| ObjectiveTo observe the effect of adult-onset hypothyroidism on the expression of sytaxin-1in dorsal hippocampus, and whether the altered protein expression could be restored by T4treatment. Further to explore the molecular mechanism of brain impairment by hypothyroidism.MethodsForty-five adult male Sprague-Dawley rats weighing250-290g were used in current experiments. The rats were divided into4groups randomly as indicated below:(1) Hypothyroid group. Twelve hypothyroid rats were induced by daily intraperitoneal injection of PTU (dissolved in saline solution,1mg/100g body weight (BW)) for six weeks;(2) T4-5group. Eleven rats were treated with PTU for six weeks as described above. But from the fifth week, they were also treated with intraperitoneally injected L-T4(dissolved in saline solution,50μg/kg BW) every day for two weeks;(3) T4-20group. Eleven rats were treated according to the same protocols as the T4-5group except increasing the dosage of L-T4to200μg/kg BW.(4) Control group. Eleven control rats were given same volume of saline solution for six weeks with hypothyroid rats. After blood collection, the brains were dissected on ice. The radioimmunoassay kits were applied to assay the levels of serum T3and T4. The optical density (OD) value representing the expression of syntaxin-1was measured using an image analysis system constituted by the MetaMorph image acquisition and processing software (JADA801D, China) and a Nikon80i microscope (Nikon, Japan) equipped with a HP computer. First, images of the dorsal hippocampal CA1, CA3and DG subfields were obtained using the optical microscope. Then, digital data.were exported into MetaMorph software for analysis and processing. The layers analyzed in the dorsal hippocampus include the stratum oriens (SO), stratum radiatum (SR) and stratum lacunosum-moleculare (SLM) in the CA1; SO, stratum lucidum (SL) and SR in the CA3; polymorphic layer (PL) and molecular layer (ML) in the dentate gyrus (DG). As a result of the different areas of analysis frame selected, the average absorbance (average optical density, AOD) was selected as the analysis index.. All statistical analysis was performed using SPSS software (version16.0). Values are expressed as mean±S.E.M. The data among the groups were compared using one-way ANOVA., followed by post hoc analysis by the least-significant difference test. Statistical significance was defined as Ps<0.05.Results1. Body weight gain At the beginning of the study, body weights of the animals were not significantly different. At the end of the experiment, body weights of the control rats were increased by48.0%. However, body weight gain in Hypothyroid, T4-5and T4-20groups were increased by only21.6%,32.6%and26.0%, respectively over the same period of time (Ps<0.05).2. Thyroid hormone levels Compared to the control group, serum levels of T3and T4were significantly lower in the hypothyroid group, were decreased by53.5%and77.6%, respectively, similar in the T4-5, and significantly higher in the T4-20group((Ps<0.001).3. Protein level of syntaxin-1in the examined layers of hippocampusThe immunoreactivity distribution of syntaxin-1was similar among these groups. Punctate spots of reaction product distributed in every layer of CA1, CA3and DG subregions. The optical density (OD) value of syntaxin-1immunoreactivity in each stratum of hippocampal subfields was also analyzed. We found that the OD values were significantly high in Hypothyroid group (Ps<0.05) and similar to controls in T4-5and T4-20group in all subareas of CA1, CA3, and DG (P>0.05). When compared to hypothyroid group, the OD values were significantly low in CA3-SO and CA3-SLM in T4-20group.ConclusionThese results suggest that adult-onset hypothyroidism caused up-regulation of syntaxin-1level in dorsal hippocampus, which could be restored by T4treatment, and the recovery degree is related to the T4dosage. |
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