Mechanism Study On Iodine Deficiency And Hypothyroidism Impairing Spatial And Conditional Memory And CaMKⅡ And MAPK Signal Pathway In The Hippocampus

ObjectiveIodine is essential for the synthesis of triiodothyronine(T₃) and thyroxine(T₄). Thyroid hormones play a vital role in the process of early growth and development of most organs,especially the brain.Hippocampus is a brain region involved in cognitive skills.Thyroid hormones deficiency during brain development leads to irreversible impairment of hippocampus,affects synaptic plasticity and cognitive functions,such as passive avoidence,spatial learning,and conditional memory.CaMKâ…¡and MAPK signal pathways are critical to generation of learning and memory.The mechanism underlying iodine deficiency and hypothyroidism impairing learning and memory and CaMKâ…¡and MAPK signal pathway in the hippocampus is unclear.Implicatios for some researchs on the effect of iodine deficiency and hypothyroidism on CA1,CA3 and DG regions of hippocampus need to be carried out.Through gestation and lactation,iodine deficient or hypothyroid darn rats were administered either an iodine-deficient diet or PTU-added drinking water.We have examineed the parameter of water maze and shutter box in pups,and tested whether iodine deficiency and hypothyroidism could regulate CaMKâ…¡and MAPK signal pathway in pups hippocampus.To figure out the mechanism that iodine deficiency and hypothyroidism impair synaptic plasticity and learning and memory.Methods 1.AnimalsFemale Wistar rats(n=28) aging 2 after pregenancy were randomly divided into four groups:control group,iodine deficient group,hypothyroid-1 group(5ppm) and hypothyroid-2 group(15ppm).The control group was fed a normal diet and tap water during the experiment.The iodine deficient group was fed an iodine-deficient diet and tap-water.The hypothyroid-1 group was fed a normal diet and had 5ppm PTU-added water and the hypothyroid-2 group nd had 15ppm PTU-added water.All the groups were from GD6 through to postnatal day(PN) 28.Dams and pups in each group were weighed frequently throughout test.Eye opening was examined by daily observation between PN15 and PN19.2.Thyroid hormonesBlood sample were collected from pups in all groups in PN14,PN21,PN28 and PN42,just before dissection of the hippocampus,and immediately centrifuged at 3000 rpm for 5 min.All the serum parameters were measured by super-sensitive chemiluminescence immunoassay(IMMULITE,Diagnostic Products Corporation,Los Angeles,CA,USA).3.Hippocampus weightPups were sacrificed in PN14,PN21,PN28 and PN42,and the hippocampus were immediately dissected out and weighted.4.ImmunohistochemistryThe rats on PN7,PN14,PN21 and PN28 were weighed,deeply anesthetized and perfused intracardially with 50～100 ml normal saline containing 0.02%heparin followed by 100～200 ml of 4%paraformaldehyde.Brains were removed and fixed overnight in the same fixative and embedded in paraffin and sectioned coronally with a microtome into 6-μm-thick sections.After incubated with the rabbit anti-total CaMKâ…¡,p-CaMKâ…¡,CaN,Ng,BDNF,total ERK1/2, p-ERK1/2,total CREB or p-CREB all sections were observated and integrated optical density total in the hippocampus CA1,CA3 and DG regions was measured using a cast-grid microscope with an image-analysis program.5.Western blotting Pups were sacrificed in PN14,PN21 and PN28,and the CA1,CA3 and DG regions of hippocampus were immediately dissected out and homogenized in 300μl of buffered isotonic cocktail containing protease and phosphatase inhibitors.The samples were subjected to SDS-PAGE,transferred protran,and incubated with rabbit/mouse antitotal CaMKâ…¡,p-CaMKâ…¡,CaM,CaN,Ng,BDNF,total ERK1/2,p-ERK1/2,total CREB or p-CREB and second antibodies.Bound antibody was bisualised using the enhanced chemiluminescence reagents and exposed to X-ray film.Densitometric analysis was performed on a computer using image program.6.Water mazeOn PN30,10-12 pups per group began training in the water maze.The task requires pups to swim to a platform.After 3 days' training,pups were placed in the distal of water maze in the 4^th day,and swam randomly.The numbers of error and latency of platform were recorded and analysised.7.Shuttle boxOn PN37,10-12 pups per group began training in the shuttle box.The numbers of shock,times of passive and active avoidance were recorded and analysised during 6 days' training.8.Effects on CaMKâ…¡and MAPK signal pathway by learning and memoryOn PN42,5 pups were perfused.Brains were fixed and stained by immunohistochemistry, and the expression of proteins on CaMKâ…¡and MAPK signal pathway was observated.3 pups were sacrificed and the CA1,CA3 and DG regions of hippocampus were immediately dissected out and homogenized.The levels of proteins on CaMKâ…¡and MAPK signal were measured by western blotting.9.StatisticsResults were expressed as mean±S.E.M.All analyses were carried out using the SPSS 11.5 software.Differences among multiple groups were analyzed using one-way analysis of variance (ANOVA) followed by Student-Newman-Keuls(SNK) test when F was significant.P values of less than 0.05 were considered statistically significant. Results1.Weights of dam,pup and hippocampus,eye openingIn all three groups of dam rats,dams from iodine-deficient groups were significantly lighter than controls in pregancy and gestation(P＜0.05).Weights of pup and hippocampus from iodine-deficient and 15ppm treatment groups were significantly smaller than controls(P＜0.05) from PN3 to PN42.Eye opening was delayed in iodine-deficient and 15ppm treatment groups, and the ratio of pups within a litter with both eyes opening was lower than controls in the same time.2.Thyroid hormones levelsIn PN14,PN21 and PN28,in 5ppm and 15ppm treatment groups,TSH was increased significantly comparing to controls(P＜0.05),whereas in iodine-deficient groups,there was no significant difference comparing to controls.In terms of serum FT₃ and FT₄ concentration, iodine-deficient and 15ppm treatment groups were significantly lower than those of controls (P＜0.05).On PN42,TSH and TH levels were returned to normal status.3.ImmunohistochemistryIn PN14,PN21 and PN28,staining for total CaMKâ…¡,Ng,BDNF,ERK1/2 and CREB in CA1 and CA3 regions of the hippocampus in iodine-deficient and 15ppm treatment groups was significantly lower than those of controls,and staining for CaN was higher than those of controls(P＜0.05).In DG region,the expression of CaMKâ…¡,CaN, BDNF and CREB in iodine-deficient and 15ppm treatment groups was significantly lower than those of controls(P＜0.05),while the expression of Ng and ERK1/2 was no significant difference in all four groups.4.Western blottingIn PN14,PN21 and PN28,protein levers of total CaMKII,p- CaMKâ…¡,Ng, BDNF,p- ERK1/2,ERK1/2,p- CREB and CREB in CA1 and CA3 regions of the hippocampus in iodine-deficient and 15ppm treatment groups was significantly lower than those of controls,and the levers of CaN was higher than those of controls(P＜0.05).In DG region, the expression of total CaMKâ…¡,CaN,BDNF,p- CREB and CREB in iodine-deficient and 15ppm treatment groups was significantly lower than those of controls(P＜0.05),while the levers of p-CaMKâ…¡,CaM,Ng,ERK1/2 and p- ERK1/2was no significant difference in all four groups.5.Behavioral performanceAfter training on water maze,the numbers of error and latency of platform of pups in iodine-deficient and 15ppm treatment groups were significantly increased compared to those of controls.From the third to 6^th of training on shuttle box,the numbers of shock,times of passive and active avoidance were in iodine-deficient and 15ppm treatment groups were significantly increased compared to those of controls(P＜0.05).6.Effects on CaMKâ…¡and MAPK signal pathway by learning and memoryAfter training of behavioral trials,in hippocampus,the expression and levers of CaN were decreased and the total CaMKâ…¡,p- CaMKâ…¡,Ng,BDNF,p- ERK1/2,ERK1/2, p- CREB and CREB were significant increased in control groups(P＜0.05).Excluding of CaN's increase,the expression and levers of all proteins were no significantly difference in iodine-deficient and 15ppm treatment groups in CA1 and CA3 regions.In DG region,the expression and levers of CaN were significantly lower than that of pre-training in iodine-deficient and 15ppm treatment groups,while p-CaMKâ…¡,CaM,Ng,ERK1/2,p-ERK1/2, CREB and p-CREB increased significantly(P＜0.05).Conclusion1.Iodine deficiency and hypothyroidism may effect pups development and lead to the weights decrease and eye opening delay.2.Iodine deficiency and hypothyroidism may increase the activity of CaN in CA1 and CA3 regions and dyregulate the CaMKâ…¡signal pathway.In DG regions,iodine deficiency and hypothyroidism can not effect the expression and levers of p-CaMKâ…¡, CaM and Ng.3.Iodine deficiency and hypothyroidism may dyregulate the MAPK signal pathway in CA1 and CA3 regions.In DG region,iodine deficiency and hypothyroidism can not effect the expression and levers of ERK1/2 and p-ERK1/2.4.Iodine deficiency and hypothyroidism may impair the generation of spatial and conditional memory,and result in increase of latency to platform,times of activative and passive invoidence,numbers of error and shock.Compared to PN28,the expression and levers of CaMKâ…¡and MAPK signal pathway failed to change in CA1 and CA3 regions of hippocampus,but the expression and levers of p-CaMKâ…¡,CaM, Ng,ERK1/2,p-ERK1/2,CREB and p-CREB increased significantly.5.The mechanism of iodine deficiency and hypothyroidism impairing spatial and conditional memory might contribute to that transient exposure to a period of iodine deficiency and hypothyroidism during a period of brain development increased the activity of CaN in CA1 and CA3 regions of hippocampus,reduced the proteins in CaMKâ…¡and MAPK signal pathway,impaired synaptic plasticity,effected the development of brain,and resulted in learning and memory deficits.