Estimation of number of follicles, volume of colloid and inner follicular surface area in the thyroid gland of rats

Abstract

Volume is an important variable in assessing the growth and involution of the thyroid gland. The functional unit in the thyroid is the follicle, which consists of thyrocytes surrounding colloid. The size of a follicle depends on the number of cells and the amount of colloid. These are interchangeable and vary according to biological activity. Direct measurements of these variables provide information on structures involved in thyroid hormone synthesis, storage and secretion, and also on changes at the morphological and functional levels. Stereological methods are developed to obtain information on three-dimensional structures from two-dimensional sections and to achieve information on an entire organ by examining a minor part of it. Full-grown male Sprague-Dawley rats were used to develop a set of methods relying on unbiased stereological principles to determine the number of follicles, the total volume of colloid and the inner follicular surface area in the thyroid gland. The total volume of colloid was positively correlated (P < 0.021) with the number of follicles and the inner follicular surface area (P < 0.002) but not to the mean volume of colloid in each follicle. Thus under physiological conditions an increase in the total volume of colloid is associated with an increased number of follicles with a constant size distribution rather than a larger volume of colloid in each follicle. This implies that under physiological conditions there is equilibrium in the size distribution of the volume of colloid in each follicle.

Fig. 1

Sampling of sections. The thyroid gland was exhaustively sectioned and sections were sampled by systematic uniform random sampling (SURS). With a random start within the first 20 sections two consecutive sections were sampled. In practice this was done for each thyroid gland by randomly choosing a number between 1 and 20, i.e. 7. Then section numbers 7 and 8, 27 and 28, 47 and 48, etc., were sampled. The first sampled section in each pair is called the primary section and the subsequent section is called the reference section.

Fig. 2

All sampled primary sections were systematically investigated as depicted in the top of the figure with a random start on each section by the microscope with a computerized microscope stage. This systematic examination was repeated four times for each thyroid gland with a new grid applied to the table each time. The grids used in this study were a point grid (A), an unbiased counting frame (B), a grid of systematically distributed points and line segments (C), and a grid of points and direction lines (D).

Fig. 3

Estimation procedures. (A) Estimation of the volume of the thyroid gland and the volume of colloid by the Cavalieri principle. The point grid was applied to the primary section. All points that touched thyroid tissue and colloid, respectively, were counted. (B) Estimation of the number of follicles by the disector. The unbiased counting frame was applied to the primary section and the corresponding field of vision was found on the reference section. All follicles present on the primary section within the counting frame and not touching the exclusion line (the black line) were identified on the reference section. Follicles present on the primary section within the counting frame but not on the reference section were counted. On this section one follicle was counted (white arrow). (C) Estimation of the volume-weighted mean volume of colloid by the PSI methods. A grid with 24 points and four parallel direction lines was applied to the primary section. Follicles were sampled if touched by a point. For all sampled follicles the colloid distance between the intersections between the direction line and the borders of the colloid surface, l₀, was measured by a normal ruler on the direction line going through the points. Two examples are shown (black arrows). (D) Estimation of the inner follicular surface area. A grid of 24 points and line segments was applied to the primary section. All points that touched thyroid tissue (black arrow) and all intersections between the line segments and the inner follicular surface trace (white arrow) were counted.

Fig. 4

The linear correlation between the total volume of colloid and the number of follicles, Y = −453 + 15.961 × X; r = 0.879, P = 0.021 (A); the total volume of colloid and the number-weighed mean volume of colloid was not significantly correlated, Y = 6.49 × 10⁻⁵ − 1.88 × 10⁻⁷ × X, r = 0.0015, P = 0.978 (B); and the linear correlation between the total volume of colloid and the inner follicular surface area, Y = 48.16 + 69.5 × X, r = 0.99, P < 0.002 (C).