BACKGROUND： Previous researches suggested that expression level of calbindin D28K mRNA decreased in substantia nigra （SN） of model rats with Parkinson disease （PD）, and this might be related to the decrease of an...BACKGROUND： Previous researches suggested that expression level of calbindin D28K mRNA decreased in substantia nigra （SN） of model rats with Parkinson disease （PD）, and this might be related to the decrease of anti-degeneration potentials of dopaminergic neurons. OBJECTIVE： To observe expression changes of calbindin D28K in SN dopaminergic neurons during their degeneration and death in midbrain of PD model rats. DESIGN： Completely randomized grouping design SETTING： Department of Neurobiology, Xuzhou Medical College MATERIALS＂ A total of 92 healthy male SD rats, with the age of 3 months, weighing 200-250 g, were selected from Experimental Animal Center of Xuzhou Medical College [certification： SCXK （su） 2003-0003]. Calbindin D28K（CB）, tyroxine hydroxylase （TH）, ABC kit, 6-hydroxydopamine （6-OHDA） and Nissl dyes were provided by Sigma Company, and sheep serum was provided by Beijing Zhongshan Company. METHODS： The experiment was carried out in the Neurobiological Center of Xuzhou Medical College from October 2003 to October 2004. ① With lot method, rats were divided into blank control group （n=28）, experimental control group （n=-28） and experimental group （n=36）. Rats in experimental group were injected with 6-OHDA at right corpus striatum for PD modeling; rats in experimental control group were injected with saline at the same site; rats in blank control group did not give any injections.② On the 7^th, 14^th, 21^st and 28^th days, SN segments on right midbrain from every 5 rats in experimental group were fixed, embedded with paraffin and cut into successively coronary pieces. Rats in other two groups were treated with the same methods and then stained with Nissl to show neuronal form. Meanwhile, CB and TH antibodies staining with immunohistochemistry were used to show CB containing dopaminergic neurons and dopaminergic neurons, and cells were calculated and observed under optic microscope. ③ On the 14^th and 28^th days, every 4 rats in experimental group and every 4 rats in control group were selected to obtain their brains and separate SN on the injured side. Western blot was used to detect expression of calbindin D28K, protein band was scanned with imaging equipment, and data were analyzed with LabWorks software. MAIN OUTCOME MEASURES：① On the 7^th, 14^th, 21^st and 28^th days, Nissl staining results of SN neurons and immunohistochemical staining results of CB and TH antibodies; ② On the 14^th and 28^th days, Western blot results of calbindin D28K in SN neurons, RESULTS; Among 92 rats, 2 rats in experimental group died after 1 day due to 6-OHDA injection and other 90 rats were involved in the final analysis. ①Nissl staining results： On the 7^th day of 6-OHDA injection, most neuronal somas on right SN pars compacta were shown as deep pycnosis or lysis breakage; on the 14^th and 21^st days, amount of neurons was decreased remarkably; on the 28^th day, most neurons in SN pars compacta disappeared. ② Results of immunohistochemical staining： Amount of positive neurons of calbindin D28K in right SN pars compacta was not changed on the 7^th day after 6-OHDA injection; on the 14^th day, the amount was higher in experimental group than that in both control groups （P 〈 0.01） and was decreased till the 21^st day, but it was still higher than that in the two control groups （P 〈 0.05）; on the 28^th day, positive neurons of calbindin D28K nearly disappeared, and the amount was lower than that in the two control groups （P 〈 0.01）. In addition, on the 7^th day after 6-OHDA injection into corpus striatum, positive TH neurons decreased 24% in right SN, and there was significant difference from that in control groups; on the 14^th, 21^st and 28^th days, positive TH neurons decreased 37%, 46% and 64%, respectively. ③ Western blot results： On the 14＇h day after 6-OHDA injection into corpus striatum, expression of calbindin D28K in right SN was higher in experimental group than that in both control groups （P 〈 0.05）; however, on the 28^th day, the expression was lower than that in the two control groups （P 〈 0.01 ）. CONCLUSION ： During degeneration and death of dopaminergic neurons, CB expression in SN pars compacta increases firstly and decreased gradually.展开更多
本实验应用原位杂交组织化学技术,利用同位素标记的寡核苷酸探针,对大鼠前脑含Calbindin-D28 K mRNA的神经元的分布状况进行了详细的观察。结果发现在不同脑区或核团中,标记神经元的数量和标记强度各不相同。某些部位含许多强阳性神经元...本实验应用原位杂交组织化学技术,利用同位素标记的寡核苷酸探针,对大鼠前脑含Calbindin-D28 K mRNA的神经元的分布状况进行了详细的观察。结果发现在不同脑区或核团中,标记神经元的数量和标记强度各不相同。某些部位含许多强阳性神经元,如:前嗅核、大脑皮质、尾壳核、缰核、下丘脑、齿状回及中脑和杏仁复合体中的部分核团;然而,在另外一些脑区中,标记细胞呈中等阳性,如:嗅球的球旁细胞、盖带、梨状区内核、海马的CA1区中的锥体细胞层以及丘脑和杏仁核复合体中的部分核团。少数脑区中的标记细胞呈弱阳性,且数量较少,如;嗅结节、隔区、斜角带核等。这些结果表明含Calbindin-D28K mRNA的神经元在大鼠前脑中具有区域特异性分布特点,从而提示Calbindin-D28K在神经系统中的某些部位可能具有重要的作用。展开更多
基金Scientific and Technological Project of State Ministry of Education, No. 204055 the Natural Science Foundation of Educational Bureau of Jiangsu Province, No. 02KJB310009
文摘BACKGROUND： Previous researches suggested that expression level of calbindin D28K mRNA decreased in substantia nigra （SN） of model rats with Parkinson disease （PD）, and this might be related to the decrease of anti-degeneration potentials of dopaminergic neurons. OBJECTIVE： To observe expression changes of calbindin D28K in SN dopaminergic neurons during their degeneration and death in midbrain of PD model rats. DESIGN： Completely randomized grouping design SETTING： Department of Neurobiology, Xuzhou Medical College MATERIALS＂ A total of 92 healthy male SD rats, with the age of 3 months, weighing 200-250 g, were selected from Experimental Animal Center of Xuzhou Medical College [certification： SCXK （su） 2003-0003]. Calbindin D28K（CB）, tyroxine hydroxylase （TH）, ABC kit, 6-hydroxydopamine （6-OHDA） and Nissl dyes were provided by Sigma Company, and sheep serum was provided by Beijing Zhongshan Company. METHODS： The experiment was carried out in the Neurobiological Center of Xuzhou Medical College from October 2003 to October 2004. ① With lot method, rats were divided into blank control group （n=28）, experimental control group （n=-28） and experimental group （n=36）. Rats in experimental group were injected with 6-OHDA at right corpus striatum for PD modeling; rats in experimental control group were injected with saline at the same site; rats in blank control group did not give any injections.② On the 7^th, 14^th, 21^st and 28^th days, SN segments on right midbrain from every 5 rats in experimental group were fixed, embedded with paraffin and cut into successively coronary pieces. Rats in other two groups were treated with the same methods and then stained with Nissl to show neuronal form. Meanwhile, CB and TH antibodies staining with immunohistochemistry were used to show CB containing dopaminergic neurons and dopaminergic neurons, and cells were calculated and observed under optic microscope. ③ On the 14^th and 28^th days, every 4 rats in experimental group and every 4 rats in control group were selected to obtain their brains and separate SN on the injured side. Western blot was used to detect expression of calbindin D28K, protein band was scanned with imaging equipment, and data were analyzed with LabWorks software. MAIN OUTCOME MEASURES：① On the 7^th, 14^th, 21^st and 28^th days, Nissl staining results of SN neurons and immunohistochemical staining results of CB and TH antibodies; ② On the 14^th and 28^th days, Western blot results of calbindin D28K in SN neurons, RESULTS; Among 92 rats, 2 rats in experimental group died after 1 day due to 6-OHDA injection and other 90 rats were involved in the final analysis. ①Nissl staining results： On the 7^th day of 6-OHDA injection, most neuronal somas on right SN pars compacta were shown as deep pycnosis or lysis breakage; on the 14^th and 21^st days, amount of neurons was decreased remarkably; on the 28^th day, most neurons in SN pars compacta disappeared. ② Results of immunohistochemical staining： Amount of positive neurons of calbindin D28K in right SN pars compacta was not changed on the 7^th day after 6-OHDA injection; on the 14^th day, the amount was higher in experimental group than that in both control groups （P 〈 0.01） and was decreased till the 21^st day, but it was still higher than that in the two control groups （P 〈 0.05）; on the 28^th day, positive neurons of calbindin D28K nearly disappeared, and the amount was lower than that in the two control groups （P 〈 0.01）. In addition, on the 7^th day after 6-OHDA injection into corpus striatum, positive TH neurons decreased 24% in right SN, and there was significant difference from that in control groups; on the 14^th, 21^st and 28^th days, positive TH neurons decreased 37%, 46% and 64%, respectively. ③ Western blot results： On the 14＇h day after 6-OHDA injection into corpus striatum, expression of calbindin D28K in right SN was higher in experimental group than that in both control groups （P 〈 0.05）; however, on the 28^th day, the expression was lower than that in the two control groups （P 〈 0.01 ）. CONCLUSION ： During degeneration and death of dopaminergic neurons, CB expression in SN pars compacta increases firstly and decreased gradually.
文摘本实验应用原位杂交组织化学技术,利用同位素标记的寡核苷酸探针,对大鼠前脑含Calbindin-D28 K mRNA的神经元的分布状况进行了详细的观察。结果发现在不同脑区或核团中,标记神经元的数量和标记强度各不相同。某些部位含许多强阳性神经元,如:前嗅核、大脑皮质、尾壳核、缰核、下丘脑、齿状回及中脑和杏仁复合体中的部分核团;然而,在另外一些脑区中,标记细胞呈中等阳性,如:嗅球的球旁细胞、盖带、梨状区内核、海马的CA1区中的锥体细胞层以及丘脑和杏仁核复合体中的部分核团。少数脑区中的标记细胞呈弱阳性,且数量较少,如;嗅结节、隔区、斜角带核等。这些结果表明含Calbindin-D28K mRNA的神经元在大鼠前脑中具有区域特异性分布特点,从而提示Calbindin-D28K在神经系统中的某些部位可能具有重要的作用。
