| Objectiven-hexane and carbon disulfide (CS2) are organic solvents widely used in many industrial processes, n-hexane is used widely in the preparation of fabrics, adhesives, lacquers and other coatings, etc. CS2 is widely used in the vulcanization of rubber and synthesis of carbon tetrachloride and the manufacture of cellophane, plywood and viscose rayon.Continued exposure to hexane or CS2 produces loss of sensory and motor function in arms and legs, which is the result of the nerve damages characterized as a central-peripheral distal axonopathy. The corresponding pathological alterations observed in this axonopathy include the accumulation of neurolfilaments (NFs) proximal to the nodes of Ranvier, a Wallerian-type degeneration consisting of accumulation of mitochondria, vesicles, microtubules (MT) and dense bodies distal to the axonal swellings, retraction of myelin from Ranvier nodes and segmental demyelination, and distal axon atrophy.The mechanisms of the axonopathy induced by hexane or CS2 are currently unclear, although the pathological alterations indicate that NF and MT are involved in this axonapathy. In the present investigation, the rat models of HD and CS2-induced axonopathy were established, and the relative levels of NF-H, NF-M, NF-L,α-tubulin,β-tubulin, andβ-actin in the tissues of sciatic nerves, spinal cords and cerebrums were determined to investigate their corresponding alterations during the axonopathy. Furthermore, the levels of gene expression of NF-H, NF-M, and NF-L subunit,α-tubulin,β-tubulin, andβ-actin mRNA were quantified using reverse transcription-polymerase chain reaction (RT-PCR). In addition, the expression and activity of calpains, ca2+-activated neutral protease responsible for the degradation of cytoskeleton, were investigated to explore the possible mechanism of neuropathy induced by n-hexane and CS2.Methods Adult male Wistar rats were treated with HD by intraperitoneal injection at dosages of 400 mg/kg/day HD respectively for 2, 4 or 8 weeks. To assess the recovery of HD-treated rats, half of 8-week treated animals continued to be observed for 16 weeks after 8-week exposure ended. Furthmore, the rats were treated with CS2 by gavage at dosages of 300 and 500 mg/kg/day respectively for continuous 12 weeks to establish the model of CS2-induced neuropathy. The onset and development of neurotoxicity were determined by neurological testing.The tissues of sciatic nerves, spinal cords and cerebrums were homogenized and then centrifuged at 100, 000×g for 30min at 4℃. The relative levels of NF-H, NF-M, NF-L,α-tubulin,β-tubulin,β-actin, m-calpain andμ-calpain in the supernatant and pellet of nervous tissues were determined by immunoblotting.The tissues of spinal cords and cerebrums were excised, and the total mRNA was isolated from flash frozen tissues using Trizol reagent. The mRNA levels of NF-H, NF-M, NF-L,α-tubulin,β-tubulin, andβ-actin genes were quantified using RT-PCR.The excised nervous tissues of the sciatic nerves, spinal cords and cerebrums were homogenized and centrifuged at 8000 rpm for 20 min at 4℃Calpain activity in the supernatant was assayed using a Calbiochem? InnoZymeTM Calpain 1 & 2 Activity Kit.Results1 The establishment of the animal modelRats exposed to HD produced progressive neurobehavioural abnormalities. After 2, 4, and 8 weeks of HD treatment at 400 mg/kg dose-rate, the rats were at three different levels of neurological defects respectively, i.e. slightly abnormal gait, moderately abnormal gait, or severely abnormal gait. After stopping the treatment, the neurological defects of treated animals displayed obvious recovery. Furthermore, CS2 intoxication could result in significant neurological defects. After 12 weeks of CS2 treatment at 300 or 500 mg/kg dose-rate, the rats were at two different levels of slightly or moderately neurological abnormalities respectively. The results above suggested that the rat model of toxicant-induced neurotoxicity were established successfully.2 The alterations of cytoskeletal proteins in rat nervous tissues2.1 HD-induced alterations of cytoskeletal proteins in rat nervous tissues 2.1.1 CerebrumCompared to the control, the contents of NF-H decreased by 31.6%, 40.3%, and 42% respectively in the supernatant, and decreased by 11.1%, 51.8%, and 53.2% respectively in the pellet at 2, 4, and 8 weeks time point of treatment with HD; NF-M decreased by 34.7%, 58.2%, and 77.2% respectively in the supernatant, and decreased by 29.4%, 50.6%, and 72.5% respectively in the pellet at 2, 4, and 8 weeks time point of HD exposure; NF-L decreased by 28%, 35.4%, and 46.3% respectively in the supernatant, and decreased by 18%, 34.7%, and 34.7% respectively in the pellet at 2,4, and 8 weeks time point.Compared to the control, the levels ofα-tubulin andβ-actin in both fractions kept unaffected during the period of HD exposure. However, the level ofβ-tubulin increased by 29% and 33% respectively in the supernatant at 4, 8 weeks time point of HD exposure, and increased by 16.3% in the pellet at 8-week time point following HD.2.1.2 Spinal cordCompared to the control, the level of NF-H in the supernatant decreased by 16%, 57%, and 58% at 2, 4, and 8 weeks time point of treatment with HD respectively, and that in the pellet decreased by 33% and 35% at 4 and 8 weeks time point of treatment respectively; Accordingly, NF-M decreased by 36%, 61%, and 65% respectively in the supernatant, and decreased by 23%, 34%, and 44% respectively in the pellet at 2, 4, and 8 weeks time point of HD exposure. Furthermore, NF-L in the supernatant decreased by 21%, 44%, and 45% respectively at 2, 4, and 8 weeks time point following HD, and that in the pellet decreased by 26% and 35% at 4 and 8-week time point respectively. However, after a 16-week recovery, the levels of three NF subunits in spinal cord of 8-week treated rats returned to normal.Compared to the control, the levels ofα-tubulin andβ-actin in both fractions remained unchangeable throughout HD exposure. However, the level ofβ-tubulin was increased by 34.8% in the supernatant at 8-week time point of HD exposure.2.1.3 Sciatic nerveIn the pellet fraction, NF-H content decreased by 19%, 22%, and 43% respectively after 2,4, and 8-week treatment of HD. Accordingly, in the supernatant fraction, the contents of NF-H in groups of 4 and 8 weeks' exposure decreased by 12% and 52%. Furthermore, after treated animals being allowed to recover for 16 weeks, the level of NF-H in sciatic nerves returned to normal.When compared to the age-matched control, the level of NF-M decreased by 15%, 24%, 88% and 83% at 2, 4, 8, and 24 weeks of the experiment in the supernatant, and accordingly decreased by 12%, 19%, 54% and 41% in the pelletWhen compared to the age-matched control, the level of NF-L in the supernatant decreased by 15%, 40%, 58% and 34% at 2, 4, 8, and 24 weeks of the experiment. Furthermore, in the pellet, a significant decrease of NF-L was observed in 4 and 8-week treated group, which decreased by 27% and 53%. However, after a 16-week recovery, the level of NF-L in the pellet returned to normal.When compared to the control, the level of a-tubulin in the pellet decreased by 40.6%, 41.2%, 34.5% and 14.2% respectively at 2, 4, 8, and 24 weeks of the experiment. However, a-tubulin content in the supernatant showed no significant alteration throughout the 24-week experiment.When compared to the control, the level ofβ-tubulin in the supernatant increased by 16%, 44%, 88.9% and 86.1% respectively at 2, 4, 8, and 24 weeks of the experiment. Accordingly, in the pellet fraction, the contents ofβ-tubulin increased by 15.3%, 9.5%, 102.1% and 99.2% respectively.When compared to the control, the level ofβ-actin in the supernatant decreased by 27.1% and 24.2% respectively at 8 and 24 weeks of the experiment. However,β-actin in the pellet increased by 24.8%, 66.6%, and 103.1% respectively at 2, 4, and 8 weeks of the experiment.2.2 Correlation relationship of NFs alteration in nerve tissues and neurobehavioral abnormality of HD-treated ratsTo investigate the contribution of NFs alteration to HD-induced neuropathy, the relationship between gait score and NFs content in rat nerve tissues was determined by multiple correlation analysis. Multiple correlation coefficients of NF-H, NF-M, or NF-L content in cerebrum with rat's gait score are 0.8872 (P<0.0001), 0.9644 (P<0.0001), and 0.8651 (P<0.0001) respectively, those of NF-H, NF-M, or NF-L content in spinal cord with rat's gait score are 0.8912 (P<0.0001), 0.9282 (P<0.0001), and 0.8981 (P<0.0001) respectively, and those in sciatic nerve are 0.9133 (P<0.0001), 0.9156 (P<0.0001), and 0.9446 (P<0.0001) respectively.2.3 CS2-induced alterations of cytoskeletal proteins in rat nervous tissues23.1 CerebrumCompared to the controls, in the supernatant fraction, NF-H levels in 300 and 500 mg/kg CS2-treated groups decreased by 31% and 33% respectively, NF-M by 52% and 58% respectively, and NF-L by 60% and 83% respectively. Furthermore, in the pellet fraction, NF-H levels in 300 and 500 mg/kg CS2-treated groups decreased by 37% and 45% respectively, NF-M by 37% and 75% respectively, and NF-L by 57% and 60% respectively.In the supernatant fraction, a -tubulin levels in 300 and 500 mg/kg CS2-treated groups increased by 20% and 27% respectively,β-tubulin increased by 63% and 71% respectively. Furthermore, the level ofβ-actin in rats treated with 500 mg/kg CS2 increase by 31%. In the pellet fraction,β-tubulin levels in 300 and 500 mg/kg CS2-treated groups decreased by 20 and 26% respectively. However, the levels of a -tubulin andβ-actin remained unchangeable in both treated groups.2.3.2 Spinal cordIn the supernatant fraction, the levels of NF-H, NF-M, and NF-L in 300 mg/kg CS2-treated group remained unaffected, however, their levels in 500 mg/kg CS2-treated group decreased by 37%, 33% and 68% respectively. In the pellet, the levels of NF-H in 300 and 500 mg/kg CS2-treated groups decreased by 20% and 24% respectively, and NF-M decreased by 16% and 65% respectively. Furthermore, the content of NF-L in 500 mg/kg CS2 group decreased by 40%.When compared to the controls,β-tubulin levels in 300 and 500 mg/kg CS2-treated groups increased by 141% and 158% respectively in the supernatant, and increased by 107% and 118% respectively in the pellet. Furthermore, in the supernatant,β-actin levels in 300 and 500 mg/kg CS2-treated groups increased by 19% and 31% respectively. However, the levels ofα-tubulin remained unchangeable in both treated groups.2.3.3 Sciatic nerveCompared to the controls, in the supernatant fraction, NF-H levels in 300 and 500 mg/kg CS2-treated groups decreased by 21.3% and 30.9% respectively, NF-L by 24.4% and 26.8% respectively. In the pellet fraction, the levels of NF-H levels in 300 and 500 mg/kg CS2-treated groups decreased by 12.6% and 21.4% respectively, and NF-L by 4.6% and 45.4% respectively. As far as NF-M was concerned, its levels in 300 and 500 mg/kg CS2-treated groups increased by 21.8% and 41.5% respectively in the supernatant fraction, and increased by 41% and 43.4% respectively in the pellet.When compared to the controls, the levels ofα-tubulin andβ-actin in 500 mg/kg CS2-treated groups increased by 74% and 17.3% respectively in the supernatant fraction. However, in the pellet, the levels ofα-tubulin andβ-actin in 500 mg/kg CS2-treated groups decreased by 19.7% and 30.5% respectively. As far asβ-tubulin was concerned, its levels in 300 and 500 mg/kg CS2-treated groups increased by 18.3% and 40.4% respectively in the supernatant. However,β-tubulin decreased by 18.4% and 26.8% respectively in the pellet.3 The alterations of cytoskeleton mRNA in rat nervous tissues3.1 HD-induced alterations of cytoskeleton mRNA in rat nervous tissues3.1.1 CerebrumWhen compared to the age-matched controls, the mRNA levels of NF-H, NF-L,α-tubulin,β-tubulin andβ-actin stayed at the level of control groups throughout the intoxication process of HD, while the mRNA levels of NF-M demonstrated obvious fluctuation, which decreased by 13.5% and 24.8% respectively at weeks 2 and 4 of HD treatment, and returned to the level of control by the end of 8 weeks treatment.3.1.2 Spinal cordWhen compared to the age-matched controls, the mRNA levels of NF-H, NF-M, NF-L,α-tubulin,β-tubulin andβ-actin showed no significant alteration throughoutthe HD exposure.3.2 CS2-induced alterations of cytoskeleton mRNA in rat nervous tissues 3.2.1 CerebrumWhen compared to the control, the mRNA level of NF-M in rats treated with 300 mg/kg CS2 decreased by 21.6%, and that in rats treated with 500 mg/kg CS2 by 21.8%. Furthermore, the mRNA levels of NF-L andβ-tubulin in rats treated with 300 mg/kg CS2 decreased by 18.1% and 13.5% respectively. However, the mRNA levels of NF-H,α-tubulin andβ-actin showed no significant alteration in both treated groups.3.2.2 Spinal cordWhen compared to the control, the mRNA levels of NF-H, NF-M and NF-L in rats treated with 300 mg/kg CS2 were elevated by 32%, 120%, and 102% respectively, and those in rats treated with 500 mg/kg CS2 by 46%, 181%, and 130% respectively. Furthermore, the mRNA levels ofβ-tubulin andβ-actin in rats treated with 300 mg/kg CS2 were elevated by 207% and 94% respectively, and those in rats treated with 500 mg/kg CS2 by 212% and 91% respectively. However, the mRNA level ofα-tubulin showed no significant alteration in both CS2-treated groups.4 The alterations of calpains content in rat nervous tissues4.1 HD-induced alterations of calpains content in rat nervous tissues4.1.1 CerebrumWhen compared to the controls, the contents of m-calpain decreased by 15.3% and 21.8% at time point of week 4 and 8 respectively in the supernatant, and decreased by 17.4% only at week 8 in the pellet,.As far asμ-calpain was concerned, in the supernatant,μ-calpain levels increased by 24.6% and 64.4% at time point of week 4 and 8 respectively. In the pellet,μ-calpain increased by 72.7% only at week 8.4.1.2 Spinal cordWhen compared to the control, the contents of m-calpain decreased by 11.6%, 19.9% and 20.4% respectively on week 2, 4 and 8 in the supernatant, and decreased by 15.8%, 35.9%, and 54.6% respectively in the pellet. After a 16-week recovery, the level of m-calpain in spinal cord of 8-week treated rats returned to normal.When compared to the control, the contents ofμ-calpain decreased by 15.4% (P>0.05) and 23% (P<0.05) respectively on week 2 and 4 in the supernatant, and decreased by 12.9% (P>0.05) and 24.3% respectively in the pellet. However, the level ofμ-calpam was elevated by 62.3% and 33.6% respectively on week 8 and 24 in the supernatant. Furthermore, the level ofμ-calpain was elevated by 25.2% on week 8 in the pellet.4.1.3 Sciatic nerveWhen compared to the controls, the level of m-calpain increased by 46% and 44% at time-point of week 8 and 24 respectively in the supernatant, and increased by 61% only at 8-week time point in the pellet fraction.As far asμ-calpain was concerned, in the supernatant fraction, the content ofμ-calpain increased by 30%, 63% and 54% at time point of week 4, 8 and 24 respectively. Correspondingly,μ-calpain increased by 31%, 59% and 34% respectively in the pellet fraction.4.2 CS2-induced alterations of calpains content in rat nervous tissues4.2.1 CerebrumWhen compared to the control, the levels of m-calpain in both fractions showed no significant alteration in both CS2-treated groups. As far asμ-calpain was concerned, the levels ofμ-calpain in rats treated with 300, 500mg/kg CS2 were elevated by 34% and 53% in the supernatant fraction respectively. In the pellet fraction, its levels increased by 74% and 49% respectively.4.2.2 Spinal cordIn the supernatant fraction, the levels of m-calpain in rats treated with 300, 500 mg/kg CS2 were decreased by 46% and 48% respectively. However, the level of m-calpain in the pellet fraction stayed at the level of the controls. As far asμ-calpain was concerned, the levels ofμ-calpain in rats treated with 300, 500 mg/kg CS2 were elevated by 39% and 47% in the supernatant fraction respectively when compared to control group. In the pellet fraction, its levels increased by 37.5% and 36.2% respectively.4.2.3 Sciatic nerveWhen compared to the control, the levels of m-calpain in rats treated with 300, 500 mg/kg CS2 were elevated by 18.3% and 42.7% in the supernatant fraction respectively. In the pellet fraction, its levels increased by 19.3% only in 500 mg/kg CS2-treated group.As far asμ-calpain was concerned, the levels ofμ-calpain in rats treated with 300 mg/kg CS2 decreased by 19.2% and 36.8% in the supernatant and pellet fractions respectively, and those in rats treated with 500 mg/kg CS2 increased by 35.9% only in the pellet fraction.5 The alteration of calpains activity in rat nervous tissues5.1 HD-induced alteration of calpains activity in rat nervous tissuesWhen compared to age-matched controls, the calpains activity of spinal cords decreased by 22.8%, 26.9% and 27.5% respectively on week 2, 4, 8 after HD exposure; that of cerebrums decreased by 4.2%, 8% and 37.2% respectively on week 2, 4, 8 after HD exposure. However, calpains activity in sciatic nerves increased by 14%, 20%, 34% and 18% respectively on week 2, 4, 8 and 24.5.2 CS2-induced alteration of calpains activity in rat nervous tissuesWhen compared to age-matched controls, the calpains activity of spinal cords decreased by 18.5% and 20.6% in 300 and 500 mg/kg CS2-treated group respectively. Furthermore, the calpains activity of sciatic nerves increased by 15.5% and 20.7% in 300 and 500 mg/kg CS2-treated group respectively. However, calpains activity of cerebrums remained unchangeable in both CS2-treated groups.Conclusions1 The intoxication of HD and CS2 resulted in significant alterations of NFs contents in nerve tissues of rats, among which the alterations of NF-M and NF-L appeared to be more noticeable than NF-H.2 HD-induced NFs alterations in rat nerve tissues were highly correlated with neurobehavioral abnormality of HD-treated rats.3 The reversibility of NF decline induced by HD is associated with the recovery of HD-treated rats.4 The intoxication of HD and CS2 resulted in significant alterations ofα-tubulin,β-tubulin andβ-actin in nerve tissues, which was mainly observed in the peripheral nerves of rats treated with HD, and observed in central and peripheral nervous tissues treated with CS2. Among them, the alterations ofβ-tubulin appeared to be more noticeable than those ofα-tubulin andβ-actin.5 The effect of HD and CS2 on NFs,β-tubulins andβ-actin showed some diversity, among which the alterations of NFs appeared to be more marked than tubulins andβ-actin.6 CS2 induced significant alterations in the gene expression of cytoskeleton in rat nerve tissues, i.e. the expression of NF-H, NF-M, NF-L,β-tubulin andβ-actin mRNA was upregulated significantly in spinal cord, and the expression of NF-H, NF-L andβ-tubulin mRNA was downregulated significantly in cerebrum. However, HD had no significant effect on the expression of cytoskeleton gene.7 HD and CS2 induced significant alterations in the expression and activity of calpains in rat nervous tissues. Among them, the activity of calpains decreased in the CNS, and increased in the PNS significantly. Calpains activation in distal axons might be responsible for the decline of NFs induced by HD and CS2.8 The alteration of cytoskeleton induced by HD and CS2 was involved in the development of HD or CS2- induced neuropathy. |
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