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Hyperproliferative Hepatocellular Alterations after Intraportal Transplantation of Thyroid Follicles

2000; Elsevier BV; Volume: 156; Issue: 1 Linguagem: Inglês

10.1016/s0002-9440(10)64710-4

1525-2191

Frank Dombrowski, Luisa Klotz, Hans Jörg Hacker, Yànhuá Lǐ, Dietrich Klingmüller, Klaudia Brix, Volker Herzog, Peter Bannasch,

Liver Disease Diagnosis and Treatment

The thyroid hormone 3,5,3′-triiodo-l-thyronine (T3) is a strong direct hepatocyte mitogen in vivo. The effects of T3 resemble those of peroxisome proliferators, which are known to induce hepatocellular tumors in rats. With the aim of studying long-term local effects of thyroid hormones on liver parenchyma, small pieces of thyroid tissue were transplanted via the portal veins into the livers of thyroidectomized male Lewis rats. At 1 week, 3 weeks, 3 months, and 18 months after transplantation, the transplants were found to proliferate, to synthesize thyroglobulin, and to release thyroxine and T3. At 3 and 18 months after transplantation, the hepatocytes of the liver acini downstream of the transplanted follicles showed an increase in cytoplasmic basophilia, a loss of glycogen, an enlargement and hyperchromasia of their nuclei, and a strong increase in cell turnover compared with unaltered liver acini. The altered hepatocytes exhibited an increase in the activities of glucose-6-phosphate dehydrogenase, glucose-6-phosphatase, malic enzyme, mitochondrial glycerol-3-phosphate dehydrogenase, cytochrome-c-oxidase, and acid phosphatase; the activities of glycogen synthase and glycogen phosphorylase were strongly decreased. The hepatocytic alterations downstream of the transplanted follicles could be explained by effects of T3. On the other hand, they resembled alterations characteristic of amphophilic preneoplastic liver foci observed in different models of hepatocarcinogenesis. The thyroid hormone 3,5,3′-triiodo-l-thyronine (T3) is a strong direct hepatocyte mitogen in vivo. The effects of T3 resemble those of peroxisome proliferators, which are known to induce hepatocellular tumors in rats. With the aim of studying long-term local effects of thyroid hormones on liver parenchyma, small pieces of thyroid tissue were transplanted via the portal veins into the livers of thyroidectomized male Lewis rats. At 1 week, 3 weeks, 3 months, and 18 months after transplantation, the transplants were found to proliferate, to synthesize thyroglobulin, and to release thyroxine and T3. At 3 and 18 months after transplantation, the hepatocytes of the liver acini downstream of the transplanted follicles showed an increase in cytoplasmic basophilia, a loss of glycogen, an enlargement and hyperchromasia of their nuclei, and a strong increase in cell turnover compared with unaltered liver acini. The altered hepatocytes exhibited an increase in the activities of glucose-6-phosphate dehydrogenase, glucose-6-phosphatase, malic enzyme, mitochondrial glycerol-3-phosphate dehydrogenase, cytochrome-c-oxidase, and acid phosphatase; the activities of glycogen synthase and glycogen phosphorylase were strongly decreased. The hepatocytic alterations downstream of the transplanted follicles could be explained by effects of T3. On the other hand, they resembled alterations characteristic of amphophilic preneoplastic liver foci observed in different models of hepatocarcinogenesis. Although T3 has been shown to be a strong mitogen on hepatocytes in vivo,1Francavilla A Carr BI Azzarone A Polimeno L Wang Z van Thiel DH Subbotin V Prelich JG Starzl TE Hepatocyte proliferation and gene expression induced by triiodothyronine in vivo and in vitro.Hepatology. 1994; 20: 1237-1241PubMed Google Scholar, 2Shinozuka H Stimulation of liver cell growth by direct mitogens.in: Bannasch P Kanduc D Papa S Tager JM Cell Growth and Oncogenesis. Birkhäuser, Basel1998: 213-225Crossref Google Scholar little is known about long-term effects of thyroid hormones on liver parenchyma.3Bayraktar M Van Thiel DH Abnormalities in measures of liver function and injury in thyroid disease.Hepato-Gastroenterol. 1997; 44: 1614-1618PubMed Google Scholar The idea to transplant thyroid follicles into the liver of thyroidectomized rats was born from a similar transplantation model, in which a long-term increase in locally secreted pancreatic islet hormones (especially insulin) induced alterations of liver acini downstream of intrahepatically transplanted islets of Langerhans in diabetic rats.4Dombrowski F Lehringer-Polzin M Pfeifer U Hyper-proliferative liver acini after intraportal islet transplantation in streptozotocin-induced diabetic rats.Lab Invest. 1994; 71: 688-699PubMed Google Scholar The hepatocellular alterations were in many respects in line with insulin effects and simultaneously similar to those observed in glycogen-storing foci, which are well known from different models of hepatocarcinogenesis.5Dombrowski F Filsinger E Bannasch P Pfeifer U Altered liver acini induced in diabetic rats by portal vein islet isografts resemble preneoplastic hepatic foci in their enzymic pattern.Am J Pathol. 1996; 148: 1249-1256PubMed Google Scholar The glycogen-storing liver acini downstream of the transplanted islets proceeded to hepatocellular neoplasms as shown in a long-term study.6Dombrowski F Bannasch P Pfeifer U Hepatocellular neoplasms induced by low-number pancreatic islet transplants in streptozotocin diabetic rats.Am J Pathol. 1997; 150: 1071-1087PubMed Google Scholar One type of preneoplastic liver focus, namely the amphophilic cell focus, which is well known to occur in different models of hepatocarcinogenesis in different species7Bannasch P Pathogenesis of hepatocellular carcinoma: sequential cellular, molecular and metabolic changes.Prog Liver Dis. 1996; 14: 161-197PubMed Google Scholar, 8Goodman DG Marenpott RR Newberne PM Popp JA Squire RA Proliferative and selected other lesions in the liver of rats.in: Streett CS Burek JD Hardisty JF Garner FM Leiminger JR Pletcher JM Moch RW Guides for Toxicologic Pathology, ch. GI-5. Society of Toxicologic Pathologists/American Registry of Pathology/Armed Forces Institute of Pathology, Washington1994: 1-24Google Scholar and which is one of the most often observed types of focal lesion in human liver cirrhosis,9Su Q Benner A Hofmann WJ Otto G Pichlmayr R Bannasch P Human hepatic preneoplasia: phenotypes and proliferation kinetics of foci and nodules of altered hepatocytes and their relationship to liver cell dysplasia.Virchows Arch. 1997; 431: 391-406Crossref PubMed Scopus (80) Google Scholar was not induced by the islet transplants.6Dombrowski F Bannasch P Pfeifer U Hepatocellular neoplasms induced by low-number pancreatic islet transplants in streptozotocin diabetic rats.Am J Pathol. 1997; 150: 1071-1087PubMed Google Scholar This type of focus of altered hepatocytes is characterized by amphophilic (basophilic and eosinophilic) cytoplasm due to a proliferation of mitochondria, which are closely associated with rough endoplasmic reticulum, and a strong decrease in glycogen.7Bannasch P Pathogenesis of hepatocellular carcinoma: sequential cellular, molecular and metabolic changes.Prog Liver Dis. 1996; 14: 161-197PubMed Google Scholar Alterations of enzyme activities in this type of preneoplastic liver focus show an energy-wasting metabolism and have been suggested to reflect thyromimetic effects of the responsible oncogenic agents.10Bannasch P Klimek F Mayer D Early bioenergetic changes in hepatocarcinogenesis: preneoplastic phenotypes mimic responses to insulin and thyroid hormone.J Bioenerg Biomembr. 1997; 29: 303-313Crossref PubMed Scopus (70) Google Scholar, 11Mayer D Metzger C Leonetti P Beier K Benner A Bannasch P Differential expression of key enzymes of energy metabolism in preneoplastic and neoplastic rat liver lesions induced by N-nitrosomorpholin and dehydroepiandrosterone.Int J Cancer. 1998; 79: 232-240Crossref PubMed Scopus (33) Google Scholar We thus decided to investigate whether intrahepatic thyroid follicle transplants are able to induce amphophilic cell foci in thyroidectomized rats. Male Lewis rats (inbred strain) weighing about 200 g were used. In the main group (MG), the animals were thyroidectomized; 2 weeks later thyroid tissue pieces were transplanted via the portal vein into the right part of the liver. The animals of control group 1 (CG1. were not thyroidectomized; thyroid tissue pieces were transplanted via the portal vein into the right part of the liver. Control group 2 animals (CG2) were thyroidectomized but did not receive a transplant. The thyroidectomy was done under an anesthesia with diethylether. Housing and treatment of the animals were in line with the guidelines of the Society for Laboratory Animals Service (GV-Solas) and the strict German animal protection law. In preliminary experiments, it was found that thyroid follicles or small pieces of thyroid tissue dissected by razor blades were too fragile for an effective transplantation. The rigidity of intrafollicular thyroglobulin was suspected to be the main problem. Therefore a pretreatment of donor animals was necessary to get follicles almost depleted of thyroglobulin. Donor animals received an iodine-poor diet (<0.04 μg iodine/g) with 0.05% propylthiouracil and tap water for 10 days, followed by an iodine-poor diet and H2O containing 1% KClO4 for 2 days; and finally an iodine-poor diet and H2O for 3 days. The following day the donor animals were killed, and the thyroid gland was prepared. The pretreatment resulted in an about fourfold enlargement of the gland with almost empty follicular lumina. The glands were washed with Hanks' solution (pH 7.2) and dissected with razor blades into small cubes of about 0.2 mm3. One gland was used for two recipient animals. The small cubes were dispersed in ice-cold Hanks' solution (pH 7.2) with 1% Dextran (Mr 35,000–50,000; Merck, Darmstadt, Germany) and were aspirated with 500 μl of this mixture into a syringe with a needle of 23G × 1″. The addition of dextran reduced the attachment of the tissue cubes to each other and to the wall of the syringe. The recipient animals were anesthetized with diethyl ether, and the thyroid tissue pieces were transplanted only into the right part of the liver as follows. The branch of the portal vein that supplies the left lobe and the left part of the middle lobe was occluded by a vessel clamp. After infusion of the thyroid transplants into the portal vein, the clamp was removed (maximal time of ischemia, 1 minute). With this procedure it was possible to infuse the thyroid tissue pieces only into the right part of the liver, ie, the right lobe, the caudal lobes, and the right part of the middle lobe (the border between the right part and the left part of the middle lobe is marked by the falciform ligament). Thus, the left part of the middle lobe and the left lobe could be taken as an internal control in the experiments of the MG and the CG1. At 1 week, 3 weeks, and 3 months after transplantation, six animals of each experimental group were killed between 10:00 a.m. and 11:30 a.m. An additional three animals of the MG were killed 18 months after transplantation. Additional MG animals were killed at days 3 (three animals) and 4 (three animals) without 5-bromo-2′-desoxyuridine (BrdU. application and without serum sampling (see below). Two hours before killing, another MG animal at 2 months after transplantation received 200 μCi 125I intravenously, and the intrahepatic thyroid transplants were prepared for autoradiography. Seven days before killing, half of the animals to be killed at 1 week, 3 weeks, and 3 months and all three animals to be killed at 18 months were anesthetized, and osmotic pumps (Alzet model 2ML1, Alza Corp., Palo Alto, CA) filled with 40 mg of BrdU (Sigma, Heidelberg, Germany) were surgically implanted subcutaneously over the dorsal thoracolumbal area. These pumps continuously delivered BrdU until the animals were sacrificed. The remaining animals to be killed at 1 week, 3 weeks, and 3 months received a single dose of 50 mg BrdU/kg body weight intraperitoneally 1 hour before sacrifice. Slices from the right part and from the left part of the middle lobe of the liver were snap-frozen and were used for enzyme histochemistry (see below). After removing the middle lobe of the liver, the animals were perfused with a mixture of 0.5% glutaraldehyde and 3% formaldehyde as described earlier.4Dombrowski F Lehringer-Polzin M Pfeifer U Hyper-proliferative liver acini after intraportal islet transplantation in streptozotocin-induced diabetic rats.Lab Invest. 1994; 71: 688-699PubMed Google Scholar Immediately after perfusion, about 40 slices per animal were cut from the fixed liver lobes. These slices were transferred into phosphate-buffered saline (PBS) and were systematically examined with a stereomicroscope. With some experience it was possible to identify transplanted thyroid tissue pieces in these unstained liver slices as well as after embedding (see Results). Corresponding slices of the same transplants were embedded in Epon and in paraffin. Additionally, pituitary gland, kidney, adrenal glands, lung, heart, spleen, and pancreas were embedded in paraffin. Four horizontal sections of the upper trachea and larynx together with surrounding tissue were embedded in paraffin to check the area of thyroidectomy (completeness of thyroidectomy) of the MG and the CG2 or the thyroid gland of CG1. From the paraffin-embedded specimen, serial sections of 2 to 3 μm in thickness were stained with hematoxylin and eosin (H&E) and with the periodic acid-Schiff reaction (PAS). Additional sections were used for immunohistochemistry. In addition, 10 small cubes (1 mm3) were cut from each liver and were embedded in Epon. Semithin sections of the Epon-embedded specimens were stained by the method of Richardson et al.13Richardson KC Jarett L Finke EH Embedding in epoxy resins for ultrathin sectioning in electron microscopy.Stain Technol. 1960; 35: 313-325Crossref PubMed Scopus (2426) Google Scholar Thin sections for electron microscopy were stained with uranyl acetate and lead citrate and were examined with a Phillips CM10 electron microscope (Einthoven, The Netherlands). After examination of the H&E and PAS stains, appropriate sections were selected and the corresponding sections were processed for immunohistochemistry. Immunostains of the liver for BrdU (monoclonal primary antibody from DAKO, Hamburg, Germany; dilution 1:100) and transforming growth factor-α (monoclonal primary antibody from Oncogene Science, Cambridge, MA; final antibody concentration 10 μg/ml) with antigen retrieval steps were performed as described earlier.4Dombrowski F Lehringer-Polzin M Pfeifer U Hyper-proliferative liver acini after intraportal islet transplantation in streptozotocin-induced diabetic rats.Lab Invest. 1994; 71: 688-699PubMed Google Scholar, 6Dombrowski F Bannasch P Pfeifer U Hepatocellular neoplasms induced by low-number pancreatic islet transplants in streptozotocin diabetic rats.Am J Pathol. 1997; 150: 1071-1087PubMed Google Scholar Glutathion S-transferase placental form (GST-P; polyclonal primary antibody from Biogenex, San Ramon, CA. dilution 1:100) was analyzed without antigen retrieval steps, using the LSAB+-Kit (DAKO) and the DAB+-Kit (DAKO). Immunostains of the hypophysis for thyroid-stimulating hormone (TSH) were done with a monoclonal antibody from DAKO (dilution 1:50) using the LSAB+ and the DAB+ Kits. Sections were counterstained with hematoxylin, dehydrated, and coverslipped with Pertex (Medite, Burgdorf, Germany). Anti-T3 and anti-T4 (polyclonal primary antibodies from ICN Biomedicals, Eschwege, Germany) were applied at a dilution of 1:250, anti-TG (polyclonal antibody, Institute of Cell Biology, University of Bonn, Bonn, Germany) and anti-calcitonin (monoclonal antibody from DAKO) were used at a dilution of 1:50. Secondary fluorescein isothiocyanate-conjugated goat anti-rabbit or goat anti-mouse antibodies (Sigma Chemical Co., Heidelberg, Germany) were used at a dilution of 1:50. In controls primary antibodies were omitted. H&E-stained paraffin sections were viewed systematically for apoptotic and mitotic cells at a magnification of ×400. Mitotic and apoptotic indices (MI and AI) were calculated as the number of mitotic figures and of apoptotic bodies, respectively, per 1000 hepatocytic nuclei. Paraffin sections immunostained for BrdU were examined at a magnification of ×400. BrdU labeling indices (BrdU-LI) were calculated as the number of BrdU-labeled nuclei per 1000 hepatocytic nuclei. 5000–15,000 hepatocytic nuclei were counted per animal. For the MG and the CG1, all indices were calculated separately for the liver acini downstream of the transplanted thyroid follicles in the right part of the liver, and in the left part of the liver which was free of transplants (intraindividual control). When no alterations were identifiable downstream of the transplants (MG at 1 week; CG1 at all times), the indices for the right part of the liver were calculated with the hepatocytes at a distance of about 1 mm from the transplants. For CG2 the indices were calculated only for the right lobe. The body weight, AI, MI, BrdU-LI at 1 hour, BrdU-LI at 7 days, serum T3, serum thyroxin (T4), and serum TSH of the different animal groups and the different times after thyroid follicle transplantation (Table 1) were compared with the Wilcoxon-Mann-Whitney test. Significance was accepted when P < 0.05.Table 1Effect of Thyroid Follicle Transplantation on Body Weight% Transplantation body weight at time of sacrificeAnimal group*MG, Main group (thyroidectomy and intrahepatic thyroid follicle transplantation); CG1, control group 1 (intrahepatic thyroid follicle transplantation and no thyroidectomy); CG2, control group 2 (thyroidectomy and no transplantation).1 week3 weeks3 months18 monthsMG101 ± 2 (6)101 ± 3 (6)†Mean was significantly different from CG1.137 ± 13 (6)221 ± 35 (3)CG199 ± 4 (6)123 ± 6 (6)145 ± 11 (6)—CG2104 ± 2 (6)107 ± 5 (6)111 ± 3 (6)†Mean was significantly different from CG1.—Body weight at the day of transplantation was set as 100% for MG and CG1. CG2 animals were matched without a transplantation. Mean values ± SEM are shown. The number of animals (n) is shown in parentheses.* MG, Main group (thyroidectomy and intrahepatic thyroid follicle transplantation); CG1, control group 1 (intrahepatic thyroid follicle transplantation and no thyroidectomy); CG2, control group 2 (thyroidectomy and no transplantation).† Mean was significantly different from CG1. Open table in a new tab Body weight at the day of transplantation was set as 100% for MG and CG1. CG2 animals were matched without a transplantation. Mean values ± SEM are shown. The number of animals (n) is shown in parentheses. Two hours before sacrifice, 200 μCi of 125I were injected into the tail vein of an animal from the MG at 2 months after transplantation. The liver tissue was fixed in 4% formaldehyde and embedded into paraffin. Autoradiography was done with 3-μm-thick paraffin sections and viewed with dark-field illumination at a Zeiss Axiophot microscope (Oberkochem, Germany). Serum samples were taken from aortal blood at sacrifice. Free T3 (fT3) and free T4 (fT4) were measured by an electrochemiluminescence immunoassay with an Elecsys analyzer (Boehringer Mannheim, Mannheim, Germany). TSH was measured with the rat TSH 125I assay system from Amersham (Amersham, Buckinghamshire, UK). Pieces from snap-frozen liver tissue of five rats were frozen onto the same tissue holder, and serial sections of all pieces were cut simultaneously in a cryostat (Jung, Nussloch, Germany).14Hacker HJ Grobholz R Klimek F Enzyme histochemistry and biochemical microanalysis of preneoplastic lesions.Progr Histochem Cyotchem. 1991; 23: 61-72Crossref PubMed Scopus (11) Google Scholar The sections were mounted onto the same slide or the same membrane and incubated by the respective histochemical reaction. With this technique it was possible not only to produce sections of the same thickness but also to treat them simultaneouslyunder identical conditions for the specific histochemical assays. The liver and the thyroid gland of one completely untreated rat were included as a normal control. The following enzymes were investigated: glycogen synthase (SYN), glycogen phosphorylase (PHO), glucose-6-phosphatase (G6Pase), glucose-6-phosphate dehydrogenase (G6PDH), pyruvate kinase (PK), succinate dehydrogenase (SDH), malic enzyme (ME), mitochondrial glycerol-3-phosphate dehydrogenase (mG3PDH), cytochrome c-oxidase (COX), acid phosphatase (AP), and γ-glutamyltransferase (GGT). Incubation conditions were essentially as previously described.11Mayer D Metzger C Leonetti P Beier K Benner A Bannasch P Differential expression of key enzymes of energy metabolism in preneoplastic and neoplastic rat liver lesions induced by N-nitrosomorpholin and dehydroepiandrosterone.Int J Cancer. 1998; 79: 232-240Crossref PubMed Scopus (33) Google Scholar, 15Lojda Z Grossau R Schiebler TH Enzyme histochemistry. Springer, Berlin/Heidelberg/New York1979Crossref Google Scholar, 16Hacker HJ Moore MA Mayer D Bannasch P Correlative histochemistry of some enzymes of carbohydrate metabolism in preneoplastic and neoplastic lesions in the rat liver.Carcinogenesis. 1982; 3: 1265-1272Crossref PubMed Scopus (154) Google Scholar, 17Bannasch P Hacker HJ Klimek F Mayer D Hepatocellular glycogenosis and related pattern of enzymatic changes during hepatocarcinogenesis.Adv Enzyme Regul. 1984; 22: 97-121Crossref PubMed Scopus (120) Google Scholar Furthermore, serial cryostat sections were stained for basophilia with toluidine blue, for the presence of neutral lipids with Fettrot B, and for the presence of glycogen with the PAS reaction. The intensities of the histochemical parameters in the serial sections were estimated semiquantitatively using five grades (no change, increase, strong increase, decrease, strong decrease) as compared with the reaction in the unaltered tissue of the same specimen (internal control). The body weight gains are shown in Table 1. At 1 week and 3 weeks after transplantation, MG and CG1 were not different from CG2, obviously because CG2 had no abdominal surgery. At 3 months, the weight gain of CG1 was higher compared with CG2. The histological examination of the heart, lung, kidney, adrenal gland, spleen, and pancreas did not reveal any unusual finding. The immunohistochemically TSH-positive cells of the hypophysis were larger in the thyroidectomized animals (MG and CG2) than in the nonthyroidectomized animals (CG1). As expected, the MG and CG2 animals had significantly decreased fT3 and fT4 and significantly increased TSH serum values compared with the CG1 animals (Table 2). It was surprising that no animal of the CG2 had completely negative fT3 or fT4 values. We made four-step sections at the anatomical site of the excised thyroid, but we did not find a thyroid remnant in any case. Between 3 and 18 months after transplantation, the fT3 and fT4 values of the MG increased, and the TSH decreased. At 18 months, the serum values (and the hypophyses. of the MG did not differ from the CG1 at 3 months.Table 2Effect of Thyroid Follicle Transplantation on SerumSerum ft3 and ft4 (pg/ml) and TSH (ng/ml) at time of sacrifice†Mean values ± SEM are shown. The number of animals (n) is shown in parentheses.Animal group*MG, Main group (thyroidectomy and intrahepatic thyroid follicle transplantation); CG1, control group 1 (intrahepatic thyroid follicle transplantation and no thyroidectomy); CG2, control group 2(thyroidectomy and no transplantation). and serum factor1 week3 weeks3 months18 monthsMGfT31.2 ± 0.2 (3)‡Significantly different from CG1 and not different from CG2.1.1 ± 0.2 (5)‡Significantly different from CG1 and not different from CG2.1.2 ± 0.2 (5)‡Significantly different from CG1 and not different from CG2.3.4 ± 0.3 (3)§Significantly different from all of the preceding times.¶Not different from data of CG1 at 3 months.fT43.9 ± 1.1 (3)‡Significantly different from CG1 and not different from CG2.4.1 ± 2.0 (5)‡Significantly different from CG1 and not different from CG2.5.9 ± 2.2 (5)‡Significantly different from CG1 and not different from CG2.20.7 ± 0.6 (3)§Significantly different from all of the preceding times.¶Not different from data of CG1 at 3 months.TSH58.7 ± 4.3 (3)‡Significantly different from CG1 and not different from CG2.61.3 ± 1.2 (5)‡Significantly different from CG1 and not different from CG2.62.1 ± 1.7 (5)‡Significantly different from CG1 and not different from CG2.9.3 ± 0.3 (3)§Significantly different from all of the preceding times.¶Not different from data of CG1 at 3 months.CG1fT33.2 ± 0.5 (3)∥Significantly different from CG2.3.2 ± 0.3 (5)∥Significantly different from CG2.3.4 ± 0.2 (5)∥Significantly different from CG2.—fT423.5 ± 2.7 (3)∥Significantly different from CG2.26.5 ± 2.6 (6)∥Significantly different from CG2.21.6 ± 4.1 (5)∥Significantly different from CG2.—TSH11.8 ± 1.4 (3)∥Significantly different from CG2.12.9 ± 1.1 (6)∥Significantly different from CG2.12.4 ± 1.6 (5)∥Significantly different from CG2.—CG2fT30.8 ± 0.1 (3)0.5 ± 0.3 (6)0.7 ± 0.0 (4)—fT40.8 ± 0.1 (3)2.4 ± 1.3 (5)2.8 ± 1.1 (4)—TSH63.1 ± 0.9 (3)62.0 ± 1.3 (6)61.1 ± 0.3 (4)—* MG, Main group (thyroidectomy and intrahepatic thyroid follicle transplantation); CG1, control group 1 (intrahepatic thyroid follicle transplantation and no thyroidectomy); CG2, control group 2(thyroidectomy and no transplantation).† Mean values ± SEM are shown. The number of animals (n) is shown in parentheses.‡ Significantly different from CG1 and not different from CG2.§ Significantly different from all of the preceding times.¶ Not different from data of CG1 at 3 months.∥ Significantly different from CG2. Open table in a new tab Stereomicroscopic examination of the unstained liver slices was a great help for finding the transplanted follicles for light and electron microscopical investigations (Figure 1, a and b). The transplants were vascularized (Figure 1, c and d) during the first 3 weeks, after which the follicles were surrounded by regularly fenestrated capillaries (Figure 2). In contrast, the transplants of the CG1 rats fibrosed during the first 3 weeks (Figure 1, e and f), probably due to low ("normal") TSH serum levels.Figure 2Three weeks after transplantation the thyroid follicle cells of the transplants of the MG showed regular organelles and microvilli. At this time the process of transplant vascularization was finished. The endothelial cell of the capillary, which is situated close to the thyroid follicle, shows regular fenestrations (arrowheads). The lumen of the follicle is at the top of the figure. Electron micrograph; original magnification, ×9500.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In the MG, the epithelial cells of the transplants formed typical follicles and showed a well-developed cytoplasm, regular organelles and microvilli at the electron microscopical level (Figure 2), and positive immunohistochemical stainings for TG, T4, or T3 (Figure 3, A–C) during the whole experiment. Only a few calcitonin-positive C cells were found by means of immunohistochemistry. In many specimens investigated electron microscopically, no C cell was present. The autoradiography after 125I application at 2 months after transplantation showed an iodine uptake by the intrahepatic thyroid follicles (Figure 4). The follicular epithelial cells of the MG exhibited a strong proliferative activity (Figure 5, a and b). A typical follicle transplant at 3 months is shown in Figure 6. At 18 months most of the transplants did not differ from those at 3 months, but single transplants grew up to 2 mm in size (Figure 7, a-c), and were then still showing mitotic figures.Figure 4This autoradiogram shows the regular uptake of 125I in the intrahepatic thyroid follicles of an animal of the MG 2 months after transplantation. Original magnification, ×250.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5The strong proliferative activity of the follicular epithelial and mesenchymal cells 3 weeks after transplantation is shown by the immunohistochemical localization of BrdU-labeled nuclei in a and b (left side in b). The first alteration of the hepatocytes within the acini downstream of the transplanted thyroid follicles was an increase in proliferative activity (b and c) 3 weeks after transplantation (compare with Table 3). c: The sharply defined border between a hyperproliferative acinus in the left part of the figure, which is under the influence of a transplant (not shown. and the nonaltered liver tissue in the right part of the figure. Immunostains for BrdU (administered via subcutaneously implanted osmotic pumps for 7 days); original magnifications, ×142 (a and b) and ×111 (c).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6PAS-stained section shows glycogen loss in the altered liver acini downstream of a thyroid transplant, which is situated in a portal triad, 3 months after transplantation (thyroid follicles are strongly PAS-positive in the center of the figure). The topography of the central veins (arrowheads) shows that the altered areas represent acini. Only animals of the MG exhibited these altered liver acini. Original magnification, ×140.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 718 months after thyroid follicle transplantation, single very large transplants were found, wh