Experimental Study On The Effects Of Iodine Excess On Morphology, Function, And Autoimmune Mechanism Of Thyroid In Iodine-deficiency NOD.H-2h4 Mice

Iodine, the essential trace element for synthesis of thyroid hormones, play an important role in maintaining normal growth and development of human body. The defect in iodine intake will result in severe damage in brain development and other harmful consequences, which are named as iodine deficiency disorders (IDD). Universal salt iodization(USI) recommended by authoritative organizations was aimed to eliminate IDD in global world. The implement of USI has improved the condition of IDD effectively, however, bring about the new problem——iodine excess. In 2001, WHO and international council of control iodine deficiency disorders (ICCIDD) put out the concrete standard level for ideal iodine intake state according to the medial urine iodine 100μg/L-199μg/L, which is named as adequate iodine. MUI 200μg/L-300μg/L is considered as more than adequate iodine, and more than 300μg/L as iodine excess. A five-year prospective epidemiological study was conducted by our group in order to elucidate the relationship between iodine excess and thyroid diseases. Our study have shown that the frequency of clinical and sub-clinical hypothyroidism enhanced with the increased iodine intake in population with iodine excess or more than iodine intake, which caused mainly by autoimmune thyroiditis. The predominant susceptible people to iodine excess were those lived in iodine excess area and those with genetically autoimmune background.Having finishing the studies on Wistar rats, we will step further into the field of susceptible autoimmune animal, which can enrich and complete our series of experimental research work about the subject of iodine excess and thyroid disease. NOD.H-2h4 mice, an good animal model of spontaneous autoimmune thyroiditis, were employed as our study objects in order to investigate the effects and its mechanism of iodine excess supplement on thyroid of susceptible people in iodine deficiency area. Firstly, the iodine-deficiency mouse model were fed with the fodder we manufactured by ourselves in professional factory to simulate the iodine nutrition state of susceptible people in the iodine-deficiency area. Secondly, 5 fold, 10 fold, 100 fold, 1000 fold iodine (equal to 5, 10, 100, 1000 fold of normal daily iodine intake respectively) were dissolved in their drinking water respectively. The corresponding changes incurred in morphology, ultrastructure, function, and autoimmunity in thyroids of NOD.H-2h4 mice were observed and the relationship between the changes and iodine supplement concentration or persisting time were studied, so that we can clarify the pathogenesis of iodine-induced clinic hypothyroidism of susceptible people in iodine-deficiency area, and afford the scientific evidence for their iodine supplement.Methods1. Group and treatment of animals(1) preparation of iodine-deficiency NOD.H-2h4 mice modelâ‘ 20 Wistar rats aged 5-week old were provided by laboratory animal center of China Medical University, and divided into 2 groups randomly, male and female were equal in each group. They were fed with iodine normal adequate sterile fodder and low-iodine sterile fodder respectively. Sterile water almost containing no iodine was afforded for them. All rats were sacrificed after 4 months.â‘¡20 NOD.H-2h4 mice aged 5-week old were conducted the same method as above-mentioned in specific pathogen free (SPF) laboratory of the center.(2) treatment of iodine supplementâ‘ Iodine supplement animals: 120 iodine-deficiency NOD.H-2h4 mice were divided randomly into 4 groups, male and female were equal in each group. Their drinking water with iodine concentrations were 2μg/mL (5 fold), 4μg/mL(10 fold), 40μg/mL(100 fold), 400μg/mL(1000 fold) respectively. 10 mice of each group were sacrificed at 1, 2 and 4 months after treatment.â‘¡Iodine-deficieny animal: 30 iodine-deficiency NOD.H-2h4 mice (male and female were equal) were continually fed with low iodine fodder. 10 mice were killed at 1, 2 and 4 months after treatment, respectively.â‘¢Non-iodine-deficiency animal: 30 NOD.H-2h4 mice that had been fed with normal fodder for 4 months from 5 week (male and female were equal), were continually fed with normal stuff. 10 mice were killed at 1, 2 and 4 months after treatment, respectively.2. Animal sample collection and preparation(1) Wistar ratsâ‘ Urine: Rats were put in metabolize cage for one day with free access to food and water in order to collect urine for 24 hours. Urine were then stored in refrigeration at -20℃.â‘¡Serum: Rats were anaesthetized by 10% chloral hydrate injected into abdominal cavity. Whole blood was collected after cardiac puncture. Serum was separated from blood by centrifuging and stored at -70^C for hormone determination.â‘¢Thyroid: Thyroid were removed quickly after anesthesia, and fixed in 10% formalin for morphological observation.(2) NOD.H-2h4 miceâ‘ Serum: Same method as above-mentioned.â‘¡Thyroid: Anesthetized as above mentioned, but were fixed in 10% formalin or 2.5% glutaraldehyde for morphological and ultrastructural observation, respectively.3. Index determinationâ‘ Urinary iodine: AS(â…¢)-Ce⁴⁺ catalytic spectrophotometry method was used.â‘¡Morphological changes: Paraffin embedded section with Haematoxylin & Eosin staining observed with light microscope.â‘¢Ultrastructural changes: Observed by electric microscope.â‘£Thyroid function: ELISA was used to determine TT₄, TT₃ and TSH, which kits were provided by ADL company in U.S.A.⑤TgAb: Indirect ELISA was used.4. Statistics analysisSPSS13.0 software was used to analyze the data. Urinary iodine was expressed in median urine iodine, others in means + standard deviation. ANOVA was used to analyze the means among the different groups, and SNK was used in further comparison. Pearson line relation and Spearman relation were used in the reIationship analysis. P＜0.05 was considered as significance.Results1. Experimental study on effects of iodine-deficiency on morphology and function of thyroid in NOD.H-2h4 mice(1) MUI: MUI maintained stably in rats of control group. However, MUI in iodine-deficiency group progressively decreased with the past time, especially significantly lower than the control group at the time point 4 month.(2) Serum TT₄,TT3,TSH determination: TT4,TT3 levels in rats of iodine-deficiency group were significantly lower than those in control, and TSH level significantly higher than control. However, in NOD.H-2h4 mice, there were no significant difference in TT₄,TT₃,TSH levels between the iodine-deficiency group and control.(3) Morphological changes in thyroid: Wistar rat: The size of follicle was moderate in thyroid of control group, and epithelial cells were cubic. Rich colloid could be observed in follicle lumen. But, thyroid in iodine-deficiency group showed the sign of hyperplasic thyroid goiter: follicle lumens diminished or even lost, and colloid in lumen decreased dramatically. The epithelial cell grew higher, and proliferation of blood vessel and fibrosis were severe.NOD.H-2h4 mouse: No proliferation of blood vessel and fibrosis could be found in iodine-deficiency group. There were no evident differences between the two groups.2. Experimental study on the effects of iodine excess on morphology, function, and autoimmune mechanism of thyroid in iodine-deficiency NOD.H-2h4 mice(1) Morphological changes in thyroidâ‘ non-iodine-deficiency group: The size of follicle was moderate, and the epithelial cells were cubical. Rich colloid could be observed in follicle lumen. The frequency ofthyroiditis was 2/10, 0, 1/10 at iodine supplement lmonth, 2 month and 4 month, respectively. The severity of inflammation was very slight.â‘¡iodine-deficiency group: Changes in iodine-deficiency groups were not obvious, and only the lumen enlarged after iodine-deficiency condition lasted for 8 months. At this time point, frequency of thyroiditis was 1/10.â‘¢iodine supplement group: Evident changes could be found in these groups, which characterized by thyroiditis and goiter concurred in the gland. No fibrosis happened at all time points. The frequency and severity ofthyroiditis increased with time and iodine intake level, and so do the colloid deposition in lumen.(2) Ultrastructural changes in thyroidâ‘ non-iodine-deficiency group: The epithelial cell were cubical or short cylindrical, and the circular or elliptical nuclei lied in the center. The mitochondria were intact, with on sign of swelling. The developed rough endoplasmic reticulum(RER) were surrounded with plenty of ribosome. Lysosomes disffused in cells, and many microvilli could be found.â‘¡iodine-deficiency group: No evident changes displayed in iodine-deficiency group.â‘¢iodine supplement group: Obvious changes became more severe progressively with the increased time and iodine intake level, such as mitochondria swelling, RER dilating, secondary lysosomes increasing, microvilli reducing. Infiltration of lymphocytes increased in the same tendency.(3) Serum TT₄,TT₃,TSH determinationâ‘ TT₄: TT₄ remained stable levels in non-iodine-deficiency group and iodine-deficiency group, and no significant difference was found among them. TT₄ in iodine supplement groups were close to the level of two control groups, and there was no difference among them at 1 month, but declined at 2 month, especially the level in 1000 fold group was significantly lower than the controls. At 4 month TT₄ increased near the controls, and no significant differences was found among them.â‘¡TT₃: No significant difference was found among all the groups.â‘¢TSH: No significant difference was found among all the groups.(4) Serum TgAb determinationTgAb level maintained stable levels in non-iodine-deficiency group and iodine-deficiency group, and no significant difference was found among them. TgAb enhanced gradually in all iodine supplement groups with time or iodine intake level increasing. TgAb levels in 100 fold, 1000 fold iodine supplement groups at 2, 4 month were significantly higher than those in controls, especially the TgAb in 1000 fold at 4 month reach the peak level, which far more significantly higher than all other groups.Conclusions1. The study based on the subjects of Wistar rat and NOD.H-2h4 mouse was aim to explore the effects of iodine-deficiency on morphology and function of thyroid in these two kinds of animals(1) The iodine-deficient animal model has been successfully made in Wistar rats with our low iodine fodder in the study, which was characterized by overt hypothyroidism and diffuse hyperplasia goiter.(2) No evident morphological and functional changes of thyroid could be induced in NOD.H-2h4 mice in the same method for Wistar rats. The susceptibility to iodine deficiency of NOD.H-2h4 mice was less than Wistar rats.2. The study was to investigate the effects of iodine excess on morphology, function and autoimmunity of thyroid in iodine-deficiency NOD.H-2h4 mice.(1) Iodine excess induces thyroiditis in iodine-deficiency NOD.H-2h4 mice, the frequency and severity of thyroiditis correlate positively with the level and exposure period of iodine excess. The occurred time point of thyroiditis is associated with the amount of iodine ingestion.(2) Iodine excess induces morphological changes of thyroid in iodine-deficiency NOD.H-2h4 mice, which is coincided with thyroiditis and diffuse hyperplasia goiter.(3) Iodine excess could lead to destruction of thyroid follicles and ultrastructural damages of epithelial cells in iodine-deficiency NOD.H-2h4 mice.(4) Iodine excess could affect the TgAb level in iodine-deficiency NOD.H-2h4 mice. TgAb correlated positively with the level and exposure period of iodine excess.(5) Iodine excess could inhibit thyroid function in iodine-deficiency NOD.H-2h4 mice. Hypothyroidism may be induced with the long exposure period to iodine excess.