OrIGINAL PAPErS - Advances in Clinical and Experimental Medicine

OrIGINAL PAPErS - Advances in Clinical and Experimental Medicine

original papers
Adv Clin Exp Med 2011, 20, 5, 553–558
ISSN 1230-025X
© Copyright by Wroclaw Medical University
Irena Kasacka, Ewa Arciszewska
The Effects of Propranolol on the Rat Magnocellular
Neurosecretory System in Experimental
Hyperthyroidism
Wpływ propranololu na układ neurosekrecyjny wielkokomórkowy
szczura w doświadczalnej nadczynności tarczycy
Department of Histology and Cytophysiology, Medical University of Bialystok, Białystok, Poland
Abstract
Background. The sympathetic nervous system – especially the adrenergic part, plays a major role in the pathogenesis of hyperthyroidism. Propranolol, a non-selective β receptor antagonist, is commonly used in the treatment of
hyperthyroidism.
Objectives. To assess the effects of propranolol on the magnocellular neurosecretory system of hyperthyroid rats.
Material and Methods. The study used 30 rats, which were assigned to one experimental group (rats with hyperthyroidism receiving propranolol at a dose of 4.0 mg/kg b.w. for seven days) and two control groups: one consisting of healthy rats, the other of hyperthyroid animals. The rats were sacrificed under anesthesia, and the brains
and pituitaries were collected. The material was fixed in Bouin’s fluid as modified by Bock. Topographic staining
was performed with hematoxylin and eosin, while paraldehyde fuchsine was used to demonstrate Gomori-positive
neurosecretory material, using Gomori’s technique.The histoenzymatic reaction for acid phosphatase activity was
performed. All the structures of the magnocellular neurosecretory system were assessed, i.e. the supraoptic nucleus
(SON), the paraventricular nucleus (PVN), the median eminence (ME) and the neural part of the pituitary.
Results. In the hyperthyroid rats receiving propranolol, there was a reduction in the supraoptic and paraventricular
nuclei in the perikarya of neurosecretory cells, as well as an increase in the neurosecretion content in the pars nervosa of the pituitary. In the perikarya of neurons there was also an increase in acid phosphatase reaction product,
which is an important indicator of disturbances in the cells’ functional activity.
Conclusions. The results of the current study indicate that propranolol affects all the chains of the magnocellular
neurosecretory system of hyperthyroid rats (Adv Clin Exp Med 2011, 20, 5, 553–558).
Key words: magnocellular neurosecretory system, hyperthyroidism, propranolol, rat.
Streszczenie
Wprowadzenie. W patogenezie nadczynności tarczycy dużą rolę odgrywa układ współczulny, a zwłaszcza jego
część adrenergiczna. Lekiem powszechnie stosowanym w leczeniu nadczynności tarczycy jest propranolol – nieselektywny antagonista receptorów β.
Cel pracy. Ocena wpływu propranololu na układ neurosekrecyjny wielkokomórkowy szczura z nadczynnością
tarczycy.
Materiał i metody. Badania przeprowadzono na 30 szczurach, które podzielono na 2 grupy porównawcze (jedną
stanowiły szczury zdrowe, drugą zwierzęta z nadczynnością tarczycy) i jedną grupę doświadczalną szczurów z nadczynnością tarczycy otrzymujących przez 7 dni propranolol w dawce 4,0 mg/kg c.c. Zwierzęta uśmiercano w narkozie i pobierano mózgowia i przysadki mózgowe. Materiał utrwalano w płynie Bouine’a w modyfikacji Bocka.
Wykonano barwienie topograficzne hematoksyliną i eozyną, barwienie na zawartość neurosekretu fuksyną paraaldehydową wg Gomoriego w modyfikacji Fischera i Haskell oraz wykonano reakcję histoenzymatyczną na aktywność fosfatazy kwaśnej. Oceniano wszystkie struktury układu neurosekrecyjnego wielkokomórkowego: jądro nadwzrokowe (SON), jądro przykomorowe (PVN), wyniosłość pośrodkową i część nerwową przysadki mózgowej.
Wyniki. U szczurów z nadczynnością tarczycy otrzymujących propranolol obserwowano zmniejszenie w perikarionach komórek neurosekrecyjnych jądra nadwzrokowego i przykomorowego oraz zwiększenie w części nerwowej przysadki mózgowej zawartości neurosekretu. Poza tym stwierdzono w perikarionach neuronów nasilenie
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I. Kasacka, E. Arciszewska
intensywności produktu reakcji fosfatazy kwaśnej, będącej ważnym wykładnikiem zaburzeń aktywności funkcjonalnej komórek.
Wnioski. Wyniki przeprowadzonych badań wskazują, że propranolol ma wpływ na wszystkie ogniwa układu neurosekrecyjnego wielkokomórkowego szczura z doświadczalną nadczynnością tarczycy (Adv Clin Exp Med 2011,
20, 5, 553–558).
Słowa kluczowe: układ neurosekrecyjny wielkokomórkowy, nadczynność tarczycy, propranolol, szczur.
Hyperthyroidism is a pathological syndrome
characterized by overactivity of the thyroid gland,
resulting in increased release of thyroid hormones
into the blood [1, 2]. The sympathetic nervous system, especially the adrenergic part, plays a major
role in the pathogenesis of hyperthyroidism.
The typical symptoms of hyperthyroidism include tachycardia, increased systolic pressure and
decreased diastolic pressure, psychomotor agitation,
enhanced glycemia and lipolysis [3]. These symptoms
depend on stimulation of the adrenergic sympathetic
pathway [4]. Increased activity of the sympathetic
system is the reason hyperthyroidism is treated with
drugs that block the adrenergic system [5].
The most commonly used β-blocker is propranolol, which by inhibiting the peripheral conversion of thyroxine (T4) to triiodine (T3) eliminates
symptoms accompanying hyperthyroidism – i.e.
tachycardia, tremor, glucose tolerance impairment
– and decreases hypercalcemia [1]. As a non-selective antagonist of the β-1 and β-2 receptors, it
inhibits the effect of catecholamine on target tissue [5, 6]. Propranolol is used in the treatment of
circulatory system diseases [7, 8], is widely used in
neuropsychiatry [9], especially in the treatment of
schizophrenia [10], and alleviates the symptoms
accompanying porphyria [11].
Thyroid hormones accumulate in the brain,
where they serve as precursors for adrenergic neurotransmitters [12]. Thus, they may affect the function of the central nervous system. The supraoptic
nucleus (SON) and the paraventricular nucleus
(PVN) have a number of nerve endings that play
an important role in the regulation of hypothalamic neurosecretion.
The β-adrenergic receptors play the most crucial role in the normal functioning of the magnocellular neurosecretory system. Since propranolol
passes through the blood-brain barrier [13], it can
also be assumed to affect the supraoptic and paraventricular nuclei.
Many authors have dealt with hyperthyroidism and beta-blockers, but a survey of the literature shows that little attention has been paid to the
effect of beta-blockers on the structure and function of the hypothalamic-pituitary system.
The objective of the current study was to assess
the effects of propranolol on the magnocellular
neurosecretory system of hyperthyroid rats.
Material and Methods
The Animals
Male Wistar rats weighing 190–200 g were
used in all the experiments. The rats were housed
at a constant room temperature in a 12-h light/12h dark cycle and given a standard laboratory diet
and water ad libitum. All the procedures were performed in accordance with the Medical University
of Bialystok’s guidelines for animal care.
The rats were divided into three groups: Group
I (the control group) consisted of five healthy rats
treated with 0.9% solution of saline at a dose of
80.0 µg/kg b. w. for 14 days via intraperitoneal
injection; Group II: ten animals with hyperthyroidism, induced by administering L-thyroxine
(Sigma Chemical Co.) at a dose of 80.0 µg/kg b. w.
for 14 days via intraperitoneal injection; Group III:
15 rats with hyperthyroidism (induced as in Group
II) which then received propranolol at a dose of
4.0 mg/kg b. w. in a daily intraperitoneal injection for seven days. The animals were decapitated
under pentobarbital anesthesia (50 mg/kg b. w.).
The brains and pituitaries were then dissected and
fixed in Bouin’s fluid as modified by Bock [14] for
two days at 37°C, and embedded in paraffin. Next,
5-µm paraffin sections were stained with hematoxylin and eosin (for general histological examination) and with paraldehyde fuchsine, using the
modification of Gomori’s technique described by
Fischer and Haskel [15] to demonstrate Gomoripositive neurosecretory material. In addition, the
histoenzymatic reaction for acid phosphatase (AP)
activity as a cytochemical marker was performed
by the Gomori method.
Results
Group I: Control Animals
Receiving Saline Only
The supraoptic nucleus showed a predominance of large polymorphic neurons with a distinct
acinar cell nucleus, most frequently lying off-center.
In most cells, relatively numerous neurosecretion
granules or follicles accumulated in the periphery
of the neuronal body as semi-lunar structures, or
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Propranolol in Hyperthyroidism
formed agglomerations around the nucleus. There
were also cells with a trace amount of neurosecretion or neurons totally devoid of Gomori-positive
substances. Neurosecretion accumulation in the
form of grains, follicles or spheres was by far more
pronounced in the neuronal processes.
In the lateral part of the paraventricular nucleus, small multipolar neurons with moderate content of neurosecretion were found to predominate.
Cells with medium content of neurosecretion located in the vicinity of the cell nucleus predominated over the remaining area. Only in few neurons, neurosecretion filled up the neuroplasm.
In the median eminence, the neurosecretory
material was seen in the fibers running from SON
and PVN neurons. Numerous neurosecretion
granules were also found within the pars nervosa
of the pituitary, where distinct cross-sections of
blood vessels of varying calibers and glial cells were
seen in addition to nerve fibers.
A positive, high-intensity reaction for AP activity
was observed. The reaction intensity in SON cells was
usually evenly distributed, and only in some neurons
the phosphatase-positive granules were medium or
small and located by the nuclear envelope or on one
of the cell poles. In the neurosecretory cells of the
PVN, small or medium granules were evenly distributed in the neuroplasm, sometimes concentrically accumulating around the cell nucleus.
Group II: Animals Receiving
L-thyroxine
areas were seen among the neurocytes. The neurosecretion content was found to increase both in
neuronal bodies and in the processes (Fig. 1). Most
cells contained large and medium amounts of neurosecretion, which, in the form of granules and
nodules, filled up the cytoplasm or accumulated at
one of the cell poles. Nerve fibers were filled with
neurosecretion. Many neurocytes were fusiform in
shape.
Within the median eminence and pituitary,
the neurosecretion content was decreased and the
blood vessels were markedly dilated (Fig. 2).
Fig. 2. Neurohypophisis. Group II. A small amount of
neurosecretory material. Staining according to FischerHaskell. Magn. ×200
Ryc. 2. Część nerwowa przysadki mózgowej, grupa
II, mała zawartość neurosekretu, barwienie metodą
Fischer-Haskell, pow. ×200
In the rats receiving L-thyroxine, neuropil
rarefaction and dilation of the vascular lumen in
both nuclei were observed. Large non-staining
Fig. 1. Supraoptic nucleus. Group II. A large amount
of neurosecretory material both in the perikaryon and
processes. Staining according to Fischer-Haskell. Magn
×200
Ryc. 1. Jądro nadwzrokowe, grupa II, duża zawartość
neurosekretu w ciałach i wypustkach komórek nerwowych, barwienie metodą Fischer-Haskell, pow. ×200
Fig. 3. Supraoptic nucleus. Group II. Neuronal perikarya showed increased acid phosphatase reaction
product. Granular reaction product seen within the
cytoplasm of the neuronal perikarya. Staining according to Gomori. Magn. ×400
Ryc. 3. Jądro nadwzrokowe, grupa II, w cytoplazmie
ciał komórek nerwowych widoczny wzrost produktu
reakcji fosfatazy kwaśnej, ziarnisty produkt reakcji
widoczny w cytoplazmie perykarionów komórek nerwowych, barwienie metodą Gomoriego, pow. ×400
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The intensity of the AP reaction in the SON
increased in comparison to that observed in the
rats receiving saline only. Phosphatase-positive
grains filled the neurosecretory cells quite evenly
(Fig. 3). In PVN cells, the intensity of AP reaction
was slightly attenuated as compared to Group I.
Group III: Animals Receiving
L-Thyroxine and Propranolol
In the SON, a number of blood vessels had
a dilated lumen and loose neuropil. Neuronal
polymorphism was observed. Among the spherical cells, which predominated, there were elongated cells with a more acidophilic cytoplasm. The
nuclear cells were usually large and acinar, but cells
with markedly smaller, pycnotic nuclei and compact nuclear chromatin were also seen. The cell
neuroplasm contained light non-staining spaces.
There was a predominance of cells with a small
amount of Gomori-positive substance accumulating mainly around the cell nucleus (Fig. 4). Only
single neurons were filled with granular neurosecretion. In the hyperthyroid rats receiving propranolol, there was far more neurosecretory material in the neuronal processes than in Group II.
In the PVN, the neuropil was rarefied and
many neurons were elongated. The amount of
neurosecretion in the neuronal bodies was lower
than in the control rats, whereas in the nerve processes it was markedly increased. A large amount
of neurosecretion was also observed in the fibrous
layer of the median eminence and in the neural pituitary in Group III (Fig. 5).
The reaction for acid phosphatase activity was
found to be very intense. Large phosphatase-positive grains, which frequently formed conglomer-
Fig. 4. Supraoptic nucleus. Group III, reduced neurosecretory content in cells. Staining according to
Fischer-Haskell. Magn. ×200
Ryc. 4. Jądro nadwzrokowe, grupa II, mała zawartość
neurosekretu w neurocytach, barwienie metodą
Fischer-Haskell, pow. ×200
I. Kasacka, E. Arciszewska
ates, were evenly distributed within the neurons of
the supraoptic and paraventricular nuclei (Fig. 6).
Discussion
Thyroid hormones – both thyroxine (T4) and
triiodothyronine (T3) – affect the hydro-electrolyte balance of the body [16, 17]. The release of
thyroid hormones is stimulated by thyreoliberin,
the thyrotropin-releasing hormone (TRH). The
Fig. 5. Neurohypophisis. Group III. A marked increase
in the neurosecretory content. Staining according to
Fischer-Haskell. Magn. ×200.
Ryc. 5. Część nerwowa przysadki mózgowej, grupa III,
zwiększona zawartość neurosekretu, barwienie metodą
Fischer-Haskell, pow. ×200.
Fig. 6. Supraoptic nucleus. Group III. Acid phosphatase activity noticeably increased. Due to the neuron cytoplasm being densely filled with the reaction
product of enzymatic activity, its granular character is
difficult to distinguish. Staining according to Gomori.
Magn. ×400
Ryc. 6. Jądro nadwzrokowe, grupa III, aktywność fosfatazy kwaśnej wyraźnie większa, ze względu na ciasne
upakowanie produktu reakcji enzymu w cytoplazmie
neuronów jego ziarnisty charakter jest trudny do
rozróżnienia, barwienie metodą Gomoriego, pow. ×400
Propranolol in Hyperthyroidism
synthesis and release of vasopressin (AVP) and
oxytocin (OT) are regulated by plasma osmolality
and volume [18].
In the current study, in rats with hyperthyroidism, an increase was observed in the neurosecretion content in the cells of the supraoptic and
paraventricular nuclei and within nerve processes
of these cells. The amount of neurosecretion in the
median eminence and in the neural part of the pituitary in these rats was lower than in the healthy
animals.
The reduced content of neurosecretion in the
neurohemal organ found in this study may be associated with high levels of neurohormones passing into blood circulation. This has been confirmed
by other authors [19, 20]. Mogulkoc and Baltaci
reported that rats with hyperthyroidism induced
by L-thyroxine administered intraperitoneally at
a dose of 0.3 mg/kg b. w. for four weeks showed an
increase in the plasma vasopressin concentration;
those authors related this to the activation of the
renin-angiotensin system observed in hyperthyroidism [19]. According to Ciosek and Drobnik,
animals injected intravenously with TRH for two
days in a daily dose of 100 ng/100 g b. w. show reduced vasopressin levels in the hypothalamus and
in the fibers of the neural pituitary, which is associated with increased concentration of this hormone
in the plasma [20]. Clinical investigations have also shown an increased release of neurohormones
from the magnocellular neurosecretory system to
the blood in patients with hyperthyroidism: Marcisz et al. observed an elevated level of AVP that
returned to normal after the termination of treatment and normalization of the thyroid gland [21].
Moreover, hyperthyroidism has been found to
have no effect on mRNA AVP expression in the
paraventricular nucleus [22], but it stimulates the
expression of mRNA OT in the hypothalamus, increasing oxytocin content in the posterior lobe of
the pituitary [23].
The results of the current study as well as those
reported by other authors demonstrate that the
increased level of thyroid hormones in hyperthyroidism most likely mobilizes the hypothalamic
nuclei (SON and PVN) to increase their activity.
This is manifested by the increased content of neu-
557
rosecretion in the neurosecretory center of the hypothalamus, which passes to the neural part of the
pituitary and then into blood circulation.
Propranolol is highly lipophilic, and thus it
readily crosses the blood-brain barrier [8, 13, 24].
Therefore it is most likely to affect the hypothalamus. Hyperthyroidism results in increased numbers of β-adrenergic receptors, which play a major
role in the normal functioning of the magnocellular neurosecretory system. Thyroid hormones
exert affect tissues via the adenylate cyclase-cyclic
AMP system, whose activity increases in hyperthyroidism [25]. Propranolol inhibits β-adrenergic
receptors, thus contributing to a decrease in adenylate cyclase reactivity and in consequence to
a reduction in the level of cyclic AMP [26]. This
mechanism leads to an attenuation of the protein
synthesis processes in the cell. The vasopressin,
oxytocin and neurophysin produced in the cells of
the supraoptic and paraventricular nuclei are protein substances. It can be thus assumed that propranolol contributes to a decrease in the synthesis
processes within SON and PVN neurons, leading
to the reduced amount of neurosecretory material observed in the present study. The increased
amount of neurosecretion in nerve processes, in
both the SON and the PVN, is most likely related
to its shift from nerve cells, which results in its
storage in the pars nervosa of the pituitary. All this
suggests that propranolol decreases the body’s demand for AVP, which causes it to accumulate in
the neurohemal organ.
Apart from functional alterations, the present
study also showed morphological changes in both
the neurocytes and neuropil within the SON and
PVN of the rats receiving propranolol. Most probably these were degenerations, as shown by the enhanced AP reaction. The apparent changes in AP
activity may suggest a neurophagial process in response to the altered metabolic conditions induced
by propranolol.
The results of the current study indicate that
propranolol, commonly used in the treatment of
hyperthyroidism, may have a significant effect on
the magnocellular neurosecretory system, inducing the changes detected in the morphological investigation.
References
[1] Cooper DS: Hyperthyroidism. Lancet 2003, 362, 459–468.
[2] Wilson GR, Curry RW: Subclinical thyroid disease. Am Fam Phys 2005, 72, 1517–1524.
[3] Bhatt P, Makwana D, Santani D, Goyal R: Comparative effectiveness of carvedilol and propranolol on glycemic
control and insulin resistance associated with L-thyroxin-induced hyperthyroidism – an experimental study. Can
J Physiol Pharmacol 2007, 85, 514–520.
[4] Słowińska-Srzednicka J: Aminy katecholowe a czynność tarczycy. Pol Tyg Lek 1984, 34, 1013–1016.
[5] Adamska-Dyniewska H: Miejsce leków beta-adrenolitycznych we współczesnej terapii. Probl Ter Monit 1998, 9,
143–156.
558
I. Kasacka, E. Arciszewska
[6] Kornischka J, Cordes J, Agelink MW: 40 years beta-adrenoceptor blockers in psychiatry. Fortschr Neurol Psychiatr
2007, 75, 199–210.
[7] Perez-Stable EJ, Halliday R, Gardiner PS, Baron RB, Hauck WW, Acree M, Coates TJ: The effect of propranolol
on cognitive function and quality of life: a randomized trial among patients with diastolic hypertension. Am J Med
2000,108, 359–365.
[8] Glasscock E, Yoo JW, Chen TT, Klassen TL, Noebels JL: Kv1.1 potassium channel deficiency reveals brain-driven
cardiac dysfunction as a candidate mechanism for sudden unexplained death in epilepsy. J Neurosci 2010, 30,
5167–5175.
[9] Haspel T: Beta-blockers and the treatment of aggression. Harv Rev Psychiatry 1995, 2, 274–281.
[10] Fischel T, Hermesh H, Aizenberg D, Zemishlany Z, Munitz H, Beniamini Y, Wejzman A: Cyproheptadine versus propranolol for the treatment of acute neuroleptic-induced akathisia: a comparative double-blind study. J Clin
Psychopharmacol 2001, 21, 612–615.
[11] Thadani H, Deacon A, Peters T: Diagnosis and management of porphyria. Br Med J 2000, 320, 1647–1651.
[12] Dratman M: Localization of triiodothyronine in nerve ending fractions of the rat brain. Proc Nat Acad Sci 1976,
73, 941–945.
[13] Anderson JL: Beta-blockers. Cardiac Electrophys Rev 2000, 4, 300–307.
[14] Bock R, Brinkman H, Marckwort W: Farberische Beobachtungen zur Frage nach dem primaren Bildungsort von
Neurosekret in supraoptico-hypophysaren. System Z Zellforsch Mikrosk Anat 1968, 87, 534–544.
[15] Fischer ER, Haskel AE: Combined Gomori methods for demonstration of pancreatic alpha and beta cells. Am
J Clin Pathol 1954, 24,1433–1438.
[16] Dakine N, Oliver C, Grino M: Thyroxine modulates corticotrophin-releasing factor but not arginine vasopressin
gene expression in the hypothalamic paravenricular nucleus of the developing rat. J Neuroendocrinol 2000, 12,
774–783.
[17] Ciosek J, Drobnik J: Vasopressin and oxytocin release and the thyroid function. J Physiol Pharmacol 2004, 55,
423–441.
[18] Chana Y, Sladek CD: Osmotic regulation of vasopressin and oxytocin release in rate sensitive in hypothalamoneurohypophysal explants. Am J Physiol 1990, 25, 492–500.
[19] Mogulkoc R, Baltaci AK: Influences of isotonic, hypertonic and hypovolemic treatments on vasopressin response
and fluid-electrolite balance in L-thyroxine-induced hyperthyroid rat. Life Sci 2006, 79, 817–821.
[20] Ciosek J: Vasopressin and oxytocin release as influenced by thyrotropin-releasing hormone in euhydrated and
dehydrated rats. J Physiol Pharmacol 2002, 53, 423–437.
[21] Marcisz C, Jonderko G, Kucharz EJ: Changes of plasma arginine-vasopressin level in patients with hyperthyroidism during treatment. Med Sci Monit 2001, 7, 409–414.
[22] Ceccatelli S, Giardino L, Calza L: Response of hypothalamic peptide mRNA to thyroidectomy. Neuroendocrinology
1992, 56, 694–703.
[23] Adan RA, Cox JJ, van Kats JP, Burbach JP: Thyroid hormone regulates the oxytocin gene. J Biol Chem 1992, 267,
3771–3777.
[24] Pélissier-Alicot AL, Gaulier JM, Dupuis C, Feuerstein M, Léonetti G, Lachâtre G, Marquet P: Post-mortem
redistribution of three beta-blockers in the rabbit. Int J Legal 2006, 120, 226–232.
[25] Malbon CC, Moreno FJ, Cabelli RJ, Fain JN: Fat cell adenylate cyclase and beta-adrenergic receptors in altered
thyroid states. J Biol Chem 1978, 253, 671–678.
[26] Lu Y, Zhang Y, Shan H, Pan Z, Li X, Li B, Xu C, Zhang B, Zhang F, Dong D, Song W, Qiao G, Yang B:
MicroRNA-1 downregulation by propranolol in a rat model of myocardial infarction: a new mechanism for ischaemic cardioprotection. Cardiovasc Res 2009, 84, 434–441.
Address for correspondence:
Irena Kasacka
Department of Histology and Cytophysiology
Medical University of Bialystok
Kilińskiego 1
15-089 Białystok
Poland
Tel: +48 85 748 54 58; tel./fax +48 85 748 55 16
E-mail: [email protected]
Conflict of interest: None declared
Received: 21.12.2010
Revised: 5.07.2011
Accepted: 5.10.2011