Effect Of Different Levels Of Iodine Intake On Brain Development In Rat Offspring

After successfully establishing animal models of iodine deficiency and iodine excess in Wistar rats, we try to explore the relationship of the iodine metabolism, thyroid function and its morphology between maternal rats and their offspring with different iodine intake. And we measured the activity of the type 2 iodothyronine deiodinase (D2) for evaluation of thyroid hormone level in brain tissue of rat pups. Furthermore, we tested mRNA and protein expression of brain development marker e.g neuron-specific enolase(NSE), growth associated protein-43(GAP-43), myelin basic protein(MBP) and synapsin Ⅰ, in order to study the effect of iodine deficiency and iodine excess on brain development.Wistar rats were randomly divided into six groups according to body weight and sex, i.e, ①low iodine(LI);②normal iodine (NI);③five-fold high iodine(5HI);④ ten-fold high iodine(10HI);⑤fifty-fold high iodine(50HI);⑥one hundred-fold high iodine(100HI). LI rats were fed with low iodine diet derived from an endemic goiter area (iodine content in diet is 20-40μg/kg) and drank deionized water;rats in other groups were fed with normal feedstuff (average iodine content is 300μg/kg).Rats in NI group drank tap water, while rats in high iodine groups drank tap water containing different doses of potassium iodate. The total iodine intake per day in diet of 6 groups was 0.6μg/d(LI), 6.15μg/d(NI), 30.75μg/d(5HI), 61.5μg/d(10HI), 307.5μg/d(50HI), 615μg/d(100HI) respectively. Wistar rats were fed for 3 months and then mated randomly. The lactating mothers and their 14 and 28 days old offspring were studied in our study. Some techniques such as ion exchange chromatography, RT-PCR techniques, immunohistochemistry techniques, and etc were used in our experiments. The urinary iodine, iodine in breast milk, serum TH and thyroid morphological changes of maternal rats and their offspring were observed;we detected body and brain weight and D2 activity in pups' brain;neuronal marker-NSE and GAP-43 mRNA and protein expression were measured while MBP and synapsin I protein expression were simply detected. Results:(1) Urinary iodine and milk iodine: In maternal rats urinary iodine level in LI group was much lower than that in all other groups (p<0.01), and the urinary iodine level NI and all HI groups were significantly parlleled with their iodine intakes of 5, 10, 50, 100 folds. However, the iodine content in mother's milk, as iodine intake of pups, was only moderately increased, not paralleled with their iodine intake. The pups' urinary iodine was also paralleled with their iodine intake (iodine level in mother's milk).(2) TH level: Serum TT4 level in LI mothers and their pups was lower than that in all other groups (p<0.01). The serum TT4 level in mothers of iodine excess groups decreased gradually with their increase of iodine intake and there was statistical significance in 10HI and 50HI groups. But the serum TT4 levels had no statisticalsignificance in 14 days pups between NI and all HI groups. However, The serum TH levels in 28 days pups of iodine excess groups decreased gradually with their increase of iodine intake and there was statistical significance in TT4 and FT4 level in 100HI group.(3) Thyroid morphological changes: The histological examination thyroid tissue of maternal rats and their offspring in NI group indicated that the size of thyroid follicles was moderate, epithelial cells were cubical or flat and colloid was abundant in the follicular lumen. In LI group, obvious follicular hyperplasia was found in maternal rats and their pups. However, in iodine excess groups some histological changes were found in thyroid tissues and the major changes included increased follicles with colloid accumulation and concurrence of follicular hyperplasia;especially in 50HI and 100HI groups of parents rats, the hyperplasia became remarkable and the thyroid appeard TSH stimulating alteration. But in their pups moderate follicular hyperplasia was only found in 100HI group.(4) Body and brain weight of pups, D2 activity in pups' cerebrum tissue: The body and brain weight of 14 and 28 days old rats in LI group were significantly lower than the NI group while there was no statistical significance between NI group and iodine excess groups. D2 activity in pups' cerebrum of LI group increased compared with the NI group, but only at 14 days there was statistical significance. In all iodine excess groups, D2 activity of 14 and 28 days old rats was moderately lower than the NI group.(5) NSE, GAP-43 mRNA expression in pups'brain: In cerebral cortex, there was no difference in NSE mRNA expression of 14 and 28 days old rats between LI and NI group. However, GAP-43 mRNA expression of 14 days obviously increasedwhile that at 28 days significantly decreased. NSE mRNA expression of iodine excess groups gradually decreased with their increase of iodine intake at both 14 and 28 days. There was no difference in GAP-43 mRNA expression of 14 and 28 days between all HI groups and NI group. In hippocampus, there was no difference in NSE mRNA expression of 14 and 28 days old rats between LI and NI group. GAP-43 mRNA expression of 28 days significantly decreased while there was no change at 14days;NSE mRNA expression of iodine excess groups gradually decreased with their increase of iodine intake and there was statistics significance at 14 days. GAP-43 mRNA expression of 14 and 28 days significantly decreased with their increase of iodine intake. Compared above results between brain regions, NSE mRNA expression was decreased in cerebral cortex than in hippocampus at 14 days, but there was no significance at 28 days. However, GAP-43 mRNA expression has no difference at 14 and 28 days. Compared between two age days, NSE mRNA expression increased in cerebral cortex and decreased in hippocampus with their ages increase. But GAP-43 mRNA expression decreased in both cerebral cortex and hippocampus with their age increase.(6) NSE, GAP-43, MBP and synapsin I protein expression in pups'brain (immunohistochemical test): In LI group, NSE and MBP positive reactivity was less and synapsin I was more than NI group at 14 and 28 days. In iodine excess groups, NSE, and synapsin I positive reactivity was stronger;MBP positive reactivity was decreased with the increase of iodine intakes, but it still much more than the LI group. Synapsin I positive reactivity increased with the increase of iodine intakes, moreover, it was much stronger in 10HI, 50HI and 100HI group than the LI group. In cerebral cortex and hippocampus of 14 days, there was no difference in GAP-43 positivereactivity between LI and NI group;but it was moderately decreased with the increase of iodine intakes in HI groups. GAP-43 positive reactivity expressed in pyramidal cell cytoplasm in LI and all HI groups of 28 days. And its positive reactivity was similar in LI and 100HI group and was less in 5HI, 10HI and 50HI group than the LI and 100HI group. In hippocampus of 28 days, GAP-43 positive reactivity was moderately less than the NI group. Conclusions:(1) The lactating mothers with high iodine intake could decrease the iodine intake of their offspring by the regulation mechanism of mammary gland. This indicated that maternal generation could protect their progeny from the iodine excessin some degree.(2) Cerebral D2 activity was all increased in rat pups with both deficiency andexcess, indicating that TH level in the brain tissue was decreased in varying degrees.(3) Iodine deficiency and iodine excess could inhibit MBP protein expression and myelination by the TH deficiency in the brain. It also could enhance Synapsin I protein expression inhibiting neurotransmitter release and synaptic transmission. Inhibition role of iodine excess on MBP is less while its promotion role on SynapsinI was much than the Iodine deficiency.(4) Based on NSE, GAP-43 mRNA and protein expression level, there was no convincible evidence to demonstrate that iodine excess affected neuron development.(5) But the date of myelination and synaptic transmission strongly indicated that iodine deficiency and iodine excess could affect brain development, but the inhibitive effect of iodine deficiency was more severe.