 15-fi frozen section stained with Sudan black B showing concentric pattern of sudanophilic material i n both the zona glomerulosa and zona
reticularis. x 50.
3
Adrenal of a control animal. A 25-p frozen section treated with a 5 0 / 5 0 mixture of
concentrated sulfuric and acetic acids, Schultz reaction, showing cholesterol material
in zona glomerulosa. X 30.
4
Adrenal of a hibernated animal. A 25-p frozen section treated as i n figure 3 showing
cholesterol material in both the zona glomerulosa and zona reticularis. X 30.
5
Adrenal of a hibernated animal. A 25-p frozen section treated as i n figure 3 showing
cholesterol material in both the zona glomerulosa and zona reticularis. x 125.
6
Medulla of adrenal from control animal. A 25-p frozen section of a gland fixed in a
2.5% solution of potassium bichromate at a pH of 5.6 showing a negative bichromate
reaction (outlined in black). X 125.
7 Medulla of adrenal from hibernated animal treated as in figure 6 showing a positive
bichromate reaction. X 125.
284
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