Age related changes in the parenchymal cells of human parathyroid gland.
Parathyroid glands have a significant place in the history of medicine, as they were the last major organ to be recognised in humans. [1,2] They were first identified by Richard Owen in 1850 in Indian Rhinoceros. [1,2] However, the detailed anatomy of parathyroid glands in humans were revealed to the world in 1880 by a Swedish Medical Student, Sandstrom. 
The parathyroid glands despite their small size, play a vital role in calcium homeostasis with the secretion of parathyroid hormone. Serum calcium plays many physiological functions including neuromuscular excitability, muscle contraction, blood coagulation and bone mineralisation.  Hence, the regulation of serum calcium level invites special attention. Chief cells synthesise and secrete PTH to correct or maintain normal blood calcium levels by sensing changes in serum calcium levels. 
The parathyroid glands are of great clinical importance as hyperparathyroidism in general is related with renal, bone and metabolic diseases. Renal calculi, nephropathy and osteoporosis are seen commonly in old age. Hence, it is worthwhile to take up a detailed microscopic study of human parathyroid glands in various age groups with special focus on parenchymal cells. As chief cells are the secretory cells, detailed study of their age related changes will be helpful in analysing the reason behind the higher occurrence of osteoporosis, nephropathy and other clinical conditions related with hyperparathyroidism with advancing age. Functional significance of oxyphil cell is still a controversy. Many researchers believed that they were degenerated forms of chief cells without specific functions. [5,6] The present study put forward an attempt to give some clarity regarding this matter. Available literature search showed a significant deficit regarding the age related studies of parathyroid gland, especially in Indian population.
The main objective of this work is to establish the quantitative and qualitative study of parenchymal cells of human parathyroid glands in different age groups and find out the relative proportion of these cells related to age.
MATERIALS AND METHODS
The present study was conducted in the Department of Anatomy, Govt. Medical College, Thiruvananthapuram; 71 specimens of human parathyroid glands were collected from the autopsies done in the Forensic Department with due regards on ethical ground. Specimens were taken from autopsies done before six hours following death to avoid autolytic changes. Crush injuries of neck region, death due to diseases of parathyroid and kidney were excluded from the study.
Persons of both sexes ranging from 3 to 93 years were divided into 8 age groups.
Superior and inferior parathyroid glands were dissected out from the posterior border of thyroid lobes near anastomotic connection between superior and inferior thyroid arteries and from the inferior pole of thyroid gland respectively. Dissected specimens were fixed in 10% formalin or Bouin's fluid and subjected to routine histological processing. The processed blocks were sectioned at 5 pm thickness and sections were stained with routine haematoxylin and eosin. Special staining methods were also used to highlight certain specific features. The general architecture of parathyroid gland was studied in detail by using binocular light microscope.
Quantitative assessment regarding number and diameter of chief cells and oxyphil cells and total number of cells were recorded. Cell countings were taken under oil immersion. A net micrometre with 10 x 10 mm square grid composed of 100 squares of 1 mm size was used for counting the cells. The number of cells in 100 squares (100 [mm.sup.2]) were counted and recorded. Five such different areas were randomly selected and the average value per 100 [mm.sup.2] area was calculated. The total number of each type of cells was calculated by multiplying the average number of cells in 100 [mm.sup.2] with number of 100 [mm.sup.2] areas in the whole gland. 
Diameter of chief cells and oxyphil cells were measured under 100x objective with a horizontal eyepiece micrometre called "graticule." The graticule was calibrated with a stage micrometre. The value of one eyepiece division is determined by calibrating it with the stage micrometre for every optical combination to be used. 
The data obtained from morphometric measurements of parathyroid gland components were subjected to Analysis of Variance (ANOVA). Regression analysis was done as the appropriate statistical method to observe the dependence of various parameters, such as number and diameter of chief cells and oxyphil cells with age. These data were plotted in regression graphs with appropriate equation.
Parathyroid glands were covered by a thin fibroelastic connective tissue capsule from which delicate septae were seen, dividing the glandular parenchyma into poorly defined lobules. Connective tissue invasion of the gland was sparse at the earlier age groups with a tendency to increase with age. Blood vessels and a few nerve fibres were seen embedded in the septae. Cords of parenchymal cells were also separated by connective tissue fibres.
Presence of fat cells in the stroma was a characteristic feature. Adipocytes were sparse in the stroma of children below seven years. They appeared late in the first decade of life and showed a steady increase until the age of 51 to 60 years. In the stroma, fat cells were arranged in groups or scattered in between the parenchymal cells.
Parenchyma showed two types of epithelial cells in adults, chief cells and oxyphil cells (Figure 1). Chief cells were the main parenchymal cell type seen in all age groups, arranged in cords or plates, channelled by capillaries and sinusoids. They were observed as polyhedral cells of 5--7 pm with poorly defined outline. They had large central vesicular nuclei almost filling the cell, leaving a thin rim of cytoplasm. Secretory granules were observed in the cytoplasm. Dark and light types of chief cells were also observed in all the age groups. Proportion of these cells varied in individuals, which showed no relation with the age. Water clear cells could be visualised in some sections other than the typical light and dark types of chief cells.
Oxyphil cells were seen as large polygonal cells about 9-12 [micro]m in size. Cytoplasm was strongly acidophilic and granular in nature with well-defined cell membrane. They had a central, circular, darkly stained nucleus, which was slightly smaller than that of chief cells. Special staining methods such as Masson's trichrome showed more clear pictures of oxyphils with abundant cytoplasmic granules. Transitional cells with features of both chief and oxyphil cells were also noticed. They were circular or ovoid in shape, larger than chief cells with cytoplasm showing uniform granules. More number of transitional cells were seen in the middle age group.
Chief cells were predominated among the cell population. Considering all the specimens, chief cells constituted about 94.9% and oxyphils 5.1% of the total cell population. Specimens collected from 3-6 years showed only chief cells. Cells were closely packed, arranged in sheets with little connective tissue stroma. In this study, typical microscopic architecture of parathyroid gland was observed from the age of seven years (Figure 2). At seven years, chief cells were arranged in anastomosing cords, separated by connective tissue septa. Vascular channels were seen in the invading septae. The size of the chief cells showed a gradual increase as age advanced. Number of chief cells also showed a significant increase until the third decade and a slight decline thereafter.
Appearance of oxyphil cell was noticed at the age of seven years. In the second age group, oxyphil cells were present but were few in numbers and arranged as single cells or pairs in between the chief cells. In third and fourth decades, their number increased and were seen in small groups. A significant increase in the number of oxyphil cells was noted after the age of 40 years and they were arranged in large groups or clumps (Figure 3).
In late ages, large nodules of oxyphil cells were observed. In a section of a 93-year-old man, 50% of the gland was occupied by oxyphil cells (Figure 4) in the form of well circumscribed and vascular nodules. Diameter of oxyphil cells also showed a tendency to increase with age. In general size and number of oxyphil cells showed a steady increase as age advanced.
Analysis of mean diameter of parenchyma cells showed significant difference between various age groups. Regarding chief cells, a high value of difference was obtained between first and second age groups and second and third age groups. Data were subjected to regression analysis with age. Coefficient of regression is found significant with a high [R.sup.2] value with appropriate regression equation. A steady rise was observed in the diameter of the chief cells up to fourth decade with not much change thereafter. Diameter of oxyphil cells showed a gradual increase from childhood to old age.
On analysis of number of chief cells, significant difference between first six age groups was noted. A high value of difference was observed between first and second and second and third age groups. On regression analysis significant coefficient of regression was obtained with a cubic equation. It was represented in a regression Graph 1, which shows a steady increase in the number of chief cells up to third decade with a slight decline thereafter until fifth decade. Since then there is not much change.
ANOVA showed significant difference in the number of oxyphil cells among different age groups. The value of difference was very high between third and fourth group as well as seventh and eighth group. Regression analysis showed significant increase in the number of oxyphils with respect to age. Coefficient of regression was found significant with a high [R.sup.2] value (0.8005) and a regression Graph 2 is plotted accordingly. Number of oxyphils showed a very high positive correlation with age.
Since 1880, when Sandstrom revealed the detailed anatomy and histology of parathyroid glands. [2,3] It has been clearly known that two types of epithelial cells of parenchyma are the chief cells and oxyphil cells. [5,7,9,10] In the present study, these parenchymal cells were found to be arranged in irregular anastomosing cords or sheets adjacent to vascular channels. As chief cells, they are secretory, they predominate in the total cell population. [1,11] In infants' early childhood chief cells were the only type of cells seen and were arranged in closely packed sheets with little connective tissue stroma channelled by capillaries as described by Gilmour.  Castleman and Mallory  described chief cells as poorly outlined polyhedral cells with a large round centrally placed nucleus. The cells measure 6-8 pm with scanty cytoplasm. Chief cell showed similar characteristics in our study also. Diameter of chief cells was detected as 5-7 pm from second decade of life.
Light and dark type of chief cells were described in previous works, [7,10,12] Munger and Roth  described that the human chief cells differ according to the level of their activity as inactive light cells and active dark cells. In the present study, light and dark types of chief cells were observed in all age groups. The proportion of these cells varied in individuals of same age group and showed no relation with age. Other Researchers [13,14] have reported that the chief cells undergo morphologic changes corresponding to different levels of secretory cycles in relation to serum calcium level. This might be the reason for the observed variations in our study with regard to the number of light and dark chief cells in different individuals of same age group.
Water clear cells, a variant of chief cell with vacuolated cytoplasm were occasionally seen (less than 1%). Castleman and Roth  observed that they might be pathological or artifacts. Huaye Chen  also pointed the presence of water clear cells in parathyroid hyperplasia or adenoma. In the present study, the number of chief cells showed a rapid increase from first to second and second to third decades. Maximum increase was observed in third decade followed by a gradual decrease until fifth decade and remained so thereafter. These finding agree well with Rother,  who noticed a rapid rise of chief cells in the second and third decades and decreases slowly until old age.
Oxyphil cells are the second type of parenchymal cells in parathyroid glands of certain animal groups and humans. [1,13] In the present study, oxyphil cells were seen to appear from the age of seven years. This is definitely earlier than the time of appearance noticed by other Researchers. [5,9,16] They were seen as polygonal cells 9-12 pm in diameter with strongly acidophilic and granular cytoplasm, well-defined margins and centrally located round nucleus. These findings are in conformity with previous works. [15,17] Cytoplasmic granules could be visualised with routine as well as special stains. Huaye Chen  pointed that they are tightly packed mitochondria in his ultra-structural studies.
In younger age groups oxyphil cells were few in number, arranged singly or in pairs between chief cells. In third and fourth decades, their number increased and seen as small groups. They significantly increased after 40 years and arranged in large groups and clumps. Large nodules of oxyphil cells were observed in late ages, occupying greater portions of the gland. These nodules appeared as well circumscribed and vascular. These findings are similar to that of Castleman and Roth. 
The number of oxyphil cells showed a gradual increase up to 40 years (0.1%--2.8%) and a rapid rise thereafter (6% 13.64%). Christi  noticed that they make up to 1.8% of the total population before 40 years and up to 9.05% after 40 years. Rother  detected their % to be only 1.5. Diameter of oxyphil cells gradually increased from childhood to old age. Statistical analysis showed a positive correlation between diameter and age as stated by Rother. 
Transitional cells with features of both chief and oxyphil cells were detected in all age groups in this study as reported in previous works. [15,17] The presence of transitional cells with features intermediate between chief and oxyphil cells supports the view of transformation of chief cells to oxyphil cells with aging. In this study number of oxyphil cells was seen to increase with age, while chief cells showed significant decrease in advancing age. This above finding that is increase in oxyphil cells with a corresponding decrease in chief cells is in favour of the postulate of transformation of chief cells to oxyphil cells in course of life.
Even though majority of Researchers [1,5,16,17,18] agree with this view, difference of opinion are there regarding the function of oxyphil cells. Roth and Trembley stated that they are degenerate forms of chief cells without specific function. Huaye Chen and Christi contradict this by pointing evidence of activity in oxyphil cells. The presence of numerous mitochondria in the cytoplasm of oxyphil cells suggests a need of energy production.  According to Christi,  oxyphil cells show histochemical reactions for phospholipids and various dehydrogenases.
Huaye Chen  also observed higher enzyme activity in oxyphil cells. In his immunostaining studies oxyphil cells show the potential to produce PTH, PTH related protein and calcitriol. Moreover, oxyphil cells express parathyroid relevant genes found in chief cells suggesting they are not simple degenerated chief cells. CaR and Vit. D receptor are highly expressed in oxyphil cells. Christi  contradicted the postulate of degeneration with the following observations that oxyphil nodules are highly vascular and cells rarely show evidence of degeneration such as vacuolation. The present study has limitations regarding detection of the functional status of oxyphil cells. But the presence of highly granular cytoplasm and the vascularity in the nodules of oxyphil cells observed in old age are in favour of activity of oxyphil cells. These findings lead to a conclusion that they might be derived from chief cells and continuing their function to maintain the calcium homeostasis in advanced age.
Chief cells show slight decline in number with advancing age. This definitely leads to a possibility of decrease in serum calcium level. Old age diseases related with hypocalcaemia, such as osteoporosis are more common. This itself indicates the possibility of function of oxyphil cells in old age like chief cells to produce parathyroid hormone.
The present work provides an opportunity to define normal histological variations in parenchymal cells of human parathyroid gland related to age. Chief cells were of constant in occurrence, whereas oxyphil cells appeared from the age of 7 years. Number of chief cells increased up to third decade with a slight decline thereafter. Number and diameter of oxyphil cell showed a positive correlation with age. A rapid increase in the number of oxyphil cells was observed after 40 years. They showed a tendency to form larger groups and nodules in old age. Analysis of relative proportion of parenchymal cells in different age groups supports the postulate of transformation of chief cells to oxyphil cells in the course of life. This study will serve as a basic guideline for future clinical studies related with its endocrine functions.
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Mini A (1), Manju S (2)
(1) Associate Professor, Department of Anatomy, Government Medical College, Paripaliy, Koiiam, Kerala.
(2) Associate Professor, Department of Anatomy, Government TD Medical College, Aiappuzha, Keraia.
Financial or Other, Competing Interest: None.
Submission 04-01-2017, Peer Review 29-01-2017, Acceptance 03-02-2017, Published 13-02-2017.
Corresponding Author: Dr. Mini A, Associate Professor, Department of Anatomy, Governmen t Medical College, Paripally, Kollam, Kerala.
Caption: Figure 1. Normal Architecture of Parathyroid Gland in a 25-year-old Male x 100. Cords of Chief Ceil Separated by Fat Cells and Connective Tissue Septae. Note Blood Vessel (BV), Sinusoid (SN) and Oxyphil Cells (Red Arrow)
Caption: Figure 2. Early appearance of Oxyphil Cell (Golden Arrow) in a 7-year-old Child
Caption: Figure 3. Section taken from 56-year-old (M). Masson's Trichrome Staining x 400. Note Connective Tissue Septae (Deep Blue) and Oxyphil Cells (Deep Pink)
Caption: Figure 4. Section of a 93-year-old (M) x 400 showing Large Oxyphil Nodules with Vascular Channels. Chief Cells significantly Reduced
Caption: Graph 1. Age Wise Distribution Number of Chief Cells
Caption: Graph 2. Age Wise Distribution Number of Oxyphil Ceils
Table 1 Sl. Age No. of No. Group Specimens 1 1-10 Yrs. 05 2 11-20 Yrs. 10 3 21-30 Yrs. 10 4 31-40 Yrs. 09 5 41-50 Yrs. 10 6 51-60 Yrs. 11 7 61-70 Yrs. 08 8 70 and Above 08 Total 71 Table 2. Mean Diameter of Parenchymal Cells in Different Age Groups Age Diameter of Diameter of Group Chief Cell ([micro]m) Oxyphil ([micro]m) 0-10 Years 3.644 [+ or -] 1.078 2.25 [+ or -] 3.38 11-20 Years 5.721 [+ or -] 0.297 9.65 [+ or -] 0.906 21-30 Years 6.410 [+ or -] 0.358 10.461 [+ or -] 0.478 31-40 Years 6.520 [+ or -] 0.3 10.573 [+ or -] 0.34 41-50 Years 6.212 [+ or -] 0.389 10.124 [+ or -] 1.1102 51-60 Years 5.912 [+ or -] 0.429 10.798 [+ or -] 0.605 61-70 Years 6.646 [+ or -] 0.407 11.305 [+ or -] 1.068 70 Above 6.478 [+ or -] 0.309 11.221 [+ or -] 0.618 CD (.05) 0.466 1.27 Table 3. Mean Number of Parenchymal Cells in Different Age Groups Age Group Number of Number of Chief Cells Oxyphil Cells 0-10 Years 3081 [+ or -] 1778 2.33 [+ or -] 4.64 11-20 Years 7218 [+ or -] 1422 68.4 [+ or -] 39.53 21-30 Years 11290 [+ or -] 1592 177.2 [+ or -] 37.68 31-40 Years 10405 [+ or -] 1104 291.6 [+ or -] 106.6 41-50 Years 8804 [+ or -] 613 660.8 [+ or -] 160.9 51-60 Years 7747 [+ or -] 659 680.3 [+ or -] 172 61-70 Years 7070 [+ or -] 376 637.7 [+ or -] 136.1 70 Above 6099 [+ or -] 1056 1063.6 [+ or -] 369.7 CD (.05) 1087.8 148.8 Age Group Total Number of Cells 0-10 Years 3083 [+ or -] 1781 11-20 Years 7286 [+ or -] 1448 21-30 Years 11468 [+ or -] 1594 31-40 Years 10696 [+ or -] 1118 41-50 Years 9368 [+ or -] 557 51-60 Years 8427 [+ or -] 604 61-70 Years 7732 [+ or -] 374 70 Above 7062 [+ or -] 550 CD (.05) 1052.7
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|Title Annotation:||Original Research Article|
|Author:||Mini, A.; Manju, S.|
|Publication:||Journal of Evolution of Medical and Dental Sciences|
|Date:||Feb 13, 2017|
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