Cryopreserved Rat Thyroid Autotransplantation in the Treatment of Postoperative Hypothyroidism

Abstract

To verify the viability and functionality of cryopreserved thyroid autotransplantation in rats who underwent total thyroidectomy in the treatment of postoperative hypothyroidism. Thirty-two Wistar rats were randomly assigned into groups (G) with eight animals each: control (CG); simulation (SG); hypothyroidism (HTG) and transplanted (TG). At the beginning and in the 13th week of the experiment, serum levels of total T3, free T4, TSH and calcium were determined. In both the first and 14th weeks, scintigraphic examinations, 99m-Tc pertechnetate radioisotope biodistribution and histopathology were performed. In the 14th week, the expression of proliferating cell nuclear antigen (PCNA) and cellular apoptosis (caspase-3) were also evaluated. In the 13th week, the transplanted animals had normal serum levels of total T3 and free T4. TSH levels showed a tendency towards normality. In the 14th week, scintigraphic exams displayed graft isotopic uptake in all animals in the TG group. Histological examinations 13 weeks after transplantation showed the viability and functionality of thyroid follicles. PCNA revealed significant immunoreactivity of the graft (p < 0.001) when the TG was compared to the CG. There was no difference between CG and TG considering the expression of activated caspase-3. The experimental study confirmed the viability and functionality of thyroid autotransplantation implanted in skeletal muscle with evidence of cell proliferation without cellular apoptosis. This surgical strategy was effective in the treatment of postoperative hypothyroidism.

Keywords: autologous transplantation; cryopreservation; endocrinology; rats; thyroidectomy.

Figure 1

At both t = 0 and 13 weeks, biochemical tests (BT), including serum levels of total T3, free T4 and TSH, and total calcium, were performed for all groups (red box with arrow). In the HTG, the thyroid hormone and calcium serum levels were also determined one week after thyroidectomy (that is, in the 2nd week after the beginning of the experiment). Total thyroidectomy (TT) and cryopreservation (CRYO) of the thyroid gland occurred in the 1st week (blue box with arrow). In the 2nd week, the autologous graft (AG) was implanted in the TG (grey box with arrow). In both the t = 0 and 14th weeks, scintigraphic examination (99mTc) was accomplished (yellow box with arrow). In the 14th week, histological evaluation (H), immunohistochemistry analysis (IHC) and radioisotope biodistribution (RB) were performed (white box with arrow).

Figure 2

There was a decrease in total T3 serum level in the HTG compared with the results of the other groups (p < 0.001), which showed no significant differences among them. A significant reduction in the free T4 serum level was also observed in the HTG compared to the TG, CG and SG. An increase in TSH levels was found in both the HTG and TG, but with higher values in the former TG (p < 0.05). Horizontal bars represent medians, boxes represent the 25th and 75th percentiles, and vertical bars represent ranges. The level of significance was set at p < 0.05.

Figure 3

(A) Whole-body scan showing isotope capture of the radioisotope in the thyroid gland topography (arrow), epigastric region (X) and bladder (*) of the CG. (B) Absence of isotope uptake of the radiotracer in the cervical topography (arrow) of the HTG. (C) Isotope uptake of the technetium by the graft (surrounded by circles), confirming its viability 12 weeks after the implant in TG. In the small image of the rat (at right), it is also seen that there is an absence of isotope uptake in the cervical location of the TG.

Figure 4

Photomicrographs of the thyroid gland in CG, SG and TG in the 14th week. In the CG and SG, it was possible to observe intact thyroid follicles, consisting of a simple epithelial layer (#), whose content is filled with colloid (*). Images of the transplanted animals (TG) showed follicles containing numerous vesicles of endocytosis (+). In TG animals, the presence of muscle tissue was observed between follicular cells (&). (H&E, magnification of x200 and x400). Scale bars = 50 for (A) Scale bars= 10 µm for (B).

Figure 5

There was a statistically significant increase (p < 0.001) in the expression of PCNA in the TG compared to the CG (brown color marking). No differences were observed caspase-3 antibody immunoreactivity between both groups. Scale bars: 50 µm.