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 pathogen-esis 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 hyper-thyroidism receiving propranolol at a dose of 4.0 mg/kg b.w. for seven days) and two control groups: one consist-ing 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 ner-vosa 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 – niese-lektywny 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 nad-czynnością tarczycy otrzymujących przez 7 dni propranolol w dawce 4,0 mg/kg c.c. Zwierzęta uśmiercano w nar-kozie 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 paraal-dehydową wg Gomoriego w modyfikacji Fischera i Haskell oraz wykonano reakcję histoenzymatyczną na aktyw-ność fosfatazy kwaśnej. Oceniano wszystkie struktury układu neurosekrecyjnego wielkokomórkowego: jądro nad-wzrokowe (SON), jądro przykomorowe (PVN), wyniosłość pośrodkową i część nerwową przysadki mózgowej.
Wyniki. U szczurów z nadczynnością tarczycy otrzymujących propranolol obserwowano zmniejszenie w peri-karionach komórek neurosekrecyjnych jądra nadwzrokowego i przykomorowego oraz zwiększenie w części ner-wowej przysadki mózgowej zawartości neurosekretu. Poza tym stwierdzono w perikarionach neuronów nasilenie
Adv Clin Exp Med 2011, 20, 5, 553–558 ISSN 1230-025X
OrIGINAL PAPErS
intensywności produktu reakcji fosfatazy kwaśnej, będącej ważnym wykładnikiem zaburzeń aktywności funkcjo-nalnej komórek.
Wnioski. Wyniki przeprowadzonych badań wskazują, że propranolol ma wpływ na wszystkie ogniwa układu neu-rosekrecyjnego 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 sys-tem, especially the adrenergic part, plays a major role in the pathogenesis of hyperthyroidism.
The typical symptoms of hyperthyroidism in-clude 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 propra-nolol, which by inhibiting the peripheral conver-sion of thyroxine (T4) to triiodine (T3) eliminates symptoms accompanying hyperthyroidism – i.e. tachycardia, tremor, glucose tolerance impairment – and decreases hypercalcemia [1]. As a non-se-lective antagonist of the β-1 and β-2 receptors, it inhibits the effect of catecholamine on target tis-sue [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 neu-rotransmitters [12]. Thus, they may affect the func-tion 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 hypotha-lamic neurosecretion.
The β-adrenergic receptors play the most cru-cial role in the normal functioning of the magno-cellular neurosecretory system. Since propranolol passes through the blood-brain barrier [13], it can also be assumed to affect the supraoptic and para-ventricular nuclei.
Many authors have dealt with hyperthyroid-ism and beta-blockers, but a survey of the litera-ture shows that little attention has been paid to the effect of beta-blockers on the structure and func-tion 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/12-h dark cycle and given a standard laboratory diet and water ad libitum. All the procedures were per-formed 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 hyperthy-roidism, 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 injec-tion 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 hema-toxylin and eosin (for general histological exami-nation) and with paraldehyde fuchsine, using the modification of Gomori’s technique described by Fischer and Haskel [15] to demonstrate Gomori-positive 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
formed agglomerations around the nucleus. There were also cells with a trace amount of neurosecre-tion 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 nucle-us, small multipolar neurons with moderate con-tent of neurosecretion were found to predominate. Cells with medium content of neurosecretion lo-cated in the vicinity of the cell nucleus predomi-nated over the remaining area. Only in few neu-rons, 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 distrib-uted in the neuroplasm, sometimes concentrically ac-cumulating around the cell nucleus.
Group II: Animals Receiving
L-thyroxine
In the rats receiving L-thyroxine, neuropil rarefaction and dilation of the vascular lumen in both nuclei were observed. Large non-staining
areas were seen among the neurocytes. The neu-rosecretion content was found to increase both in neuronal bodies and in the processes (Fig. 1). Most cells contained large and medium amounts of neu-rosecretion, 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. 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 nerwow-ych, barwienie metodą Fischer-Haskell, pow. ×200
Fig. 2. Neurohypophisis. Group II. A small amount of neurosecretory material. Staining according to Fischer-Haskell. Magn. ×200
Ryc. 2. Część nerwowa przysadki mózgowej, grupa II, mała zawartość neurosekretu, barwienie metodą Fischer-Haskell, pow. ×200
Fig. 3. Supraoptic nucleus. Group II. Neuronal per-ikarya showed increased acid phosphatase reaction product. Granular reaction product seen within the cytoplasm of the neuronal perikarya. Staining accord-ing to Gomori. Magn. ×400
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 spheri-cal cells, which predominated, there were elon-gated cells with a more acidophilic cytoplasm. The nuclear cells were usually large and acinar, but cells with markedly smaller, pycnotic nuclei and com-pact 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 accumulat-ing mainly around the cell nucleus (Fig. 4). Only single neurons were filled with granular neuro-secretion. In the hyperthyroid rats receiving pro-pranolol, there was far more neurosecretory mate-rial 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 pro-cesses it was markedly increased. A large amount of neurosecretion was also observed in the fibrous layer of the median eminence and in the neural pi-tuitary in Group III (Fig. 5).
The reaction for acid phosphatase activity was found to be very intense. Large phosphatase-pos-itive grains, which frequently formed
conglomer-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-electro-lyte balance of the body [16, 17]. The release of thyroid hormones is stimulated by thyreoliberin, the thyrotropin-releasing hormone (TrH). The
Fig. 4. Supraoptic nucleus. Group III, reduced neu-rosecretory 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
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 phos-phatase activity noticeably increased. Due to the neu-ron cytoplasm being densely filled with the reaction product of enzymatic activity, its granular character is difficult to distinguish. Staining according to Gomori. Magn. ×400
synthesis and release of vasopressin (AVP) and oxytocin (OT) are regulated by plasma osmolality and volume [18].
In the current study, in rats with hyperthy-roidism, an increase was observed in the neurose-cretion 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 pi-tuitary 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 as-sociated with high levels of neurohormones pass-ing 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 hyperthy-roidism [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 re-duced vasopressin levels in the hypothalamus and in the fibers of the neural pituitary, which is associ-ated with increased concentration of this hormone in the plasma [20]. Clinical investigations have al-so shown an increased release of neurohormones from the magnocellular neurosecretory system to the blood in patients with hyperthyroidism: Mar-cisz et al. observed an elevated level of AVP that returned to normal after the termination of treat-ment 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, in-creasing 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 hyperthy-roidism most likely mobilizes the hypothalamic nuclei (SON and PVN) to increase their activity. This is manifested by the increased content of
neu-rosecretion in the neurosecretory center of the hy-pothalamus, 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 hypothala-mus. Hyperthyroidism results in increased num-bers of β-adrenergic receptors, which play a major role in the normal functioning of the magnocel-lular neurosecretory system. Thyroid hormones exert affect tissues via the adenylate cyclase-cyclic AMP system, whose activity increases in hyperthy-roidism [25]. Propranolol inhibits β-adrenergic receptors, thus contributing to a decrease in ade-nylate 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 tein substances. It can be thus assumed that pro-pranolol contributes to a decrease in the synthesis processes within SON and PVN neurons, leading to the reduced amount of neurosecretory mate-rial 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 de-mand 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 prob-ably these were degenerations, as shown by the en-hanced AP reaction. The apparent changes in AP activity may suggest a neurophagial process in re-sponse 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, induc-ing the changes detected in the morphological in-vestigation.
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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
