The Toxic Damage Of Cadmium And Protective Effect Of NAC On Rat's Cortical Neuron Cultured In Vitro

To study cadmium (Cd) neurotoxicity mechanism on rat's cerebral cortex neuron, the model of experimental neurons which were obtained from the neonate Sprague-Dawley rats in 24h was established successfully. Toxic effect of different concentrations (5μmol/L Cd, 10μmol/L Cd, 20μmol/L Cd) of cadmium on neuron's morphology, viability, apoptosis rate, mitochondrial membrane potential, [Ca²⁺]_i, content of ROS intra-cellular and the enzymatic activity changes of lipid peroxidation and protective effect of N-Acetyl Cysteine (NAC) were evaluated by the inverted light microscope, transmission electron microscope (TEM), the fluorescence microscope, the flow cytometry (FCM), adopting the Nissl's staining method, microculture tetrazolium assay (MTT) and related kits, and the expresstion of MT in neurons stimulated by cadmium was detected with immumohistochemistry technique. The results were as follows: (1) The results of morphological analysis and viability measurement showed that the cortical neuron's viability was meridian, and they formed an integrated neural network on day 6 post-beginning of culturing. (2) The morphological changes indicated a very significant decrease in pericaryons and processes after incubating 12h and 24h (P﹤0.01). The mitochondria were bloated, cristae was deformed and blurred, and the matrix was out-flowed. With prolonging of the culturing time and increasing, concentrations of cadmium acetate, the inhibitory effect was enhanced gradually. (3) Then they were lucifugal incubated with Hoechst 33258 in 5 min. The results revealed that the nucleu??impled, crescent liked and chromatin condensed, even nucleus disintegrated. The apoptosis rate was higher than the control group. (4) Neuron's mitochondrial membrane potential, descended with different concentrations of cadmium acetate after culturing 12h. And the 20μmol/L group was a very significantly decreased compare with the control group (P﹤0.01). (5) Neuron's [Ca²⁺]_i intra-cellular descended with different concentrations of cadmium acetate after culturing 12h, which was a very significantly decreased compare with the control group (P﹤0.01). (6) Neuron's containt of ROS, increased with different concentrations of cadmium acetate after culturing 12h. And the 10μmol/L, 20μmol/L groups were a very significantly increased compare with the control group (P﹤0.01); The activities of GSH-Px treated by cadmium acetate of different concentrations for 12h were decreased, the 20μmol/L group was significantly decreased compare with the control group(P﹤0.05). However, the activity of CAT and the content of MDA in neurons were very significantly increased than that of the control group, (P﹤0.01). The activity of SOD in neurons was increased than that of the control group as well, but not significantly. (7) Compared NAC antagonistic groups with the poisoning groups, The apoptosis rate of neurons, the content of ROS, the activities of CAT and [Ca²⁺]_i was degraded relative to the poisoning groups. Meanwhile, the activities of GSH-Px increased accordingly, but the content of SOD and MDA was decreased compared with the poisoning groups. (8) The amount of MT-â…¢expression stimulated by cadmium increased significantly.Conclusion: (1) Cadmium can inhibit cortical neuron differentiation, which led to the pericaryon diminutus and process decurtation than the control group, It also destroy the normal morphous of mitochondria. (2) Neuron apoptosis what cadmium induced is one of possible mechanisms and displays a dose effect in comparison with the control group, apoptosis cells increased, mitochondrial membrane potential decreased, and [Ca²⁺]_i in neurons rose. (3) Cadmium can result in oxidative stress and modify antioxidative enzyme activity. Therefore, lipid peroxidation is also one of mechanisms of cadmium neurotoxicity. (4) NAC can rivalry neuron toitoxity induced by cadmium. It reduce apoptosis rate and the contant of [Ca²⁺]_i in neurons. It also inhibits the oxidative damage of cadmium. The data indicated that NAC could increase antioxidative effects and reduce lipid pe??dation in neurons of SD rats in vitro. (5) Cadmium concentration induces the amount of MT-â…¢expression increased.