| ObjectiveThe hypothyroid rats were induced by daily intraperitoneal injection of 6-n-propyl-2-thiouracil (PTU) 1 mg/100g body weight. The expression of SNAP-25 (synaptosomal-associated protein of 25 kDa) in hippocampus of adult onset hypothyroidism rats and the effect of Levothyroxin (L-T4) treatment were detected by immunohistochemistry. Further to explore the molecular mechanism of brain impairment by hypothyroidism.MethodsAdult male Sprague-Dawley rats weighing 250~300g were used in current experiments. Fouty-five rats were divided into 4 groups randomly as indicated below: (1) Hypothyroid group. Twelve hypothyroid rats were induced by daily intraperitoneal injection of PTU (dissolved in saline solution, 1 mg/100g body weight (BW)) for six weeks; (2) T4-5 group. 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) T4-20 group. Eleven rats were treated according to the same protocols as the T4-5 group except increasing the dosage of L-T4 to 200μg/kg BW. (4) Control group. Eleven control rats were given same volume of saline solution for six weeks with hypothyroid rats. The radioimmunoassay kits were applied to assay the levels of serum T3 and T4, and immunohistochemical method was used to determine the level of SNAP-25 in dorsal hippocampus. All statistical analysis was performed using SPSS software (version 16.0). Values are expressed as mean±S.E.M. The data among the groups were compared using one-way ANOVA. 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 (F(3,42) = 0.21, P = 0.93). The normal growth of body weight in the controls was increased by 49.0% at the time of the experiment. However, body weight gain in Hypothyroid, T4-5 and T4-20 groups increased only by 21.5%, 32.5% and 26.1%, respectively, over the same period of time, which is significantly lower than the controls (Ps < 0.05).2. Thyroid hormone levels The average levels of serum T3 and T4 were significantly low in the Hypothyroid group (Ps < 0.02), but high in the T4-20 group (Ps < 0.001) comparing with the Control group, the average levels of serum T3 and T4 in the T4-5 group is similar to the Control group.3. Protein level of SNAP-25 in the examined layers of CA1, CA3 and DG subfields of hippocampus The immunoreactivity distributions of SNAP-25 was similar among these groups. Punctate spots of reaction product distributed in every layer of CA1, CA3 and DG subregions. The optical density (OD) value of SNAP-25 immunoreactivity in each stratum of hippocampal subfields was also analyzed. We found that the OD values were significantly high in Hypothyroid group (Ps < 0.039) and similar to controls in T4-5 and T4-20 group in all subareas of CA1, CA3, and DG. When compared to Hypothyroid group, the OD values were significantly low in CA3-SR, DG-PL and DG-ML in T4-5 group, as well as in all examined layers in T4-20 group (Ps < 0.048).ConclusionThese results suggest that adult-onset hypothyroidism caused up-regulation of SNAP-25 level in dorsal hippocampus, which could be restored by T4 treatment, and the recovery degree is related to the T4 dosage. |
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