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The effect of thiouracil excess thyroxine and thyroidectomy on the ependymal cells with special reference to the subcommissural organ.

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The Effect of Thiouracil, Excess Thyroxine a n d
Thyroidectomy on the Ependymal Cells with
Special Reference to the Subcommissural Organ ’
Department of Anatomy and Embryology, College of Veterinary Medicine,
Helsinki, Finland
The effects of cxccss thyroxine, thiouracil and thyroidcctomy upon the
ependymal cells of the subcommissural organ, the choroid plexus and the wall of the
caudal part of the third cerebral ventricle in the adult male albino rats were studied.
A total of 203 rats were used. Thiouracil and thyroxine werc administered orally,
thyroidectomy was performed by means of radioiodine. Thiouracil was seen to diminish the ependymal nuclear volume especially i n the subcommissural organ and i n the
wall of the third ventricle. Similar effects were observed by thyroidectomy, though
the change was significant only in the ependyma covering the wall of the third
ventricle. Excess thyroxine, again, increased nuclear volume of all ependymal cells
studied. The changes could be observed over a period of 4-18 days. The signs of a
return to normal after withdrawal of thyroxine arid thiouracil were perceived after
four days. Visual estimation revealed no clear change in aldehyde-fuchsin-positive
“secretory material” in the subcommissural organ of any test group. The functional
significance o f the results is discussed.
The ependymal cells cover the ~vallsof
the cerebral ventricles. There is also a layer
of ependymal cells on the surface of the
choroid plexus. In some regions the ependymal cell system is specialized. An example of this is in the subcornmissural organ (SCO), where the ependymal cells
differ both structurally and presumably
also functionally from other ependymal
The current concept is that the site of
formation of cerebrospinal fluid is the choroid plexus (Fleischhauer, ’65). The ependymal cells of the choroid plexuses
maintain a characteristic electrolyte structure in the cerebrospinal fluid; in other
words these cells actively regulate the
transfer of ions and apparently also the
other components of the liquor. The production of cerebrospinal fluid is, thus, labelled a process of “secretion.” The physiological significance of the ependymal
layer covering thP wall of the cerebral ventricle itself is still largely unclarified. It
appears to play a role in the metabolism
of the tissues of the central nervous system (Oksche, ’61) and very possible also
acts as a secretory cell system.
A N A ~ Rrrc.,
159: 379-366.
The SCO is today generally regarded as
a n independent structural entity, possibly
with its own function. It is believed to be
a secreting gland; its secretion taking place
mainly in the ependyma and possibly also
in the underlying hypendyma (Olsson, ’58;
Talanti, ’58; Oksche, ’61; Lenys. ’65;
Palkovits, ’65). It is possible to demonstrate within the ependymal cells the presence of a secretion which stains selectively
in the same manner as hypothalamic neurosecretory material. The secretion is
thought to function in thc control of fluid
balance. It would thus be a n auxiliary
neurosecretory system more primitive in
character than the hypothalamic system
(Landau, ’60).
The SCO is apparently reciprocally associated with certain endocrine glands.
It has been shown (Lenys, ’65; Palkovits,
’65) that changes suggestive of altered activity take place in the structure of the
adrenal cortex under the influence of SCO
manipulation, However, the results of experiments endeavouring to establish the
aldosteronotropic effect are contradictory.
I Supported by grants from the Sigrid. Jusdlius
Foundation, Helsinki, Finland and the Kational Research Council for Medical Sciences, Finland.
Many investigations, particularly morphological studies, suggest the existence of
a SCO-pineal system, but functional investigations in this regard have also been
conflicting and indefinite.
Little work has been reported on the
relationship between the thyroid gland and
the SCO. Yamada (’61) observed that
electrical ablation of the nuclei habenulae,
the pineal body and the SCO of the rat
had no effect on the thyroid gland. Earlier
it was observed that changes in thyroid
function affect both secretory ganglion
cells of the hypothalamus and the amount
of secretory material in the neurosecretory
system (see for ref. DAngelo, ’63). ‘The
object of the present study was to discover
whether similar changes are observed in
the morphology of the ependymal secretion
of the SCO. As a parallel investigation a
karyometric study was made of changes
in the ependymal cells of the choroid
plexus and the walls of the cerebral ventricle.
The animals used for the experiment
were 203 adult male albino rats of the
LongEvans strain. Their weight ranged
between 202 and 245 gm. All animals were
kept under the same conditions and in the
same room, receiving the same basic pellet diet with tap water ad libitum.
Thyroxine (Na-I-thyroxine 0.01% ) and
thiouracil (4-methyl-2-thiouracil 1 % ) were
administered orally with the diet. Thyroidectomy was performed, giving 13’1 intraperitoneally in a quantity equivalent to
1.5 mC per animal. The animals were
divided into three groups. Each series had
its own control animals, but because of
practical reasons, it was not possible to
carry out the tests simultaneously.
In the first test series there were 108
rats of which 36 were control animals.
The experimental rats were divided into
two equal subgroups, the first receiving
thyroxine, the second thiouracil. These
subgroups were further subdivided into
six groups of six rats each. “he period of
treatment in the case of group I was 4
days, group I1 8 days, group I11 12 days,
group IV 18 days, group V 24 days and
group VI 30 days. The animals in each
group were killed at the termination of the
test period and simultaneously control animals of equal size were also killed.
In the second test series there were 75
animals of which 25 were controls. The
experimental animals were divided into
two equal groups, of which the first received initially eight days treatment with
thyroxine and the second, eight days thiouracil treatment. Both test groups were
subdivided into groups of five. Those in
group I were killed upon termination of
treatment, group I1 4 days, group I11 8 days,
group IV 14 days and group V 21 days
after withdrawal of drugs. Correspondingly, control animals were killed simultaneously to the experimental rats.
In the third series there were 20 animals
of which 10 were control rats. The 10
experimental animals received radioiodine
treatment. A11 the animals in this series
were killed after eight weeks of treatment.
All rats were killed by rapid decapitation and without anaesthesia. The brains
and thyroid glands were embedded in paraffin after fixation in 10% formalin. The
brains were sectioned serially at 10 11 in
the sagittal plane. Every other section
was retained of which alternate ones were
stained with aldehyde-fuchsin (Landing,
IIall and West, ’56) and with hematoxylineosin. Sections were taken of each thyroid
gland to ascertain the effects of the thyroxine, thiouracil and radioiodine treatment. The linear measurement technique
(Uotila and Kannas, ’52) was used to
determine the amount of the epithelium,
colloid and stroma of the thyroid.
For determination of the nuclear crosssection area of the ependymal cells, an
image of the nucIeus was projected at
1700 X on a drawing board with the aid
of a camera lucida, and its contour traced
onto paper. The areas were then measured
by planimetry. The nuclear area of about
50 ependymal cells of the SCO, the choroid plexus and the wall of the caudal part
of the third ventricle was measured in each
animal. In all about 30750 cells were
The amount of aldehyde-fuchsin-positive “secretory material” in the SCO was
recorded on the basis of visual assessment
and a scoring system of 0-5 was employrd.
Sections from the different groups which
were to be cornpared were always stained
simultaneously and in the same reagents
and staining solutions.
From the series of observations obtained
the means and their standard errors were
calculated. Student's t-test was applied in
comparing two means (Fisher, '50).
The epithelium of the thyroid gland was
found to have strongly increased in the thiouracil-treated rats and to have gone down
slightly in those treated with thyroxine
(figs. 1, 2, 3 ) . E.g. the average epithelium
percentage was in the rats of the thiouracil
groups 111-VI in the first test series 81.6,
in the rats of thyroxine groups 111-VI in
the first test series 40.4 and in the correspmding control rats 45.2. Microscopical
examination revealed that complete radiothyroidectomy had been achieved in all
animals treated with radioiodine.
Karymetric nzeasurements of the
ependymal cells
The first test series: Results are shown
in table 1. Thyroxine caused an increase
in the nuclear cross-section area in all
types of ependymal cells. This increase was
particularly short-lived in the SCO, and
returned to control levels within 12 days.
The increase in nuclear area in choroid
plexus cells was the most clearly perceptible, and the difference from the control
material was maintained at a highly significant level throughout the entire test
Thiouracil decreased the nuclear cross
section area. The weakest effect of thiouracil was observed in the choroid plexus.
Representative nuclei of the ependymal
cells of the first test series are shown in
figures 4-15.
The second test series: Results appear
in table 2. Treatment over eight days gave
essentially the same result as the first series. In all cell types the difference between the thyroxine-treated animals and
the control group was highly significant.
In those treated with thiouracil the reduction in the area of the nuclear cross section was significant only in the sco.
Withdrawal of treatment resulted in a
tendency for the nuclear size to return to
normal in all types of cells studied. Recovery was rapid and generally perceived
four days after the termination of treat-
First test series. Influence of excess thyroxine and thiouracil o n nuclear cross section areas
of the ependymal cells. Areas expressed i n squure microns
Subcommissural organ
Mean 2: S.E.
+ S.E.
Group I
Group I1
Group 111
Group 1V
Group V
Group V I
16.152 0.20
15.99 C0.21
16.18 -? 0.28
16.20 '0.26
15.962 0.18
Group I
Group I1
Group I11
Group I V
Group V
Group V I
17.71 t0.27
16.03 = 0.16
16.34 ~ 0 . 1 5
16.22 t0.35
> 0.1
16.65 C 0.20
18.72 f0.20
16.34 & 0.19
17.47 .f- 0.23
Group I
Group I1
Group I11
Group IV
Group V
Group VI
16.16 t0.31
15.56 -k 0.29
15.17 t0.23
15.91 L0.20
14.74 t0.15
> 0.1
> 0.1
< 0.01
> 0.1
< 0.001
< 0.01
15.37 0.23
14.43 + 0.29
15.02 t0.23
14.35 0.20
13.92 f0.15
15.60 + 0.13
1 As
compared with controls.
Wall of the third ventricle
Choroid plexus
20.59 -+ 0.23
20.61 C 0.25
20.34 2 0.27
20.84 0.22
20.89t: 0.26
14.86 0.16
14.90 '-0.14
1 4 . 6 0 0.18
< 0.001
< 0.001
> 0.1
> 0.1
< 0.01
Mean -1- S.E.
< 0.001
20.90 rt 0.27
19.54 r t 0.23
20.51 20.25
22.11 :t 0.26
22.23 2 0.20
> 0.1
< 0.01
< 0.1
< 0.001
< 0.001
< 0.01
< 0.1
> 0.1
> 0.1
> 0.1
< 0.001
< 0.001
19.89 r': 0.23
19.58 150.24
17.90 2 0.21
< 0.1
< 0.01
< 0.001
< 0.001
> 0.1
< 0.001
< 0.001
< 0.001
< 0.001
< 0.001
< 0.001
Second test series. Influence of withdruwul of thyroxine und thiouracil treatment on nuclear
cross section areas of the ependymal cells. Areas expressed in square microns
Subcommissural organ
Mean 5 S.E.
Group I
Group I1
Group I11
Group IV
Group V
16.58 0.23
15.672 0.20
Group I
Group I1
Group 111
Group IV
Group V
Group I
Group I1
Group 111
Group V
Choroid plexus
17.632 0.23
16.38 0.27
Wall of the third ventricle
15.60~0.19 0.001
16.50f0.23 < 0.1
16.7720.27 < 0.001
17.43:k 0.27
16.38zk 0.26
20.71= 0.23
20.25-L 0.19
20.70= 0.24
20.712 0.23
16.11-C 0.22
15.203 0.17
< 0.1
< 0.001
< 0.001
< 0.001
> 0.1
+ S.E.
< 0.001
< 0.001
> 0.1
< 0.01
< 0.1
22.15C 0.27
20.48& 0.24
< 0.001
< 0.001
> 0.1
> 0.1
> 0.1
21.26C 0.23
> 0.1
< 0.001
> 0.1
> 0.1
< 0.001
15.72C0.19 > 0.1
As compared with controls.
Third test series. Influence of thyroidectomy on nuclear C T O S S section areas o n the ependymal
cells. Areas expressed i n square microns
Subcommissural organ
Mean & S.E.
15.45& 0.16
Choroid plexus
Wall of the third ventricle
Mean f S.E.
14.86C 0.11
< 0.1
> 0.1
< 0,001
As compared with controls.
ment. After the restoration stage, differences from control levels were slight with
the exception of the SCO, where the recovery phase was followed by a ten days
period during which the nuclear size remained increased in animals treated with
The third test series: Results are given
in table 3 . Thyroidectomy brought about a
reduction in nuclear size in the ependymal
cells of the wall of the third ventricle and
of the SCO. In the ependyma covering the
wall of the third ventricle a significant difference between treated animals and control animals was observed. This was almost significant in the SCO.
material” in the SCO
No significant differences were observed
among animals in any experimental group
and controls regarding the aldehyde-fuchsin-positive material. The amount of “secretory material” showed a tendency to
diminish as thyroid activity decreased.
though the differences from control animals were not significant.
Karyometry has been held to be useful
in the assessment of changes in the activity
of cells. If a cell is affected in some way,
its metabolism will change and this can
be seen as changes in cell structure (Caspersson, ’50) and also in the nuclear volume of the cell. A change in nuclear volume observed in karyometric tests thus
indicates a change in the metabolic activity of the cell. Karyomctry can tell US
nothing of the quality of the changes in
activity. Nevertheless a change in the nuclear volume of a secretory cell might be
considered a sign of a change also in the excessive administration of thyroxine may
secretory activity of that cell. Generally an be connected among other things with inincrease in nuclear volume is associated creased cerebrospinal fluid secretion in the
with increased activity, while a decrease cerebral ventricles. On the other hand a
in volume corresponds to diminished ac- fall in the activity of the thyroid gland
tivity (Benninghoff, ' 5 3 ; Szentilgothai et seems not to diminish its secretion.
The karyometric results obtained are
al., '62).
of earlier observations on ganOn the basis of the results obtained in
the present study it would appear that glion cells of the hypothalamus (Talanti,
changes produced experimentally in thy- '67a, '67b). It seems possible that variaroid function have their effects on the nu- tions in thyroid activity might exert a simiclear volumes of all ependymal cells ex- lar influence on the nuclear volume of the
amined. Provided that the above concep- cells in even wider areas of the central
tion of the influence of functional activity nervous system. The mechanism of this
upon nuclear volume is correct, it would influence is problematic. There may be
seem that the activity of the ependymal direct effect of thyroid hormone in the
cells is dependent also upon the thyroid cells, or it may take place indirectly. A
activity. An overdose of thyroid hormone general metabolic effect of thyroxine may
brought about an increase in nuclear vol- be involved.
ume, so that it may be presumed to have
caused increased activity in the ependymal Benninghoff, A. 1953 Das funktionelle Kerncells. On the other hand thyroidectomy
odem als Indicator der Zelltatigkeit. Mon. 2001.
and the administration of thiouracil led
Ital., 61 suppl.: 84-124.
to a reduction in nuclear size of the epen- Caspersson, T. 0. 1950 Cell Growth and Cell
Function. W. W. Norton, New York.
dyma in the SCO and in the wall of the
S. A. 1963 Central nervous regulathird ventricle, in other words to a decrease DAngelo,
tion of the secretion and release of thyroid
in cell activity. Hypoactivity of the thystimulating hormone. In: Advance i n Neuroroid had a surprisingly weak effect on the
endocrinology. Edited by A. V. Nalbandov.
Urbana: Univ. of Illinois Press, pp. 158-205.
karyometry of the choroid plexus.
Although the reactions of all three types Fisher, R. A. 1950 Statistical methods for research workers. Oliver & Boyd, Edinburgh.
of ependymal cell investigated were alike, Fleischhauer,
K. 1965 Uber Physiologie und
there were considerable quantitative difPharmakologie des Ventrikelliquors. Wiener Z.
Nervenheilk., suppl. 1: 10-26.
ferences among them. There was also considerable quantitative variation in the rate Landau, E. 1960 L'organe sous-commissural.
Acta anat., 41: 156-160.
of change in each cell type. These variaLanding, B. H., H. E. Hall and C. I). West 1956
tions derive in part from individual
Aldehyde-fuehsin-positive material of the postediffences in both nuclear volume and
rior pituitary. Lab. Investig., 5: 256-261.
in reactive capacity of the nuclei. In Lenys, R. 1965 Contribution a l'etude de la
structure et du role de l'organe sous-commispart also, however, these differences may
sural. These, Nancy, pp. 163-189.
be attributable to differences in the nature Oksche, A. 1961 Der histoehemisch nachweisof activity, that is differences in the type
bare Glykogenaufbau und-abbau in den Astrozyten und Ependymzellen als Beispiel einer funkof cell activity may have differing phystions-abhangigen Stoffwechselaktivitat der Neuiological significances.
Zellforsch., 54: 307-361.
It appears that ependymal secretion of -_ roglia. 2.
1961 Vergleichende Untersuehuiigen
the SCO is related in some way at least on
uber die sekretorische Aktivitiit des Subkommisthe thyroid gland. This relationship apsuralorgans und den Gliacharakter seiner
Zellen. 2. Zellforsch., 54: 549-612.
pears to be concerned with the intensity of
secretory activity. Secretion increases with Olsson, R. 1958 Studies on the subcommissural
organ. Acta Zml. (Stockh.), 39: 71--102.
a rise in thyroid activity and decreases Palkovits, M. 1965 Morphology and Function
with its hypoactivity. The physiological
of the Subcommissural Organ. Budapest:
Akademiai Kiado, pp. 67-89.
significance of this, however, is impossible
to determine on the basis of the results of SzentLgothai, J., €3. Flerko, B. Mess and B.
IIalasa 1962 Hypothalamic Control of the
a morphological study.
Anterior Pituitary. Akademiai Kiado, Budapest.
The increase in ependymal activity in Talanti, S. 1958 Studies on the subcommissural
the choroid plexus in association with an
organ in some domestic animals with refer-
ence to sccretory phenomena. Ann. Med. Exp.
Biol. Fenn., 36 suppl. 9: 1-98.
1967a The effect of thyroidectomy on
the hypothalamus of the rat with special reference to the hypothalamic-hypophyseal neurosecretory system. Acta Physiol. Scand., 70: 8087.
1967b The effect of thiouracil and excess thyroxine on the hypothalamus of the rat
with special reference to neurosecretory phcnoniena. Z. Zellforsch., 79: 92-109.
Uotila, U., and 0. Kannas 1952 Quantitative
histological method of determining the proportions of the principal components of thyroid
tissue. Acta Endocrinol., 11 : 49-60.
Yamada, T. 1961 The effect of electrical ablation of the nuclei habenulae, pineal body and
subcommissural organ on endocrine function,
with special referencc to thyroid function.
Endocrinology, 69: 706-711.
All figures show sections stained with hematoxylin-eosin.
Thyroid of a control rat.
x 630.
3 Thyroid of a rat treated for 24 days with thyroxine. x 630.
Thyroid of a rat treated for 24 days with thiouracil.
Ependymal nuclei of the subcommissural organ of a control rat.
x 1500.
Ependymal nuclei of the subcommissural organ of a rat treated for
eight days with thyroxine. X 1500.
Ependymal nuclei of the subcommissural organ of a rat treated for
12 days with zhiouracil. X 1300.
Ependymal nuclei of the subcommissural organ of a thyroidectomized
rat. >( 1500.
Ependymal nuclei of the choroid plexus of a control rat.
Ependymal nuclei OP the choroid plexus of a rat treated for 8 days
with thyroxine. X 1500.
10 Ependymal nuclei of the choroid plexus of a rat treated for 24 days
with thiouracil. X 1500.
11 Ependymal nuclci of the choroid plexus of a thyroidectomized rat.
x 1500.
12 Ependymal nuclci of the wall of the third ventricle of a control rat.
x 1500.
13 Ependymal nuclei of the wall of the third ventricle of a rat treated
for 18 days with thyroxine. X 1500.
14 Ependymal nuclei of the wall of the third ventricle of a rat treated
for 18 days with thiouracil. X 1500.
15 Epcndymal nuclei of the wall of thc third ventricle of a thyroidectomized rat. X 1500.
S. Talanti
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effect, ependymal, excess, thiouracil, references, subcommissural, thyroxine, special, organy, cells, thyroidectomy
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