Ultrastructural morphometry of parathyroid cells in rats of different ages

Summary Parathyroid glands of newborn to 1-year-old male Sprague-Dawley rats were fixed by perfusion or by immersion, and prepared for electron microscopy. Parathyroid glands fixed by immersion exhibited parenchymal cells with variable ultrastructure, indicating that these cells were in different stages of the proposed secretory cycle in parathyroid cells. In contrast, parathyroid cells of glands fixed by perfusion were uniform in ultrastructure, suggesting that all cells were in the same stage of secretory activity. Parathyroid glands of 3-, 4-, 6-, 8- and 10-week-old rats also were fixed by perfusion and analysed by electron-microscopic morphometry. These data demonstrated an increase in cell volume and in surface area of the membrane compartments concerned with parathyroid hormone secretion: these changes were not related to variations in serum calcium concentration. Both the qualitative observations and the quantitative data do not favour the idea of a secretory cycle in rat parathyroid cells.     

Intestinal calcium transport: parathyroid hormone and adaptation to dietary calcium.

Thyroparathyroidectomy prevents the elevation of intestinal calcium transport in response to low dietary levels of calcium. Removal of the thyroparathyroid glands reduces elevated intestinal calcium transport of rats on low calcium diets to the levels found in rats fed high calcium diets. This reduction took place 4 days after surgery. The chronic administration of a constant exogenous source of parathyroid hormone to thyroparathyroidectomized rats fed either a high or low calcium diet resulted in high rates of intestinal calcium transport independent of dietary calcium. Since 1,25-dihydroxyvitamin D₃ supplementation eliminates adaptation in a similar manner, these results strongly support the idea that parathyroid glands mediate intestinal adaptation to low dietary calcium presumably by the stimulation of 1,25-dihydroxyvitamin D₃ biosynthesis by secreted parathyroid hormone.     

Parathyroid cell variants may be provoked during immersion fixation

Parathyroid glands of cattle, dogs, cats, mice and rats were immersed in glutaraldehyde or mixtures consisting of glutaraldehyde, formaldehyde and acrolein in either Na-phosphate, Na/K-phosphate or Na-cacodylate buffer, and postfixed with OsO4 in the same buffers or, alternatively, in s-collidine. Excellent preservation of bovine, feline and murine parathyroid glands was achieved with fixation mixtures containing 1% glutaraldehyde, 1.5-2% formaldehyde and 2.5-5% acrolein in 0.1 M Na-cacodylate with or without Ca2+ and Mg2+, Na-phosphate or Na/K-phosphate at 4 degrees C followed by postfixation with 1% OsO4 in the same buffers or in s-collidine containing sucrose, Ca2+ and Mg2+. This procedure largely abolished the occurrence of parathyroid cell variants. Bovine parathyroid glands were also satisfactorily preserved with 1% glutaraldehyde and 2% formaldehyde whereas 1% glutaraldehyde and 2.5 or 5% acrolein, lower or higher buffer osmolarity, or immersion at room temperature led to vacuolization of RER and to breakdown of membranes. In contrast, all fixation protocols led to the formation of dark and light cell variants and to multinucleated syncytial cells in dog and rat parathyroids. The results thus show that parathyroid cell variants arise during immersion fixation and that aldehydes, buffers and temperature are important factors for provoking parathyroid cell variants.     

Biosynthetic activity of parathyroid cells during different function of the parathyroid glands

By means of autoradiograph c and morphometric methods parathyroid glands of 31 rats after single injection of 3H-leucin (2,5 mc Cu/g of body mass 25 min. before killing) were investigated. Parathyroid glands were inhibited by multiple injections of calcium gluconate solution or by a diet with increased content of calcium and vitamin D2. Parathyroids were stimulated by multiple injections of Trilon B solution, by a diet with increased content of phosphates or by subtotal parathyroid resection. Straight correlation between parathyroid function, average section area of parathyrocytes and average number of silver granules per one parathyrocyte was revealed. Hence, average section area of parathyrocytes is objective morphometric criterion of parathyroid function.     

Activation of calcium-sensing receptor accelerates apoptosis in hyperplastic parathyroid cells

Calcimimetic compounds inhibit not only parathyroid hormone (PTH) synthesis and secretion, but also parathyroid cell proliferation. The aim of this investigation is to examine the effect of the calcimimetic compound NPS R-568 (R-568) on parathyroid cell death in uremic rats. Hyperplastic parathyroid glands were obtained from uremic rats (subtotal nephrectomy and high-phosphorus diet), and incubated in the media only or the media which contained high concentration of R-568 (10(-4)M), or 10% cyclodextrin, for 6h. R-568 treatment significantly suppressed medium PTH concentration compared with that of the other two groups. R-568 treatment not only increased the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay-positive cells, but also induced the morphologic changes of cell death determined by light or electron microscopy. These results suggest that CaR activation by R-568 accelerates parathyroid cell death, probably through an apoptotic mechanism in uremic rats in vitro.     

Pre-conditioning cryosurgery: cellular and molecular mechanisms and dynamics of TNF-α enhanced cryotherapy in an in vivo prostate cancer model system

Cryosurgery is increasingly being used to treat prostate cancer; however, a major limitation is local recurrence of disease within the previously frozen tissue. We have recently demonstrated that tumor necrosis factor alpha (TNF-α), given 4h prior to cryosurgery can yield complete destruction of prostate cancer within a cryosurgical iceball. The present work continues the investigation of the cellular and molecular mechanisms and dynamics of TNF-α enhancement on cryosurgery. In vivo prostate tumor (LNCaP Pro 5) was grown in a dorsal skin fold chamber (DSFC) on a male nude mouse. Intravital imaging, thermography, and post-sacrifice histology and immunohistochemistry were used to assess iceball location and the ensuing biological effects after cryosurgery with and without TNF-α pre-treatment. Destruction was specifically measured by vascular stasis and by the size of histologic zones of injury (i.e., inflammatory infiltrate and necrosis). TNF-α induced vascular pre-conditioning events that peaked at 4h and diminished over several days. Early events (4-24 h) include upregulation of inflammatory markers (nuclear factor-κB (NFκB) and vascular cell adhesion molecule-1 (VCAM)) and caspase activity in the tumor prior to cryosurgery. TNF-α pre-conditioning resulted in recruitment of an augmented inflammatory infiltrate at day 3 post treatment vs. cryosurgery alone. Finally, pre-conditioning yielded enhanced cryosurgical destruction up to the iceball edge at days 1 and 3 vs. cryosurgery alone. Thus, TNF-α pre-conditioning enhances cryosurgical lesions by vascular mechanisms that lead to tumor cell injury via promotion of inflammation and leukocyte (esp. neutrophil) recruitment.     

A vasopressor factor partially purified from human parathyroid glands.

Recently, a parathyroid hypertensive factor was postulated to play a role in the pathogenesis of hypertension in genetically hypertensive rats. Therefore it was examined, whether in human parathyroid glands a vasopressor substance can be detected. For this purpose, homogenates of hyperplastic parathyroid glands from 20 patients with tertiary hyperparathyroidism were deproteinized and fractionated by gel chromatography. The fractions obtained were tested for vasopressor activity in isolated perfused rat kidneys. A vasopressor fraction containing substances of 0.6-2.5 kDa was identified in the parathyroid glands. The responsible product was heat sensitive, peptidase-, trypsin- and carboxypeptidase y- sensitive and hydrophilic, as it did not bind to hydrophobic reversed-phase gel. These results suggest that parathyroid glands contain a hydrophilic peptide-like vasopressor substance different from the parathyroid hormone.     

1,25(OH)2D3 only affects long-term levels of plasma Ca2+ but not the rapid minute-to-minute plasma Ca2+ homeostasis in the rat.

Results from our lab have shown previously that parathyroid hormone (PTH) is not the key factor in the rapid regulation of plasma Ca2+. The possible role of 1,25(OH)2D3 in the rapid minute-to-minute regulation of plasma Ca2+, as addressed by a possible rapid non-genomic action of 1,25(OH)2D3, was therefore studied in vivo in rats. The rapid calcemic recovery from induction of hypocalcemia by a brief EGTA infusion was examined in vitamin D-depleted rats with intact parathyroid glands and in vitamin D depleted rats 1 h after parathyroidectomy (PTX). The influence of different levels of plasma 1,25(OH)2D3 on the rapid calcemic recovery from hypocalcemia was examined in PTX rats treated with 1,25(OH)2D3 for two days at two different doses of 0.2 microg/day, 0.05 microg/day or vehicle, and in PTX rats being BNX for two days, as well. Additionally, the long-term effect of 1,25(OH)2D3 on plasma Ca2+ homeostasis was examined. Plasma Ca2+ recovered significantly (P<0.05) 10 min after discontinuing EGTA in vitamin D-depleted rats with or without parathyroid glands. Plasma Ca2+ increased significantly (P<0.05) and at the same rate after induction of hypocalcemia in PTX rats with different levels of plasma 1,25(OH)2D3. The final levels of plasma Ca2+ obtained were set by 1,25(OH)2D3 in a dose-related manner. 1,25(OH)2D3 did not affect the rapid calcemic recovery from EGTA induced hypocalcemia, but only had an effect on the long-term plasma Ca2+ homeostasis in the rat.     

The influence of a single ethanol injection on normal thyroid tissue of the rat

Macro- and microscopic changes in the normal thyroid gland of rats, and in the surrounding tissues 2 and 4 weeks after a single intrathyroidal ethanol injection (IEI), together with the influence of such treatment on the function of the recurrent laryngeal nerves and of the parathyroid glands, were assessed. The intraoperative macroscopic evaluation at 2 weeks (20 rats) and 4 weeks (20 rats) after IEI revealed the presence of a scar at the site of the IEI-treated lobe in seven (35%) and six (30%) rats, respectively, and the reduction of lobe dimensions in thirteen (65%) and fourteen (70%) rats, respectively. The microscopic evaluation of the lobe after IEI showed coagulative necrosis, reduction in thyroid follicle volume, disturbance of follicle structure, haemorrhage, haemosiderin deposits, inflammatory infiltration and fibrosis. No microscopic changes were observed in the tissues surrounding the thyroid, nor in the parathyroid glands located extrathyroidally or in the second thyroid lobe. No vocal cord dysfunction or significant changes in serum calcium levels after IEI were detected.     

Parathyroid ultrastructure after aldehyde fixation, high-pressure freezing, or microwave irradiation

Parathyroid cell variants, commonly observed in parathyroid glands fixed by immersion in glutaraldehyde, are believed to be the result of cyclic changes in the course of parathyroid hormone secretion. Immersion of bovine parathyroid glands in a mixture consisting of 1% glutaraldehyde, 1.5% formaldehyde, and 2.5% acrolein, followed by post-fixation in 1% osmium tetroxide, resulted in high uniformity with only one cell variant, whereas the same fixation procedure led to disruption of cell membranes and formation of cell variants in rat parathyroids. Parathyroid glands of both cattle and rats prepared by high-pressure quick-freezing and subsequent freeze-substitution contained only one cell variant. Excellent preservation of the ultrastructure of bovine and rat parathyroids, also exhibiting only one cell variant, was achieved by microwave irradiation in the presence of 2.5% glutaraldehyde in Na-cacodylate followed by post-fixation with OsO4 in Na-cacodylate or s-collidine, both containing Ca2+ and Mg2+. Use of the appropriate buffer, as well as osmication, is essential for successful fixation utilizing microwave energy. The main effects are considered to be heating specimens within sufficient short periods and enhancement of subsequent osmium fixation. The results support the idea, arising after examination of perfusion-fixed parathyroid tissue, that parathyroid cell variants occur during improper aldehyde fixation rather than that they express functional diversity.     

Parathyroid cell variants may be provoked during immersion fixation

Summary Parathyroid glands of cattle, dogs, cats, mice and rats were immersed in glutaraldehyde or mixtures consisting of glutaraldehyde, formaldehyde and acrolein in either Na-phosphate, Na/K-phosphate or Na-cacodylate buffer, and postfixed with OsO₄ in the same buffers or, alternatively, in s-collidine.Excellent preservation of bovine, feline and murine parathyroid glands was achieved with fixation mixtures containing 1% glutaraldehyde, 1.5–2% formaldehyde and 2.5–5% acrolein in 0.1 M Na-cacodylate with or without Ca²⁺ and Mg²⁺, Na-phosphate or Na/K-phosphate at 4°C followed by postfixation with 1% OsO₄ in the same buffers or in s-collidine containing sucrose, Ca²⁺ and Mg²⁺. This procedure largely abolished the occurence of parathyroid cell variants. Bovine parathyroid glands were also satisfactorily preserved with 1% glutaraldehyde and 2% formaldehyde whereas 1% glutaraldehyde and 2.5 or 5% acrolein, lower or higher buffer osmolarity, or immersion at room temperature led to vacuolization of RER and to breakdown of membranes. In contrast, all fixation protocols led to the formation of dark and light cell variants and to multinucleated syncytial cells in dog and rat parathyroids. The results thus show that parathyroid cell variants arise during immersion fixation and that aldehydes, buffers and temperature are important factors for provoking parathyroid cell variants.     

Pre-conditioning cryosurgery: Cellular and molecular mechanisms and dynamics of TNF-α enhanced cryotherapy in an in vivo prostate cancer model system

Cryosurgery is increasingly being used to treat prostate cancer; however, a major limitation is local recurrence of disease within the previously frozen tissue. We have recently demonstrated that tumor necrosis factor alpha (TNF-α), given 4 h prior to cryosurgery can yield complete destruction of prostate cancer within a cryosurgical iceball. The present work continues the investigation of the cellular and molecular mechanisms and dynamics of TNF-α enhancement on cryosurgery. In vivo prostate tumor (LNCaP Pro 5) was grown in a dorsal skin fold chamber (DSFC) on a male nude mouse. Intravital imaging, thermography, and post-sacrifice histology and immunohistochemistry were used to assess iceball location and the ensuing biological effects after cryosurgery with and without TNF-α pre-treatment. Destruction was specifically measured by vascular stasis and by the size of histologic zones of injury (i.e., inflammatory infiltrate and necrosis). TNF-α induced vascular pre-conditioning events that peaked at 4 h and diminished over several days. Early events (4–24 h) include upregulation of inflammatory markers (nuclear factor-κB (NFκB) and vascular cell adhesion molecule-1 (VCAM)) and caspase activity in the tumor prior to cryosurgery. TNF-α pre-conditioning resulted in recruitment of an augmented inflammatory infiltrate at day 3 post treatment vs. cryosurgery alone. Finally, pre-conditioning yielded enhanced cryosurgical destruction up to the iceball edge at days 1 and 3 vs. cryosurgery alone. Thus, TNF-α pre-conditioning enhances cryosurgical lesions by vascular mechanisms that lead to tumor cell injury via promotion of inflammation and leukocyte (esp. neutrophil) recruitment.     

Effect of enkephalins on the function of the calcium-regulating endocrine glands in shock

The experiments on white rats weighing 180-220 g have shown that in traumatic and hemorrhagic shock the initial increase in parathyroid hormone blood concentration is followed by the decrease of functional activity of parathyroid glands. Calcitonin concentration is found to increase during the first hours of shock. The changes in calcium-regulating gland function result in significant disturbances of calcium exchange during shock. The injection of synthetic leu-enkephalin analogs to rats with shock leads to normalization of calcium-regulating endocrine glands function.     

Cryosurgery and rhTNF-α play synergistic effects on a rat cortex C6 glioma model

Glioma, a type of brain tumor originating from glioma cells, varies widely in aggressiveness and causes serious symptoms, but the treatments are limited. Studies have shown that cryosurgery has multiple effects on tumor treatments, and administration of human tumor necrosis factor-alpha (rhTNF-α) arguments the anti-tumor effect of cryotherapy in breast and prostate cancers. To test the hypothesis that cryosurgery and rhTNF-α play synergistic effects against brain tumors, we established a brain glioma model on rat cortex regions following different treatments: the G1 group was sham-operated; the G2 group was treated with cryosurgery; the G3 group was treated with rhTNF-α; and G4 group received combined treatment with cryosurgery and rhTNF-α. Tumor sizes were measured by magnetic resonance imaging; DNA fragmentation was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL assay); P21(WAF1/CIP1) and proliferating cell nuclear antigen (PCNA) expression levels were scored using immunohistochemical staining. G2 and G4 rats had significantly longer survival time than did G1 rats. Tumor sizes in each group were significantly decreased as compared with those in G1 rats. PCNA-positive cells were significantly decreased in G2, G3 and G4 rats as compared with G1 rats. In contrast, DNA fragmentation and P21(WAF1/CIP1)-positive cells were significantly increased in each treatment group. Importantly, a combined treatment enhanced the effects of cryosurgery. Combined treatment with cryosurgery and rhTNF-α may have a synergistic effect on glioma tumor therapy, enhancing the inhibition of proliferation and the induction of apoptosis.     

Ultrastructural observations on the mechanism of secretion in the rat parathyroid after fluoride ingestion

Summary The secretory mechanism of the parathyroid glands of fluoride treated rats is evaluated ultrastructurally and compared to that of control rats. The principal difference between the two groups of rats concerns the rate of activity of the chief cells of the gland. In the control animals, these cells are predominantly inactive. In the fluoride-treated rats, they exhibit a more active stage of the secretory cycle. The active chief cells in rats treated with fluoride contain increased numbers of secretory granules. These granules are released into the perivascular spaces within cytoplasmic projections suggesting an apocrine-like mechanism for the secretion of parathyroid hormone. Secretory granules are observed free in the perivascular spaces and within the cytoplasm of capillary endothelial cells in the parathyroid glands.     

Adaptation of intestinal calcium absorption: parathyroid hormone and vitamin D metabolism.

It has already been demonstrated that the adaptation of intestinal calcium absorption of rats on a low calcium diet can be eliminated by thyroparathyroidectomy plus parathyroid hormone administration. This treatment elevates intestinal and plasma levels of 1,25-dihydroxyvitamin D₃ in rats on a high calcium diet while producing no change in rats on a low calcium diet. It therefore appears likely that the modulation of intestinal calcium absorption by dietary calcium is mediated by the parathyroid glands and the renal biogenesis of 1,25-dihydroxyvitamin D₃. Changes in the other unknown vitamin D metabolite levels as a result of dietary calcium are also modified by thyroparathyroidectomy and parathyroid hormone administration, but the effect of these metabolites on intestinal calcium transport is unknown.     

Thyroid cryotherapy in an experimental rat model

In recent years cryotherapy has been more and more frequently used for the treatment of tumors of different organs. Until now, the use of cryotherapy for the treatment of thyroid lesions, as well as histopathologic changes in thyroid tissue after cryotherapy, has not been described. Nitrous oxide cryotherapy of one thyroid lobe in twenty 12-week male Wistar rats was performed. After 2 and 4 weeks, the cryotreated thyroid lobe and the second lobe along with a part of the trachea, esophagus, and the subhyoid muscles adhering to the thyroid were excised and assessed macro- and microscopically. The macroscopic evaluation, performed 2 and 4 weeks postcryotherapy, revealed atrophy of the cryotreated lobe in 4 and 3 rats, respectively, and reduction of the cryotreated lobe dimensions in 6 and 7 rats, respectively. In the specimens of the lobes excised 2 weeks following cryotherapy, examined microscopically, necrosis, granulomatous inflammation, hemorrhages, and hemosiderin deposits were found most often, whereas in the specimens of the lobe excised after 4 weeks lymphocytic inflammation and fibrosis were mainly observed. No microscopic changes were observed in the thyroid lobes that were not frozen or in the parathyroid glands located inside these lobes or extrathyroidally, either ipsilaterally or contralaterally to the cryotreated thyroid lobes. There was no microscopic damage to other tissues adjacent to the thyroid gland. No rat developed vocal cord dysfunction after cryotherapy and no significant changes in serum calcium level before and after cryotherapy were observed. The results obtained show that it is possible to cryoblate thyroid tissue without damaging the tissues adjacent to the thyroid, as well as to spare function of the recurrent laryngeal nerves and parathyroid glands.     

Phase‐sensitive fluorescence detector for parathyroid glands during thyroidectomy: A preliminary report

Despite advances in medical technology, the parathyroid glands are still damaged during thyroid surgery. Our previous studies exploring methods for locating the parathyroid glands using autofluorescence have limitations, such as turning off the surgical light or requiring additional matching between the autofluorescence image and real‐surgical field‐of‐view. We developed a probe‐type parathyroid autofluorescence detector using a phase‐sensitive process and optical filtering to overcome these limitations. A preliminary clinical trial was performed on eight parathyroid glands in four patients. The normalized mean signal of the normal parathyroid glands was 332% stronger than that of the thyroid, and 384%, 459% and 286% stronger than the signal of the muscle, trachea and fat, respectively. Additionally, the device also detected fluorescence from indocyanine green.     

Participation of tissue basophils of the parathyroid glands in the regulation of the morphofunctional state of the organ

By means of cytometry estimation of mitotic index and nucleo-cytoplasmic relation of parathyrocytes, parathyroid glands have been studied in 52 white rats and 28 white mice under conditions of intal inhibition (30 mg/kg of body mass twice a day intraperitoneally) of tissue basophils (TB) secretory activity. For stimulation of the glands, repeated injections of trilon B and hemiparathyroidectomy are used. The experiment lasts for 3.5 days. In the rat parathyroid glands, containing a considerable amount of TB, intal does not produce any important effect of quantitative parameters of the organ's structure in intact animals, nevertheless it prevents development of hypertrophy of the parathyrocytes in the stimulated glands, as well as makes weaker the proliferative response of the cells to hemiparathyroidectomy. In mice, their parathyroid glands containing single TB, under conditions of stimulation of the parathyroid function, intal does not produce any inhibitory effect to growth of middle size parathyrocytes. The results obtained demonstrate that the parathyroid TB actively participate in regulation of the organ's morphofunctional state, intensifying the stimulating effect of hypocalcaemia to the parathyroid parenchyma.     

Ultrastructural alterations in mammalian parathyroid glands induced by fixation

The influence of fixation methods, buffers and ions on the ultrastructure of parathyroid cells was studied in dogs, cats, rats and mice. Parathyroids fixed by immersion showed 3 chief cell variants referred to as cells in active, intermediate and resting stages, multinucleated syncytial cells, atrophic cells and, only in 1 feline parathyroid, a few oxyphil cells. Parathyroid glands fixed by perfusion, however, consisted only of 1 cell type. Satisfactory preservation was achieved by perfusion with 2.5% glutaraldehyde in 0.1 M Na cacodylate containing 0.25 mM CaCl2 and 0.5 mM MgCl2, and postfixation with 1% OsO4 in 0.1 M s-collidine containing 0.5 mM CaCl2 and 1.0 mM MgCl2. Good preservation was also obtained using Na phosphate during prefixation and postfixation. Other combinations of buffers led to shrinkage, dilation of rough endoplasmic reticulum cisternae, disruption of membranes or loss of matrix and secretory granules. The results demonstrate that the variants of parathyroid chief cells, multinucleated syncytial cells and atrophic cells arise during fixation.