Maternal marginal iodine deficiency affects the expression of relative proteins during brain development in rat offspring

in Journal of Endocrinology
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  • 1 Department of Endocrinology and Metabolism, The Liaoning Provincial Key Laboratory of Endocrine Diseases, The First Hospital of China Medical University, Department of Geriatrics, Shenyang Northern Hospital, No.155 Nanjing Bei Street, Hepig District, Shenyang 110001, People's Republic of China

Marginal iodine deficiency is a major health problem in pregnant women, but its impact on nerve and intelligence development in offspring has been rarely reported. Our study aimed to investigate the effects of maternal marginal iodine deficiency on nerve and cognitive development in offspring and the related mechanisms. Marginal iodine-deficient rats were given 3 μg iodine per day, while normal control rats were given 4 μg iodine daily. Western blot was used to detect the amounts of brain-derived neurotropic factor (BDNF) and early growth response protein 1 (EGR1) in the hippocampus of each group. Immunohistochemistry was used to measure c-jun and c-fos expression in the hippocampal CA1 region. Finally, the water maze method was used to measure spatial performance. Free thyroxine (FT4) levels in marginal iodine-deficient rats decreased by about 30%. Seven days after birth, EGR1 and BDNF protein levels significantly decreased in the hippocampus of marginal iodine deficiency rats compared with the normal control group. In addition, c-jun and c-fos expression in the hippocampus of 40-day-old rats was decreased in marginal iodine-deficient rats, compared with control. The spatial learning and memory ability of 40-day-old marginal iodine-deficient rats had a downward trend compared with the normal control group. FT4 significantly decreased after pregnancy in rats with marginal iodine deficiency, affecting the expression of related proteins in the brain of offspring.

Abstract

Marginal iodine deficiency is a major health problem in pregnant women, but its impact on nerve and intelligence development in offspring has been rarely reported. Our study aimed to investigate the effects of maternal marginal iodine deficiency on nerve and cognitive development in offspring and the related mechanisms. Marginal iodine-deficient rats were given 3 μg iodine per day, while normal control rats were given 4 μg iodine daily. Western blot was used to detect the amounts of brain-derived neurotropic factor (BDNF) and early growth response protein 1 (EGR1) in the hippocampus of each group. Immunohistochemistry was used to measure c-jun and c-fos expression in the hippocampal CA1 region. Finally, the water maze method was used to measure spatial performance. Free thyroxine (FT4) levels in marginal iodine-deficient rats decreased by about 30%. Seven days after birth, EGR1 and BDNF protein levels significantly decreased in the hippocampus of marginal iodine deficiency rats compared with the normal control group. In addition, c-jun and c-fos expression in the hippocampus of 40-day-old rats was decreased in marginal iodine-deficient rats, compared with control. The spatial learning and memory ability of 40-day-old marginal iodine-deficient rats had a downward trend compared with the normal control group. FT4 significantly decreased after pregnancy in rats with marginal iodine deficiency, affecting the expression of related proteins in the brain of offspring.

Introduction

It is well known that iodine is a trace element essential for the synthesis of triiodothyronine (T3) and thyroxine (T4), which play a crucial role in the process of early growth and development of most organs, especially the brain (Sethi & Kapil 2004), through genomic and nongenomic actions in neurons (Bernal 2005, Davis et al. 2008). During gestation, the mother is the only source of T4 before midgestation and the major source afterward for the developing brain as the fetal thyroid gland is not functional and is unable to produce thyroid hormone (TH) until the second trimester (Pop et al. 2003). So maternal inadequate intake of iodine leads to insufficient production of thyroid hormones, which is potentially damaging for neurodevelopment of the fetus throughout pregnancy (Morreale et al. 2004) and results in permanent and profound effects on neurological functions that contribute to cognitive and neurological impairments (Bernal et al. 2003, Tremont et al. 2003). The hippocampus is a brain region involved in cognitive skills such as learning and memory (Gerges & Alkadhi 2004) and the CA1 area in the hippocampus is the most important region for spatial learning (Huang et al. 1995) where memory is encoded, consolidated, and stored (Vara et al. 2003). The maturation and function of the hippocampus are dependent on thyroid hormone (Gerges & Alkadhi 2004). Congenital hypothyroidism can reduce the expression of many proteins in the hippocampus of neonatal rats (Dong et al. 2005) and affect its function.

In 1996, China implemented the Universal Salt Iodization (USI) regulation. Since then, the iodine nutrition condition of Chinese people has been at an appropriate or excessive level. Marginal iodine deficiency is a major health problem for pregnant women. According to the evaluation criteria set for pregnant women by the World Health Organization (WHO) in 2007, marginal iodine deficiency is defined as a urinary iodine concentration between 100 and 150 μg/l (WHO/UNICEF/ICC IDD 2007). From our survey in Shenyang, almost half of the pregnant women were in a state of marginal iodine deficiency (Shi et al. 2009), similar to survey results from other areas (Li et al. 2004, Zhang et al. 2005). It has been shown that the cognitive performance of offspring is severely affected by moderate-to-severe iodine deficiency in pregnant women. However, studies investigating the impacts of marginal iodine deficiency in pregnant women on nerve and cognitive performance of their offspring have been rarely reported. According to the characteristics of marginal iodine deficiency obtained from epidemiological surveys, we established an animal model of marginal iodine deficiency in Wistar rats that were relatively sensitive to iodine. With this established rat model, we investigated the influences and mechanisms of marginal iodine deficiency on nerve and cognitive performance in offspring.

Materials and methods

Experimental animals and grouping

For the first part, 80 female specific pathogen-free (SPF) Wistar rats that had been weaned for 1 month and weighed 100–110 g were purchased from Beijing Vital River Experimental Animal Technology LLC, Beijing, China. After adaptive feeding for 1 week, the rats were assigned to one of the five treatment groups and counterbalanced for weight (mean weight, 107 g). The groups were divided as follows: i) low-iodine group (L group, n=20), ii) marginal iodine deficiency group 1 (2 μg iodine, n=10), iii) marginal iodine deficiency group 2 (3 μg iodine, n=20), iv) normal control group (N group, n=20), and v) normal control group 1 (N1 group, n=10). Rats were fed for 3 months and then mated with normal male rats with a female-to-male ratio of 2:1. And after pregnancy, the above five groups were named LP, 2μgP, 3μgP, NP, and N1P group respectively.

Based on the findings of first part, for the second part, after adaptive feeding for 1 week, 90 female SPF Wistar rats were divided into three groups (n=30 for each group) and counterbalanced for weight as follows: i) low-iodine group (L group), ii) marginal iodine deficiency group (M group), and iii) normal control group (N group). Rats were raised and mated according to the methods used in the first experiment and the offspring were fed the same diet as their mothers.

Feeding

The L and N1 groups drank deionized water, while the marginal iodine deficiency and N groups drank deionized water with dissolved potassium iodate of different concentrations. The low-iodine chow was made by mixing low-iodine foods (corns, soybeans, and millets) supplemented with inorganic salts and trace elements necessary for animal survival. The iodine content in the chow was 60 μg/kg, as measured by the Shenyang Academy of Agricultural Sciences. The N1 group was fed a normal rat diet (Institute of Endocrinology, China Medical University, Shenyang, China), but the food composition and the proportions were the same as the low-iodine diet. The average iodine content in the normal diet was 200 μg/kg. According to the daily food intake (20 mg) and daily water intake (30 ml) for rats, the daily iodine intakes were 1.2 μg/day (L group), 2 μg/day (2μg group), 3 μg/day (3μg group), 4 μg/day (N group), and 4 μg/day (N1 group) respectively.

Collection and measurement of urinary iodine

Chemicals were purchased from John Chem Co. Ltd, Linyi, China for the measurement of urinary iodine excretion using ammonium persulfate–arsenic cerium catalytic spectrophotometry (WHO/UNICEF/ICCIDD 2007). Urine was collected with metabolic cages every 3 days after 2 months of feeding.

Observation of fertilization and procreation conditions in pregnant rats

In the next morning after mating, the vaginal smears of the female rats were examined. A positive record of pessus and vaginal smears were defined as embryonic day 0 (E0). The pregnancy reaction, fertilization condition, and stillbirth condition were observed on a daily basis. Also, the total number and death number were observed and recorded. The following calculations were used for each group: Pregnancy rate=number of pregnant rats/number of female rats×100%; mortality of pregnant rats=death number of pregnant rats/total number of pregnant rats×100%; and newborn rat mortality=total death number of newborn rats/total number of newborn rats.

Hormone determination

We used a solid-phase chemiluminescence enzyme immunoassay to measure TSH, total T4 (TT4), free T4 (FT4), total T3 (TT3), and free T3 (FT3) (Diagnostic Products Corporation, Los Angeles, CA, USA). The functional sensitivity of the TSH assay was 0.02 mIU/l. The intra-assay coefficients of variation (CV) of serum for TSH, TT4, FT4, TT3, and FT3 were 1.57–4.12, 1.26–3.20, 2.24–6.33, 2.42–5.63, and 0.57–4.31% respectively. The inter-assay CV values were 1.26–5.76, 3.58–6.67, 4.53–8.23, 3.50–5.19, and 5.23–8.16% respectively.

Measurement of hormone and iodine content in the thyroid

Thyroid tissues were precisely weighed, sheared into pieces, added in cold homogenate, and prepared into tissue homogenate, which was then centrifuged at 1000 g for 15 min. Supernatant was removed and diluted into 3 ml/mg for measurement. The hormone was measured using solid-phase chemiluminescence enzyme immunoassay, and iodine content in the thyroid was measured with the ammonium persulfate–arsenic cerium catalytic spectrophotometry (WHO/UNICEF/ICC IDD 2007).

Immunohistochemistry

Rat brains were fixed in 4% paraformaldehyde and embedded in paraffin. Coronal sections (5 μm) were collected on Super Frost glass slides (Hehyglass, Haimen, China). All sections were deparaffinized, rehydrated, and then treated for endogenous peroxidase with 3% H2O2 for 10 min. After washing with PBS, all sections were incubated with a rabbit anti-c-fos or anti-c-jun antibody (Santa Cruz Biotechnology, 1:100 dilution) at 37 °C for 30 min. Sections were then washed three times in PBS followed by additional incubation with biotinylated secondary antibodies (Maixin, Fuzhou, China) for 30 min at 37 °C. Then, the sections were incubated for 10 min in avidin–biotin–peroxidase (Maixin) and washed three times in PBS. Sections were then reacted with a solution of 3,3-diaminobenzidine (DAB, Sigma). Finally, the sections were counterstained with Mayer's hematoxylin, rinsed, and mounted in neutral gum (China National Medicines, Shanghai, China).

Image analysis

The Microscopic Image Analysis System (MetaMorph/DP10/Bx41/UIC/OLYMPUS, US/JP) was used to analyze the c-fos and c-jun immunoreaction products in the bilateral dorsal hippocampal CA1 region (400× magnification).

Western blotting

On postnatal day 7 (PND7), pups in each group were deeply anesthetized and killed by ether. The brain was removed and the hippocampus was dissected on ice rapidly, frozen in liquid nitrogen, and stored at −70 °C until use.

Hippocampi dissected from pups at PND7 were homogenized in ice-cold solubilizing buffer containing protease inhibitors. Homogenate was centrifuged at 12 000 g for 15 min at 4 °C. The supernatant was collected for protein concentration measurement. Samples (50 μg for early growth response protein 1 (Egr1) and 60 μg for brain-derived neurotropic factor (BDNF)) were separated by SDS–PAGE and then transferred onto PDVF membranes (Millipore, Billerica, MA, USA) by electrophoresis. Blots were blocked for 2 h at room temperature with 5% BSA and then incubated overnight at 4 °C with antibodies to Egr1 (1: Signal, Danvers, MA, USA) and BDNF (1:1000 Millipore). Blots were then incubated with HRP-conjugated secondary antibodies (Zhongshan Goldenbridge, Beijing, China). Finally, the blots were visualized with an ECL western blot detection system (Alphaview1.3, Proteinsimple Inc, CA, USA).

Morris water maze

The Morris water maze experiments were conducted in 40-day-old rats. On the first day, the rats were put into a water maze for 120 s to swim freely and adapt to the environment of the water maze. On the second day, a platform was set randomly in the middle of one quadrant. The rats were trained with two procedures per day – one in the morning and one in the afternoon. In each procedure, four separate training times were performed. In the first procedure, one quadrant was chosen. The rats were placed in the water at the center of the pool wall and the procedure was repeated in the other three quadrants. A computer was used to monitor and record the path, time (escape latency, EL), swimming length, swimming speed, and the strategy used to locate the transparent platform. The procedure was immediately stopped if a rat found the platform within 120 s. Rats were allowed to stay on the platform for 10 s for memory strengthening. If the rats did not find the platform within 120 s, then the EL was recorded as 120 s. At this point, the researchers subsequently guided the rats to the platform and allowed them stay on the platform for 10 s before removing the rats from the platform. After resting for 30–60 s, the rats proceeded to the next round of training. The interval of every training session was 10 s. The test environment and position stayed the same during training. The rats were trained eight times per day.

Statistical analysis

Data were analyzed using SPSS 17.0 software and expressed as mean±s.d. A two-sample t-test was used to compare between two groups, and ANOVA was used for multigroup comparisons. A P<0.05 was considered statistically significant.

Results

Body weight, thyroid weight-to-body weight ratio, and urinary iodine level

After being fed for 3 months, the average weight of the 2μg group was 270.60±20.72 g (P<0.05 vs N group), which did not meet the standards (Glinoer et al. 1995, Mun et al. 2006) for marginal iodine deficiency, and thus the following experiments were discontinued for the 2μg group. The average body weight of the L group was significantly decreased compared with the other three groups (P<0.05), but the average body weight between the 3μg iodine, N, and N1 groups was similar (P>0.05). The thyroid weight-to-body weight ratio increased significantly in the L group (P<0.05) than the 3μg, N, and N2 groups, while there was no difference among the 3μg, N, and N2 groups (P>0.05). The urinary iodine value in the L group (63.50±13.16 μg/l) and the 3ug group (107.00±24.40 μg/l) was significantly lower than the other two groups (P<0.01 and P<0.05 respectively), while the urinary iodine value in the N group (202.75±31.60 μg/l) and the N1 group (210.30±23.19 μg/l) did not change (P>0.05).

The thyroid function of rats before pregnancy and on E17

All indexes of thyroid function between the N and N1 groups were similar before pregnancy (P>0.05; Table 1). Serum TSH levels in the L group were significantly higher than in the other groups (P<0.01), and no obvious difference was observed among the other groups (P>0.05). After pregnancy (E17: 17 days after their pregnancy), the TSH levels had a downward trend in all the groups. In addition, on E17, FT4 had a downward trend in all the groups, and FT4 in the L group was significantly lower than the 3μg and N groups (P<0.05 and P<0.01 respectively) after pregnancy for 17 days. The FT4 of 3μg group decreased by 30%, while FT4 in the N group only decreased by 4%. TT4 in the L group was significantly lower when compared with the N group (P<0.01), while TT4 in the 3μg group was decreased compared with the N group, but not statistically significant. FT3 was not significantly different among all groups, but the 3μg group had slightly higher FT3 levels than the N group and the increased trend continued after pregnancy. TT3 decreased after pregnancy in all the groups. TT3/TT4 and FT3/FT4 levels in the L group were significantly lower than the low-iodine pregnancy group (LP) (P<0.05), 3μg, and N groups. No obvious differences were observed among the other groups. TT3/TT4 and FT3/FT4 levels in the 3μg and the marginal iodine deficiency pregnancy (3μgP) groups were slightly higher than the N and normal iodine pregnancy (NP) groups, but the differences were not statistically significant (P>0.05).

Table 1

The levels of various hormones reflecting the thyroid function of rats before pregnancy and on E17 (mean±s.e.m.). The P labeling represents the results for the groups in their 17-day pregnancy period.

GroupnTT4 (μg/dl)FT4 (pmol/l)TT3 (ng/dl)FT3 (pmol/l)TSH (mIU/l)FT3/FT4TT3/TT4
L72.41±0.70*18.82±4.45*89.34±5.945.40±0.330.92±0.29*0.32±0.0630.04±0.012
LP61.56±0.55*9.28±0.12*72.35±5.964.71±0.870.23±0.03*0.53±0.080.075±0.011
3μg74.35±0.6031.03±4.1783.40±10.485.67±0.350.039±0.040.215±0.0430.0210±0.003
3μgP72.50±0.2522.46±1.5164.36±5.245.02±0.550.016±0.0010.228±0.0160.0284±0.004
N74.59±0.3327.28±2.7790.82±8.915.30±0.350.032±0.0050.206±0.0230.0203±0.002
NP73.05±0.5327.12±3.5659.17±5.884.43±0.220.017±0.030.213±0.0240.0271±0.007
N174.61±0.2723.07±1.8691.00±7.675.41±0.430.033±0.003

*Compared with N group, P<0.05.

T3/T4 and iodine content in thyroid

In the L group, the levels of T3 and T4 were too low to be measured (Table 2). In addition, the iodine content in the thyroid of the L group was significantly lower than that in the other groups (P<0.05), and there was no obvious difference among other groups (P>0.05). T3/T4 levels in the 3μg group were slightly higher than the N and N1 groups but were not statistically significant (P>0.05). At E17, the iodine content in the thyroids of the 3μgP and NP groups was lower than in the non-pregnancy groups. There was no significant difference between the above two groups (P>0.05).

Table 2

T3/T4 and iodine content in thyroid (mean±s.e.m.). 3μgP and NP groups measured on rats from M group and N group 17 days after their pregnancy

GroupnT3/T4Iodine content in thyroid (μg/100 mg)
L786.53±15.91*
3μg734.97±9.15178.21±10.10
N732.63±6.49180.86±13.24
N1732.94±5.45178.00±15.07
3μgP7169.49±18.09
NP7169.13±12.81

*Compared with N group, P<0.05.

State of pregnancy, delivery, and thyroid function of rat offspring on P7

Fertilization rate, mortality of pregnant rats, and mortality of newborn rats in the low-iodine group (1.2μg iodine intake daily) were higher than those in the 3μg and N groups (P<0.05), and the number of newborn rats in the low-iodine group was less than the other two groups (P<0.05) (Table 3). No difference was observed between the 3μg and N groups. At P7, serum TSH levels in the offspring of the low-iodine group were significantly higher than those of the normal control group (P<0.05). Serum TT4 and FT4 levels of the low-iodine group were also significantly lower than the N group. A significant difference was not observed between the 3μg and N groups (P>0.05).

Table 3

Thyroid function of PND7 rats (mean±s.e.m.)

GroupnTT4 (μg/dl)FT4 (pmol/l)TSH (mIU/l)
L121.12±0.10*6.62±0.45*0.138±0.02*
M122.45±0.3810.22±0.580.077±0.048
N122.44±0.0710.47±0.600.089±0.004

*Compared with N group, P<0.05.

Index analysis of Morris water maze behaviors of 40-day-old rats

On the first day of adaptive training, the time to find the platform was long and a difference in the average EL among the three groups was not obvious (Table 4). On the second day, rats in all groups adapted to the pool environment and most of the rats actively searched for the platform. The average EL of all groups continuously decreased, but the rate differed among groups, indicating that rats in each group had partial memory loss between training sessions. ANOVA indicated that a difference was not observed in EL between the N and M groups, while the EL of the N and M groups was significantly shorter than that for the L group. The results of the third day were similar to the second day. On the fourth day, the EL in the N group was still significantly shorter than the L group. In addition, the EL of the M group was longer than N group but was not statistically significant. The results of the fifth day were similar to the fourth day.

Table 4

Escape latency (s) in the Morris water maze experiment (mean±s.e.m.)

GroupnSecond dayThird dayFourth dayFifth day
L886.688±8.02*,64.566±6.41*,27.22±5.06*28.725±4.65*
M859.038±7.8637.656±7.3120.806±5.1319.819±3.41
N856.728±7.1937.434±6.8112.656±2.0310.970±1.44

*Compared with N group, P<0.05, compared with M group, P<0.05.

The expression of c-fos and c-jun proteins

As observed from the results of immunohistochemistry, the expression levels of c-fos and c-jun in the M group were significantly lower than that in the N group (P<0.05) (Fig. 1). However, when compared with the low-iodine group, c-fos and c-jun expression in the M group was higher but not statistically significant.

Figure 1
Figure 1

Immunohistochemistry pictures of c-fos and c-jun in newborn rat hippocampi in each group (P40, ×400). (A) c-fos and (B) c-jun. (C and D) IHC IOD values of c-fos and c-jun respectively in newborn rat hippocampi in each group on P40. *Compared with N group, P<0.05. Full colour version of this figure available via http://dx.doi.org/10.1530/JOE-12-0410.

Citation: Journal of Endocrinology 217, 1; 10.1530/JOE-12-0410

Hippocampus EGR1 and BDNF protein expression in PND7 rats

EGR1 and BDNF protein expressions were measured by western blot (Fig. 2). The results showed that the L and M groups had significantly lower Egr1 expression than the N group (P<0.05). Similarly, the BDNF protein expression in the L and M groups were significantly lower than the N group (P<0.05).

Figure 2
Figure 2

Western blot of postnatal day 7 rat hippocampi of EGR1 and BDNF protein levels. (A) EGR1 and (B) BDNF. (C and D) Western blot gray values for EGR1 and BDNF respectively. *Compared with normal control group (N), P<0.05. Full colour version of this figure available via http://dx.doi.org/10.1530/JOE-12-0410.

Citation: Journal of Endocrinology 217, 1; 10.1530/JOE-12-0410

Discussion

The main focus of previous studies regarding iodine deficiency was to establish severe iodine deficiency models and investigate the impact of iodine deficiency on offspring development. Our study established a marginal iodine deficiency model in rats based on the following principles: in the normal physiological status, there were no symptoms of iodine deficiency, but during pregnancy, the requirement for iodine increased, resulting in iodine deficiency. This study also investigated the changes in iodine metabolism and thyroid function, as well as the influences of maternal marginal iodine deficiency on offspring neural intelligence development and the related molecular mechanisms.

The marginal iodine deficiency in our study was very mild, and therefore before pregnancy the thyroid was auto-regulated and had no functional changes. However, after pregnancy the iodine requirement increased significantly, which resulted in over stimulation of the thyroid and changes in thyroid function. We found that to synthesize 100 g thyroid hormone, which is physiologically required by a Wistar rat, 0.68 μg iodine was needed. The average body weight of our experimental rats was <300 g, so a daily iodine intake of 2 μg only met the physiological requirements (Escobar-Morreale et al. 1996). According to a literature report (Heninger & Albright 1975), when the daily iodine intake of a Wistar rat was 3 μg, the T4 concentration reached most of the tissues. Furthermore, if the daily iodine intake of a Wistar rat was 4 μg, all the physiological requirements were satisfied. According to the ANI93 standard of animal diets, the iodine content of the diet used in our study was 200 μg/kg. Assuming the daily food intake was 20 g, the daily iodine intake requirement was 4 μg.

In our study, we chose daily iodine intakes of 2 and 3 μg as the marginal iodine deficiency. We evaluated the model according to the results of epidemiological survey. The epidemiological characteristics of people in marginal iodine-deficient areas are prevalent before and after pregnancy: before pregnancy, the autoregulatory mechanism of the thyroid is active and serum TSH does not increase. Serum FT4 has a downward trend and T3 has an upward trend. Additionally, the urinary iodine is lower than the normal but has no influences on normal growth. After pregnancy, obvious symptoms of thyroid over stimulation appear, and TSH increases within the normal range, the iodine content of the thyroid decreases, the T3/T4 value further increases, and the number of newborn rats and their survival rate stays the same (Glinoer et al. 1995, Glinoer 1997a,b, Lavado-Autric et al. 2003, Morreale de Escobar & Escobar del Rey 2003, Mun et al. 2006, Pedraza et al. 2006).

In our study, growth, thyroid function, iodine content in the thyroid, and urinary iodine showed no difference between the N group (iodine intake was 4 μg/day) and the N1 group (normal diet), indicating that low-iodine foods with water containing iodine have no deficiency of nutritive elements and can be used as an alternative for a normal diet. After 3 months of feeding, the body weight of rats in the 2μg group (iodine intake was 2 μg/day) was significantly lower than the N group, suggesting that the iodine intake affected the growth of rats and thus did not meet our standard for marginal iodine deficiency. Before pregnancy, the urinary iodine of rats in the 3μg group (iodine intake was 3 μg/day) was significantly lower than the normal control groups (N group and N1 group), indicating a lower iodine excretion rate and iodine intake. However, the iodine content in the thyroid, hormone content in the thyroid, thyroid function, pregnancy status, and the procreation conditions were not different between these two groups, indicating that the rats in the 3μg group were not iodine deficient but only marginally deficient.

After pregnancy, the iodine content in the thyroid of the 3μg and N groups decreased, but the rate of decrease was similar. Before and after pregnancy, the T3/T4 value of the 3μg group was slightly higher than the N group and the rate of increase after pregnancy was slightly higher than the N group. After pregnancy, the FT4 levels of the 3μg group decreased by about 30%, while the value in the N group only decreased by 4%, which was similar to the result of the epidemiological survey (Pedraza et al. 2006). In the survey, the decrease of FT4 did not exceed 15% after pregnancy in subjects with sufficient iodine. In our study, the 3μg group met the requirements for marginal iodine deficiency.

The hippocampus is the part of the brain that is related to cognitive ability, including learning and memory ability (Gerges & Alkadhi 2004). The CA1 area in the hippocampus is important for spatial learning. The memories are coded and incorporated and then stored plastically by synapses. The hippocampus is also a sensitive area regulated by the thyroid hormone. The abnormal expression of related proteins in the hippocampus affects brain intelligence. In this study, although there was no difference in the water maze test between the M and N groups, there was increased EL in the M group, indicating impairment in learning and memory abilities.

BDNF expression can be directly regulated by thyroid hormone (Fujinoto et al. 2004). Some studies have shown that thyroid hormone deficiency results in a significant decrease in BDNF content in the brain of newborn rats (Lasley & Gilbert 2011). However, if the rats are supplied with thyroid hormone, the BDNF content will increase rapidly (Camboni et al. 2003). BDNF receptor binding with TrkB activates intracellular signal transduction. MARK plays an important role in the differentiation and migration of nerve cells, the reconstruction of synaptic plasticity, and the consolidation of learning and memory. Furthermore, MARK induces hippocampus granular cells to produce continuous long-term potentiation (LTP) (Kobayashi et al. 2006), which is currently recognized as the neural basis for learning and memory. The influences of BDNF on learning and memory are mainly due to the regulatory effects of BDNF on LTP (Jia et al. 2010). In our study, BDNF expression in the PND7 newborn rats in the M group was significantly lower than the N group, which suggests that the relative thyroid hormone deficiency in female rats with marginal iodine deficiency has a negative impact on BDNF and can further influence the brain development of offspring.

Egr1 belongs to the early growth reactive protein family and is also referred to as krox24 or Zif268. The expression of Egr1 is mainly regulated by thyroid hormone through TRE. Egr1 is related to the connectivity of regulatory nerves and the adaptability of synapses. Thyroid hormone deficiency can result in the decrease in Egr1 content in the cortex and hippocampus of the CNS (Kobayashi et al. 2009). Synaptic activation can induce the generation of EGR1 and regulate the synaptic plasticity related to learning and memory, which is critical for the maintenance of late LTP. The formation of long-term memory depends on the hippocampus and the consolidation of early constructed memory. During the process of LTP induction by the dentate gyrus of the hippocampus, the transcription of Egr1 is quick and robust. Studies have shown that the expression of Egr1 is a critical genetic mechanism for the maintenance of late LTP and the stability of long-term memory (Ranieri et al. 2012). In our study, the Egr1 expression of PND7 newborn rats in the low-iodine group and the marginal iodine deficiency group were significantly lower than the normal control group, indicating that marginal iodine deficiency in female pregnant rats has a great influence on Egr1 expression of offspring, which in turn affects learning and memory ability.

Immediate early genes (IEGs) can couple the short-term function of the extracellular signal and the long-term change in cell function, suggesting that IEGs are tightly related to learning and memory regulation (Herdegen et al. 1997, Guzowski 2002). c-fos and c-jun are the most important characteristic genes in the IEG families. Additionally, if cells have been stimulated by a series of physicochemical and biological factors, c-fos and c-jun can be rapidly expressed and encoded to generate the Fos and Jun proteins, which regulate neuronal plasticity. Neuronal plasticity is considered to play an important role in memory formation, especially long-term memory formation (French et al. 2001). In our study, the expression of c-fos and c-jun in the hippocampus CA1 area decreased significantly in the marginal iodine deficiency group. Immunoreaction products were mainly distributed in the cytomembrane and cytoplasm. In the nucleus, the expression of c-fos and c-jun was significantly lower than the N group but higher than the low iodine group, suggesting that the changes in c-fos and c-jun expression and their distribution during marginal iodine-deficient conditions play a role in the learning and memory abilities of newborn rats.

Expression of C-fos and c-jun protein in the M group was significantly lower than that of normal control group but higher than that of low-iodine group. The above result indicates that in MWM, the learning and memory capacity of the M group was on a downward trend compared with the normal control group, while it was still different from the low-iodine group, which has significantly decreased learning and memory capacity.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This work was supported by Department of Education Innovation Team of Liaoning Provincial (Grant number LT2010108) and the Science and Technology Program of Liaoning Province (Grant number 2010225019); Doctoral Program Fund Higher Education (Fund for the Doctoral) (Grant number 20102104110005); special funds of clinical research of Chinese Medical Association – Endocrine disease research ([2010]60 Grant number 10020030225).

References

  • Bernal J 2005 Thyroid hormones and brain development. Vitamins and Hormones 71 95122.

  • Bernal J, Guadano-Ferraz A & Morte B 2003 Perspectives in the study of thyroid hormone action on brain development and function. Thyroid 13 10051012. (doi:10.1089/105072503770867174)

    • Search Google Scholar
    • Export Citation
  • Camboni D, Roskoden T & Schwegler H 2003 Effect of early thyroxine treatment on brain-derived neurotrophic factor mRNA expression and protein amount in the rat medial septum/diagonal band of Broca. Neuroscience Letters 350 141144. (doi:10.1016/S0304-3940(03)00880-2)

    • Search Google Scholar
    • Export Citation
  • Davis PJ, Leonard JL & Davis FB 2008 Mechanisms of nongenomic actions of thyroid hormone. Frontiers in Neuroendocrinology 29 211218. (doi:10.1016/j.yfrne.2007.09.003)

    • Search Google Scholar
    • Export Citation
  • Dong J, Yin H, Liu W, Wang P, Jiang Y & Chen J 2005 Congenital iodine deficiency and hypothyroidism impair LTP and decrease c-fos and c-jun expression in rat hippocampus. Neurotoxicology 26 417426. (doi:10.1016/j.neuro.2005.03.003)

    • Search Google Scholar
    • Export Citation
  • Escobar-Morreale HF, del Rey FE, Obregón MJ & de Escobar GM 1996 Only the combined treatment with thyroxine and triiodothyronine ensures euthyroidism in all tissues of the thyroidectomized rat. Endocrinology 137 24902502. (doi:10.1210/en.137.6.2490)

    • Search Google Scholar
    • Export Citation
  • French PJ, O'Connor V, Jones MW, Davis S, Errington ML, Voss K, Truchet B, Wotjak C, Stean T & Doyère V 2001 Subfield-specific immediate early agene expression associated with happocampal long-term potention in vivo. European Journal of Neuroscience 13 968976. (doi:10.1046/j.0953-816x.2001.01467.x)

    • Search Google Scholar
    • Export Citation
  • Fujinoto N, Jinno N & Kitamura S 2004 Activation of estrogen response element dependent transcription by thyroid hormone with increase in estrogen receptor levels in a rat pituitary cell line, GH3. Journal of Endocrinology 181 7783. (doi:10.1677/joe.0.1810077)

    • Search Google Scholar
    • Export Citation
  • Gerges NZ & Alkadhi KA 2004 Hypothyriodism impairs late LTP in CA1 region but not in dentate gyrus of the intact rat hippocampus: MAPK involvement. Hippocampus 14 4045. (doi:10.1002/hipo.10165)

    • Search Google Scholar
    • Export Citation
  • Glinoer D 1997a Maternal and fetal impact of chronic iodine deficiency. Clinical Obstetrics and Gynecology 40 102116. (doi:10.1097/00003081-199703000-00011)

    • Search Google Scholar
    • Export Citation
  • Glinoer D 1997b The regulation of thyroid function in pregnancy: pathways of endocrine adaptation from physiology to pathology. Endocrine Reviews 18 404433. (doi:10.1210/er.18.3.404)

    • Search Google Scholar
    • Export Citation
  • Glinoer D, De Nayer P, Delange F, Lemone M, Toppet V, Spehl M, Grün JP, Kinthaert J & Lejeune B 1995 A randomized trial for the treatment of mild iodine deficiency during pregnancy: maternal and neonatal effects. Journal of Clinical Endocrinology and Metabolism 80 258269. (doi:10.1210/jc.80.1.258)

    • Search Google Scholar
    • Export Citation
  • Guzowski JF 2002 Insights into immediate-early gene function in hippocampal memory consolidation using antisense oligonucleotide and fluorescent imaging approaches. Hippocampus 12 86104. (doi:10.1002/hipo.10010)

    • Search Google Scholar
    • Export Citation
  • Heninger RW & Albright EC 1975 Alteration in tissue and serum concentrations of TSH, iodide, T4 and T3 induced by various dietary iodide levels. Proceedings of the Society for Experimental Biology and Medicine 150 137141.

    • Search Google Scholar
    • Export Citation
  • Herdegen T, Skene P & Bähr M 1997 The c-Jun transcription factor – bipotential mediator of neuronal death, survival and regeneration. Trends in Neurosciences 20 227231. (doi:10.1016/S0166-2236(96)01000-4)

    • Search Google Scholar
    • Export Citation
  • Huang YY, Kandel ER, Varshavsky L, Brandon EP, Qi M, Idzerda RL, McKnight GS & Bourtchouladze R 1995 A genetic test of the effects of mutations in PKA on mossy fiber LTP and its relation to spatial and contextual learning. Cell 83 12111222. (doi:10.1016/0092-8674(95)90146-9)

    • Search Google Scholar
    • Export Citation
  • Jia Y, Gall CM & Lynch G 2010 Presynaptic BDNF promotes postsynaptic long-term potentiation in the dorsal striatum. Journal of Neuroscience 30 1444014445. (doi:10.1523/JNEUROSCI.3310-10.2010)

    • Search Google Scholar
    • Export Citation
  • Kobayashi K, Tsuji R, Yoshioka T, Mino T & Seki T 2006 Perinatal exposure to PTU delays switching from NR2B to NR2A subunits of the NMDA receptor in the rat cerebellum. Neurotoxicology 27 284290. (doi:10.1016/j.neuro.2005.11.008)

    • Search Google Scholar
    • Export Citation
  • Kobayashi K, Akune H, Sumida K, Saito K, Yoshioka T & Tsuji R 2009 Perinatal exposure to PTU decreases expression of Arc, Homer 1, Egr 1 and Kcna 1 in the rat cerebral cortex and hippocampus. Brain Research 1264 2432. (doi:10.1016/j.brainres.2008.12.029)

    • Search Google Scholar
    • Export Citation
  • Lasley SM & Gilbert ME 2011 Developmental thyroid hormone insufficiency reduces expression of brain-derived neurotrophic factor (BDNF) in adults but not in neonates. Neurotoxicology and Teratology 33 464472. (doi:10.1016/j.ntt.2011.04.001)

    • Search Google Scholar
    • Export Citation
  • Lavado-Autric R, Ausó E, García-Velasco JV, Arufe Mdel C, Escobar del Rey F, Berbel P & Morreale de Escobar G 2003 Early maternal hypothyroxinemia alters histogenesis and cerebral cortex cytoarchitecture of the progeny. Journal of Clinical Investigation 111 10731082.

    • Search Google Scholar
    • Export Citation
  • Li JS, Deng JY & Zhou DY 2004 Analysis on iodine nutrition of important people in Sichuan provincel. Journal of Preventive Medicine Information 20 104107.

    • Search Google Scholar
    • Export Citation
  • Morreale D, Escobar G, Obregon MJ & Escobardel RF 2004 Role of thyroid hormone during early brain development. European Journal of Endocrinology 151 U25U37. (doi:10.1530/eje.0.151U025)

    • Search Google Scholar
    • Export Citation
  • Morreale de Escobar G & Escobar del Rey F 2003 Consequences of iodine deficiency for brain development. In The Thyroid and the Brain, pp 33–56. Eds J deVijlder & G Morreale de Escobar. Stuttgart, Germany: Schattauer Verlag.

  • Mun RM, Qin LX & Zhang Y 2006 Iodine deficiency during pregnancy and thyroid disease. Journal of Practical Obstetrics and Gynecology 22 585589.

  • Pedraza PE, Obregon MJ, Escobar-Morreale HF, Escobar del Rey F & Morreale de Escobar G 2006 Mechanisms of adaptation to iodine deficiency in rats: thyroid status is tissue specific. Its relevance for man. Endocrinology 147 20982210. (doi:10.1210/en.2005-1325)

    • Search Google Scholar
    • Export Citation
  • Pop VJ, Brouwers EP, Vader HL, Vulsma T, van Baar AL & de Vijlder JJ 2003 Maternal hypothyroxinaemia during early pregnancy and subsequent child development: a 3-year follow-up study. Clinical Endocrinology 59 282288. (doi:10.1046/j.1365-2265.2003.01822.x)

    • Search Google Scholar
    • Export Citation
  • Ranieri F, Podda MV, Riccardi E, Frisullo G, Dileone M, Profice P, Pilato F, Di Lazzaro V & Grassi C 2012 Modulation of LTP at rat hippocampal CA3-CA1 synapses by direct current stimulation. Journal of Neurophysiology 107 18681880. (doi:10.1152/jn.00319.2011)

    • Search Google Scholar
    • Export Citation
  • Sethi V & Kapil U 2004 Iodine deficiency and development of brain. Indian Journal of Pediatrics 71 325329. (doi:10.1007/BF02724099)

  • Shi XG, Teng XC & Shan ZY 2009 An epidemiological study of the relationship between iodine intake levels and thyroid function during early pregnancy. Chinese Journal of Practical Internal Medicine 29 520522.

    • Search Google Scholar
    • Export Citation
  • Tremont G, Stern RA, Westervelt HJ, Bishop CL & Davis JD 2003 Neurobehavioral functioning in thyroid disorders. Medicine and Health, Rhode Island 86 318322.

    • Search Google Scholar
    • Export Citation
  • Vara H, Munoz-Cuevas J & Colino A 2003 Age-dependent alterations of long-term synaptic plasticity in thyroid-deficient rats. Hippocampus 13 816825. (doi:10.1002/hipo.10132)

    • Search Google Scholar
    • Export Citation
  • WHO/UNICEF/ICC IDD 2007 Assessment of Iodine deficiency disorders and Monitoring their Elimination. A Guide for Programme Managers. 3rd edition, pp 1–108.

  • Zhang ZZ, Guo HY & He LL 2005 Iodine nutritive surveillance of typical population in partial areas of the inner mongolia autonomous region. Chinese Journal of Endemiology 24 305307.

    • Search Google Scholar
    • Export Citation

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    Immunohistochemistry pictures of c-fos and c-jun in newborn rat hippocampi in each group (P40, ×400). (A) c-fos and (B) c-jun. (C and D) IHC IOD values of c-fos and c-jun respectively in newborn rat hippocampi in each group on P40. *Compared with N group, P<0.05. Full colour version of this figure available via http://dx.doi.org/10.1530/JOE-12-0410.

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    Western blot of postnatal day 7 rat hippocampi of EGR1 and BDNF protein levels. (A) EGR1 and (B) BDNF. (C and D) Western blot gray values for EGR1 and BDNF respectively. *Compared with normal control group (N), P<0.05. Full colour version of this figure available via http://dx.doi.org/10.1530/JOE-12-0410.

  • Bernal J 2005 Thyroid hormones and brain development. Vitamins and Hormones 71 95122.

  • Bernal J, Guadano-Ferraz A & Morte B 2003 Perspectives in the study of thyroid hormone action on brain development and function. Thyroid 13 10051012. (doi:10.1089/105072503770867174)

    • Search Google Scholar
    • Export Citation
  • Camboni D, Roskoden T & Schwegler H 2003 Effect of early thyroxine treatment on brain-derived neurotrophic factor mRNA expression and protein amount in the rat medial septum/diagonal band of Broca. Neuroscience Letters 350 141144. (doi:10.1016/S0304-3940(03)00880-2)

    • Search Google Scholar
    • Export Citation
  • Davis PJ, Leonard JL & Davis FB 2008 Mechanisms of nongenomic actions of thyroid hormone. Frontiers in Neuroendocrinology 29 211218. (doi:10.1016/j.yfrne.2007.09.003)

    • Search Google Scholar
    • Export Citation
  • Dong J, Yin H, Liu W, Wang P, Jiang Y & Chen J 2005 Congenital iodine deficiency and hypothyroidism impair LTP and decrease c-fos and c-jun expression in rat hippocampus. Neurotoxicology 26 417426. (doi:10.1016/j.neuro.2005.03.003)

    • Search Google Scholar
    • Export Citation
  • Escobar-Morreale HF, del Rey FE, Obregón MJ & de Escobar GM 1996 Only the combined treatment with thyroxine and triiodothyronine ensures euthyroidism in all tissues of the thyroidectomized rat. Endocrinology 137 24902502. (doi:10.1210/en.137.6.2490)

    • Search Google Scholar
    • Export Citation
  • French PJ, O'Connor V, Jones MW, Davis S, Errington ML, Voss K, Truchet B, Wotjak C, Stean T & Doyère V 2001 Subfield-specific immediate early agene expression associated with happocampal long-term potention in vivo. European Journal of Neuroscience 13 968976. (doi:10.1046/j.0953-816x.2001.01467.x)

    • Search Google Scholar
    • Export Citation
  • Fujinoto N, Jinno N & Kitamura S 2004 Activation of estrogen response element dependent transcription by thyroid hormone with increase in estrogen receptor levels in a rat pituitary cell line, GH3. Journal of Endocrinology 181 7783. (doi:10.1677/joe.0.1810077)

    • Search Google Scholar
    • Export Citation
  • Gerges NZ & Alkadhi KA 2004 Hypothyriodism impairs late LTP in CA1 region but not in dentate gyrus of the intact rat hippocampus: MAPK involvement. Hippocampus 14 4045. (doi:10.1002/hipo.10165)

    • Search Google Scholar
    • Export Citation
  • Glinoer D 1997a Maternal and fetal impact of chronic iodine deficiency. Clinical Obstetrics and Gynecology 40 102116. (doi:10.1097/00003081-199703000-00011)

    • Search Google Scholar
    • Export Citation
  • Glinoer D 1997b The regulation of thyroid function in pregnancy: pathways of endocrine adaptation from physiology to pathology. Endocrine Reviews 18 404433. (doi:10.1210/er.18.3.404)

    • Search Google Scholar
    • Export Citation
  • Glinoer D, De Nayer P, Delange F, Lemone M, Toppet V, Spehl M, Grün JP, Kinthaert J & Lejeune B 1995 A randomized trial for the treatment of mild iodine deficiency during pregnancy: maternal and neonatal effects. Journal of Clinical Endocrinology and Metabolism 80 258269. (doi:10.1210/jc.80.1.258)

    • Search Google Scholar
    • Export Citation
  • Guzowski JF 2002 Insights into immediate-early gene function in hippocampal memory consolidation using antisense oligonucleotide and fluorescent imaging approaches. Hippocampus 12 86104. (doi:10.1002/hipo.10010)

    • Search Google Scholar
    • Export Citation
  • Heninger RW & Albright EC 1975 Alteration in tissue and serum concentrations of TSH, iodide, T4 and T3 induced by various dietary iodide levels. Proceedings of the Society for Experimental Biology and Medicine 150 137141.

    • Search Google Scholar
    • Export Citation
  • Herdegen T, Skene P & Bähr M 1997 The c-Jun transcription factor – bipotential mediator of neuronal death, survival and regeneration. Trends in Neurosciences 20 227231. (doi:10.1016/S0166-2236(96)01000-4)

    • Search Google Scholar
    • Export Citation
  • Huang YY, Kandel ER, Varshavsky L, Brandon EP, Qi M, Idzerda RL, McKnight GS & Bourtchouladze R 1995 A genetic test of the effects of mutations in PKA on mossy fiber LTP and its relation to spatial and contextual learning. Cell 83 12111222. (doi:10.1016/0092-8674(95)90146-9)

    • Search Google Scholar
    • Export Citation
  • Jia Y, Gall CM & Lynch G 2010 Presynaptic BDNF promotes postsynaptic long-term potentiation in the dorsal striatum. Journal of Neuroscience 30 1444014445. (doi:10.1523/JNEUROSCI.3310-10.2010)

    • Search Google Scholar
    • Export Citation
  • Kobayashi K, Tsuji R, Yoshioka T, Mino T & Seki T 2006 Perinatal exposure to PTU delays switching from NR2B to NR2A subunits of the NMDA receptor in the rat cerebellum. Neurotoxicology 27 284290. (doi:10.1016/j.neuro.2005.11.008)

    • Search Google Scholar
    • Export Citation
  • Kobayashi K, Akune H, Sumida K, Saito K, Yoshioka T & Tsuji R 2009 Perinatal exposure to PTU decreases expression of Arc, Homer 1, Egr 1 and Kcna 1 in the rat cerebral cortex and hippocampus. Brain Research 1264 2432. (doi:10.1016/j.brainres.2008.12.029)

    • Search Google Scholar
    • Export Citation
  • Lasley SM & Gilbert ME 2011 Developmental thyroid hormone insufficiency reduces expression of brain-derived neurotrophic factor (BDNF) in adults but not in neonates. Neurotoxicology and Teratology 33 464472. (doi:10.1016/j.ntt.2011.04.001)

    • Search Google Scholar
    • Export Citation
  • Lavado-Autric R, Ausó E, García-Velasco JV, Arufe Mdel C, Escobar del Rey F, Berbel P & Morreale de Escobar G 2003 Early maternal hypothyroxinemia alters histogenesis and cerebral cortex cytoarchitecture of the progeny. Journal of Clinical Investigation 111 10731082.

    • Search Google Scholar
    • Export Citation
  • Li JS, Deng JY & Zhou DY 2004 Analysis on iodine nutrition of important people in Sichuan provincel. Journal of Preventive Medicine Information 20 104107.

    • Search Google Scholar
    • Export Citation
  • Morreale D, Escobar G, Obregon MJ & Escobardel RF 2004 Role of thyroid hormone during early brain development. European Journal of Endocrinology 151 U25U37. (doi:10.1530/eje.0.151U025)

    • Search Google Scholar
    • Export Citation
  • Morreale de Escobar G & Escobar del Rey F 2003 Consequences of iodine deficiency for brain development. In The Thyroid and the Brain, pp 33–56. Eds J deVijlder & G Morreale de Escobar. Stuttgart, Germany: Schattauer Verlag.

  • Mun RM, Qin LX & Zhang Y 2006 Iodine deficiency during pregnancy and thyroid disease. Journal of Practical Obstetrics and Gynecology 22 585589.

  • Pedraza PE, Obregon MJ, Escobar-Morreale HF, Escobar del Rey F & Morreale de Escobar G 2006 Mechanisms of adaptation to iodine deficiency in rats: thyroid status is tissue specific. Its relevance for man. Endocrinology 147 20982210. (doi:10.1210/en.2005-1325)

    • Search Google Scholar
    • Export Citation
  • Pop VJ, Brouwers EP, Vader HL, Vulsma T, van Baar AL & de Vijlder JJ 2003 Maternal hypothyroxinaemia during early pregnancy and subsequent child development: a 3-year follow-up study. Clinical Endocrinology 59 282288. (doi:10.1046/j.1365-2265.2003.01822.x)

    • Search Google Scholar
    • Export Citation
  • Ranieri F, Podda MV, Riccardi E, Frisullo G, Dileone M, Profice P, Pilato F, Di Lazzaro V & Grassi C 2012 Modulation of LTP at rat hippocampal CA3-CA1 synapses by direct current stimulation. Journal of Neurophysiology 107 18681880. (doi:10.1152/jn.00319.2011)

    • Search Google Scholar
    • Export Citation
  • Sethi V & Kapil U 2004 Iodine deficiency and development of brain. Indian Journal of Pediatrics 71 325329. (doi:10.1007/BF02724099)

  • Shi XG, Teng XC & Shan ZY 2009 An epidemiological study of the relationship between iodine intake levels and thyroid function during early pregnancy. Chinese Journal of Practical Internal Medicine 29 520522.

    • Search Google Scholar
    • Export Citation
  • Tremont G, Stern RA, Westervelt HJ, Bishop CL & Davis JD 2003 Neurobehavioral functioning in thyroid disorders. Medicine and Health, Rhode Island 86 318322.

    • Search Google Scholar
    • Export Citation
  • Vara H, Munoz-Cuevas J & Colino A 2003 Age-dependent alterations of long-term synaptic plasticity in thyroid-deficient rats. Hippocampus 13 816825. (doi:10.1002/hipo.10132)

    • Search Google Scholar
    • Export Citation
  • WHO/UNICEF/ICC IDD 2007 Assessment of Iodine deficiency disorders and Monitoring their Elimination. A Guide for Programme Managers. 3rd edition, pp 1–108.

  • Zhang ZZ, Guo HY & He LL 2005 Iodine nutritive surveillance of typical population in partial areas of the inner mongolia autonomous region. Chinese Journal of Endemiology 24 305307.

    • Search Google Scholar
    • Export Citation