Mechanism Study On Iodine Deficiency And Hypothyroidism Impair Neural Development And Expression Of Related Proteins Of Caveolin-1 And Doublecortin In Rat Hippocampus

Iodine is an essential trace element that is critical for the synthesis of thyroid hormone (TH). The maintenance of thyroid function directly depends on adequate availability of dietary iodine. Iodine deficiency is one of the most common, preventable causes of brain damage in the world. Sufficient levels of TH, thyroxine (T4), and 3,5,3-triiodothyronine (T3) are indispensable to mammalian brain development and metabolic homeostasis, and they play a vital role in hippocampal formation; the hippocampus expresses receptors for TH. It is clear that hypothyroidism results in stunted growth and impairs brain development. Caveolin-1 plays an important role in the formation of plasma membrane caveolae and anchors them to the actin cytoskeleton, regulates cell interactions with the extracellular matrix, pulls together and modulates signaling molecules, and transports cholesterol. Moreover, caveolin-1 may participate in neuronal differentiation and maturation because it is localized to the growth cones and dendrites of hippocampal neurons. Synaptophysin is an integral protein of small pre-synaptic vesicles that is involved in the release of neurotransmitter vesicles and present in virtually all nerve terminals.The levels of synaptophysin protein expression parallel the numbers of synapses and the density and quantity of nerve terminals. Doublecortin could regulate the migration of multiple classes of cortical neurons, facilitating migration via actions at the distal ends of neurites that promote neurite extension. Neural cell adhesion molecule (NCAM)-180 has been implicated in critical morphogenetic processes during CNS development such as neuronal migration and layering, and axonal growth, guidance and fasciculation. Importantly, researches have determined that iodine deficiency-induced developmental defects of the CNS are irreversible in fetuses and children, and hypothyroidism alteres synaptic development and function. However, the underlying mechanisms are not known. Thus, caveolin-1, synaptophysin, doublecortin and NCAM-180 may be involved in the deficits in neural development that follow iodine deficiency and hypothyroidism. The present study provides an important complement to the existing literature regarding alterations in hippocampal development induced by iodine deficiency and PTU-induced hypothyroidism.Methods1,AnimalsWistar rats (250-280 g) were obtained from the Center for Experimental Animals at China Medical University (Shenyang, China). Rats were housed at an environmental temperature of 24±1℃on a 12/12-h light/dark cycle and had access to food and water ad libitum. The day the vaginal plug was discovered was considered to be gestational day (GD) 0. The pregnant rats were randomly assigned into four treatment groups (n= 7 per dose group):control group, iodine-deficient group,5 ppm PTU-treated group, or 15 ppm PTU-treated group. The iodine-deficient group was administered with an iodine-deficient diet (iodine content:14.11±1.96 ng/g) and tap water from GD6 until postnatal day (PN) 28. Rats of the PTU-treated groups were administered 5 ppm or 15 ppm PTU in the drinking water and fed with a normal diet (iodine content: 470.50±46.52 ng/g) from GD6 to PN28. Each litter was culled to 9-10 pups on PN4 (equal numbers of males and females in each group when possible). On PN14, PN21, PN28, or PN42,8 pups in each group were weighted and anesthetized by etherization.2,Observation of body weight and measurement of FT3, FT4, and TSH During growth and development, all the pups were observed carefully on the body weight. Heart blood samples were obtained from 8 pups in each group at each time point, and serum was separated and stored at-70℃for subsequent measurement of free triiodothyronine (FT3), free thyroxine (FT4), and thyroid stimulating hormone (TSH) by a supersensitive chemiluminescence immunoassay.3,Measurement of surviving neuronsOn PN14, PN21, PN28, and PN42, the brains of 5 rats per group were preserved via intracardiac perfusion with 50-100 ml normal saline containing 0.02% heparin, followed by 200-400ml 4% paraformaldehyde in 0.1 M potassium phosphate buffer (pH 7.4). Then, the brains were quickly removed from the skull and fixed overnight in the same fixative. The fixed brains were embedded in paraffin and sectioned into 6-μm-thick coronal sections. Brains were sectioned in a serial manner when intact structure of hippocampus was observed in the slices. Every fifth/sixth slice was collected per animal on gelatin-coated microscope slides. Three sections of each rat brain were selected randomly for Nissl staining. After deparaffinization in xylene for 10 min followed by 100% ethanol, slides were washed in deionized water. Then, slides were performed with routine Nissl staining based on the thionine technique and then analyzed under a microscope. The hippocampal subregions of interest were selected: CA1, CA3, and dentate gyrus (DG). Imaging-Pro-Plus (Media Cybernetics, Inc., Silver Spring, MD) was used to perform quantitative analysis of cell number. Total number of surviving cells was counted in a blinded manner only if structures were identified clearly. Three different fields were selected from CA1, CA3, or DG regions per section respectively and three sections per animal were evaluated to obtain a mean value. Five rats per group per time point were used to obtain an overall mean value for subsequent statistical analysis.4,Analysis of nerve fibersOn PN14, PN21, PN28, and PN42, the brains of 5 rats per group were preserved via intracardiac perfusion with 50-100 ml normal saline containing 0.02% heparin, followed by 200-400ml 4% paraformaldehyde in 0.1 M potassium phosphate buffer (pH 7.4). Then, the brains were quickly removed from the skull and fixed overnight in the same fixative. The fixed brains were embedded in paraffin and sectioned into 6-μm-thick coronal sections. Brains were sectioned in a serial manner when intact structure of hippocampus was observed in the slices. Every fifth/sixth slice was collected per animal on gelatin-coated microscope slides. Three sections of each rat brain were selected, and then they were stained by silver staining. The sections were microscopically analyzed at 40×magnification for the hippocampal sub-regions of interest:CA1, CA3, and DG. The boundaries for the sub-regions were determined according to the Paxions-Wastson atlas of the rat brain. Then images were obtained at a magnification of 400×to analyze the nerve fibers and cell bodies.5,Detection of related proteinsThe brains of the 3 pups per group per time point were rapidly dissected from the skull and immediately submerged in ice-cold artificial cerebrospinal fluid. Then, the hippocampus was removed and dissected on ice rapidly. With the help of a dissecting microscope, the hippocampus was bisected and the dorsomedial half was divided into four slabs cut perpendicular to the long axis of the hippocampus. According to the Paxions-Wastson atlas of the rat brain, each slab was dissected into CA1, CA3 and DG regions. The protein was estimated by coomassie brilliant blue staining. Proteins were transferred onto nitrocellulose membranes. After blocking the nonspecific sites with PBS containing 0.1% Tween 20 and 5% defatted dried milk, membranes were washed and incubated with primary antibody, rabbit anti-caveolin-1, rabbit anti-synaptophysin, rabbit anti-doublecortin, rabbit anti-NCAM and rabbit anti-β-actin. Blots were developed with the Easy Enhanced Chemiluminescence Western Blot Kit. For each blot, the P-actin lanes were analyzed as a quality control sample. The signals from target bands on each blot were normalized to the mean signal for the quality control sample bands in order to simplify the comparisons across the blots and reduce inter-blot variability. 6,Statistical analysisAll analyses were carried out using SPSS software, and the analyzers were blind to the treatment of each group. Data are presented as mean±standard deviation (SD) and considered statistically significant at P＜0.05. To verify consistent protein loading among the gels, some blots were probed for P-actin and then the ratio with caveolin-1 or synaptophysin or doublecortin or NCAM was determined. There was no statistical difference between the density percent control and the ratio with P-actin; therefore, the analyses for the percent control are presented for each blot. At each time point, comparisons of the pups'body weights, FT3, FT4 and TSH levels, surviving cell numbers, protein levels of caveolin-1, synaptophysin, doublecortin and NCAM-180 were made using one-way ANOVA. When the F-value indicated significance, least-significant difference post hoc comparisons were made as appropriate to correct for multiple comparisons. All P-values were two-tailed.Results1,Changes of TH and TSHReductions in TH with concomitant elevations in TSH were observed in hypothyroid offspring by the researcher. Interestingly and in line with this statement, our data showed that the offspring displayed hypothyroxinemia in iodine-deficient group (reduced FT4 with no significant increase in TSH) and hypothyroidism in 5ppm and 15ppm PTU-treated groups. The iodine-deficient and 15ppm PTU-treated groups had significantly lower serum FT3 and FT4 than the controls on PN14, PN21, and PN28 (P＜0.05). TSH levels were increased significantly in 5ppm and 15ppm PTU-treated offspring than controls (P＜0.05). On PN42, the concentrations of serum FT3, FT4, and TSH in iodine-deficient and PTU-treated groups were restored.2,Alterations of body weightsMany studies have shown that PTU treatment reduces offspring body weights. Our data shown that the offspring's body weights in iodine-deficient,5ppm and 15ppm PTU-treated groups were statistically significant lower than those of controls on PN14, PN21, PN28, and PN42 (P＜0.05)3,Impairment of neuronsIodine deficiency and hypothyroidism reduce hippocampal surviving cells. To investigate whether or not hippocampal neuronal survival was impaired by iodine deficiency and hypothyroidism, histological examination of hippocampal neurons was performed on Nissl-stained sections. The results reveal lower surviving cells in the hippocampal neurons of offspring with low circulating TH levels in the CA1, CA3, and DG regions on PN21, PN28, and PN42(P＜0.05). The mean number of surviving cells in the hippocampus of the iodine-deficient,5 ppm PTU-treated, and 15 ppm PTU-treated rats was reduced compared to controls. On PN28 and PN42, compared with the control group, nerve fibers were found injured in the treated groups of regions CA3 and DG, as well as reduced in number. Injury appeared as fibers broken, disordered, bundled together, or fused with other fibers. In the control group, healthy nerve fibers were observed in all regions. The control nerve fibers were arranged in a good order with clear processes. Unlike regions CA3 and DG, nerve fibers in region CA1 were not yet damaged on PN28.4,Effects on the related proteinsOn PN14, PN21, PN28, and PN42, caveolin-1, synaptophysin, doublecortin and NCAM-180 were expressed in the CA1, CA3, and DG regions of the hippocampus of all groups, respectively. Significant upregulation of caveolin-1 was observed in rats exposed to the iodine-deficient,5 ppm PTU-adulterated, or 15 ppm PTU-adulterated diet relative to controls (P＜0.05). Synaptophysin was significantly down-regulated in rats exposed to the iodine-deficient,5 ppm PTU-adulterated, or 15 ppm PTU-adulterated diet relative to the controls (P＜0.05). A significant downregulation of doublecortin was observed in rats exposed to iodine-deficient and 15 ppm PTU-treated groups in CA1, CA3, and DG regions (P＜0.05). Upregulation of NCAM-180 in CA1, CA3, and DG regions were observed in iodine-deficient and 15 ppm PTU-treated rats from PN14 on (P＜0.05). Especially on PN42, we observed doublecortin decrease and NCAM-180 increase when TH levels were restored to a normal state, suggesting irreversible hippocampus impairment.Conclusion1,Iodine deficiency or PTU-induced hypothyroidism led to morphological damage in the hippocampus. Lower surviving cells in the hippocampal neurons of offspring with low circulating TH levels in the hippocampal subregions and nerve fibers were impaired.2,Developmental iodine deficiency or PTU-induced hypothyroidism led to downregulation of doublecortin and synaptophysin and upregulation of Caveolin-1 and NCAM-180 in the hippocampal subregions at each time point.3,The alterations of hippocampal morphology and expression of related proteins were still obvious on PN42 despite the recovery of TH to normal status.