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The cytology of the parathyroid and thyroid glands of rats with experimental rickets.

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THE CYTOLOGY O F THE PARATHYROID AND
THYROID GLANDS O F RATS WITH
EXPERIMENTAL RICKETS
E. DE ROBERTIS *
Depurtment of Anatomy, The University of Cluicayo, Illinois
FOUR PLATES (NINE
FIGURES)
From Erdheim 's studies ( '11-'14) on spontaneous rickets
in rats it has been known that rickets is related to hypertrophy
of the parathyroid glands. This has been confirmed in human
and in experimental rickets of different species of many
authors. (Literature in Goldblatt, '31, and Shelling, '35.)
Because there is a lack of cytological studies on the parathyroid glands of rachitic animals, Prof. W. Bloom suggested
this research. This study of the cytological modifications of
the parathyroid cell in hyperfunction complements my previous study of the cytology of the hypofunctional gland (De
Robertis, '40).
In the course of these observations on the parathyroid gland
it was found that in low phosphorus rickets there is a great
activation of the thyroid gland in contrast to the normal
picture seen in low calcium rickets.
MATERIAL AND TECHNIQUE
Twenty-eight rats, aged 25 days at the beginning of the
experimental period, were used. Thirteen were fed a low
phosphorus, nine a low calcium rachitogenic diet, and six
were normal controls. I n each experiment rats of the same
litter were used, and in each case litter-mate controls were
fed Purina fox-chow as a normal diet. The rats were kept in
Fellow of the Rockefeller Foundation.
417
T H E ANATOMICAL RRCORD, VOL.
APRIL,
1941
79,N O . 4
darkness during the experimental periods, in each case of
4 weeks.
The diets fed were based on the Steenbock-Black rachitogenic diet no. 2965 a s modified by Hess, Weinstock, Rivkin
and Gross ( '29). The values used for the calcium and phosphorus content of the diets are from analyses by Shohl and
Wolbach ('36).
I. Low phosphorus diet. (Ca. = 1.25% ; P = 0.12%)
Corn meal
76%
Wheat gluten
20%
NsCl
1%
CnC03
3%
11. Low calcium diet. (Ca = 0.05% ; P = 0.80%)
Same as diet I, but with 3% KH2P0, substituted for CaCO,.
The animals were killed by a blow on the head and thyroid
and parathyroid were fixed in Zenker-formol f o r the general
histological study and in Regaud and Champy for the cytological one. The glands fixed in Zenker-formol were embedded
in nitrocellulose, sectioned serially at 7 p and stained with
hematoxylin-eosin-azure I1 and hfallory-axan. Those fixed in
Regaud were embedded in nitrocellulose, sectioned serially
at 5 p and stained with Bensley's acid fuchsin methyl-green
method. The material fixed in Champy was impregnated according to Kolatchev-Nassonow for the Golgi apparatus, embedded according to Peterfi's method with methyl benzoatecelloidin and paraffin and sectioned at 4 p.
OBSERVATIONS
As the histology and cytology of the parathyroid gland of
young normal rats have been previously described (De
Robertis, '40) here will be described only the changes found
in the glands following the action of rachitogenic diets.
The parathyroid glarcd
iqz
low-phosphorus rickets
The parathyroid gland appears to have increased slightly in
volume (fig. 2) in comparison with that of the normal control
(fig. 1); this increase is not as prominent as in the low
PARATHYIlOID A N D THYROID G L A N D CYTOLOGY
419
ualciuni rickets (fig. 3 ) . Two glands show prolongations of the
epithelium, going through the capsule and penetrating deeply
between the thyroid follicles (figs. 4 and 5). In tw70 cases
little islands of parathyroid tissue were found, one case near
the gland and the other f a r from it, deeply in the thyroid
tissue (fig. 4).
Similar prolongations o r islands were not seen in the controls or in the low calcium rickets studied.
The parathyroid gland in these rickets animals has, in
general, a more compact structure than the controls. The
epithelial cords are separated by thin connective tissue spaces
(fig. 2 ) and in some cases parts of the gland have a diffuse
structure (compact, uniform type of 3larine, '32). With ordinary histological methods parathyroid cells of the rat are of
a single type, comparable to the principal cells of other species.
Their volume seems, in general, to be slightly increased. The
cytoplasm is abundant and relatively homogeneous. The nucleus is ovoid with one or two folds in the membrane, sometimes it appears larger than in the normal but retains its
normal structure. In spite of the increased number of cells
over the control glands, no increase of mitosis or signs of
amitosis were found. This suggests that the hyperplastic
action occurred in the first stages of tlic action of the rachitogenic diet and not after rickets is marked.
The slides impregnated for the demonstration of the Golgi
apparatus yield more information and suggest that most of the
parathyroid cells are functioning actively. With this method
it is possible t o differentiate between two types of cells: clear
cells and dark osmiophilic ones described by Rosof ( '34),
which are characterized by heavier impregnation of the nucleus
and cytoplasm. The dark cells appear increased in number.
The Golgi apparatus is more heterogeneous and complex than
in normal parathyroid cells. I n this case the aspect is directly
opposed to that observed in hypofunctional cells (De Robertis,
'40,figs. 8,9). I n most of the cells the Golgi apparatus appear
fragmented into coarse pieces (figs. 7, 8). The filamentous or
simple reticular forms frequently observed in normal and
420
E. DE ROBERTIS
in most of the hypofunctional cells is rarely seen here. The
pieces of Golgi apparatus are widely scattered in the cytoplasm and frequently have bleb-like formations (fig. 8 B).
I n many cells, in addition to the Golgi apparatus, there
are a great number of small vacuoles with osmiophilic borders
t o which portions of Golgi apparatus are frequently attached
(fig. 7). The number of these cells is increased in relation to
the normal glands (De Robertis, '40, fig. 7). These vacuoles
are often situated in the apical cytoplasm and particularly
near the nucleus, but they may be also found at the basal pole,
that is, near the connective tissue strand (fig. 7). I n some
cells the polarity usually observed appears inverted ; the
nucleus is situated within the trabecula, and most of the
cytoplasm with the vacuoles toward the connective tissue
space and capillaries.
I n other cells the cytoplasm and the nucleus become darkly
stained (osmiophile) and it is difficult, and sometimes impossible, t o differentiate the Golgi apparatus from the vacuoles
with osmiophile borders.
Another kind of dark cell is shown in figure 8 C. The cytoplasm and the nucleus are shrunken laterally and the sides are
concave. These cells seem to have lost part of their cytoplasmic
content because they show only few vacuoles near the nucleus
and some pieces of Go16 filaments.
The study of the slides fixed in Regaud and stained with
Bensley 's acid fuchsin-methyl-green shows that in some cells
the mitochondria appear in long and undulated filaments
homogeneously distributed in the cytoplasm. I n other cells
they are spindle shaped or vesicular modifications of the filaments, while in still other cells the mitochondria diminish in
number and vacuole-like formations with pale stained borders
appear. Finally there are cells with dark stained cptoplasm
where no mitochondria can be seen.
The parathyroid gland i w low calciurn rickets
The parathyroid gland appears mucli larger than in the
controls (fig. 3). Thyroid follicles adjacent t o the parathyroid
PARATHYROID A N D THYROID GLAND CYTOLOGY
421
are compressed and collapsed and sometimes the gland bulges
out of the space that it normally occupies and pushes aside the
adjacent muscles (fig. 3 ) . The parathyroid gland has a compact structure ; narrow strands of connective tissue separate
the epithelial cords formed by several rows of cells.
With ordinary histological methods the cells closely resemble
the normal ones although, some of them have more cytoplasm
and larger nuclei. The hypertrophy of the gland is principally
due to a n increase in number of the cells (hyperplasia) and
to a less extent to an increase in volume of the cells (hypertrophy). At this time, however, the number of mitoses is
normal and there is no sign of amitosis. It is probable that
the hyperplasia is produced during the earlier stages of the
action of the rachitogenic diet.
The slides impregnated with osmic acid show the clear and
dark osmiophilic cells, between which are all intermediary
stages. Dark cells are greatly increased in number. I n some
cases as is shown in figure 9, the proportion of dark cells is
50% in contrast to 15 to 25% given by Rosof as normal.
The Golgi apparatus is also more complex and heterogeneous than in the normal; there are only a few cells with Golgi
apparatus reduced to a filament or simple reticulum. I n
some cells it is a complex reticulum widely spread and with
bleb-like formations. I n others the reticulum is broken up
and there appear vacuoles with osmiophilic borders. Tn still
other cells .the cytoplasm becomes osmiophilic (dark cell)
and sometimes the cells are so blackened that all the cytological
detail is lost. Finally, there are some dark cells smaller and
flattened laterally, with diminished cytoplasmic content. I n
most of the cells the mitochondria appear in long and slender
rods; spindle or vesicular forms are also seen. There are
some dark cells with shrunken cytoplasm which do not show
mitochondria. These cells are present in small numbers and
seem to be similar to the last type of dark osmiophilic cells
described above,
422
E. DE ROBXRTIS
l h c tliyroid g l a d iir; low phosphorus rickets
In contrast to the normal cytological picture found in the
low calcium rickets, the thyroid gland in the low phosphorus
rickets shows signs of activation. Nost of the follicles, especially those of the center of the gland, appear collapsed,
with little o r 110 colloid (fig. 6). The colloid stains with different colors with the azan method. Generally it has a great
number of vacuoles produced by the fixing-agent. The epithelium is highly cylindrical arid frequently the cells show one
or two collojd droplets in the apical pole which a r e generally
surrounded by a clear space. These spaces described by
Anderson a s chromophobic colloid a r e an artefact of fixation,
according to the results given by the freezing-drying method
(De Robertis, '41).
I n the apical and basal zones of the cells there are many
clear vacuoles. The hyperplasia of the gland is evidenced by
many mitoses (fig. 6, &I).I n general the blood vessels appear
greatly dilated (fig. 6) and there is some oedema. Frequently
red blood cells a r e found inside the follicles.
The Golgi apparatus also shows signs of activation; it
appears as a complex and wide reticulum at the apical zone
sending prolongations to the pole and sides of the nucleus.
The mitochondria appear in long rods oriented in basoapical direction and a r e more abundant near the apical pole
and in the sides of the cytoplasm. A t the apical pole mitochondria a r e parallel to this border of the cell.
All these data a r e in opposition to those observed in controls and i n low ealcium rickets in which the follicular epithelium is cuboidal and without mitosis, the intrafollicular colloid
abundant (fig-. l ) , and the Golgi apparatus much less developed (fig. 9).
DISCUSSION AND SUMMARY
The parathyroid g1aw.l iiz cxperirnetatal rickets. Erdheim
('llL'l4) found in the spontaneous rickets of the r a t a hypertrophy of the gland related to a hypertrophy and hyperplasia
of the cells. Pappenheimer and Minor ('21) in human rickets
PA1:ATEIYBOID AND T H Y R O I D GLAND CYTOLOGY
423
arrived at approximately the same conclusion, although Ritter
('20) found that the increase in size of the gland, in similar
material, was due to liyperplasia of the connective tissue,
hyperemia and oedema. Erdheim 's findings were confirmed
experimentally by Sorour ('23) i n rats kept in darkness with
a low calcium diet, by Luce ( '23), also in r a t s m7ith a calciumdeficient diet, and by Stoltenberg ('29) in experimental rickets
of rabbits. Recently Bauman and Spriiison ( '39) in rabbits
with chronic rickets produced by a calcium-deficient diet
found a n increase i n volume of the parathyroid gland to two
or three times the normal. Using the test of Hamilton and
Schwartz ('32) they found a n increase of the parathyroid
hormone in the blood stream which corresponded to fifteen to
twenty-five Hansen units. On the other hand i n low phosphorus rickets Hamilton and Schwartz ( '33) had previously
found a n increase of the hormone in the blood. Ham et a1.
( '40) concluded that parathyroid hypertrophy occurs in low
calcium rickets, but not in low phosphorus rickets.
Our observations lead to the conclusion that in both low
calcium and low phosphorus rickets there is a hypertrophy of
the gland with hypertrophy and, especially, hyperplasia of
the cells. This hyperplasia was greater in low calcium rickets
than in low phosphorus rickets, but in the latter there were
found i n two cases prolongations of the parathyroid gland
inside the thyroid and in two other small parathyroid tissue
islands outside of the gland.
I n both kinds of rickets the cytological picture indicates that
cells a r e greatly activated in their function. Osmiopliilic cells
appear increased in number, particularly in some cases of low
calcium rickets in which there were 50% of dark cells instead
of 15 to 25% which is the normal proportion according to
Rosof ( '34).
I n a previous paper it was shown that in the functional
atrophy of the parathyroid gland produced by injection of
parathyroid extract, dark cells disappear and the Golgi apparatus becomes reduced to a thick filament o r simple reticulum. I concluded that this was related to a hypofunctional or
resting stage of the cell. The greater complexity and hetero-
424
E. DE ROBERTlS
geneity of the Golgi apparatus in the normal cells a r e probably
related to digerent functional stages.
I n the rachitic rats, in most of the cells, the Golgi apparatus
is more complex than i n the normal gland. It appears as a
complicated reticulum with bleb-like formations, or broken
up through the cytoplasm loaded of small vacuoles surrounded by osmiophilic borders (fig. 7). In other cells the
whole cytoplasm becomes osmiophilic (dark cells) with the
Golgi apparatus less conspicuous. Some of the dark cells
become flattened and shrunken and apparently lose some of
their cytoplasmic content because they only show very few
vacuoles with osmiophilic borders (fig. 8, C). As in normal
glands, no granules that could be interpreted as secretion were
found. I think that the cytological picture above described
can be interpreted a s the manifestation of a n active or
increased function of the cells as a response t o the inadequate
calcium or phosphorus in the diet, in addition to the lack of
vitamin D.
The thyroid gland in experimental rickets. Several authors
have described changes of the thyroid gland in experimental
rickets. Murray ( '23) in pups found an acute hyperplasia of
the thyroid gland. Sorour ( '23) in rats describes hypertrophy
and a microscopical aspect similar to that seen in Basedow 's
disease. Stoltenberg ('29) in rachitic rabbits found hyperfunction of the thyroid gland, with a high cylindrical epithelium and a small quantity of colloid in the follicles. I n the
thyroid of our rachitic r a t s we observed a sharp difference
between * the low calcium and the low phosphorus rickets.
While in the former the gland appeared normal, in the latter
there was a great hyperplasia and activation evidenced by increase i n the number of mitoses, higher epithelium with
increase in number of intracellular colloid droplets and
vacuoles, loss of intrafollicular colloid and the more complex
and scattered Golgi apparatus.
As in both of the diets used there was the same lack of
vitamin D, and a s in some other rats on a normal diet to which
extra calcium was added (which I am now studying) there
PAL4THYROID AND T H Y R O I D G L A S D CYTOLOGY
425
is not activation of the gland, it is probable that the activation
of the thyroid in the low phosphorus rickets map be related
to the phosphorus deficiency.
CONCLUSIONS
1. I n both low calcium and low phosphorus rickets of the
rat there is hypertrophy of the parathyroid gland due to
hypertrophy and especially t o hyperplasia of the parathyroid
cells. This hypertrophy is more marked in the low calcium
rickets.
2. I n both low calcium and low phospliorus rickets the parathyroid cells seem greatly activated in their function as shown
by an increase in the number of dark osmiophilic cells, greater
complexity of the Golgi apparatus, more numerous cells with
vacuoles with osmiophilic borders and the presence of vesicular
mitochondria. This cytological picture looks like an exaggeration of that observed in normal glands, and is the opposite
of that observed in hypofunctional glands.
3. While the thyroid of low calcium rickets seems normal,
the thyroid of low phospliorus rickets appears hypertrophic
and greatly activated.
I wish to acknowledge my indebtedness to Profs. W. Bloom
and F. C. &Lean f o r their advice and criticism during the
accomplishment of this work.
LITERATURE CITED
BAUMAN,
E. J., AND D. B. SPRINSON
1939 Hyperparathyroidism produced by
diet. Am. J. Physiol., vol. 125, pp. 741-747.
DE ROBERTIS,E. 1940 The cytology of the parathyroid gland of rats injected
with liarathyroid extract. Anat. Rec., rol. 78, pp. 473-495.
1941 The intracellular colloid of normal and activated thyroid gland
of the rat studied by the freezing-drying method. Am. J. Anat.,
( I n press.)
ERDHEIM,
J. 1911 hlorphologische Studien uber Beziehungen der Epithelkorperchen zuni Kalkstoffwechsel. Frankfurt Ztschr. f . Path., vol. 7,
pp. 175-230.
1914 Rachitis and Epithelkorperchen. Wieu.
GOLDBLATT,
H. 1931 Die neure Richtung der experimentellen Rachitisforscliung.
Ergeb. allg. Pathol., vol. 35, pp. 58-492.
426
E. DE ROBERTIS
HAM,A. W., N. LITTNER,
T. G. H. DRAKE,E. C.
ROBERTSON AND F. F. TISDALL
1940 Physiological hypertrophy of the parathyroids, its cause and its
relation to rickets. Am. J. Path., vol. 16, pp. 877-286.
HAMILTON,
B., AND C. SCHWARTZ
1932 A method for the determination of
parathyroid hormone. J. Pharm. and Exp. Ther., vol. 46, pp. 285-292.
1933 Hickets and hyperparathyroidism. Am. J. Dis. Children,
V O ~ , 46, pp. 775-785.
HESS, A . F., h1. WEINSTOCK,
H. RIVKIN AND J. GROSS 1929 Observations suggesting a local factor in pathogenesis and healing of rickets. Proc.
8oc. Exper. Biol. Med., vol. 27, pp. 140-142.
LUCE,E. M. 1923 The size of the parathyroids of rats and the effect of a diet
deficiency of Ca. J. Path., vol. 26, pp. 200-207.
MURRAY,T. 1923 The thyroid gland in experimental rickets. Brit. J. Exp.
Path., vol. 4, pp. 335-339.
PAPPENHEIMER,
A. M., AND J. M. MINOR 1921 Hyperplasia of the parathyroid
in human rickets, J. Med. Res., vol. 42, pp. 391-403.
RIT’PER, C. 1920 Oher Epithelkorperchenbefunde bei Rarhitis und anderen
Knochennerkrankungrn. Frankfurt. Ztsch. f. Path., vol. 24, pp. 137177.
ROSOF, J. 1934 An experimental study of the histology and cytology of the
parathyroid glands in the albino rat. J. Exp. Zool., vol. 68, pp. 121-167.
SHELLING,
D. H. 193.5 The parathyroids in health and disease. C. V. Mosby
Co., St. Louis.
SOROUR,
M. F. 1923 Versuche iiber Einfluss von Nalirung, Licht und Bewegung
auf Knochenentwicklung und endokrine Driise junger Ratten mit
besonderer Beriicksirhtigung der Rachitis. Beitr. z. Path. Anat.,
V O ~ . 71, pp. 467-481.
STOLTENBERG,
L. 1929 Inneresekretorische Driisen bei Rachitis und Spasmophilie.
Norsk Mag. f. Laegevidcnskapen, vol. 90, pp. 729-744.
SHOHL,
A. T., A N D S. B. WOLBACH1936 The effect of low calcium-high phosphorus diets a t various levels and ratios upon the production of rickets
and tetany. J. Nutrition, vol. 11, pp. 275-291.
STEENROCX,
H., AND A. BLACK 1925 Fat-soluble vitamins. XXIII. The induction
of growth promoting and calcifying properties in f a t s and their unsaponifiable constituents by exposure to light. J. Biol. Chem., vol. 64,
pp. 263-298.
PLATE 1
EXPLANATION O F FIGURES
1 Section through the largest diameter of the parathyroid gland of a control
rat. Normal thyroid follicles are also seen. Zenker-formol, Iiematoxylin-eosinazure 11. X 145.
2 Section through the largest diameter of the parathyroid gland of a r a t with
low phosphorus rickets. Incarease of the volume of the gland, with hypertrophy
and hyperplasia of the cells, is apparent. The thyroid gland nppears activated.
Zenker-formol, heniatoxylin-eosin-azure 11. X 145.
PARATHYROID AX1) TlIYROID GL.\ND CYTOLOGY
E. L)I: RORRRTIS
PLATE I
PLATE 2
EXPLANATION OF FIGURES
3 Section through the largest diameter of the parathyroid g l a d of a rat
with low calcium rickets. The gland is greatly increased in volume. Thyroid
gland :ippears normal but the follicles situated near the parathyroid gland are
compressed. Zenker-formol, hcniatoxylin-eosin-azureIT. X 145.
4 Palathyroid gland of ;I rat with low phosphorus rickets; p.p.a., prolongation
of parathyroid tissue betwren the thyroid follicles and an island of parathyroid
tissue. p i . Zenker-forrnol, hematoxylin-eosin azure TI. X 120.
428
PARATHYROID AX1) TlIYROID GL.\ND CYTOLOGY
E. L)I: RORRRTIS
429
PLATE 2
PLATE 3
ESPL.4h’A‘lIOX O F FIGTJRES
5 I’ar:itliyroid gland of n rat, with n low phosphoius riekcts, estcnding between
follicles of the thyroid. p . 1 ~Zenker-forniol, Ileniatoxylin cosin-azure 11. x 360.
6 Thyroid gland of n r a t with a low phosphorus rickets. Thyroid follicles
appcnr collapsed (c.f.) \\-it21 loss of colloid and high rylindrieal epithelium;
blood vessels a r e distended. F o u r illituses of thyroid cells are seen (m.) Zenkerformol, hcniatox~lin-eosiii-~zuie
11. X 630.
PARATHYROID ASI) T I I Y R O I D G l A N I ) CYl’OLOGY
1. DE I l O U E E T T S
431
PLATE ::
PLATE 4
EXPLANATION OF FIGLTRES
Figures 7 arid 8 arc drawings made with help of t h r camera lucida with 2 mm.
apoch. obj. and 15 X comp. oc. a t table level. Figures 7, 8, 9 a r e from parathyroid
glands of rats impregnated with the Kolatscliov-Nassonow method.
7 Parathyroid gland of a rat with a low phosphorus rickets. The Golgi
apparatus (G.a.) appears broken up and scattered through the cytoplasm. Many
vacuoles with osrniophilic borders ( v.o.) are seen.
8 Parathyroid gland of a r a t with a low phosphorus rickets. Capillary a t c.
A . Cell with Golgi apparatus (G.a.) broken up and with many vacuoles with
osmiophilie borders (v.o.).
R. Cell with Golgi apparatus broken up, but without vacuoles.
C. Dark, osmiophilic cell shrunken and compressed laterally.
Only a few vavuoles are seen near thv nucleus.
9 Parathyroid and thyroid g h n d of a low Ca rickets. The parathyroid gland
consists of about 50% dark osrniophilie cells. The thyroid gland shows normal
follicles with cuboidal cells and the Golgi apparatus (G.a.). X 550.
432
PARATHYROID AND THYROID G L A N D CYTOLOGY
E. DE lIOIIEI1TIS
433
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