The Effect Of Hashimoto’s Thyroiditis On Emotional Behavior In Mice And Its Neuroinflammatory Mechanisms

Objective Hashimoto’s thyroiditis(HT),also called chronic lymphocytic thyroiditis,is the most common cause of hypothyroidism.It is now well-recognized that hypothyroidism may affect the mood of patients.However,recently,increasing clinical studies have reported that even within normal thyroid function,HT is associated with abnormal emotional behaviors,especially depression and anxiety,the mechanisms involved remain unclear.Neuroinflammation may play a key role in the etiology of mood disorders in humans and behavioral disturbances in rodents.Therefore,this study established a euthyroid HT model in mice and investigated the effect of HT itself on emotional behavior in mice.Meantime,we focused on the frontal cortex and set out to examine the cellular and molecular events associated with neuroinflammation,such as the activation status of microglia and astrocytes as well as the expression of pro-inflammatory cytokines.In a word,this study aimed to investigate whether HT itself was capable of triggering neuroinflammation accompanied by emotional alterations.Methods After 7 days of acclimatizing,female NOD mice were randomly divided into two groups,namely the control group(Con group)and Hashimoto’s thyroiditis group(HT group).Experimental HT was induced by immunizing NOD mice with thyroglobulin(Tg)and adjuvant twice.Mice treated with adjuvant at the same time served as controls.Four weeks after the last challenge,mice were tested for anxiety-like behavior in the open field(OFT)and elevated plus maze tests(EPM)and depression-like behavior in the forced swimming(FST)and tail suspension tests(TST).On the last day following the TST,mice were deeply anesthetized and randomly sacrificed in the morning.The blood,the thyroid,and the frontal lobe were collected immediately.Electrochemiluminescence immunoassay(ECLIA)and enzyme-linked immunosorbent assay(ELISA)were performed to measure the serum concentrations of thyroiditis-related parameters.HE staining was used to evaluate the pathological changes of thyroids in animals.Immunohistochemistry was performed to assess the activation state of microglia and astrocytes in the frontal cortex.Transmission electron microscopy(TEM)was used to observe ultrastructures of microglia,astrocytes,and neurons.Real-time RT-PCR was used to detect the m RNA expression of glial markers(Iba1 and GFAP)and glial-derived inflammatory factors,such as IL-1β,TNF-α,and IL-6.TUNEL staining was used to evaluate neuronal apoptosis.ELISA and Real-time RT-PCR were performed to investigate the changes in components of frontal serotonin(5-HT)signaling.Results 1.Building a euthyroid HT model in mice:(1)Body weights and weight gain: There was no significant difference in body weights and weight gain between groups during the whole experiment.(2)Evaluation of thyroiditis: Control mice had intact thyroid follicles with an even distribution,and monocyte infiltration was hardly found in thyroid tissues.In contrast,HT mice displayed significant thyroid enlargement with disorderly and destroyed thyroid follicles,and monocyte infiltration was found more or less in thyroid tissues.Further quantitative analysis revealed that the severity of thyroiditis in HT mice was significantly higher than that in controls(P < 0.001)(3)Serum levels of thyroiditis-related parameters: Serum levels of thyroid autoantibodies(Tg-Ab and TPO-Ab)in the HT group were apparently higher than those in the Con group(P < 0.001,P < 0.01),while no differences in serum T3,T4,or TSH levels was detected between groups.2.Behavioral results:(1)OFT: HT mice made significantly fewer entries into and spent less time in the central zone than did control mice(P < 0.05,P < 0.001),while motor function examined by total distance and mean speed did not differ between groups.Additionally,rearing and grooming actions were markedly reduced in HT mice compared to those in control mice(P < 0.05,P < 0.05).(2)EPM: HT mice tended to spend less time in and made fewer entries into the open arms relative to control animals(P < 0.05,P < 0.05).Additionally,HT mice exhibited a significant decrement in the percentage of open-arm time and the number of open-arm entries(P < 0.01,P< 0.05).(3)FST: HT mice showed a significant elevation in immobility time compared to control mice(P < 0.01).(4)TST: HT mice spent more time in the immobility state than did control mice(P < 0.001).3.Activation states in microglia :(1)Iba1 immunolabeling: In HT mice,there was an apparent increase in frontal microglia activation in HT mice compared to that in controls,as demonstrated by the greater numbers of activated microglia and higher percentages of Iba1-stained areas(P < 0.05,P < 0.05).(2)RT-PCR: The frontal Iba1 m RNA levels in HT mice were significantly higher than that of the control mice(P < 0.01).(3)TEM: The control group showed an irregular nucleolus with chromatin condensation beneath the nuclear membrane and a primary lysosome.In contrast,the HT group showed an enlarged nucleolus with increased lysosomes including primary lysosomes and secondary lysosomes.4.Activation states in astroglia :(1)GFAP immunolabeling: In HT mice,the soma of astroglia became larger.Further quantitative analysis revealed that there was an apparent increase in astroglia activation in HT mice compared to that in controls,as demonstrated by the greater numbers of activated astroglia and higher percentages of GFAP-stained areas(P < 0.01,P < 0.001).(2)RT-PCR: The frontal GFAP m RNA levels in HT mice were significantly higher than that of the control mice(P < 0.05).(3)TEM: The control group showed the classical appearance of astrocyte with a narrow rim of chromatin beneath the nuclear membrane.The cytoplasm was pale,contained an endoplasmic reticulum and a few mitochondria.In contrast,the astroglia in the HT group had an organelle-rich cytoplasm that included a well-developed Golgi apparatus,many mitochondria,and some endoplasmic reticulum.5.Pro-inflammatory cytokine expression: Frontal expressions of IL-1β and TNF-α were significantly upregulated in HT mice compared to the expression in controls(P < 0.05,P < 0.05).In line with this,there was a tendency(albeit not significant)for higher IL-6 expression in the frontal cortex of HT mice than in that of controls(P = 0.08).6.Neuronal ultrastructure: Ultrastructure of frontal neurons in HT mice was similar to controls with the nucleus with intact nuclear membranes and evenly distributed chromatin as well as with the rich cytoplasm containing many ribosomes,mitochondria,and endoplasmic reticulum.7.Neuronal apoptosis:(1)TEM: There were no apoptotic features,such as chromatin margination,nuclear condensation,or apoptotic bodies in the neurons examined from both groups.(2)TUNEL staining: There was no changes in TUNEL-positive neurons in the frontal cortex between groups.8.Alterations in 5-HT signaling: The m RNA levels of tryptophan-degrading enzyme indoleamine-2,3-dioxygenase(IDO1)and 5-HT transporter(SERT)were significantly higher in HT mice than those of controls(P < 0.05,P < 0.01).As expected,the level of 5-HT showed a significant reduction in the frontal cortex of HT mice compared to that in controls(P < 0.05).Conclusions In a word,our results suggested that HT induced frontal neuroinflammation and altered related serotonin signaling in the euthyroid state,which may underlie the deleterious effects of HT itself on emotional function.