| ObjectiveAlthough recent clinical researches have reported an increased risk for cognitive disorders in Hashimoto’s thyroiditis(HT)patients,even in the euthyroid state,the mechanisms involved remain unclear.The hippocampus is a classic brain region associated with cognitive function,among which the formation of long-term potentiation(LTP)in the Schaffer collateral-CAl pathway plays an important role in the process of learning and memory.Therefore,this study established an euthyroid HT model in mice to investigate the effect of HT itself on cognitive function.At the same time,we evaluated the alterations of synaptic plasticity in Schaffer collateral-CAl pathway and explored the possible reasons for these changes.MethodsForty NOD female mice aged 6-7 weeks and weighing 18-20 g were randomly divided into two groups(control group:20 mice and HT group:20 mice)after acclimatizing for one week.Experimental HT was established by subcutaneously immunizing mice twice with porcine thyroglobulin(Tg)combined with Freund’s adjuvant.Control mice were injected with phosphate-buffered saline(PBS)in adjuvant at the same time intervals as porcine Tg-injected animals.Four weeks after modeling,the cognitive function of both groups of mice was measured by Morris water maze test.After behavioral studies,6 mice from each group were randomly selected to measure the basic synaptic transmission efficiency and induce LTP in the hippocampal Schaffer collateral-CA1 pathway by recording field potential in vivo.When the electrophysiological experiment was done,all the mice were sacrificed under deep anesthesia.Blood and thyroid tissue were collected,and brain tissue was taken on ice to isolate the hippocampus.Blood was centrifuged to collect serum,electrochemiluminescence immunoassay method and enzyme-linked immunosorbent assay were used to measure thyroiditis-related parameters in the serum of mice in each group.The score of thyroiditis was evaluated by histopathological examination with Hematoxylin and eosin staining.The ultrastructural changes of synapses as well as the neurons and astrocytes were observed by transmission electron microscopy in hippocampal CAl area of the two groups of mice,TUNEL staining was used to observe neuronal apoptosis.The distribution of astrocyte marker GFAP was determined by immunohistochemistry and immunofluorescence in paraffin section of brain tissue.The contents of glutamate,a neurotransmitter closely related to cognition,in the hippocampus were analyzed by high performance liquid chromatography combined with mass spectrometry.Real time fluorescence quantitative PCR and Western Blot were used to evaluate the expression of glutamate cycle-related proteins in the hippocampus.Results1.Establish a euthyroid HT model in mice.①Body weight,hippocampus weight and hippocampal coefficient.During the experiment,there was no significant difference in the weight of the two groups of mice.The hippocampus was isolated and measured weight after sacrifice to calculate the hippocampus coefficient.The results showed that there was no statistical difference in body weight(p=0.66),hippocampal weight(p=0.97)and hippocampal coefficient(p=0.92)between the two groups.②Thyroid histopathology.Observed by naked eyes,the thyroid of mice in HT group was larger than that in control group.HE staining showed that the thyroid follicles in the control group were intact and evenly distributed,and there was no obvious lymphocyte infiltration between the follicles.Meanwhile,the thyroid follicles in the HT group were damaged and arranged disorderly,and a large number of lymphocytes infiltrated between the follicles.According to the degree of lymphocyte infiltration,the thyroiditis score of HT group was significantly higher than that of the control group,and the difference was statistically significant(p<0.01).③The levels of thyroid hormones and thyroid autoantibodies in serum.Compared with the control group,the levels of T3,T4 and TSH in the serum of mice in the HT group were not statistically different,while the levels of TPOAb and TgAb were significantly increased,and the differences were statistically significant(p<0.01).④The levels of thyroid hormones and thyroid autoantibodies in the hippocampus.Compared with the control group,there were no significant differences in the levels of T3 and T4 in the hippocampus of the HT group,but the levels of TgAb were significantly increased(p<0.01).2.Morris water maze test results.①Orientation navigation test.The escape latency time of two training groups became shorter as training days increased.Meanwhile,there was no statistically significant difference between the control group and the HT group in swimming speed(p>0.05).Compared with the control group,the mean escape latency and swimming distance in the HT mice were longer than those of the control group on days 2-5 of the training,and the differences were statistically significant(p<0.01).② Spatial probe test.Compared with the control group,the percentage of swimming time and distance in the target quadrant of HT group decreased,and the difference was statistically significant(p<0.01)3.In vivo electrophysiology.① The basic synaptic transmission efficiency of hippocampal Schaffer collateral-CA1 pathway(I/O curve).Compared with the control group,the I/O function of hippocampal CA1 region in HT group was significantly decreased(p<0.05).②Long-term potentiation in hippocampal Schaffer collateral-CA1 pathway.In this study,LTP was induced in hippocampal CA1 region by high frequency stimulation.Compared with the control group,the LTP amplitude of mice in the HT group was reduced,and the difference was statistically significant(p<0.05)4.Observation of synaptic ultrastructure in hippocampal CA1 region.Transmission electron microscopy showed that the ultrastructure of the hippocampal synapses revealed lower synaptic density(p<0.01),shorter active zone length(p<0.05),thinner PSD(p<0.05),and decreased synaptic curvature(p<0.01)in the HT mice compared with those in the control mice.The other synaptic interface parameters,such as synaptic cleft,did not show differences between groups5.Observation of neuron ultrastructure in hippocampal CA1 region.Transmission electron microscopy showed that in the control group,the nuclear membrane is smooth and complete,chromatin is evenly distributed,and organelles such as ribosomes,mitochondria,and endoplasmic reticulum can be seen in the cytoplasm The ultrastructure of the neuron in the HT group was similar to that in the control group.Neither of the two group was observed the typical morphological characteristics of apoptosis,such as chromatin condensation,nuclear pyknosis and apoptotic corpuscular formation.Further TUNEL staining showed no significant difference in TUNEL positive neurons in the hippocampus between the two groups6.Observation of astrocyte ultrastructure in hippocampal CA1 region.Transmission electron microscopy showed the classical appearance of astrocyte with a narrow rim of chromatin beneath the nuclear membrane in the control group.The cytoplasm was pale and sparse,contained an endoplasmic reticulum(arrows)and a few mitochondria.In the HT group,the chromatin in the nucleus in the astrocyte was clumped and coagulated,the cytoplasm was vacuolated,accompanied by swelling of mitochondria,expansion of endoplasmic reticulum,and partial exfoliation of ribosomes in endoplasmic reticulum.GFAP,a marker of astrocytes,was further detected by immunohistochemistry and immunofluorescence staining.Real-time fluorescence quantitative PCR and Western Blot were used for quantitative analysis of GFAP.Compared with the control group,the average optical density of GFAP and the number of GFAP labeled positive cells in the hippocampus in HT group were significantly decreased,and the relative expression levels of GFAP mRNA and protein were also decreased,the differences were statistically significant(p<0.01)7.Evaluation of glutamate-glutamine cycle function in hippocampus.HPLC-tandem mass spectrometry was used to analyze the changes of glutamate content in the hippocampus.The results showed that,compared with the control group,the content of glutamate in the hippocampus of HT mice was significantly higher(p<0.01).GLT-1 and GLAST,which are located in astrocytes,are responsible for glutamate uptake in the synaptic cleft.GS can convert glutamate which enters astrocyte into glutamine Compared with the control group,the mRNA levels and protein expressions of GLT-1,GLAST and GS in the hippocampus of HT group were significantly decreased(p<0.01),and the expression of NR2B subunit in the NMDAR receptor was also decreased,the difference is statistically significant(p<0.01)ConclusionOur research confirms that euthyroid Hashimoto’s thyroiditis can also cause hippocampal-dependent learning and memory dysfunction in mice.The reason can be at least partially attributed to astrocyte damage,which results in a decrease in the number of synapses,damage to the synaptic structure and dysfunction of glutamate-glutamine cycle in the hippocampus and ultimately impair the hippocampal Schaffer collateral-CA1 pathway LTP. |
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