The major findings of this study with rats exposed to developmental iodine deficiency or PTU-induced hypothyroidism were that (1) nerve fibers in the hippocampal CA1, CA3, and DG regions were impaired, (2) the levels of doublecortin were lower than controls, and (3) the levels of NCAM-180 were increased. We have previously shown that the concentrations of serum TH in the iodine-deficient and PTU-treated groups gradually returned to control levels by PN42 . However, the recovery of TH on PN42 did not rescue the alterations in doublecortin and NCAM-180 in the hippocampus regions. These findings are in consistent with the irreversible hippocampal injuries following developmental iodine deficiency and PTU-induced hypothyroidism in rats [47, 48].
Iodine deficiency is the most frequent cause of preventable brain damage in the world. However, remedies to eliminate iodine deficiency disorders are slow to develop . Fetuses and infants among the population at high risk for iodine deficiency and its associated reduced intellectual ability . It is generally accepted that the rapid rate of brain growth in the late gestation and early postnatal stages make the immature brain susceptible to iodine and subsequent thyroid deficiency . Our previous study has shown that iodine deficiency is still a serious public health problem in China . The present study is a follow-up project to explore neuronal degeneration in two rat models following developmental exposure to low levels of TH.
TH is essential for brain differentiation processes, including dendritic and axonal growth, synaptogenesis, neuronal migration, myelination, and expression of specific mature neuron proteins . Inadequate supply of TH to the developing brain leads to several histological changes in the brain, such as decreased axonal density, reduced numbers of dendrites and dendritic spines, and delayed synaptogenesis [52, 53]. Our previous study revealed that low circulating TH levels induce deleterious hippocampal morphology changes in the CA1, CA3, and DG regions on study days PN21, PN28, and PN42 . In the current study, observation of the silver-stained neurons of the CA1, CA3, and DG regions showed that nerve fiber injuries were observed at later stages of brain development. In the silver-staining tissue sections there were far fewer healthy nerve fibers in the treatment groups compared to controls. It deserves noting that there was no visible nerve fiber injury in the early stages on PN14 and PN21. Currently the authors do not have evidence to explain this time delay. It could be due to the fact that most of the brain structures examined in these rats are formed by the third postnatal week . Indeed, a delayed damage was also observed in the CA1 following transient ischemia . Taken together with our previous analysis , we speculate that iodine deficiency and hypothyroidism may lead to deficits in neural development, degeneration of hippocampal nerve fibers, and thus an impairment of neuronal function.
It is clear that hypothyroidism results in stunted growth and impairs brain development [56, 57]. A recent study has concluded that the hypothyroidism-induced developmental damages of the hippocampus take place in CA1, CA3, and DG regions . In the hippocampus, DG granule cells project to the dendrites of pyramidal cells of CA3 via mossy fibers. At the same time, these cells contribute a major input to CA1 (the Schaffer collaterals). This circuit is implicated in different functions of hippocampus . The neuronal progenitor cells in the dentate subgranular zone (SGZ) can differentiate into neurons and glial cells [59, 60]. Some of the newborn neurons migrate to the granular layer and extend mossy fiber axons, indicating that they are integrated into the functional circuitry of the hippocampus [61–64]. Given that doublecortin is a marker of new neurons and expresses in differentiating and migrating neurons , a reduction of doublecortin by dietary iodine deficiency and PTU-induced hypothyroidism in the present study is in agreement with a reduction in nerve fibers and newborn neurons. Our data suggest a connection between hippocampal morphology injuries and iodine deficiency and hypothyroidism. Considering that a reduction of newly born granule cells may influence mossy fiber innervations in the CA3 region, it is conceivable that iodine deficiency and hypothyroidism may subsequently impair neuronal connectivity, pyramidal cell excitability and memory formation [65–67] by reducing newborn granule cells and nerve fiber innervations in the hippocampus.
NCAM is a cell adhesion molecule that mediates homophilic adhesion between cells to regulate CNS development [22, 23]. The NCAM isoform, NCAM-180, plays an important role in synaptic remodeling and LTP . Hence, NCAM plays an important role in structural remodeling of the nervous system, and one of these important functions is to hold developing neurites together during neuron outgrowth and formation of neural connections . It has been shown that both doublecortin and NCAM are regulated by TH via a classical genomic molecular mechanism . Indeed, this finding is in agreement with our observations of the implications of the two proteins in the structural injuries in the hippocampus following developmental iodine deficiency and hypothyroidism in rat pups. In addition, our data of upregulation of NCAM expression by iodine deficiency and hypothyroidism is in consistent with the literature where hypothyroidism disrupts cell migration [69, 70]. As we know that cell adhesion molecules serve as guidance clues for migration and axonal growth , via which the migrating neurons interact with extracellular matrix proteins to find the migration path. Therefore, NCAM can hold developing neurites together during neuron outgrowth and formation of neural connections . So, hypothyroidism-induced NCAM-upregulation may alter the normal neuronal development, including the nerve fiber injuries. However, so far, the authors do not know the mechanisms underlying hypothyroidism-upregulated NCAM expression in the hippocampus. It could be due to NCAM gene upregulation by hypothyroidism . There is also another piece of evidence supporting the transcriptional upregulation of NCAM in the neonatal rat brain by hypothyroidism . Furthermore, it cannot be excluded that iodine deficiency and PTU-induced hypothyroidism may fail to suppress the activation of NCAM-180, leading to the up-regulation of NCAM-180 expression .
Concerning NCAM expression, we did see a discrepancy with the literature, where Gilbert and colleagues have shown that PTU starts to upregulate NCAM 1 genes at 3 ppm . In contrast, in the present study NCAM-180 protein expression is not sensitive to PTU at 5 ppm. The authors do not have an answer for this difference, but it could be due to protein expression changes being less sensitive than the gene regulations. In addition, it should also be taken in account that this paper used a different test method, sampled at a different time point, and investigated different hippocampus regions from the Gilbert study .
Interestingly, we observed doublecortin decrease and NCAM-180 increase on PN42 when TH levels were restored to a normal state, suggesting irreversible hippocampus impairment. This is in line with many pieces of evidence, where maternal iodine deficiency during pregnancy and lactation are confirmed as one of the causes of irreversible CNS damage in offspring [43, 45, 47, 48, 71, 72]. In summary, we used two rat models to show that developmental iodine-deficiency and hypothyroidism causes irreversible mal-regulation of doublecortin and NCAM-180 a few weeks before the obvious injury of nerve fibers in the hippocampus, demonstrating that the developmental iodine deficiency and hypothyroidism impair the expression of doublecortin and NCAM-180, leading to nerve fiber malfunction and thus the impairment in hippocampal development. A detailed mechanism is still lacking by which developmental iodine deficiency and hypothyroidism regulate doublecortin and NCAM proteins and deserves further investigation.