Thyroid glands have two different endocrine cell populations – follicular cells that secrete thyroid hormones and, second, C cells or parafollicular cells. With regard to the latter, apart from its role in calcium homoeostasis, they are probably also involved in the intrathyroidal regulation of follicular cells. This hypothesis was supported by different features, such as their characteristic ‘parafollicular’ position, their predominance in the central region of the thyroid lobe – the so-called C-cell region – and their implication in the secretion of many different regulatory peptides 1,2.
Changes in metabolism and body development were observed in patients with thyroid dysfunctions and are based on hyperstimulation of follicular cells using a goitrogenic substance [propylthiouracil (PTU)] 1,3.
Wexler and Sharretts 4 aimed to determine the molecular and hormonal mechanisms regulating bone function. They declared interactions between the thyroid gland and the skeleton in the normal state and in disorders of thyroid function. The hypothalamic–pituitary–thyroid axis plays a key role in skeletal development and regulation of adult bone turnover 5.
Previous studies 3,5 describing C-cell behaviour in different pathological states of the thyroid gland are conflicting. Therefore, this work was conducted to study the probable relationship between follicular and parafollicular cells in drug-induced hypothyroidism caused by PTU and consequently its effects on bone.
Materials and methods
Thirty male albino rats were included in this study carried out in Kasr El-Aini Hospital. Their ages ranged from 5 to 8 months and their average weight was 180 g.
Rats were divided into three groups of 10 rats each. Animals of each group were housed in two separate hygienic cages and kept in a clean well-ventilated room according to the guidelines of the Animal Ethics Committee. They were maintained on a standard pellet diet and allowed free access to water.
Group I: The control group consisted of 10 rats. They received 0.5 ml saline solution once daily orally for 4 weeks.
Group II: The 10 rats of this group received PTU (Thyrocil, Amoun Phamaceutical Co.) at a single daily dose of 50 mg/kg, as given to rabbits 6, for 4 weeks; this was adjusted using Paget’s equation to 16.875 mg/rat of an average weight of 180 g 7. The dose was introduced orally using an insulin syringe.
Group III (the recovery hypothyroid group): This group included 10 rats that received PTU at the same dose as used in group II for 4 weeks. Animals of this group were sacrificed 1 month after cessation of drug administration.
Before scarification retro-orbital blood samples were collected in capillary tubes. Serum thyroxin (T4) levels were analysed by radioimmunoassay in the Chemical Pathology Department of Kasr El-Aini Hospital. Rats were sacrificed by chloroform inhalation; right-sided thyroid lobes were fixed in 10% formol saline, embedded in paraffin and cut at 7 μm thickness. Sections were subjected to H&E staining 8 and immunohistochemical staining.
Thyroid sections were stained using calcitonin antibody-2 (Rabbit Polyclonal Antibody) 1 (DAKO A-576; Dako, Glostrup, Denmark). The secondary antibody was a biotinylated antiserum to rabbit/mouse immunoglobulins (Life Trade, Egypt), and 3,3-diaminobenzidine tetrahydrochloride was used as a chromogen (Sigma). Tissue sections were counterstained with Mayer’s haematoxylin. Negative controls were obtained by skipping the application of the primary antibody.
Left-sided thyroid lobes were fixed in glutaraldehyde, processed for semithin sections and stained with toluidine blue 9. From all the studied groups, 0.5 cm specimens from the midshaft of right-sided femur bones were decalcified using 10% EDTA for 5 days and processed into paraffin blocks, from which 7-μm-thick sections were cut and stained with H&E.
The morphometric study was carried out using a Leica Qwin 500 C image analyzer computer system (Leica Imaging System Ltd, Cambridge, UK). Slides were examined under a light microscope. The following measurements were taken in 10 nonoverlapping fields for each rat:
- Height of follicular cells from each H&E section at magnification ×400.
- Height of parafollicular cells (C cells) in thyroid sections stained with calcitonin for immunohistochemical studies at magnification ×400.
- Area% of calcitonin immunoreactive C cells at magnification ×100.
- Number of calcitonin immunoreactive C cells at magnification ×100.
- Cortical bone thickness from each H&E section at magnification ×40.
- Diameter of Haversian canals from each H&E section at magnification ×100.
Data were tabulated and statistically analysed using SPSS software, version 9 (SPSS Inc., Chicago, Illinois, USA). The statistical analysis included the arithmetic mean and SD. Comparison between different groups was made using one-way analysis of variance followed by the post-hoc Tukey test. Data were expressed as mean±SD. P value was considered statistically significant when less than 0.05 10.
Examination of the thyroid sections of all groups was performed using a light microscope.
Histological results of thyroid sections
Thyroid sections revealed thyroid follicles of different sizes; their cavities contained acidophilic colloid with peripheral vacuolations. Thyroid follicles were lined by cubical follicular cells that exhibited rounded nuclei. Minute blood capillaries were extended between thyroid follicles (Figs 1 and 2). The follicular epithelium exhibited cubical cells. These cells showed spherical vesicular nuclei (Fig. 3).
This group recorded a positive, brownish, cytoplasmic reaction in C cells that were adjacent to the basal membrane of the thyroid follicles and present in interfollicular tissue. The surrounding follicular cells were nonreactive (Fig. 4).
Group II thyroid sections revealed an apparent increase in follicle size, which exhibited peripheral colloidal vacuolations. Groups of follicles were lined by vacuolated proliferating follicular cells. In some follicles, multiple layers of follicular cells were obviously seen. Presence of dilated congested blood vessels and congested infiltrating capillaries was recorded (Figs 5 and 6). The mean follicular cell height was significantly increased (P<0.05) compared with that of the control group (Table 1).
Examination of semithin sections revealed some thyroid follicles with papillary in-growth projecting into the lumen, and other follicles exhibited multiple follicular cells with pale nuclei and vacuolated cytoplasm that nearly obliterated their lumen. Mast cells filled with coarse metachromatic granules appeared in the interfollicular tissue (Fig. 7).
Large C cells bordering the follicles or present in interfollicular tissue were observed (Fig. 8). The mean values of their height and mean area% and number were significantly increased (P<0.05) compared with the control group (Table 1).
In the hypothyroid recovery group, thyroid follicles were variable in size and were partially filled with colloid. Most of the thyroid follicles were lined by cubical cells; a few of them were lined by more than one layer of follicular cells. Minimal congestion was still observed (Figs 9 and 10). Statistically, a significant decrease in mean follicular cell height compared with group II but a significant increase in comparison with the control group (P<0.05) was recorded (Table 1). Semithin sections revealed thyroid follicles lined mostly with cubical follicular cells that occasionally exhibited a vacuolated cytoplasm and pale vesicular rounded nuclei (Fig. 11).
Clusters of immunoreactive C cells were observed either adjacent to the basal membrane of the thyroid follicles or occupying the interfollicular tissue (Fig. 12). A nonsignificant difference in the mean value of C-cell height was recorded compared with groups I and II. However, regarding the mean area% and mean number of calcitonin immunoreactive C cells, a significant increase (P<0.05) was found compared with the control group with a significant decrease (P<0.05) compared with group II (Table 1).
Thyroid function test result
The mean value of T4 in group II and group III was significantly decreased compared with the control group. The mean T4 of group III was significantly increased compared with the hypothyroid group (Table 1).
Histological results of bone sections
Histological examination of H&E-stained sections from the midshaft of the control femur revealed a normal compact bone with the covering periosteum and cellular bone marrow cavity (Fig. 13). Haversian systems formed of osteocytes embedded in the lacunae arranged concentrically around Haversian canals were noted (Fig. 14).
Sections of femur in group II revealed a significant thickening (P<0.05) of the cortical bone and a significant narrowing (P<0.05) of the Haversian canals when compared with the control group (Figs 15 and 16 and Table 2).
The hypothyroid recovery group revealed a compact bone with the covering periosteum and cellular bone marrow cavity (Fig. 17). The mean value of cortical bone thickness was statistically nonsignificant compared with the control group but significantly decreased (P<0.05) compared with group II (Table 2). The Haversian systems formed of osteocytes embedded in the lacunae were arranged concentrically around the Haversian canals (Fig. 18). The mean value of the Haversian canal diameter was statistically nonsignificant compared with the control group and group II (Table 2).
The thyroid gland is a unique endocrine gland; it is the largest, is superficially located and is specialized in the production, storage and release of the thyroid hormones thyroxine (T4) and triiodothyronine (T3) 11,12, which are required for normal cell growth and development 13,14.
Humes and colleagues 15,16 stated that one of the most frequent thyroid disorders in humans is hypothyroidism, a condition when the production of the thyroid hormones decreases as a result of dysfunction of the thyroid gland, which disrupts the synthesis and secretion of hormones.
In the present study, hypothyroidism was artificially induced in healthy and metabolically stable rats. The study aimed at investigating the behaviour of follicular and C cells in an experimentally induced hypothyroid condition, which may point to a functional interaction between follicular and parafollicular cells of the thyroid gland. Moreover, this work aimed to study the changes occurring in the histological structure of the midshaft of the femur bone as a result of altered thyroid function under a hypothyroid condition.
The current study recorded that follicular and parafollicular cells showed signs of hyperactivity under induced hypothyroid conditions. It could be concluded that C cells evolve at the same pace as follicular cells when thyroidal status changes. This conclusion could be supported by the assumption that both cells are functionally coordinated, which might be attributed to the direct influence of PTU, or to the indirect influence of thyroid-stimulating hormone (TSH) on parafollicular cell activity.
The related changes in the thyroid glands were investigated in three groups of rats: group I (the control group), group II (the hypothyroid group) and group III (the recovery hypothyroid group).
In the present study the mean level of T4 was significantly decreased in group II and group III in relation to the control group, whereas the level in group III was significantly increased compared with the hypothyroid group. This was in accordance with the study by Sokkar and colleagues 17,18, who pointed to the significant reduction in serum triiodothyronine (T3) and thyroxine (T4) after the administration of a goitrogenic substance (PTU), which interfered with the iodination of tyrosine. Finkel et al. 19 also attributed this to inhibition of the oxidative processes required for iodination of tyrosyl groups, inhibition of coupling of iodotyrosine to form T3 and T4 and to blocking of the conversion of T4 to T3. It could be added that, upon PTU discontinuation, all its effects were reversed, resulting in increased serum T4 levels.
H&E-stained thyroid sections of group II revealed an increase in the length of the follicular epithelium, which was confirmed statistically by a significant increase in the follicular cell height compared with the control group. This was concomitant with the results of other researchers 3 who confirmed a significant increase in the height of the follicular epithelium in the hypothyroid group. Further, in the current study there were increases in follicular sizes, which appeared to be lined by multiple layers of follicular cells. This could be attributed to the low level of T4 that led to increased TSH levels, which was responsible for the proliferative activity of follicular cells. The investigators 3,18,20 confirmed the previous suggestion and added that intrafollicular adenomatosis consisted of an increase in the number of epithelial cells in the follicles, forming in some instances papillary projections into the lumen, which occasionally divided the follicle in the middle or even completely obliterated the lumen, giving an appearance of adenomatous solidification.
Also, some researchers 12,21 clarified that thyroid gland activity is regulated by the hypothalamic–pituitary–thyroid axis, including the negative feedback loop. The authors pointed to TSH as a major growth factor for the thyroid. The thyroid gland under TSH undergoes enlargement, hyperplasia, neovascularization and morphological alterations of thyrocytes related to their involvement in the production, processing and release of thyroid hormones.
In the present study, some thyroid follicles exhibited peripheral colloidal vacuolations that were more obvious when compared with the control group. On the basis of the fact that PTU has no effect on the iodinated thyroglobulin already stored in the gland and that clinical effects of this drug may be delayed until thyroglobulin stores are depleted 19, it could be suggested that follicular cells increase their activity in taking up and releasing thyroid hormones into the circulation to compensate for the increased demand. Gartner and Hiatt 22 confirmed this and reported that, during great demand for the thyroid hormone, follicular cells extend pseudopods into the follicular lumen to envelop and absorb the colloid. Further, the hypothyroid group showed an extensive network of dilated congested blood capillaries heavily infiltrating the thyroid follicles upon PTU administration, which could be attributed to the high level of TSH. This finding was concordant with those of the authors 12, who reported that better vascularization of the thyroid gland after methimazole (competitive inhibitor of thyroxine peroxidase as PTU) treatment was manifested by widened elongated capillaries that followed the margins of the follicles. Endothelial cells were in close contact with the base of thyrocytes, sometimes seemingly pushing them towards the follicular lumen.
In addition, Ramsden 23 attributed the vascularization to growth factors and vasoactive factors produced in the thyroid. They included the fibroblast growth factor and the vascular endothelial growth factor, which were potent angiogenic proteins. These growth factors, in cooperation with high concentrations of TSH in response to low concentrations of triiodothyronines, might regulate the growth and function of follicular and endothelial cells. Hence, it could be speculated that prolonged high levels of circulating TSH induced follicular cell hypertrophy and increased stromal vascularity as reported by Standring 24.
The morphological changes observed in semithin sections of group II revealed tall columnar follicular cells; some of the cells were filled with cytoplasmic vacuolations and had pale nuclei. The previous findings might be due to fluid accumulation and glandular overstimulation. Rubin and Strayer 25 specified that hypertrophy occurs as a result of an increase in cell size and functional capacity when trophic signals or functional demand increases; adaptive changes to satisfy these needs lead to increased cellular size (hypertrophy) and, in some cases, increased cellular number (hyperplasia). Further, the authors explained cellular hyperplasia by stimulating resting (G0) cells to enter the cell cycle (G1) to start multiplication. This may be a response to the altered endocrine milieu, increased functional demand or chronic injury.
Similar observations were described by Underwood 26, who attributed these changes to the accumulation of fluid. Kuma et al. 27 reported that microscopic examination of follicular cells revealed small, clear vacuoles within the cytoplasm and attributed this finding to hydropic and vacuolar degeneration.
In addition, some authors 25,27 explained that hydropic swelling results from impairment of cellular volume regulation, a process that controls ionic concentrations in the cytoplasm. They added that injurious agents may interfere with the membrane-regulated process by increasing the permeability of the plasma membrane to sodium, as a result of which the capacity of the pump to extrude sodium is exceeded, damaging the pump directly, or interfering with the synthesis of ATP, thereby depriving the pump of its fuel. The authors concluded that the accumulation of sodium in the cell led to an increase in water content to maintain isosmotic conditions and consequently caused cell swelling.
In the current study, semithin sections from group II revealed the presence of mast cells in the vicinity of blood capillaries, which might explain the process of angiogenesis. The investigators 28,29 demonstrated that the number of mast cells increased near blood capillaries. They were closely associated with connective tissue remodelling and localized at the thyroid tissue regenerative site where both thyroid folliculogenesis and angiogenesis take place.
Furthermore, Hiromatsu and Toda 30 stated that several mast cell mediators that regulate endothelial cell proliferation and function are angiogenic. The authors added that such mediators included the stem cell factor, the vascular endothelial growth factor, the epidermal growth factor, the basic fibroblast growth factor, and the platelet-derived growth factor that induce chemotactic migration of mast cells to sites of neovascularization. Mast cell products such as tryptase also degrade the connective tissue matrix to provide space for neovascular sprouts. Csaba and Pállinger 31 demonstrated that rat and mouse mast cells, together with other cells of the immune system, contain T3, and that mast cell T3 concentration seemed to be regulated by TSH. These facts point to the thyroid hormone as a possible candidate playing a regulatory/modulatory role in mast cell dynamics.
With regard to the C-cell changes associated with the thyroid status, Martín-Lacave et al. 1 suggested that the possible mechanisms involved in C-cell changes with thyroid status were in line with changes in follicular cells. Considering that TSH serum levels were increased in hypothyroid rats and decreased in T4-treated rats, three possible explanations were partially related to thyrotropin functions: TSH directly regulates C cells; follicular cells somehow regulate the C-cell activity; and C cells regulate follicular cells. The first hypothesis is sustained by different reports 32 describing the appearance of a reactive C-cell hyperplasia when TSH levels were increased in rats. Some investigators 33–36 clarified the second hypothesis and reported that the regulation of C cells by follicular cells could be carried out by either a local elevation of T3 and T4, or through the release of regulatory substances. For example, growth factors such as insulin-like growth factors or fibroblast growth factor, and other products such as thyroglobulin, play a decisive role in the autocrine regulation of TSH-stimulated follicular cell growth, differentiation and synthesis of thyroid hormones. According to this hypothesis, those substances could also exert a possible paracrine influence on C cells. Moreover, Sawicki 34 explained the third hypothesis in which C cells were probably involved in the intrathyroidal regulation of secretion and growth processes by secreting numerous regulatory peptides usually defined as ‘paracrine factors’ that were found in C cells exclusively. Some regulatory peptides like calcitonin, somatostatin and katacalcin seemed to be involved as inhibitors of thyroid hormone secretion, whereas gastrin-releasing peptide and helodermin were seen to be involved as stimulators of thyroid hormone secretion.
With regard to sections stained with anti-calcitonin antibody in group II, the present study revealed a significant increase in the height, mean area% and number of calcitonin immunoreactive C cells compared with the control group. This might be due to the high level of TSH as a result of goitrogen (PTU) administration leading to hyperplasia and hypertrophy of immunopositive C cells. These findings coincided with those of Martín-Lacave and colleagues 1,37,38, who reported that the hyperactive thyroid showed the presence of enlarged C cells distributed either in small groups or singly. These observations might point to the possible relationship between the functional state of the thyroid gland and the activity of C cells.
With regard to the bone results of the present study, the effect of artificially induced hypothyroidism on the midshaft of the femur was studied by light microscopy to clarify the related histological changes in bone in such a condition. In the current study, H&E-stained sections of group II (the hypothyroid group) showed a significant increase in bone thickness and a significant decrease in the diameter of Haversian canals compared with the control group. This could be related to the decreased bone resorption due to hyperplasia and hypertrophy of C cells under the effect of increased levels of TSH.
These findings were in accordance with the results of the studies by Gogakos and colleagues 5,39, who found that hypothyroidism reduced bone turnover and prolonged the remodelling cycle: the duration of the osteoclastic resorption phase was extended two-fold, whereas the time taken for osteoblastic bone formation and secondary mineralization was prolonged four-fold. These changes resulted in low bone turnover and an overall gain in bone mass and mineralization that led to elevated bone mineral density. Moreover, Hase et al. 40 reported that TSH seems to be an inhibitor of bone turnover and inhibits osteoclast formation, resorption and survival. Other investigators 41,42 added that calcitonin had an inhibitory effect on osteoclast activity by suppressing bone resorption. In addition, osteoblasts were stimulated to increase osteoid synthesis and calcium deposition.
The current study recorded a significant increase in T4 level in group III (the recovery hypothyroid group) compared with group II. This finding might be related to the recovering levels of TSH that exerted no further stimulation on the thyroid epithelium. In H&E-stained and semithin sections of group III, the follicular cell height showed a significant regression compared with group II. Most thyroid follicles were lined by vacuolated cubical follicular cells that exhibited pale vesicular rounded nuclei. Minimal congested blood vessels continued to be present. These recovering observations could be explained by the ability of some cells to regain their normal appearance upon drug discontinuation. Kuma et al. 27 mentioned that, when stress abates, involution occurs, the height of the epithelium falls, colloid accumulates and follicular cells resume their normal size and architecture. The present study recorded that the height of parafollicular cells showed a nonsignificant change compared with the control group and group II. However, the mean area% and number of calcitonin immunoreactive C cells revealed a significant increase compared with the control group and a significant decrease compared with group II. In addition, Rubin and Strayer 25 described that hypertrophy and hyperplasia were reversible on discontinuation of stress. The recovery group of this study revealed a partial decrease in cortical bone thickness and an increase in the width of Haversian canals upon PTU discontinuation.
The current study concluded that the hypothyroid condition points to a functional inter-relation between follicular cells and parafollicular cells of the thyroid gland. In hypothyroid rats, both follicular and C cells displayed signs of hyperactivity. Calcitonin is an essential indicator of C cells and is considered a marker in C-cell lesions. Thyroid hormones are essential for normal skeletal development. Experimentally induced hypothyroidism resulted in increased C-cell number, consequently decreasing bone resorption and increasing cortical bone thickness as determined by histomorphometry. Thyroid dysfunction affects both follicular and calcitonin-producing cells and consequently affects the bone.
Conflicts of interest
There is no conflict of interest to declare.
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