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 7th, 14th, 21st and 28th 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 14th and 28th 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. West 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 7th, 14th, 21st and 28th days, Nissl staining results of SN neurons and immunohistochemical staining results of CB and TH antibodies; ② On the 14th and 28th days, West 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. ① Nissl staining results: On the 7th day of 6-OHDA injection,most neuronal somas on right SN pars compacta were shown as deep pycnosis or lysis breakage; on the 14th and 21st days, amount of neurons was decreased remarkably; on the 28th 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 7th day after 6-OHDA injection; on the 14th day,the amount was higher in experimental group than that in both control groups (P ＜ 0.01) and was decreased till the 21st day, but it was still higher than that in the two control groups (P ＜ 0.05); on the 28thday, 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 7th 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 14th, 21st and 28th days, positive TH neurons decreased 37%, 46% and 64%, respectively. ③ West blot results: On the 14th 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 28th 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.