Ultrastructure of Thyroid and Parathyroid Bodies in Case of Hypocalcemia and Hypercalcemia

Aims: To analyze ultrastructural transformations of parafollicular cells of thyroid body and main cells of Parathyroid body in case of experimental blood hypocalcemia and hypercalcemia. Place and Duration of Study: Tashkent Methodology and Study Design: White outbreed male rats with the mass of 130-140 g were split by 3 groups based on content of the free calcium ions in the blood serum: normal (benchmark, n=10); low (hypocalcemia, n=40); high (hypercalcemia, n=40). Experimental intraperitoneal administration of 2,5% ethylenediominotetraacetic acid and 10% Calcium gluconate during 1, 3, 6 and 10 days resulted in hypocalcemia and hypercalcemia consequently, in elevation and reducing the parathormone contained in the blood. Results: In case of the normal content of the calcium ions in the blood, the main, oxyphilic, intermediate and cambial cells were identified. The main cells as the most numerous ones are characterized with oblong form and small size. The nucleus is large, located eccentrically, the nucleolem even, sulcated, with invagination, and nucleoplasm is rich with euchromatin. Conclusion: Hypocalcemia after ethylenediominotetraacetic acid introduction in 1, 3, 6 and 10 days is activating B type cells. Hypercalcemia after calcium gluconate introduction in 1, 3, 6 and 10 days causes hy Parathyroid body po-function.


INTRODUCTION
According to the numerous research works, about half of calcium in the blood plasma is associated with organic phosphates, therefore its free ions amount to about 1.2 mM/l. As for the cell internal content, the calcium ion concentration in its cytoplasm is by 10,000 lessabout 100 nM/l. Despite it is actively involved in the neurotransmission, segmentation, an astalsis, control many metabolic processes, its concentration both in the blood and in the cell cytoplasm may fluctuate with small amounts [1][2][3][4][5]. It is achieved due to the existence of sophisticated enzymatic and transmission structures in the cell membrane, reverse correlation in the functional system, comprising the main cells (CC) of Parathyroid body (PB) and parafolicular cells (PC) of thyroid body (TB), absorbing cells of intestine and kidney, osteoblastic cells and osteoclasts of bony tissues [3,6,7]. Increase of the PB parathormones (PH) is causing several effects oriented to the keeping the phosphorus and calcium balance: both elements increase in the bony tissues in case of blood hypomineralization in different correlation, phosphorus reabsorbtion in the kidney decreases and calcium reabsorbtion increases, and, to the contrary, the phosphorus absorption in the narrow intestine increases, and the calcium absorption decreases [8]. Given this fact, we have set a GOAL: to analyze ultrastructural transformations of parafollicular cells of thyroid body and main cells of Parathyroid body in case of experimental blood hypocalcemia and hypercalcemia.

MATERIALS AND METHODS
White outbreed male rats with the mass of 130-140 g were split by 3 groups based on content of the free calcium ions in the blood serum: normal (benchmark, n=10); low (hypocalcemia, n=40); high (hypercalcemia, n=40).
Hypocalcemia was achieved through the daily intraperitonial introduction of 2.5% disodium salt water solution of ethylenediominotetraacetic acid (EDTA 1.0 ml per 100 g of the animal's mass). EDTA with the calcium ions of the blood serum is forming insoluble salt and decreases calcium concentration. On the contrary, hypercalcemia was achieved through the daily intraperitonial introduction of 10% solution of calcium gluconate (1.0 g per 100 g of the animal's mass). Removal of the animals from experiment was accomplished in line with the European Convention for the Protection of Animals Used in the Scientific Research (1984), within 1, 3, 6 and 10 days. Calcium ion concentration in the blood serum was identified using atomic sorption spectrophotometer (Beckman, USA). For the mother solution preparation СаСОз was dissolved in the minimal amount of hydrochloric acid in an amount of О.5 g of СаСОз in 1 ml. The mother solution of lanthanum chloride was received through solution of 58.65 g of salt in 250 ml of concentrated hydrochloric acid. After that solution was amended to 1,000 ml with distilled water. The blood serum was diluted in correlation 1:25. The serum solutions were compared with the reference solution of the calcium ions through spectrophotometry.
The pieces of PB and TB with the size 1 mm3 for electronic and microscopic examination were fixed in the buffered 2.5% solution of gluteraldehyde (20 min.) with the post-fixation in 1% solution of osmic acid (1.5 hours). After generally accepted follow-through the spirits with increasing concentration the tissue was introduced in araldite. Ultrathin sections were received on LKB-4800 ultramicrotome (Sweden) and observed on IEOL-100S microscope (Japan). PH was identified with radioimmunoassay technique with the help of the bull PH and set of antiserums produced by RIA-RTG-100 Company (Belgium).

RESULTS AND DISCUSSION
In case of the normal content of the calcium ions in the blood (Table 1), the main, oxyphilic, intermediate and cambial cells were identified.
The main cells as the most numerous ones are characterized with oblong form and small size. The nucleus is large, located eccentrically, the nucleolem may be even, sulcated, with invagination, and nucleoplasm is rich with In case of hypercalcemia from period to period the number of LGs in PB CC cytoplasm increases and the length of SER membranes decreases; Golgi apparatus reduces, mitochondria becomes smaller (Fig. 2), the high dense particles, presumably, calcium appear in their matrix [4]. The concentration of the large number of electron dense secretory granules is typical of cytoplasm near the cell membrane. The cell membrane almost does not form the lines and interdigitations. Therefore, hypercalcemia causes CC hypo-function and main cell A type domination.
TB is a vital organ producing not only triiodothyronine (Т3), thyroxin (Т4), but also calcitonin playing certain role in regulation of the calcium homeostasis in the blood [5]. In physiological content of the calcium ions (3.48+0.48 mEq/L; Table 1) tireocytes and parafollicular cells can be found in the wall of each follicular gland, which in terms of ultrastructure can be of two types: a) similar to tireocytes and b) parafollicular cells (PCs). PCs in the wall of the follicular gland or among follicular glands are located singularly or in a form of small groups of 2-3 cells. These cells with round, oblong or cubic shapes in the wall of follicular gland may contact with colloid located in the follicular gland gap; they form dense contact, thin desmosomes and low-grade interdigitations with adherent cells. PC's base is constantly contacting with 1-3 blood channels through the thin homogenic basal lamina. PC's nucleus with 1-2 compact plasmosomes are excentrically located and are rich with euchromatin. SER profiles in cytoplasm are forming 2-3 flattened cisterns, Golgi apparatus is identified near the nucleus and occupies rather large area with all structures. The substance with moderately dense matrix and number of crista is located inside some of them. Mitochondria are not numerous, small with moderately dense matrix and number of crista. Free ribosomes and polysomes are evenly disseminated along entire cytoplasm. The existence of significant number of single type round electron dense SGs with thin light ring in cytoplasm near cytolemme adherent to the blood capillary is typical of PC. In case of hypocalcemia (Table 1) PCs become larger and receive mainly conical shape. The nucleus has uneven surface and unevenly expanded perinumain space; nucleoplasm contains approximately equal number of eumatine and heteromatine. SER profiles are in moderate quantity, they form the different size vacuoles with the moderate density substance. Mitochondria are small with dense matrix and some crista; Golgi apparatus is with hypoplasia and contains the moderate number of vesicoles and single vacuoles (Fig. 3). In case of hypocalcemia SHs are large, have high density and much greater quantity, compared the rats with the normal concentration of calcium ions in the blood.  In case of hypercalcemia (Table 1) PCs have the cubic shape (Fig. 4), they are located as groups of 2-3 cells, are lightened and almost do not contain SHs. Single germinating cells can be found in the hyperplastic structures of Golgi apparatus closely interacting with unstriated reticulum and nuclear membrane. The nucleus has a round shape, relatively large and contains mainly euchromatine. Perinumain space is unevenly expanded. SER profiles have significant length and are unevenly expanded. Mitochondria have round and oblong shape and are in the functional stress condition. PCs almost do not form interdigitations with adherent cells. The nerve terminals, blood channels sent by the flattened endothelin and penetrated with the large number of pores are permanently identified under PCs. PCs' basal membranes and channels endothelin are lying close to each other and separated just with narrow and almost invisible interstitium.
Therefore, in case of hypocalcemia and hypercalcemia PCs in the blood serum are in the condition of hypo-function and hyper-function, respectively. In hyper-functional condition the process of SH removal is more accelerated, compared to it generation at the stages of intracellular synthesis and aging. In hypofunctional condition SH removal is hardly slowed down while the stages of its synthesis and aging do not experience visible modifications. Though the secretory cycle presents the chain of consequent and interrelated processes simultaneously taking place in the cytoplasm nucleus and cell organ, in condition of hypofunction and hyper-function in PC its final stage when the mature product is synthesized from the cell and interstitium and blood should be considered as the most reactive.
According to the researches, extracellular concentration of the calcium ions [1,4,5,8], plays an important role in the blood coagulation, bony tissue remodeling, neural impulse transmission, etc. It is under the strict control by calcitonin, parathormone and calcitriol hormones which have the reverse correlation among them. Receptors of the calcium ions are membrane proteins and activated in case of increase of its concentration in the blood and interstitium. They "include" the secondary messengersinozitoltriphosphate (IP3) and diacylglycerol (DAG), which are causing increase of the calcium concentration inside PCs and exocytosis of SHs containing calcitonin. From the other hand, in the main cells of PB its high level decreases secretion of PH increasing calcium in the blood. This activity is mediated by DAG and protein kinase C, and, probably, by decrease of cAMP concentration in the result of G-protein activation. Receptors of the calcium ions are also localized in nephritic and intestinal epithelium [4,5,8]. The mechanisms facilitating keeping calcium ion homeostasis in the blood are controlling protein -receptor on membrane of various cells of internals.

CONCLUSION
1. Hypocalcemia after EDTA introduction in 1, 3, 6 and 10 days is activating B type cells, facilitates secretory material removal from them, and increase of PH in the blood. At that, retention of the secretory product removal is observed in PC. 2. Hypercalcemia after calcium gluconate introduction in 1, 3, 6 and 10 days causes PB hypo-function: secretion removal from the gland cells is slowed, the share of the main A type cells increases and PH content in the blood is decreased. Secretion process in TB PC is accelerated. 3. In case of hypocalcemia and hypercalcemia the structural changes of TB PC and main cells of PB are the morphological equivalent of existence of the reverse correlation among them, their functioning association in regulation of the calcium ion concentration in the blood.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.