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The reduction of osmic acid as an indicator of adrenal cortical activity in the rat.

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THE REDUCTION O F OSMIC ACID AS AN INDICATOR
O F ADRENAL CORTICAL ACTIVITY
IN THE RAT
LOUIS B. FLEXNER AND ARTHUR GROLLMAN
Department of Anatomy and the Department of Pharmacology and Experimental
Therapeutics, T h e Jolzns Hopkins University, Baltimore, Marylmd
T W O PLATES (SIXTEXX FIGURES)
INTRODUCTION
Despite several attempts to correlate one or another cytological change in the cells of the adrenal cortex with its state
of activity, there is no well-defined method whereby the activity of the gland can be judged by its histological appearance.
The practical importance of such a procedure is obvious. The
observations presented below are concerned with the variation of those elements of the cortical cells which reduce osmic
acid under conditions known to depress (administration of
cortical hormone) o r to stimulate (cold, heat, drugs) the activity of the gland. These observations have made it possible
to define cytological changes characteristic of different degrees of activity of the adrenal cortex of the rat.
MATERIAL AND METHODS
Except for a series of guinea pigs which were injected with
thyrotropic hormone, the white rat was used as the experimental animal. Litter mates matched for weight and sex
were used for each series of experiments. The animals were
sacrificed by decapitation or by a sharp blow on the head.
Tinless otherwise mentioned, the adrenals were then removed,
cut in two, and immediately placed in fixative.
207
208
LOUIS B. FLEXNER AND ARTHUR GROLLMAN
R.Iaximow's osmic acid fixative as modified by Gersh ('39)
was used in the dark. The small pieces of adrenal were placed
in about 5 cc. of a solution consisting of 1part of 25% neutral
formalin, 9 parts of Zenker's fluid without acetic acid, and
1.5 parts of 2% osmic acid. This solution was renewed after
24 hours. At the end of the next 24 hours the tissue was
rinsed well in six changes of distilled water. It was then
transferred to 2% osmic acid for 24 hours, washed in twelve
changes of distilled water over a period of 3 hour, and dehydrated by immersion in SO%, 95% and absolute alcohol
successively for 3 hour. From the absolute alcohol it was
transferred to a mixture of equal parts of absolute alcohol
and xylol for 15 minutes; then to xylol for 15 minutes and
finally embedded in 62°C. paraffin. Sections were cut at 3 or
9 p. They were passed through iodine-alcohol. Some blocks
were fixed in Zenker 's formol and stained with hematoxylin
and eosin.
I n all the experiments, except where noted, animals had free
access to the standard McCollum diet and water. They were
maintained at a uniform room temperature of 80 t 5".
RESULTS
The reduction o f osrnic acid b y the normal adrevzal gland.
On the basis of their reduction of osmic acid five zones (plate
2, fig. 1) can be distinguished in the adrenal cortex of the
normal white rat. The most peripheral of these consists of
the glomerulosa plus a thin layer of cells, varying slightly
in its thickness, of the underlying fasciculata. This zone will
be referred to as the peripheral zone. Immediately beneath
this peripheral zone and lying i n the fasciculata, in the normal animals of the ages studied here, is a zone almost free of
substances reducing osmic acid; this zone will be designated
the clear zone. The remaining fascicular zone will be divided
into two portions : approximately the outer two-thirds contains considerable reducing substance aEd will be referred
to as the outer fascicular zone; the inner portion of the fasciculata, normally poor i n reducing substances, will be desig-
ADRENAL CORTICAL ACTIVITY
209
nated as the inner fascicular zone. Finally, there is the thin
reticular zone lying next t o the medulla.
Adrend cortical hormone series. The most certain method
for reducing the activity of an endocrine gland is to administer the hormone elaborated by that gland. To avoid the
incidental effects, inevitable when one injects a crude extract
into animals, we gave the hormone by mouth, exclusively.
Animals 1 month of age were used in order to minimize the
quantities of hormone necessary for the experiment. The
adrenal cortical hormone was assayed on adrenalectomized
rats (Grollman, '36) and incorporated with a weighed amount
of the animal's food. Either a charcoal adsorbate, or a purified concentrate prepared by the methods described elsewhere
(Grollman, '36, '39) or commercially available forms of these
products were uti1ized.l
The results of the administration of the cortical hormone
are given in table 1 and figures 2, 3 and 4 of plate 1. I n a
dosage of 0.5 unit a day f o r 3 days the hormone produced
the first definitive change from normal. This change consisted of a diminished reduction of osmic acid in the peripheral and clear zones. A dosage of 2 units a day (plate 1,
fig. 3) f o r 3 days practically abolished reduction in the peripheral and clear zones and in addition decreased the reducing
substances in the outer fascicular zone. Six units given daily
for 4 days (plate 1, fig. 4) almost completely eliminated reducing substance from all zones except the outer fascicular
where, however, it was definitely diminished.
It appeared that the zones in order of sensitiveness to the
cortical hormone, as measured by changes in the osmic acid
reducing substances, were first the peripheral and clear zones ;
second, the fascicular and reticular zones. Complete disWe are indebted to Dr. Ernest Blanchard of Schiefflin and Company, and to
Dr. George Cortland of the Upjohn Company, f o r a supply of p a r t of the hormone used in the present investigation.
a The term unit as used here is t h a t amount which suffices t o maintain normal
growth in a n adrenalectomized month old rat. This amount of hormone is equivalent, when tested in this manner, to 1 mg. of desoxycorticosterone acetate or
0.01 mg. of the most potent crystalline product obtainable (Grollman, '39).
0
TABLE I
+,
1-month-old control
Cortin, 0.25 unit f o r 3 days
Cortin, 0.5 unit for 3 days
Cortin, 2 units f o r 3 days
Cortin, 6 units f o r 4 days
4-month-old control
Cold (10°C.) for 0.5 hour
Cold f o r 1 hour
Cold f o r 2 hours
Cold f o r 4 hours
Cold f o r 2 2 hours
4-month-old control
Heat (39°C.) f o r 0.5 hour
Heat for 1 hour
Heat for 3 hours
Heat (39") f o r 6 hours and
(37") f o r 4 hours
4-month-old control
Hypertrophy, 1 day
Hypertrophy, 4 days
Hypertrophy, 8 days
Hypertrophy, 13 days
TREATMENT
+
z+
4+
3+
0 to 2+
4+
2+
0 to
4+
24- to 443+ t o 4+
4+
a+
0 to
0
0
+
2+
P.Z.
t r to
0 to
4+
tr
3+
+
+
2+
3+
2+ to 3+
tr
+
0
tr
0
tr
tr
+
C.Z.
+0 t o tr
2+
3+
4+
2+
2+ to 3 +
3+
3+ to 4+
3+ to 4+
t r t o 2+
+
+
2+ t o 3 +
2+
2+ t o 3+
OUTER F.Z.
+
+
+
+
+
t r to
tr to
2+
to 3+
3 + to 4+
tr to 3+
3+
tr
t r to
2+
+
+
0 to
0 to tr
0 to
0 to t r
0
INNER F . Z .
+
+
+
+
t r t o -t
tr to
2+
3+
3+ t o 4 $
0
+
tr to
2+
3+
2+
0
+
+
+
+ to 2+
Oto+
0
t r to
to 2+
0 to
Oto+
0 to
R.2
T h e variation i n the reduction of osmic acid b y the varioas zones of the adrenal cortex of the white rat under several experinzental
conditions. P.Z., C.Z., P.Z.,and R.Z., refer respectively t o peripheral zone, clear zone, fascicular zone and reticular zone.
0, indicates no reduction of osmic acid; tr, a trace of reduction,
a+, 3+ and 4 + , increasing
degrees of reduction, respectively
ADRENAL CORTICAL ACTIVITY
211
appearance of reducing substances occurred last in the outer
fasciculata.
Cold series. Litter mates, 4 months old, were placed in a
room at 10°C. for periods up to 22 hours. The effect of this
environment on the content of substances reducing osmic acid
is shown in table 1 and in the photomicrographs of plate 2
(figs. 2 to 4). Cold of this degree produced an effect obvious
a t the end of 4 hour. The most constant change consisted of
an increase in reduction by the peripheral and clear zones;
there was also, in some instances, a definite increase in the
inner fascicular and reticular zones. Exposure to cold for
1hour further increased the reducing substances in the peripheral, clear and outer fascicular zones without producing constant changes in the other zones. Additional cold, up to a
period of at least 4 hours (plate 2, fig. 3) produced its most
pronounced change by an increase in reduction by the fascicular and the reticular zones. Animals in the cold for
periods approaching 1day (fig. 4) showed a very marked loss
of reducing substance. This was evident in all zones ; but the
largest quantity of reducing substance was retained by the
peripheral zone.
It appeared that the zones in order of sensitiveness to cold
as measured by changes in substances reducing osmic acid
were the peripheral and clear, the outer fascicular, and finally
the inner fascicular and reticular zones.
Heat series. Litter mates, 4 months old, were exposed t o
temperatures from 37 to 40°C. for various periods up to
10 hours. This elevation of temperature produced a marked
effect a t the end of hour (table 1 and plate 2, fig. 5). Substances reducing osmic acid increased in all zones with the
most marked effect evident in the peripheral and clear zones.
Exposure to heat for 1hour produced no further noteworthy
difference beyond an intensification of reduction in the outer
fasciculata. A marked change was, however, noted in animals which were kept at these high temperatures f o r from
3 to 5 hours (fig. 6). This change consisted of almost complete loss of reducing substance from all portions of the
+
THE ANATOMICAL RECORD, VOL. 75, NO. 2
212
LOUIS B. FLEXNER. A N D ARTHUR GROLLMAN
cortex except the peripheral zone and the inner portion of
the outer fasciculata. The adrenals (figs. 7 and 8) from a
prostrate animal kept at high temperature f o r 10 hours
showed either no reducing substance or reducing substance
limited to the peripheral zone.
The different zones reacted to heat in essentially the same
order as t o cold excepting that, at the temperatures used, the
reaction to cold took relatively longer to manifest itself.
Hypertrophy series. A series of 4-month-old animals was
unilaterally adrenalectomized and the remaining adrenal
removed at various periods after the adrenalectomy. The
results are shown in table 1 and plate 1 (figs. 5 to 7). The
earliest change noted, 1 day following adrenalectomy, was an
increase in reducing substance in the peripheral and clear
zones of the remaining adrenal. This had become striking
by 8 days (fig. 6). Increase of reducing substance in these
zones was followed by definite increase in the outer fascicular,
reticular and inner fascicular zones in the order named. All
zones showed an almost uniformly great increase of reducing
substance within 2 weeks (fig. 7). The different zones reacted
t o hypertrophy in the same order as to heat and cold.
Other observations. Variations, usually slight but at times
marked, were observed between the two adrenal cortices of
an experimental animal and in the reaction of different parts
of the same cortex in response to cortin, cold, heat and hypertrophy. When the two glands showed differences in reaction,
at times the right, and at other times the left gland was the
more reactive. The cortices of several normal animals older
than a year were examined; they showed considerable variation in reducing substance; in most instances there was less
reducing substance in the peripheral and outer fascicular
zones than in the 4-month-old animals. The clear zone in
animals 1month old contained somewhat more reducing substance than that of animals 4 months old (fig. 1, plates 1
and 2).
Substances reducing osmic acid were also studied in several
other circumstances without any attempt to produce other
ADRENAL CORTICAL ACTIVITY
213
than a marked effect. Thyrotropic hormone3 produced a
general increase in the reducing substance of the cortex of
the guinea pig adrenal ; injection of tetrahydro-p-naphthylamine and inanition (plate 1, fig. 8) due to starvation gave
large increases in the reducing substances in the cortex of
the rat.
The histological picture of the adrenal was not affected if
the animal, after death and before fixation, was kept at room
temperature for 2 hours and then for 20 hours in the ice box.
DISCUSSION
The experimental conditions described above have been
chosen with the view of reducing or increasing adrenal cortical activity from the normal. The adrenal cortex of an animal given cortical hormone will be inhibited, for in the presence of an extraneous supply of its hormone, the cortex will
deliver less hormone to the animal. The other procedures
utilized in the above-described experiments all stimulate adrenal cortical activity. I n the case of unilateral adrenalectomy, the second gland hypertrophies and maintains a normal
supply of the vital hormone. Extremes of temperature also
stimulate the activity of the adrenal cortex. This has been
demonstrated by 1) the hypertrophy of the glands which
follows subjecting an animal t o these conditions, 2 ) the additional hormone required to maintain animals following adrenalectomy as compared t o that necessary when such animals
are maintained at the normal room temperature and 3) the
short survival period of adrenalectomized animals maintained
at abnormal temperatures (Grollman, '36). Inanition, also,
as judged by the same criteria stimulates adrenal cortical
activity although conditions here are probably complicated
by the incidental metabolic disturbances. The injection of
tetrahydro-P-naphthylamine,which is a sympathetic stimulant, o r of the thyrotropic hormone of the anterior pituitary
We are indebted to Ayerst, McKenna and Harrison for some of the thyrotropic hormone used. The remainder was prepared in the laboratory by alkaline
extraction of the anterior pituitary gland of cattle.
214
LOUIS B. FLEXNER AND ARTHUR GROLLMAN
gland, also cause hypertrophy of the adrenal and increase
cortical activity secondary, probably, to the increased metabolism which these substances induce (Grollman, '36).
Stimulation of cortical activity causes a n increase of osmic
acid-reducing substances ; reduction of activity, a decrease in
these substances. Ingle, Higgins and Kendall ( ' 3 8 ) ,who gave
rats massive but unassayed doses of cortical extract, also
noted the complete disappearance of cortical lipoid under
these conditions. The change in concentration of reducing
substance, moreover, is rather accurately proportional to the
extent of exposure to the experimental condition so that the
histological picture gives a measure of the degree of reduction or increase of activity.
The different zones of the cortex of the white rat vary in
their response to a n altered environment. The most reactive
zones a r e the peripheral and clear; they show a large increase of reducing substances with adequate stimulation and
a complete loss of reducing substance with adequate inhibition. The next most reactive zone is the outer fascicular and
it is followed by the less reactive reticular and inner fascicular zones which respond about alike. Though their response
is delayed, the reticular and inner fascicular zones can, with
adequate stimulus, show a magnitude of reaction almost as
great as the other zones. This marked response of the reticularis makes doubtful the validity of the generally accepted
view that it is an area of the cortex made up of dying cells
(Grollman, '36).
Prolonged periods of exposure to heat or cold produce a
decrease of substances reducing osmic acid which is interpreted as indicative of exhaustion. I n the one prostrate animal exposed to high temperature for 10 hours, for example,
one adrenal was practically without reducing substance and
the other showed reduction only in the peripheral zone.
It is clear, consequently, that stimulation or inhibition of
cortical activity produces characteristic changes not to be confused with one another except in extreme cases. These extreme cases involve complete loss of reducing substances due
ADRENAL CORTICAL ACTIVITY
215
to exliaustion of the stimulated cortex or complete loss of
reducing substances due to suppression of activity by large
doses of cortical hormone. Confusion may, however, arise
from changes due to metabolic disturbances which may give
a histological picture like that found in a highly stimulated
gland.
The substances responsible for the reduction of osmic acid
in the adrenal cell are of two varieties. The unsaturated
constituents of the visible droplets of lipoid will reduce osmic
acid and deposit the lower oxides of osmium on the surface
o r within the interior of these droplets. Besides these visible
droplets other reducing substances are present in the cytoplasm of the cell which also cause the deposition of reduced
osmic acid (Hoerr, '36). These reducing substances comprise such compounds as ascorbic acid and glutathione, which
are present in large quantities in the adrenal, the cortical
hormone, and probably other, as yet unidentified, reducing
substances.
I t should be emphasized that the definite correlation which
we have shown to exist in the rat between the reduction of
osmic acid and cortical activity cannot yet be transferred t o
other species. For example, in the mouse, experiments (Gersh
and Crollman, '39) undertaken from a point of view different
from that presented here, may at first glance present a contradiction t o our findings on the rat. I n the mouse, small doses
of cortical hormone produce apparently inconstant clianges
in the cortical reducing substances. It is well known (Whitehead, '33), however, that the lipoid content of the adrenal in
the mouse is highly variable. Correlation of concentration
of reducing substances with state of activity, consequently,
must in the case of the mouse involve a large series to be
treated by statistical methods o r a smaller series composed of
highly uniform individuals. Moreover, since lipoid and other
reducing substances are not conspicuous cytological inclusions
in the adrenal cortex of all species (Elliott, '14), it is realized
that the results obtained in the rat are probabIy not applicable to all other mammals. Undoubtedly, the degree of uni-
216
LOUIS B. FLEXNER AND ARTHUR GROLLMAN
formity of our own experiments is attributable in part to the
maintenance of constant experimental conditions and the use
of a highly standardized experimental animal.
SUMMARY
The cytological changes occurring in the adrenal cortex of
the rat as revealed by the reduction of osmic acid have been
correlated with changes in the activity of the gland. Reduction in adrenal activity was induced by the administration
of various amounts of the cortical hormone; activity was
stimulated by unilateral adrenalectomy, heat, cold, the injection of tetrahydro-p-naphthylamine,thyrotropic hormone, or
inanition. The changes in reduction of osmic acid by the
cortical cells occurring under these various conditions are
described as they occur in the various zones of the adrenal.
I n general, depression of activity is correlated with a decrease in the reduction of osmic acid; stimulation of activity,
with an increase in the reduction of osmic acid.
LITERATURE CITED
ELLIOTT,
T. R. 1814 Pathological changes i n the adrenals.
Quart. J. Med.,
pp. 47-90.
GERSH, I. 1939 The structure and function of the parenchymatous glandular
cells in the neurohypophysis of the rat. Am. J. Anat., vol. 64, pp.
V O ~ . 8,
407-444.
GFXSH, I.,
AND A. GROLLMAN 1939 The nature of the X-zone of the adrenal
gland of the mouse. Anat. Rec., in press.
GROLLMAN, A. 1936 The Adrenals. Baltimore and London.
1939 The comparative activity of desoxyeorticosterone and other
crystalline derivatives and of purified extracts of the adrenal cortex.
J. Pharm. Exp. Therap., in press.
I I O E m , N.
1936 A cytological analysis of the liposomes i n the adrenal cortex
of the guinea pig. Anat. Rec., vol. 66, pp. 317-342.
INGLE, J., G. M. HIGGINS
AND E. C. KENDALL1938 Atrophy of the adrenal
cortex in the rat produced by administration of large amounts of
cortin. Anat. Rec., vol. 71, pp. 363-372.
WHITEHEAD,R. 1933 Variations in the cortical lipoid of the mouse suprarenal
with sex and age. J. Anat., 701. 67, pp. 393-397.
PLATES
217
PLATE 1
EXPLANATION 0% FIGURES
Pliot~)iiiicrograplisof sections of the adrenal gland t o show the effects of the
continuous administration b y mouth of the adrenal cortical hormone, of hypertrophy of the remaining gland following unilateral adrenalectomy, and of inanition. The preparations were fixed in osmic acid, sectioned a t 9 P and photographed
unstained. X 60.
1 Untreated control. Age 4 weeks. The peripheral, clear, outer fascicular,
inner fascicular and reticular zones are indicated respectively by a, b, c, d and e.
The medulla is indicated by m.
2 Adrenal cortical hormone (4 unit) administered f o r 3 days. Age 4 weeks.
3 Adrenal cortical hormone (2 units) administered f o r 3 days. Age 4 weeks.
4 Adrenal cortical hormone (6 units) administered for 4 days. Age 4 weeks.
5 Effect of unilateral adrenalectoniy on thc rcmaining adrenal, 4 days subsequently. Age 4 months.
6 As in figure 5 after 8 days.
7 As in figure 5 a f t e r 13 days.
8 Effect of 2 days inanition on a 4-month-old rat.
218
219
THE A N A T O X I C A L RECIORD, I O L . 7 5 , N O
2
I ’ I J ~ ~ T2E
ESPLAXATION OF FIGURFS
1 Norinn1 4-month old control.
a , 11, c, d, e, a n d in 1i:ire the same indications
as i n fiyuie 1, plate 1.
EWert of cold ( I O O C . ) for 0.5 hour. Age 4 months.
Saine :is figure 2 but f o r 1 hours.
4 Snnir a s fignrc L‘ h u t f o r 22 hours.
.? Effect of lieat ( 3 7 ° C . ) f o r 0.5 hour. Age 4 inoutlie.
G Same as figure 5 but f o r 3 hours.
7 Saiiic a s figure 5 hut f o r 1 0 hours. (Right adrenal.)
8 Same :IS figurr 5 hut f o r 1 0 hours. (Lcft adrrnnl.)
2
3
220
B D R E K A L CORTICAL ..\CTIVITP
LOUIS 11. PLPXKIIR AND A R T H C K GROI.LM.4h-
PLATE 2
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