| Objectives Carbon disulfide (CS2) is an important chemical material and caninduce toxic peripheral neuropathy in human. The exact molecular mechanism remains far from elucidated. To elucidate the mechanism of neuropathy induced by CS2, CS2-subchronical toxic rat model was designed. The changes of contents of Glutathione (GSH), Malondialdehyde (MDA), and the activities of reactive oxygen species (ROS), Superoxide dismutase (SOD), Glutathione peroxidase (GSH-Px), Total antioxidative capacity (T-AOC) and Catalase (CAT) in cerebrum, cerebellum, sciatic nerve and spinal cord were detected, as well as the alterations of behaviour indices, to offer some gists to the mechanism of peripheral neuropathies induced by CS2.α-tubulin,β-tubulin, andβ-actin in spinal cords were determined to investigate the mechanisms of the axonopathy. In addition, the levels of gene expression of a-tubulin,p-tubulin, andβ-actin mRNA, was quantified using reverse transcription-polymerase chain reaction (RT-PCR) to investigate the mechanisms from which the alterations might occur.Methods1. 60 adult male Wistar rats were randomly classified into control, low-dose, high-dose groups. The rats in low-dose, high-dose groups were respectively administrated 300mg/kg and 500 mg/kg carbon disulfide dissolved in vegetable oil by gavage for continuous twelve weeks (five times per week). The control group rats were administered equivalent volume of vegetable oil.2. The rats were weighed every week and the changes of behaviour indices were observed, including the changes of heat sensory sensitivity, pain threshold, the forelimbs grip strength and the hindlimbs bracing index.3. The contents of Glutathione (GSH), Malondialdehyde (MDA), and the activities of reactive oxygen species (ROS), Superoxide dismutase (SOD), Glutathione peroxidase (GSH-Px), Total antioxidative capacity (T-AOC) and Catalase (CAT)were investigated in cerebrum, cerebellum, sciatic nerve and spinal cord.4. The excised nerve tissues of spinal cords were homogenized and then centrifuged at100, 000×g for 60min. The relative levels ofα-tubulin,β-tubulin, andβ-actin in the supernatant and pellet of central and peripheral nervous tissue homogenates were determined by immunoblotting.5. The tissues of spinal cords and cerebrum were excised, and the total mRNA was isolated from flash frozen tissues using Trizol reagent. The levels of gene expression ofα-tubulin,β-tubulin, andβ-actin mRNA was quantified using RT-PCR.Results1. Weight and neurobehavioral function testsThe body weights and neurobehavioral indexes were affected after CS2 exposure, exhibiting a dose-time-effect relationship. At the end, the body weights decrased by 21% (P < 0.01) in lower-dose rate group and by 37% (P < 0.01) in higher-dose rate group compared with control group. It was from the tenth week that the threshold of sensory nerve to heat begain to increase in lower-dose rate group, while in higher-dose rate group it was from Week 8. At the end, in the two groups, the threshold of sensory nerve to heat respectively increased remarkablely by 28% (P < 0.01) and 70% (P < 0.01) compared with the control group. The pain threshold began to increase respectively from Week 8 and Week 6 in lower- and higher-dose rate group.At the end the pain threshold was increased significantly by 33% (P < 0.01) in lower-dose rate group and 60% (P < 0.01) in higher-dose rate group. The forelimbs grip strength was decreased by 21% (P < 0.01) in lower-dose rate group and 24% (P < 0.01) in higher-dose rate group. The hindlimbs bracing index was enhanced by 35% (P < 0.01) in lower-dose rate group and 42% (P < 0.01) in higher-dose rategroup, as compared to control.2. Effects of CS2 on lipid peroxidation and antioxidative status in nerve tissuesIn cerebrum, the contents of MDA and ROS respectively increased by 33% (P<0.01) and 35% (P<0.01) in higher-dose rate group, and 20% (P<0.01) and 20%(P<0.01) in lower-dose rate group. The contents of GSH, GSH-Px, SOD, T-AOC and CAT respectively decreased by 27%(P<0.01), 12%(P<0.01), 30%(P<0.01), 21% and 40% (P<0.01) in higher-dose rate group, while in lower-dose rate group the contents of GSH, T-AOC and CAT decreased by 18%(P<0.01), ll%(P<0.01) and 18%(P<0.01), and the changes of GSH-Px and SOD were too little to make sense.In cerebellum, the contents of MDA and ROS respectively increased by 23% (P<0.01) and 19% (P<0.01) in higher-dose rate group, and by 13% (P<0.05) and 9% (P<0.05) in lower-dose rate group. The contents of GSH, GSH-Px, SOD, T-AOC and CAT respectively decreased by 27%(P < 0.01), 15%(P<0.01), 26%(P<0.01), 21%(P <0.01) and 25%(P<0.01) in higher-dose group, while in lower-dose rate group the content of GSH decreased by 10%(P<0.01), the changes of the other indexes had no significant difference.In sciatic nerve, the contents of MDA and ROS respectively increased by 23% (P<0.01) and 53% (P<0.01) in higher-dose rate group, and the content of MDA increased by 23% (P<0.01) in lower-dose rate group, the change of SOD had no signidicant difference compared with control group. The contents of GSH, GSH-Px, SOD, T-AOC and CAT respectively decreased by 20%(P<0.01), 21%(P<0.01), 30%(P<0.01), 42%(P<0.01) and 37%(P<0.01) in higher-dose rate group, while in lower-dose rate group it decreased by 15%(P<0.01), 17%(P<0.01), 17%(P<0.01), 36% and 17%(P<0.01).In spinal cord, the contents MDA and ROS respectively increased by 23% (P<0.01) and 14% (P<0.01) in higher-dose rate group, and the content of MDA increased by 18% (P<0.05) in lower-dose rate group. The contents of GSH, GSH-Px, SOD, T-AOC and CAT respectively decreased by 31% (P<0.01), 32% (P<0.01), 25% (P<0.01), 28% (P<0.05) and 31% (P<0.01) in higher-dose rate group, while in lower-dose rate group it decreased by 26% (P<0.01), 17% (P<0.01), 12% (P<0.01), 14% and 25% (P<0.01).3. The alterations inα-tubulin,β-tubulin,β-actin of spinal cordIn the supernatant fraction, the contents ofβ-tubulin andβ-actin in both treated groups increased significantly (P<0.01): the content ofβ-tubulin increased by 141% and 158% respectevely, and the content ofβ-actin increased by 19% and 32% respectively. In the pellet fraction, the content ofβ-tubulin in both groups increased by 107% (P<0.01) and 118% (P<0.01) respectively, and the others keep unaffected.4. The mRNA expression ofα-tubulin,β-tubulin,β-actin of spinal cordThe levels of of mRNA expression ofβ-tubulin andβ-actin gene were elevated consistently in CS2-treated groups (P<0.01): the levels of mRNA expression ofβ-tubulin increased by 207% and 212% respectively, and the levels of mRNA expression ofβ-actin increased by 94% and 91% respectively.Conclusions1. CS2 can significantly affect the sensory and motor functions of nervous system, providing evidence for the neurotoxicity of CS2.2. CS2 intoxication is associated with elevation of lipid peroxidation and reduction of antioxidative status in Wistar rats nerve tissues and may be served as one of mechanisms of toxic neuropathy induced by CS2.3. CS2 intoxication results in alterations of mictotubule and microfilament of spinalcord, and the increase ofβ-tubulin is the greatest.4. CS2 intoxication results in alterations of mictotubule and microfilament expression, and the alterations might be related to its neurotoxicity. |
