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Some cytological and hormonal changes in the posterior lobe of the rat's pituitary after water deprivation and stalk section.

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SOME CYTOLOGICAL AND HORXONAL CHANGES I N
THE POSTERIOR LOBE O F THE RAT’S
PITUITARY A F T E R WATER DEPRIVATION
AND STALK SECTION
ROBERT C. HICKEY, KENDRICK HARE AND RUTH S. HARE
Department of Anatomy, Cornell University Medical College, New Pork City
SIX TEXT FIGURES AND THREE PLATES (SEVENTEEN FIGURES)
That the posterior lobe of the pituitary exerts an important
influence on water exchange is now widely accepted, while
much less is known about the influence of water exchange upon
the posterior lobe. The few experiments which have been done
show that the hormonal content decreases after water deprivation. Simon (’34) demonstrated a decrease in the pressor
and oxytocic potency of the rat hypophysis after a deprivation
of drinking water of 5 to 7 days’ duration. Simon and Kardos
(’34) obtained the same results with the rabbit and guinea
pig, but found no change in the pressor or oxytocic potency
of the cat hypophysis after 14 days on a similar regime.
Gersh ( ’39) reported that assays on the posterior lobes of five
rats deprived of water for 5 days showed about a 90% diminution in the pressor, oxytocic, and antidiuretic potencies. His
paper, however, was conccrned chiefly with the demonstration
of intracellular osmiophilic droplets peculiar to the neurohypophvsis. When rats were deprived of water for 1 to 8
days, the intracellular content of osmiophilic material increased rapidly and remained high throughout the period.
Gersh reasoned from this that the osmiophilic content of the
posterior lobe is an index to its activity, and concluded
that “in the adult, cytological changes in these (parenchymatous) cells coincide with physiological fluctuations i n the
Presented before the American Physiological Society, Chicago, Ill. 1941.
319
320
ROBERT C. HICKEY A N D 0THER.S
rate of secretion of the antidiuretic substance into the blood
stream” (p. 427).
In the following experiment we have assayed the antidiuretic
potency of the posterior lobes of normal rats, of rats deprived
of water for 2 to 13 days, and of rats with a permanent
diabetes insipidus. We have also studied these posterior lobes
after fixation and staining by Gersh’s technique.
1. NORMAL AND DEHYDRATED RATS
Methods
Adult rats in groups of five or seven were completely deprived of water and fed only dried food (water content 5%)
for periods varying from 2 to 13 days. Normal rats were
kept under identical conditions except that they had free
access to water. All rats were males of the same age. Just
before an assay the animals of one normal and of one dehydrated group were killed by decapitation under Nembutal
anaesthesia. The pituitaries were removed and the posterior
lobes dissected out. Each group was treated in the following
manner : two lobes were fixed in the same bottle of Maximov’s
fluid, according to Gersh; the remaining lobes were macerated
with sand in 0.9% saline, and the resulting extract diluted
with saline to such a volume that 1 cc. of solution contained
0.001 of one posterior lobe. For assay this solution was injected intravenously, since rates of absorption after subcutaneous injection are variable.
The method of assay is described in detail elsewhere (Hare,
Hickey and Hare, ’41). The antidiuretic potency of the material assayed was determined by its effect on the urine concentration of the diabetes insipidus dog. The technique was
formerly used to demonstrate only the presence or absence
of antidiuretic material. Since in these experiments we wished
t o distinguish between different amounts of antidiuretic substance, it was necessary to estimate the response quantitatively,
and to establish both the reproducibility of the response to
a given dose and the distinction between different doses of the
same material. A given antidiuretic response was measured
CHANGES IN RAT PITUITARY AFTER DEHYDRATION
321
by the increase in urine concentration (increase in creatinine
U/P ratio) multiplied by the duration of the increase. If this
criterion is used, figure 1 demonstrates that different doses
of pitressin elicit correspondingly different responses, within
a small dosage range. Figure 2 shows that the magnitude of
the responses in figure 1is directly related to the dose.
When identical doses of the same material were assayed,
there was some variation in response. It was therefore necessary, in assaying dehydrated and normal lobes, to repeat the
injections as a check. When the responses were not reproduced, the assay was discarded.
Results
Typical assays are shown in figures 3 and 4. The response
to the dehydrated posterior lobe was compared with that to
the normal control, and the potency of the dehydrated lobe
expressed as per cent of normal. I n figure 5 the number of
days of dehydration is plotted against the potency of the
posterior lobe. There is a continuous decline in potency from
the second t o the thirteenth day of dehydration.
A careful study was made of complete serial sections ( 5 p )
of the posterior lobes stained with osmic acid according to
Gersh. Two to ten lobes for each day of dehydration, and
thirty normal controls were studied. No correlation was found
between intracellular osmiophilic droplet content and days
of dehydration or antidiuretic potency (plate 2). The normal
variation was enormous (plate l), and all variations found in
the dehydrated lobes fell within the normal limits. As Gersh
pointed out, in a given lobe the number of droplets varied so
greatly from one region to another, that it is difficult to
select a representative section from the series. Sections made
in the sagittal plane were more easily evaluated than cross
sections.
2. DIABETES INSIPIDTTS RATS
Although the potency of the posterior lobe was found to
decline during dehydration, and although no consistent varia-
CREATININE
UfP
m1
I
POTENCY
WX1
I
OO
2
:*
5
501
DOSE
A
:
5
*
,
10
*
,
15
DEHYDRATION TIME
*
Fig. 1 Different antidiuretic responses t o different doses of pitressin. Creatinine
U/P ratio is plotted against time in minutes. Arrows show time of injection, and
dosage is given in milliunits of pitressin.
Fig. 2 Correlation of dose and response. Areas of responses ( t A U / P ) in
figure 1 are plotted against the dose in nlilliunits of pitressin. Between 0.4 and
2 milliunits the relationship is linear, but since 4 milliunits elicited no greater
response than 2, doses greater than 2 milliunits cannot be assayed by this method.
Fig. 3 Assay of posterior lobe from normal and 6-day dehydrated rats. Coordinates similar to figure 1. Arrows indicate time of injection; dosage is given
as fraction of oiie posterior lobe. Average of the two responses to &day dehydrated
lobe was 25% of the response to normal lobe.
Fig. 4 Assay of the posterior lobes from normal and 13-day dehydrated rats.
Similar to figure 3. Since there was no response to 0.001 of the 13-day dehydrated
lobe, the dose was increased fifty times. The response then obtained was slightly
larger than the response to 0.001 of the normal lobe; the potency was therefore
about 2% of normal.
Fig. 5 Effect of dehydration o n the antidiuretic potency of the posterior lobe.
The abscissa is the time of dehydration in days, the ordinate is the potency in per
cent of normal. The triangle is the assay reported by Gersh ('39).
F'ig. 6 Assay of diabetes insipidus posterior lobes. Similar to figures 3 and 4.
There was no response to the injection of the diabetes insipidus lobes, although
a relatively enormous dose was given.
32"
CHANGES IN RAT PITUITARY AFTER DEHYDRATION
323
tioii in osmiophilic droplet content accompanied the decrease
in potency, these data afford no evidence on the activity of
the lobe. It is Gersh’s contention that the osmiophilic droplet
content is an index of the activity of the cells secreting the antidiuretic substance, rather than of the potency of the lobe.
The greatly increased water exchange of the diabetes insipidus animal is evidence of the diminished secretion of antidiuretic hormone by the posterior lobe. We have therefore
assayed posterior lobes from diabetes insipidus rats and
studied them histologieally according to Gersh’s technique
Methods
The pituitary stalks of four rats were cut through a retropharyngeal approach by Dr. R. A. Phillips, and the animals
obtained from him 4 months after stalk section. Water exchange was followed for several days before assay, t o verify
the persistence of the polyuria. The day of the assay the
animals were killed, and the brain was removed and fixed in
formalin. The pituitary was split longitudinally in situ; one
half was fixed in Maximov’s fluid, and the other extracted for
assay. A normal rat was prepared in the same way.
Results
The pituitaries of the diabetes insipidus rats had no antidiuretic effect (fig.6) (Fisher and Ingram, ’36). However,
the posterior lobes, although smaller than the normals, contained many intracellular osmiophilic droplets (plate 3). The
same results were obtained in the two diabetes insipidus rats
not shown in figure 6 and plate 3. Although microscopic examination of the pituitary stalk was obviously impossible,
evidence of complete transection is based upon study of the
hypothalamus. I n each case the scar involved the median
eminence, and the supraoptic nuclei were atrophied (plate 3)
(Magoun and Rnnson, ’39).
324
ROBERT C. HICKEY AND OTHERS
DISCUSSION
It has been shown that animals excrete an increased amount
of antidiuretic substance during dehydration (Gilman and
Goodman, '37, and Ingram, Ladd and Benbow, '39). We have
shown that the antidiuretic potency of the posterior lobe
diminishes during dehydration. These two phenomena are
apparently coincident, but their interdependence has yet to
be demonstrated.
We have confirmed Gersh's cytological method f o r demonstrating intracellular lipid material peculiar to cells in the
neurohypophysis. I n a series of assays on posterior lobes
of dehydrated rats we have found a progressive diminution of
the antidiuretic potency; this agrees with the one assay, on
rats dehydrated for 5 days, reported by Gersh (fig. 5). However, we have been unable to confirm his correlation of dehydration and intracellular lipid material. Gersh and Brooks
( '41) described occasional cells containing osmiophilic droplets in the posterior lobes of rats after section of the pituitary
stalk. They thought these cells might be related to some
residual hormonal content since Fisher and Ingram ( '36)
reported that the hormonal content of the posterior lobes of
diabetes insipidus cats, while enormously reduced, was not
entirely eliminated. We found a large amount of intracellular
lipid material, apparently just like that in the normals, in
the posterior lobes of rats with a permanent diabetes insipidus,
which had an antidiuretic potency of far less than 0.5% of
normal. We therefore cannot subscribe t o Gersh's theory of
the correlation of cytological changes in the posterior lobe
with fluctuations in secretory activity.
CONCIAUSTONS
1. Dehydration diminishes the antidiuretic potency of the
posterior lobe.
2. The number of intracellular osmiophilic droplets in the
posterior lobe is independent of the antidiuretic potency or
secretory activity of the gland, and of the degree of dehydration of the animal.
CHANQES IN RAT PITUITARY
AFTER DEHYDRATION
325
LITERATURE CITED
FISHER,
c., AND w. R. INGRAM
1936 The effect of interruption of the supraoptico-hypophyseal tracts on the antidiuretic, pressor, and oxytocic
activity of the posterior lobe of the hypophysis. Endocrin., vol. 20,
pp. 762-768.
GERSH,I. 1939 The structure and function of the parcnchymatous glandular
cells i n the neurohypophysis of the rat. Am. J. Anat., vol 64, pp.
407-444.
AND c. Mcc. BRUOKS1941 Correlation of physiological and cytological changes in the neurohypophysis of rats with experimental
diabetes insipidus. Endocrin., vol. 28, pp. 6-19.
GILMAN,A., A N D L. GOODMAN1937 The secretory response of the posterior pituitary to the need for water conscrvation. J. Physiol., vol. 90, pp. 113124.
HARE,KENDRICK,
R. C. HICKFXAND R. 5. HARE 1941 Renal excretion of antidiuretic substance by the dog. Am. J. Physiol., vol. 134, pp. 240-244.
INGRAM,
W. R., L. LADD
AND J. T. BENBOW 1939 The excretion of antidiuretic
substance and its relation to the hypothalamico-hypophyseal system
in cats. Am. J. Physiol., vol. 127, pp. 544-551.
MAGOUN,H. W., AND S. W. RANSON 1939 Retrograde degeneration of the
supraoptic nuclei after section of the infundibular stalk in the monkey.
Anat. Rec., vol. 75, pp. 107-123.
SIMON,ALEXAKDER1934 The pressor and oxytocic content of the hypophysis of
rats under various conditions. Am. J. Physiol., vol. 107, pp. 220-226.
SIMON,
ALEXANDER,
AND ZOLTAN
KARDOS1934 Uber den Gehalt der Hypophysenhinterlappen normaler und durstender Tiere an blutdruclr- und Uteruswirksamen Stoffen. Arch. f. exp. Path., Bd. 176, S. 238-242.
GERSH, I.,
PLATE 1
EXPLANATION OF FIGURES
7 and 8 Normal variation in osmiophilie droplet content. Two lobes shown are
from litter mate males, kept under the same conditions. They were fixed and
stained in the same bottle according t o Gersh’s technique. Transverse sections
were cut at 5 B. Tlie sections shown are the most deeply stained from each. X 77.
9 High power magnification showing intracellular position of the osmiophilic
droplets.
386
C IIANGES I N RAT 1’1TVITARY AFTER URHY IIRATION
ROBERT
(’
HICKEY A N D O T l l L R S
32i
PLATE 1
PLATE 2
l?SPl,ANATION OF FIGURES
Siigittal sect ions of nornial m c l dehydratcd lolxu.
43 months old, killrd on th(1 same day.
10 and 11 Noriiinl rats.
12,13 and 14 Rats dehydratrd for 4 days.
15,lG and li Rits dehydrated for 7 dnys.
338
X 45. All rats wrrc males,
CHANGES I X RAT PITUITARY AFTER DEHYDRATION
ROBGICT C . HICKEY A N D O T H E R S
329
PLATE 2
I’TAl’E 3
F..\PL.\Nd’I‘ION
O F PIGURLS
Postcrior 1ol)r :ind su1ir:iojitic* iiuclcus f i o m diabetes insipidus 1:its a i i d ~iormal
control, w-liosc ass:iy is shown iii figure 6 . Trnnsvcrsc sections of t h e Iiypothalanius
w w e cut :it 50 LL and sti~ined with e ~ e s y l violet. x 58. Sagittal sections of
postcrior lobe, trcwted awnrdiiig to Gcrsli, W ~ ~ I ’cCu t at 5 fi. x 48.
18 and 1 9 Normal.
20 and 2 1 1h:ibctes insipidus no. 23
22 and 23 Di:,l)etcis insipidus 110. 36.
330
CIIASGES 1X RAT J’ITIJITARY AFTER DEIITURATION
KOBERT 0. HICKEY A N D OTliEHS
331
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