有机溶剂中毒性神经病细胞骨架的损伤及其损伤机制
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摘要
研究目的
正己烷(n-hexane)和二硫化碳(carbon disulfide，CS_2)是常用的工业溶剂。正已烷主要应用于粘胶配制、除污、干洗、植物油提取、制鞋、印刷、油漆、制药、家具制造及电子元件制造等行业，而CS_2则广泛应用于粘胶纤维、玻璃纸、四氯化碳和杀虫剂等生产过程。
长期职业接触正己烷、CS_2可引起以感觉运动型周围神经病为主要临床特征的慢性中毒。早期表现为手足部感觉异常，痛觉、触觉减退呈手套、袜套样分布，进一步发展可表现为四肢无力、行走困难以至瘫痪。主要的病理改变发生在肢体远端最长、最粗的感觉和运动神经轴索，并在郎飞氏节附近形成巨大的充满神经丝(neurofilament，NF)的肿胀，郎飞氏节解剖结构扭曲且髓鞘从肿胀处回缩，有时伴有节段性脱髓鞘，最后轴索发生变性。
正己烷、CS_2中毒周围神经病的发生机制尚不清楚，但由于二者在神经行为学改变和病理改变上都非常相似，提示它们可能分享相同或相似的分子机制。同时，病理形态学研究提示NF、微管(microtubule，MT)等骨架蛋白可能参与了中毒性轴索病变的发生。国内外的研究已经报道，2，5-己二酮(2，5-hexanedione，HD)、CS_2亚慢性染毒可引起大鼠神经组织中NF含量的减少，但对这种变化发生的原因以及在它们在中毒性神经病中的作用没有进行深入探讨。因此，细胞骨架在中毒性周围神经病中的作用并没有真正阐明。
本文通过建立亚慢性中毒动物模型，观察正己烷、CS_2中毒性神经病发生过程中NF-H、NF-M、NF-L、α-tubulin、β-tubulin和β-actin等细胞骨架蛋白相对含量的变化，在此基础上进一步研究毒物对细胞骨架蛋白的基因转录、蛋白降解的影响，以探讨有机溶剂中毒性周围神经病的发病机制。
研究方法
1雄性Wistar大鼠用400mg／kg．bw的HD腹腔注射染毒，染毒时间分别为2、4和8周，建立正己烷中毒性周围神经病模型，并取一半8周染毒动物继续观察至24周。雄性Wistar大鼠分别用300、500mg／kg．bw的CS_2灌胃染毒，连续12周，建立CS_2中毒性周围神经病模型。
2取大鼠大脑、脊髓和坐骨神经组织制备匀浆，100，000×g、4℃离心60min。利用Western-Blotting技术研究大鼠大脑、脊髓、坐骨神经上清和沉淀中NF-H、NF-M、NF-L、α-tubulin、β-tubulin和β-actin等骨架蛋白以及钙激活的中性蛋白酶m-calpain和μ-calpain含量的变化。
3取大鼠大脑和脊髓组织，提取总RNA，利用RT-PCR技术研究HD和CS_2染毒对大鼠中枢神经组织中NF-H、NF-M、NF-L、α-tubulin、β-tubulin和β-actin mRNA表达水平的影响。
4取大鼠大脑、脊髓和坐骨神经组织制备匀浆，8，000×g，4℃离心30min。利用Calbiochem(?) InnoZymeTM Calpain 1&2 Activity Kit检测大鼠大脑、脊髓、坐骨神经组织中calpains总活性的变化。
研究结果
1动物模型的建立以及神经行为学指标的变化
给予HD、CS_2染毒后，动物逐渐出现肌肉松弛、肌张力降低、步态异常以至瘫痪等症状，神经行为学测试表明染毒大鼠呈现典型的中毒性周围神经病特征，这提示正己烷、CS_2中毒性周围神经病模型建立成功。同时HD染毒停止后，染毒大鼠的感觉运动指标能够逐渐改善。
2 HD、CS_2染毒大鼠神经组织中细胞骨架蛋白含量的变化
2．1 HD染毒大鼠神经组织中细胞骨架蛋白含量的时间变化趋势
2．1．1大脑
NF-H：与对照相比，大脑上清中NF-H的含量在HD染毒第2、4和8周时分别下降31.6％、40.3％和42％(P＜0.01)，大脑沉淀中NF-H的含量分别下降11.1％(P＜0.05)、51.8％和53.2％(P＜0.01)。
NF-M：与对照相比，大脑上清中NF-M的含量在HD染毒第2、4和8周时分别下降34.7％、58.2％和77.2％(P＜0.01)，大脑沉淀中NF-M的含量分别下降29.4％、50.6％和72.5％(P＜0.01)。
NF-L：与对照相比，大脑上清中NF-L的含量在HD染毒第2、4和8周时分别下降28％、35.4％和46.3％(P＜0.01)，大脑沉淀中NF-L的含量分别下降18％、34.7％和34.7％(P＜0.01)。
β-tubulin：与对照相比，大脑上清中β-tubulin的含量在HD染毒4周和8周时分别升高29％和33％(P＜0.01)，大脑沉淀中β-tubulin的含量在HD染毒8周时也升高16.3％(P＜0.05)。
α-tubulin和β-actin：与对照相比，大脑上清和沉淀中α-tubulin和β-actin的含量在HD染毒后未发生明显变化。
2．1．2脊髓
NF-H：与对照相比，脊髓上清中NF-H的含量在HD染毒第2、4和8周时分别下降16％、57％和58％(P＜0.01)，脊髓沉淀中NF-H的含量在HD染毒第4和8周时分别下降33％和43％(P＜0.01)。停药16周后，NF-H的含量恢复正常。
NF-M：与对照相比，脊髓上清中NF-M的含量在HD染毒第2、4和8周时分别下降36％、61％和65％(P＜0.01)，脊髓沉淀中NF-M的含量分别下降23％、34％和64％(P＜0.01)。停药16周后，NF-M含量又恢复正常。
NF-L：与对照相比，脊髓上清中NF-L的含量在HD染毒第2、4和8周时分别下降21％、44％和45％(P＜0.01)，脊髓沉淀中NF-L含量在第4和8周时分别下降26％和42％(P＜0.01)。停药16周后，NF-L含量又恢复正常。
α-tubulin、β-tubulin和β-actin：除HD染毒第8周时脊髓上清中β-tubulin的含量升高34.8％(P＜0.01)外，其余均未发生明显变化。
2．1．3坐骨神经
NF-H：与对照相比，坐骨神经上清中NF-H的含量在HD染毒第2、4和8周时分别下降19％、22％和43％(P＜0.01)，坐骨神经沉淀中NF-H含量在HD染毒第4和8周时分别下降12％和51％(P＜0.01)。停药16周后，NF-H含量又恢复正常。
NF-M：与年龄匹配的对照相比，坐骨神经上清中NF-M的含量在HD染毒第2、4、8周以及停止染毒16周时分别下降15％、24％、88％和83％(P＜0.01)，坐骨神经沉淀中NF-M的含量分别下降12％、19％、54％和41％(P＜0.01)。
NF-L：与年龄匹配的对照相比，坐骨神经上清中NF-L的含量在HD染毒第2、4、8周以及停止染毒16周时分别下降15％、40％、58％和34％(P＜0.01)；坐骨神经沉淀中NF-L的含量在HD染毒第4和8周时分别下降27％和53％(P＜0.01)，停药16周后恢复至正常水平。
a-tubulin：与对照相比，坐骨神经上清中a-tubulin的含量在HD染毒2、4、8周以及停药16周时均无显著性差异(P＞0.05)，坐骨神经沉淀中a-tubulin的含量在HD染毒2、4、8周以及停药16周后分别下降40.6％，41.2％、34.5％和14.2％(P＜0.01)。
β-tubulin：与对照相比，坐骨神经上清中β-tubulin的含量在HD染毒2、4、8周以及停药16周后β-tubulin的含量分别升高16％、44％、88.9％和86.1％(P＜0.01)，坐骨神经沉淀中β-tubulin的含量在HD染毒8周及停药16周后分别升高102.1％和99.2％(P＜0.01)。
β-actin：与对照相比，坐骨神经上清中β-actin的含量在染毒8周以及停药16周后分别下降27.1％和24.2％(P＜0.01)；坐骨神经沉淀中β-actin的含量在HD染毒2、4、8周时β-actin的含量分别升高24.8％、66.6％和103.1％(P＜0.01)，停药16周后恢复正常。
2．2 HD染毒大鼠神经组织中NFs含量变化与神经行为学改变的相关性分析
HD染毒诱导的大鼠神经组织NFs含量变化与其神经行为学改变之间存在明显的相关性。大脑中NF-H、NF-M和NF-L的含量与步态改变之间的复相关系数分别为0.8651(P＜0.0001)、0.9644(P＜0.0001)和0.8872(P＜0.0001)，脊髓中NF-H、NF-M和NF-L的含量与步态改变之间的复相关系数分别为0.8912(P＜0.0001)、0.9282(P＜0.0001)和0.8981(P＜0.0001)，坐骨神经中NF-H、NF-M和NF-L的含量与步态改变的复相关系数分别为0.9133(P＜0.0001)、0.9156(P＜0.0001)和0.9446(P＜0.0001)。
2．3 CS_2染毒大鼠神经组织中细胞骨架蛋白含量的变化
2．3．1大脑
与对照相比，300、500mg／kg CS_2染毒组大脑上清中NF-H含量分别下降31％和33％(P＜0.01)，大脑沉淀中分别下降37％和45％(P＜0.01)；300、500mg／kgCS_2组大脑上清中NF-M含量分别下降52％和58％(P＜0.01)，大脑沉淀中分别下降37％和75％(P＜0.01)；300、500mg／kg CS_2组大脑上清中NF-L的含量分别下降60％和83％(P＜0.01)，大脑沉淀中分别下降57％和60％(P＜0.01)。
与对照相比，α-tubulin的含量在300、500mg／kgCS_2染毒组大脑上清中分别升高20％和27％(P＜0.01)，大脑沉淀中无显著性变化；β-tubulin的含量在300、500mg／kgCS_2染毒组大脑上清中分别升高63％和71％(P＜0.01)，大脑沉淀中分别下降20％、26％(P＜0.01)；此外，β-actin的含量在500mg／kgCS_2染毒组大脑上清中升高31％(P＜0.01)。
2．3．2脊髓
与对照相比，NF-H含量在500 mg／kg CS_2染毒组脊髓上清中下降37％(P＜0.01)，而在300、500mg／kg CS_2染毒组脊髓沉淀中分别下降20％、24％(P＜0.01)；NF-M含量在500mg／kg CS_2染毒组脊髓上清中下降33％(P＜0.01)，而在300、500mg／kgCS_2染毒组脊髓沉淀中分别下降16％、65％(P＜0.01)；NF-L含量在500mg／kgCS_2染毒组脊髓上清中下降68％(P＜0.01)，脊髓沉淀中下降40％(P＜0.01)。
与对照相比，β-tubulin的含量在300、500mg／kgCS_2组脊髓上清中分别升高141％、158％(P＜0.01)，脊髓沉淀中分别升高107％、118％(P＜0.01)；β-actin的含量在300、500mg／kgCS_2组脊髓上清中分别升高19％、31％(p＜0.01)，脊髓沉淀中无显著性变化；此外，α-tubulin的含量均无显著性变化。
2．3．3坐骨神经
与对照相比，300、500mg／kgCS_2染毒组坐骨神经上清中NF-H的含量分别下降21.3％和30.9％(P＜0.01)，沉淀中分别下降12.6％(P＞0.05)、21.4％(P＜0.01)；300、500 mg／kgCS_2染毒组坐骨神经上清中NF-M的含量分别升高21.8％、42.5％(P＜0.01)，沉淀中分别升高41％、43.4％(P＜0.01)；300、500mg／kgCS_2染毒组坐骨神经上清中NF-L的含量分别下降24.4％、26.8％(P＜0.01)，沉淀中分别下降4.6％(P＞0.05)、45.4％(P＜0.01)。
与对照相比，300、500 mg／kgCS_2组坐骨神经上清中β-tubulin含量分别升高18.3％、40.4％(P＜0.01)，沉淀中分别下降18.4％和26.8％(P＜0.01)；此外，500 mg／kg CS_2组坐骨神经上清中α-tubulin、β-actin含量分别升高74％和17.3％(P＜0.01)，坐骨神经沉淀中分别下降19.7％和30.5％(P＜0.01)。
3 HD和CS_2染毒大鼠神经组织中细胞骨架mRNA表达的变化
3．1 HD染毒大鼠神经组织中细胞骨架mRNA表达的时间变化趋势
3．1．1大脑
与对照相比，大脑组织中NF-L、NF-H、α-tubulin、β-tubulin、β-actin的mRNA水平在HD染毒2、4和8周时均无显著性改变，但NF-M的mRNA水平在HD染毒2、4周时分别下降13.5％和24.8％(P＜0.01)，
3．1．2脊髓
与对照相比，脊髓组织中NF-L、NF-M、NF-H、β-actin、α-tubulin、β-tubulin的mRNA水平在HD染毒2、4和8周时均无显著性变化。
3．2 CS_2染毒大鼠神经组织中细胞骨架mRNA表达的变化
3．2．1大脑
与对照相比，NF-M的mRNA水平在300、500 mg／kg染毒组中分别下降了21.6％和21.8％(p＜0.01)，NF-L的mRNA水平仅在500mg／kg染毒组中下降18.1％(p＜0.01)。然而，NF-H的mRNA表达未发生显著性变化。
与对照相比，α-tubulin和β-actin的mRNA水平无显著性变化，而β-tubulin的mRNA水平在500 mg／kg染毒组下降13.5％(p＜0.01)。
3．2．2脊髓
与对照相比，300、500 mg／kg CS_2染毒组NF-H的mRNA水平分别升高33.3％和46.2％(P＜0.01)，NF-M的mRNA水平分别升高119.8％和181.1％(P＜0.01)，NF-L的mRNA水平分别升高101.7％和130.3％(P＜0.01)。
与对照相比，300、500 mg／kg CS_2染毒组β-tubulin的mRNA水平分别升高207％和212％(P＜0.01)，β-actin的mRNA水平分别升高94％和91％(P＜0.01)，而α-tubulin的mRNA水平无显著性变化。
4．HD和CS_2染毒大鼠神经组织中calpains表达的变化
4．1 HD染毒大鼠神经组织中calpains表达的时间变化趋势
4．1．1大脑
m-calpain：与对照相比，大脑上清中m-calpain的含量在HD染毒第4、8周时分别下降15.3％和21.8％(P＜0.01)，大脑沉淀中m-calpain的含量仅在HD染毒第8周时下降17.4％(P＜0.01)。
μ-calpain：与对照相比，大脑上清中μ-calpain的含量在HD染毒第4、8周时分别升高24.6％和64.4％(P＜0.01)，大脑沉淀中μ-calpain的含量仅在HD染毒第8周时升高72.7％(P＜0.01)。
4．1．2脊髓
m-calpain：与对照相比，HD染毒第2、4和8周时脊髓上清中m-calpain含量分别下降11.6％(P＞0.05)、19.9％(P＜0.05)和20.4％(P＜0.05)，脊髓沉淀中分别下降15.8％、35.9％和54.6％(P＜0.01)。停止染毒16周后，脊髓中m-calpain的含量又恢复正常。
μ-calpain：与对照相比，脊髓上清中μ-calpain含量在HD染毒第2、4周时分别下降12.9％和24.3％(P＜0.01)，而染毒第8周时却升高62.3％(P＜0.01)，停止染毒16周后仍然升高33.6％(P＜0.01)；脊髓沉淀中μ-calpain含量在HD染毒第2、4时分别下降15.4％(P＞0.05)和23％(P＜0.01)，而染毒第8周时却升高25.2％(P＜0.01)，停止染毒16周后恢复正常。
4．1．3坐骨神经
m-calpain：与对照相比，坐骨神经上清中m-calpain的含量在HD染毒第8周以及停药16周时分别升高46％和44％(P＜0.01)；坐骨神经沉淀中m-calpain的含量在HD染毒第8周时升高61％(P＜0.01)，停药16周后恢复正常。
μ-calpain：与对照相比，坐骨神经上清中μ-calpain的含量在HD染毒第4、8周以及停止染毒16周时分别升高30％、63％和54％(P＜0.01)，而坐骨神经沉淀中分别升高31％、59％和34％(P＜0.01)。
4．2 CS_2染毒大鼠神经组织中calpains表达的变化
4．2．1大脑组织
m-calpain：与对照相比，300、500 mg／kg CS_2染毒组大脑组织中m-calpain的含量均无显著变化。
μ-calpain：与对照相比，300、500 mg／kg CS_2染毒组大脑上清中μ-calpain的含量分别升高34％、53％(P＜0.01)，大脑沉淀中分别升高74％、49％(P＜0.01)。
4．2．2脊髓组织
m-calpain：与对照相比，300、500 mg／kgCS_2染毒组脊髓上清中m-calpain的含量分别下降46％和47.8％(P＜0.01)，脊髓沉淀中无显著变化。
μ-calpain：与对照相比，300、500 mg／kg CS_2染毒组脊髓上清中μ-calpain含量分别升高39.3％和47.2％(P＜0.01)，脊髓沉淀中分别升高37.5％和36.2％(P＜0.01)。
4．2．3坐骨神经
m-calpain：与对照相比，300、500 mg／kg CS_2染毒组坐骨神经上清中m-calpain的含量分别升高18.3％和42.7％(P＜0.01)，500 mg／kg CS_2染毒组坐骨神经沉淀中m-calpain的含量升高19.3％(P＜0.05)。
μ-calpain：与对照相比，300 mg／kg CS_2染毒组坐骨神经上清中μ-calpain的含量下降19.2％(P＜0.01)，500 mg／kg染毒组坐骨神经上清中无变化；300mg／kgCS_2染毒组坐骨神经沉淀中μ-calpain的含量下降36.8％(P＜0.01)，而500mg／kg染毒组中升高35.9％(P＜0.01)。
5．HD和CS_2染毒大鼠神经组织中calpains总活性的变化
5．1 HD染毒大鼠神经组织中calpains总活性的变化
与对照相比，大脑组织中calpains总活性仅在HD染毒第8周时下降37.2％(P＜0.01)；脊髓组织中calpains总活性在HD染毒第2、4和8周时分别下降22.8％、26.9％和27.5％(P＜0.01)，停止染毒16周后calpain活性恢复正常；坐骨神经中calpains总活性在HD染毒第2、4和8周以及停止染毒第16周后分别升高14％、20％(P＜0.05)、34％(P＜0.01)和18％(P＜0.05)。
5.2 CS_2染毒大鼠神经组织中calpains总活性的变化
与对照相比，300、500 mg／kgCS_2染毒组脊髓组织中calpains总活性分别下降18.5％和20.6％(P＜0.01)，坐骨神经组织中calpains总活性分别上升15.5％和20.7％(P＜0.01)，而大脑中calpains总活性未发生显著变化。
结论
1、HD、CS_2染毒引起大鼠中枢和周围神经组织中3种NF亚单位含量发生明显变化，其中NF-M和NF-L的变化较NF-H更加显著。
2、HD诱导的大鼠神经组织NF亚单位含量变化与其神经行为学改变之间存在高度相关性。
3、HD诱导的神经组织NF含量下降具有可逆性，并与HD中毒性神经病症状的恢复有关。
4、HD、CS_2染毒对不同神经组织中α-tubulin、β-tubulin和β-actin亚单位的影响不同。HD主要影响坐骨神经中α-tubulin、β-tubulin和β-actin的含量，而CS_2对中枢和周围神经组织中α-tubulin、β-mbulin和β-actin的含量均产生明显影响，其中β-tubulin的变化较α-tubulin和β-actin更加显著。
5、HD、CS_2对细胞骨架的六种成分的影响程度不同，其中NFs含量的变化比tubulins、β-actin显著。
6、HD染毒对神经组织中六种细胞骨架蛋白的mRNA表达未产生明显影响，而CS_2染毒影响了神经组织中六种细胞骨架蛋白的mRNA表达水平，表现为大脑组织中NF-L、NF-H和β-tubulin mRNA的转录水平下调，而脊髓组织中NF-H、NF-M、NF-L、β-tubulin和β-actin mRNA的转录水平上调。
7、HD、CS_2染毒影响了神经组织中calpains表达和活性，表现为中枢神经组织中calpains活性下降，坐骨神经中calpains活性升高。末梢轴索中calpains的激活可能与HD、CS_2诱导的神经丝含量下降有关。
8、毒物引起的神经组织中骨架蛋白改变参与了正己烷和CS_2中毒性周围神经病的发生。
Objective
n-hexane and carbon disulfide (CS_2) 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. CS_2 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 CS_2 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 CS_2 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 CS_2-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, ca~(2+)-activated neutral protease responsible for the degradation of cytoskeleton, were investigated to explore the possible mechanism of neuropathy induced by n-hexane and CS_2.
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 CS_2 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~(?) InnoZyme~(TM) Calpain 1 & 2 Activity Kit.
Results
1 The establishment of the animal model
Rats 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, CS_2 intoxication could result in significant neurological defects. After 12 weeks of CS_2 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 tissues
2.1 HD-induced alterations of cytoskeletal proteins in rat nervous tissues
2.1.1 Cerebrum
Compared 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 cord
Compared 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 nerve
In 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 pellet
When 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 rats
To 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 tissues
23.1 Cerebrum
Compared to the controls, in the supernatant fraction, NF-H levels in 300 and 500 mg/kg CS_2-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 CS_2-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 CS_2-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 cord
In the supernatant fraction, the levels of NF-H, NF-M, and NF-L in 300 mg/kg CS_2-treated group remained unaffected, however, their levels in 500 mg/kg CS_2-treated group decreased by 37%, 33% and 68% respectively. In the pellet, the levels of NF-H in 300 and 500 mg/kg CS_2-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 CS_2 group decreased by 40%.
When compared to the controls,β-tubulin levels in 300 and 500 mg/kg CS_2-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 CS_2-treated groups increased by 19% and 31% respectively. However, the levels ofα-tubulin remained unchangeable in both treated groups.
2.3.3 Sciatic nerve
Compared to the controls, in the supernatant fraction, NF-H levels in 300 and 500 mg/kg CS_2-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 CS_2-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 CS_2-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 CS_2-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 CS_2-treated groups decreased by 19.7% and 30.5% respectively. As far asβ-tubulin was concerned, its levels in 300 and 500 mg/kg CS_2-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 tissues
3.1 HD-induced alterations of cytoskeleton mRNA in rat nervous tissues
3.1.1 Cerebrum
When 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 cord
When 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 CS_2-induced alterations of cytoskeleton mRNA in rat nervous tissues
3.2.1 Cerebrum
When compared to the control, the mRNA level of NF-M in rats treated with 300 mg/kg CS_2 decreased by 21.6%, and that in rats treated with 500 mg/kg CS_2 by 21.8%. Furthermore, the mRNA levels of NF-L andβ-tubulin in rats treated with 300 mg/kg CS_2 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 cord
When compared to the control, the mRNA levels of NF-H, NF-M and NF-L in rats treated with 300 mg/kg CS_2 were elevated by 32%, 120%, and 102% respectively, and those in rats treated with 500 mg/kg CS_2 by 46%, 181%, and 130% respectively. Furthermore, the mRNA levels ofβ-tubulin andβ-actin in rats treated with 300 mg/kg CS_2 were elevated by 207% and 94% respectively, and those in rats treated with 500 mg/kg CS_2 by 212% and 91% respectively. However, the mRNA level ofα-tubulin showed no significant alteration in both CS_2-treated groups.
4 The alterations of calpains content in rat nervous tissues
4.1 HD-induced alterations of calpains content in rat nervous tissues
4.1.1 Cerebrum
When 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 cord
When 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 nerve
When 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 tissues
4.2.1 Cerebrum
When compared to the control, the levels of m-calpain in both fractions showed no significant alteration in both CS_2-treated groups. As far asμ-calpain was concerned, the levels ofμ-calpain in rats treated with 300, 500mg/kg CS_2 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 cord
In the supernatant fraction, the levels of m-calpain in rats treated with 300, 500 mg/kg CS_2 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 nerve
When compared to the control, the levels of m-calpain in rats treated with 300, 500 mg/kg CS_2 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 CS_2-treated group.
As far asμ-calpain was concerned, the levels ofμ-calpain in rats treated with 300 mg/kg CS_2 decreased by 19.2% and 36.8% in the supernatant and pellet fractions respectively, and those in rats treated with 500 mg/kg CS_2 increased by 35.9% only in the pellet fraction.
5 The alteration of calpains activity in rat nervous tissues
5.1 HD-induced alteration of calpains activity in rat nervous tissues
When 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 CS_2-induced alteration of calpains activity in rat nervous tissues
When compared to age-matched controls, the calpains activity of spinal cords decreased by 18.5% and 20.6% in 300 and 500 mg/kg CS_2-treated group respectively. Furthermore, the calpains activity of sciatic nerves increased by 15.5% and 20.7% in 300 and 500 mg/kg CS_2-treated group respectively. However, calpains activity of cerebrums remained unchangeable in both CS_2-treated groups.
Conclusions
1 The intoxication of HD and CS_2 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 CS_2 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 CS_2 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 CS_2 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 CS_2.
8 The alteration of cytoskeleton induced by HD and CS_2 was involved in the development of HD or CS2- induced neuropathy.
正己烷(n-hexane)和二硫化碳(carbon disulfide，CS_2)是常用的工业溶剂。正已烷主要应用于粘胶配制、除污、干洗、植物油提取、制鞋、印刷、油漆、制药、家具制造及电子元件制造等行业，而CS_2则广泛应用于粘胶纤维、玻璃纸、四氯化碳和杀虫剂等生产过程。
长期职业接触正己烷、CS_2可引起以感觉运动型周围神经病为主要临床特征的慢性中毒。早期表现为手足部感觉异常，痛觉、触觉减退呈手套、袜套样分布，进一步发展可表现为四肢无力、行走困难以至瘫痪。主要的病理改变发生在肢体远端最长、最粗的感觉和运动神经轴索，并在郎飞氏节附近形成巨大的充满神经丝(neurofilament，NF)的肿胀，郎飞氏节解剖结构扭曲且髓鞘从肿胀处回缩，有时伴有节段性脱髓鞘，最后轴索发生变性。
正己烷、CS_2中毒周围神经病的发生机制尚不清楚，但由于二者在神经行为学改变和病理改变上都非常相似，提示它们可能分享相同或相似的分子机制。同时，病理形态学研究提示NF、微管(microtubule，MT)等骨架蛋白可能参与了中毒性轴索病变的发生。国内外的研究已经报道，2，5-己二酮(2，5-hexanedione，HD)、CS_2亚慢性染毒可引起大鼠神经组织中NF含量的减少，但对这种变化发生的原因以及在它们在中毒性神经病中的作用没有进行深入探讨。因此，细胞骨架在中毒性周围神经病中的作用并没有真正阐明。
本文通过建立亚慢性中毒动物模型，观察正己烷、CS_2中毒性神经病发生过程中NF-H、NF-M、NF-L、α-tubulin、β-tubulin和β-actin等细胞骨架蛋白相对含量的变化，在此基础上进一步研究毒物对细胞骨架蛋白的基因转录、蛋白降解的影响，以探讨有机溶剂中毒性周围神经病的发病机制。
研究方法
1雄性Wistar大鼠用400mg／kg．bw的HD腹腔注射染毒，染毒时间分别为2、4和8周，建立正己烷中毒性周围神经病模型，并取一半8周染毒动物继续观察至24周。雄性Wistar大鼠分别用300、500mg／kg．bw的CS_2灌胃染毒，连续12周，建立CS_2中毒性周围神经病模型。
2取大鼠大脑、脊髓和坐骨神经组织制备匀浆，100，000×g、4℃离心60min。利用Western-Blotting技术研究大鼠大脑、脊髓、坐骨神经上清和沉淀中NF-H、NF-M、NF-L、α-tubulin、β-tubulin和β-actin等骨架蛋白以及钙激活的中性蛋白酶m-calpain和μ-calpain含量的变化。
3取大鼠大脑和脊髓组织，提取总RNA，利用RT-PCR技术研究HD和CS_2染毒对大鼠中枢神经组织中NF-H、NF-M、NF-L、α-tubulin、β-tubulin和β-actin mRNA表达水平的影响。
4取大鼠大脑、脊髓和坐骨神经组织制备匀浆，8，000×g，4℃离心30min。利用Calbiochem(?) InnoZymeTM Calpain 1&2 Activity Kit检测大鼠大脑、脊髓、坐骨神经组织中calpains总活性的变化。
研究结果
1动物模型的建立以及神经行为学指标的变化
给予HD、CS_2染毒后，动物逐渐出现肌肉松弛、肌张力降低、步态异常以至瘫痪等症状，神经行为学测试表明染毒大鼠呈现典型的中毒性周围神经病特征，这提示正己烷、CS_2中毒性周围神经病模型建立成功。同时HD染毒停止后，染毒大鼠的感觉运动指标能够逐渐改善。
2 HD、CS_2染毒大鼠神经组织中细胞骨架蛋白含量的变化
2．1 HD染毒大鼠神经组织中细胞骨架蛋白含量的时间变化趋势
2．1．1大脑
NF-H：与对照相比，大脑上清中NF-H的含量在HD染毒第2、4和8周时分别下降31.6％、40.3％和42％(P＜0.01)，大脑沉淀中NF-H的含量分别下降11.1％(P＜0.05)、51.8％和53.2％(P＜0.01)。
NF-M：与对照相比，大脑上清中NF-M的含量在HD染毒第2、4和8周时分别下降34.7％、58.2％和77.2％(P＜0.01)，大脑沉淀中NF-M的含量分别下降29.4％、50.6％和72.5％(P＜0.01)。
NF-L：与对照相比，大脑上清中NF-L的含量在HD染毒第2、4和8周时分别下降28％、35.4％和46.3％(P＜0.01)，大脑沉淀中NF-L的含量分别下降18％、34.7％和34.7％(P＜0.01)。
β-tubulin：与对照相比，大脑上清中β-tubulin的含量在HD染毒4周和8周时分别升高29％和33％(P＜0.01)，大脑沉淀中β-tubulin的含量在HD染毒8周时也升高16.3％(P＜0.05)。
α-tubulin和β-actin：与对照相比，大脑上清和沉淀中α-tubulin和β-actin的含量在HD染毒后未发生明显变化。
2．1．2脊髓
NF-H：与对照相比，脊髓上清中NF-H的含量在HD染毒第2、4和8周时分别下降16％、57％和58％(P＜0.01)，脊髓沉淀中NF-H的含量在HD染毒第4和8周时分别下降33％和43％(P＜0.01)。停药16周后，NF-H的含量恢复正常。
NF-M：与对照相比，脊髓上清中NF-M的含量在HD染毒第2、4和8周时分别下降36％、61％和65％(P＜0.01)，脊髓沉淀中NF-M的含量分别下降23％、34％和64％(P＜0.01)。停药16周后，NF-M含量又恢复正常。
NF-L：与对照相比，脊髓上清中NF-L的含量在HD染毒第2、4和8周时分别下降21％、44％和45％(P＜0.01)，脊髓沉淀中NF-L含量在第4和8周时分别下降26％和42％(P＜0.01)。停药16周后，NF-L含量又恢复正常。
α-tubulin、β-tubulin和β-actin：除HD染毒第8周时脊髓上清中β-tubulin的含量升高34.8％(P＜0.01)外，其余均未发生明显变化。
2．1．3坐骨神经
NF-H：与对照相比，坐骨神经上清中NF-H的含量在HD染毒第2、4和8周时分别下降19％、22％和43％(P＜0.01)，坐骨神经沉淀中NF-H含量在HD染毒第4和8周时分别下降12％和51％(P＜0.01)。停药16周后，NF-H含量又恢复正常。
NF-M：与年龄匹配的对照相比，坐骨神经上清中NF-M的含量在HD染毒第2、4、8周以及停止染毒16周时分别下降15％、24％、88％和83％(P＜0.01)，坐骨神经沉淀中NF-M的含量分别下降12％、19％、54％和41％(P＜0.01)。
NF-L：与年龄匹配的对照相比，坐骨神经上清中NF-L的含量在HD染毒第2、4、8周以及停止染毒16周时分别下降15％、40％、58％和34％(P＜0.01)；坐骨神经沉淀中NF-L的含量在HD染毒第4和8周时分别下降27％和53％(P＜0.01)，停药16周后恢复至正常水平。
a-tubulin：与对照相比，坐骨神经上清中a-tubulin的含量在HD染毒2、4、8周以及停药16周时均无显著性差异(P＞0.05)，坐骨神经沉淀中a-tubulin的含量在HD染毒2、4、8周以及停药16周后分别下降40.6％，41.2％、34.5％和14.2％(P＜0.01)。
β-tubulin：与对照相比，坐骨神经上清中β-tubulin的含量在HD染毒2、4、8周以及停药16周后β-tubulin的含量分别升高16％、44％、88.9％和86.1％(P＜0.01)，坐骨神经沉淀中β-tubulin的含量在HD染毒8周及停药16周后分别升高102.1％和99.2％(P＜0.01)。
β-actin：与对照相比，坐骨神经上清中β-actin的含量在染毒8周以及停药16周后分别下降27.1％和24.2％(P＜0.01)；坐骨神经沉淀中β-actin的含量在HD染毒2、4、8周时β-actin的含量分别升高24.8％、66.6％和103.1％(P＜0.01)，停药16周后恢复正常。
2．2 HD染毒大鼠神经组织中NFs含量变化与神经行为学改变的相关性分析
HD染毒诱导的大鼠神经组织NFs含量变化与其神经行为学改变之间存在明显的相关性。大脑中NF-H、NF-M和NF-L的含量与步态改变之间的复相关系数分别为0.8651(P＜0.0001)、0.9644(P＜0.0001)和0.8872(P＜0.0001)，脊髓中NF-H、NF-M和NF-L的含量与步态改变之间的复相关系数分别为0.8912(P＜0.0001)、0.9282(P＜0.0001)和0.8981(P＜0.0001)，坐骨神经中NF-H、NF-M和NF-L的含量与步态改变的复相关系数分别为0.9133(P＜0.0001)、0.9156(P＜0.0001)和0.9446(P＜0.0001)。
2．3 CS_2染毒大鼠神经组织中细胞骨架蛋白含量的变化
2．3．1大脑
与对照相比，300、500mg／kg CS_2染毒组大脑上清中NF-H含量分别下降31％和33％(P＜0.01)，大脑沉淀中分别下降37％和45％(P＜0.01)；300、500mg／kgCS_2组大脑上清中NF-M含量分别下降52％和58％(P＜0.01)，大脑沉淀中分别下降37％和75％(P＜0.01)；300、500mg／kg CS_2组大脑上清中NF-L的含量分别下降60％和83％(P＜0.01)，大脑沉淀中分别下降57％和60％(P＜0.01)。
与对照相比，α-tubulin的含量在300、500mg／kgCS_2染毒组大脑上清中分别升高20％和27％(P＜0.01)，大脑沉淀中无显著性变化；β-tubulin的含量在300、500mg／kgCS_2染毒组大脑上清中分别升高63％和71％(P＜0.01)，大脑沉淀中分别下降20％、26％(P＜0.01)；此外，β-actin的含量在500mg／kgCS_2染毒组大脑上清中升高31％(P＜0.01)。
2．3．2脊髓
与对照相比，NF-H含量在500 mg／kg CS_2染毒组脊髓上清中下降37％(P＜0.01)，而在300、500mg／kg CS_2染毒组脊髓沉淀中分别下降20％、24％(P＜0.01)；NF-M含量在500mg／kg CS_2染毒组脊髓上清中下降33％(P＜0.01)，而在300、500mg／kgCS_2染毒组脊髓沉淀中分别下降16％、65％(P＜0.01)；NF-L含量在500mg／kgCS_2染毒组脊髓上清中下降68％(P＜0.01)，脊髓沉淀中下降40％(P＜0.01)。
与对照相比，β-tubulin的含量在300、500mg／kgCS_2组脊髓上清中分别升高141％、158％(P＜0.01)，脊髓沉淀中分别升高107％、118％(P＜0.01)；β-actin的含量在300、500mg／kgCS_2组脊髓上清中分别升高19％、31％(p＜0.01)，脊髓沉淀中无显著性变化；此外，α-tubulin的含量均无显著性变化。
2．3．3坐骨神经
与对照相比，300、500mg／kgCS_2染毒组坐骨神经上清中NF-H的含量分别下降21.3％和30.9％(P＜0.01)，沉淀中分别下降12.6％(P＞0.05)、21.4％(P＜0.01)；300、500 mg／kgCS_2染毒组坐骨神经上清中NF-M的含量分别升高21.8％、42.5％(P＜0.01)，沉淀中分别升高41％、43.4％(P＜0.01)；300、500mg／kgCS_2染毒组坐骨神经上清中NF-L的含量分别下降24.4％、26.8％(P＜0.01)，沉淀中分别下降4.6％(P＞0.05)、45.4％(P＜0.01)。
与对照相比，300、500 mg／kgCS_2组坐骨神经上清中β-tubulin含量分别升高18.3％、40.4％(P＜0.01)，沉淀中分别下降18.4％和26.8％(P＜0.01)；此外，500 mg／kg CS_2组坐骨神经上清中α-tubulin、β-actin含量分别升高74％和17.3％(P＜0.01)，坐骨神经沉淀中分别下降19.7％和30.5％(P＜0.01)。
3 HD和CS_2染毒大鼠神经组织中细胞骨架mRNA表达的变化
3．1 HD染毒大鼠神经组织中细胞骨架mRNA表达的时间变化趋势
3．1．1大脑
与对照相比，大脑组织中NF-L、NF-H、α-tubulin、β-tubulin、β-actin的mRNA水平在HD染毒2、4和8周时均无显著性改变，但NF-M的mRNA水平在HD染毒2、4周时分别下降13.5％和24.8％(P＜0.01)，
3．1．2脊髓
与对照相比，脊髓组织中NF-L、NF-M、NF-H、β-actin、α-tubulin、β-tubulin的mRNA水平在HD染毒2、4和8周时均无显著性变化。
3．2 CS_2染毒大鼠神经组织中细胞骨架mRNA表达的变化
3．2．1大脑
与对照相比，NF-M的mRNA水平在300、500 mg／kg染毒组中分别下降了21.6％和21.8％(p＜0.01)，NF-L的mRNA水平仅在500mg／kg染毒组中下降18.1％(p＜0.01)。然而，NF-H的mRNA表达未发生显著性变化。
与对照相比，α-tubulin和β-actin的mRNA水平无显著性变化，而β-tubulin的mRNA水平在500 mg／kg染毒组下降13.5％(p＜0.01)。
3．2．2脊髓
与对照相比，300、500 mg／kg CS_2染毒组NF-H的mRNA水平分别升高33.3％和46.2％(P＜0.01)，NF-M的mRNA水平分别升高119.8％和181.1％(P＜0.01)，NF-L的mRNA水平分别升高101.7％和130.3％(P＜0.01)。
与对照相比，300、500 mg／kg CS_2染毒组β-tubulin的mRNA水平分别升高207％和212％(P＜0.01)，β-actin的mRNA水平分别升高94％和91％(P＜0.01)，而α-tubulin的mRNA水平无显著性变化。
4．HD和CS_2染毒大鼠神经组织中calpains表达的变化
4．1 HD染毒大鼠神经组织中calpains表达的时间变化趋势
4．1．1大脑
m-calpain：与对照相比，大脑上清中m-calpain的含量在HD染毒第4、8周时分别下降15.3％和21.8％(P＜0.01)，大脑沉淀中m-calpain的含量仅在HD染毒第8周时下降17.4％(P＜0.01)。
μ-calpain：与对照相比，大脑上清中μ-calpain的含量在HD染毒第4、8周时分别升高24.6％和64.4％(P＜0.01)，大脑沉淀中μ-calpain的含量仅在HD染毒第8周时升高72.7％(P＜0.01)。
4．1．2脊髓
m-calpain：与对照相比，HD染毒第2、4和8周时脊髓上清中m-calpain含量分别下降11.6％(P＞0.05)、19.9％(P＜0.05)和20.4％(P＜0.05)，脊髓沉淀中分别下降15.8％、35.9％和54.6％(P＜0.01)。停止染毒16周后，脊髓中m-calpain的含量又恢复正常。
μ-calpain：与对照相比，脊髓上清中μ-calpain含量在HD染毒第2、4周时分别下降12.9％和24.3％(P＜0.01)，而染毒第8周时却升高62.3％(P＜0.01)，停止染毒16周后仍然升高33.6％(P＜0.01)；脊髓沉淀中μ-calpain含量在HD染毒第2、4时分别下降15.4％(P＞0.05)和23％(P＜0.01)，而染毒第8周时却升高25.2％(P＜0.01)，停止染毒16周后恢复正常。
4．1．3坐骨神经
m-calpain：与对照相比，坐骨神经上清中m-calpain的含量在HD染毒第8周以及停药16周时分别升高46％和44％(P＜0.01)；坐骨神经沉淀中m-calpain的含量在HD染毒第8周时升高61％(P＜0.01)，停药16周后恢复正常。
μ-calpain：与对照相比，坐骨神经上清中μ-calpain的含量在HD染毒第4、8周以及停止染毒16周时分别升高30％、63％和54％(P＜0.01)，而坐骨神经沉淀中分别升高31％、59％和34％(P＜0.01)。
4．2 CS_2染毒大鼠神经组织中calpains表达的变化
4．2．1大脑组织
m-calpain：与对照相比，300、500 mg／kg CS_2染毒组大脑组织中m-calpain的含量均无显著变化。
μ-calpain：与对照相比，300、500 mg／kg CS_2染毒组大脑上清中μ-calpain的含量分别升高34％、53％(P＜0.01)，大脑沉淀中分别升高74％、49％(P＜0.01)。
4．2．2脊髓组织
m-calpain：与对照相比，300、500 mg／kgCS_2染毒组脊髓上清中m-calpain的含量分别下降46％和47.8％(P＜0.01)，脊髓沉淀中无显著变化。
μ-calpain：与对照相比，300、500 mg／kg CS_2染毒组脊髓上清中μ-calpain含量分别升高39.3％和47.2％(P＜0.01)，脊髓沉淀中分别升高37.5％和36.2％(P＜0.01)。
4．2．3坐骨神经
m-calpain：与对照相比，300、500 mg／kg CS_2染毒组坐骨神经上清中m-calpain的含量分别升高18.3％和42.7％(P＜0.01)，500 mg／kg CS_2染毒组坐骨神经沉淀中m-calpain的含量升高19.3％(P＜0.05)。
μ-calpain：与对照相比，300 mg／kg CS_2染毒组坐骨神经上清中μ-calpain的含量下降19.2％(P＜0.01)，500 mg／kg染毒组坐骨神经上清中无变化；300mg／kgCS_2染毒组坐骨神经沉淀中μ-calpain的含量下降36.8％(P＜0.01)，而500mg／kg染毒组中升高35.9％(P＜0.01)。
5．HD和CS_2染毒大鼠神经组织中calpains总活性的变化
5．1 HD染毒大鼠神经组织中calpains总活性的变化
与对照相比，大脑组织中calpains总活性仅在HD染毒第8周时下降37.2％(P＜0.01)；脊髓组织中calpains总活性在HD染毒第2、4和8周时分别下降22.8％、26.9％和27.5％(P＜0.01)，停止染毒16周后calpain活性恢复正常；坐骨神经中calpains总活性在HD染毒第2、4和8周以及停止染毒第16周后分别升高14％、20％(P＜0.05)、34％(P＜0.01)和18％(P＜0.05)。
5.2 CS_2染毒大鼠神经组织中calpains总活性的变化
与对照相比，300、500 mg／kgCS_2染毒组脊髓组织中calpains总活性分别下降18.5％和20.6％(P＜0.01)，坐骨神经组织中calpains总活性分别上升15.5％和20.7％(P＜0.01)，而大脑中calpains总活性未发生显著变化。
结论
1、HD、CS_2染毒引起大鼠中枢和周围神经组织中3种NF亚单位含量发生明显变化，其中NF-M和NF-L的变化较NF-H更加显著。
2、HD诱导的大鼠神经组织NF亚单位含量变化与其神经行为学改变之间存在高度相关性。
3、HD诱导的神经组织NF含量下降具有可逆性，并与HD中毒性神经病症状的恢复有关。
4、HD、CS_2染毒对不同神经组织中α-tubulin、β-tubulin和β-actin亚单位的影响不同。HD主要影响坐骨神经中α-tubulin、β-tubulin和β-actin的含量，而CS_2对中枢和周围神经组织中α-tubulin、β-mbulin和β-actin的含量均产生明显影响，其中β-tubulin的变化较α-tubulin和β-actin更加显著。
5、HD、CS_2对细胞骨架的六种成分的影响程度不同，其中NFs含量的变化比tubulins、β-actin显著。
6、HD染毒对神经组织中六种细胞骨架蛋白的mRNA表达未产生明显影响，而CS_2染毒影响了神经组织中六种细胞骨架蛋白的mRNA表达水平，表现为大脑组织中NF-L、NF-H和β-tubulin mRNA的转录水平下调，而脊髓组织中NF-H、NF-M、NF-L、β-tubulin和β-actin mRNA的转录水平上调。
7、HD、CS_2染毒影响了神经组织中calpains表达和活性，表现为中枢神经组织中calpains活性下降，坐骨神经中calpains活性升高。末梢轴索中calpains的激活可能与HD、CS_2诱导的神经丝含量下降有关。
8、毒物引起的神经组织中骨架蛋白改变参与了正己烷和CS_2中毒性周围神经病的发生。
Objective
n-hexane and carbon disulfide (CS_2) 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. CS_2 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 CS_2 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 CS_2 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 CS_2-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, ca~(2+)-activated neutral protease responsible for the degradation of cytoskeleton, were investigated to explore the possible mechanism of neuropathy induced by n-hexane and CS_2.
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 CS_2 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~(?) InnoZyme~(TM) Calpain 1 & 2 Activity Kit.
Results
1 The establishment of the animal model
Rats 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, CS_2 intoxication could result in significant neurological defects. After 12 weeks of CS_2 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 tissues
2.1 HD-induced alterations of cytoskeletal proteins in rat nervous tissues
2.1.1 Cerebrum
Compared 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 cord
Compared 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 nerve
In 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 pellet
When 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 rats
To 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 tissues
23.1 Cerebrum
Compared to the controls, in the supernatant fraction, NF-H levels in 300 and 500 mg/kg CS_2-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 CS_2-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 CS_2-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 cord
In the supernatant fraction, the levels of NF-H, NF-M, and NF-L in 300 mg/kg CS_2-treated group remained unaffected, however, their levels in 500 mg/kg CS_2-treated group decreased by 37%, 33% and 68% respectively. In the pellet, the levels of NF-H in 300 and 500 mg/kg CS_2-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 CS_2 group decreased by 40%.
When compared to the controls,β-tubulin levels in 300 and 500 mg/kg CS_2-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 CS_2-treated groups increased by 19% and 31% respectively. However, the levels ofα-tubulin remained unchangeable in both treated groups.
2.3.3 Sciatic nerve
Compared to the controls, in the supernatant fraction, NF-H levels in 300 and 500 mg/kg CS_2-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 CS_2-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 CS_2-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 CS_2-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 CS_2-treated groups decreased by 19.7% and 30.5% respectively. As far asβ-tubulin was concerned, its levels in 300 and 500 mg/kg CS_2-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 tissues
3.1 HD-induced alterations of cytoskeleton mRNA in rat nervous tissues
3.1.1 Cerebrum
When 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 cord
When 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 CS_2-induced alterations of cytoskeleton mRNA in rat nervous tissues
3.2.1 Cerebrum
When compared to the control, the mRNA level of NF-M in rats treated with 300 mg/kg CS_2 decreased by 21.6%, and that in rats treated with 500 mg/kg CS_2 by 21.8%. Furthermore, the mRNA levels of NF-L andβ-tubulin in rats treated with 300 mg/kg CS_2 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 cord
When compared to the control, the mRNA levels of NF-H, NF-M and NF-L in rats treated with 300 mg/kg CS_2 were elevated by 32%, 120%, and 102% respectively, and those in rats treated with 500 mg/kg CS_2 by 46%, 181%, and 130% respectively. Furthermore, the mRNA levels ofβ-tubulin andβ-actin in rats treated with 300 mg/kg CS_2 were elevated by 207% and 94% respectively, and those in rats treated with 500 mg/kg CS_2 by 212% and 91% respectively. However, the mRNA level ofα-tubulin showed no significant alteration in both CS_2-treated groups.
4 The alterations of calpains content in rat nervous tissues
4.1 HD-induced alterations of calpains content in rat nervous tissues
4.1.1 Cerebrum
When 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 cord
When 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 nerve
When 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 tissues
4.2.1 Cerebrum
When compared to the control, the levels of m-calpain in both fractions showed no significant alteration in both CS_2-treated groups. As far asμ-calpain was concerned, the levels ofμ-calpain in rats treated with 300, 500mg/kg CS_2 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 cord
In the supernatant fraction, the levels of m-calpain in rats treated with 300, 500 mg/kg CS_2 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 nerve
When compared to the control, the levels of m-calpain in rats treated with 300, 500 mg/kg CS_2 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 CS_2-treated group.
As far asμ-calpain was concerned, the levels ofμ-calpain in rats treated with 300 mg/kg CS_2 decreased by 19.2% and 36.8% in the supernatant and pellet fractions respectively, and those in rats treated with 500 mg/kg CS_2 increased by 35.9% only in the pellet fraction.
5 The alteration of calpains activity in rat nervous tissues
5.1 HD-induced alteration of calpains activity in rat nervous tissues
When 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 CS_2-induced alteration of calpains activity in rat nervous tissues
When compared to age-matched controls, the calpains activity of spinal cords decreased by 18.5% and 20.6% in 300 and 500 mg/kg CS_2-treated group respectively. Furthermore, the calpains activity of sciatic nerves increased by 15.5% and 20.7% in 300 and 500 mg/kg CS_2-treated group respectively. However, calpains activity of cerebrums remained unchangeable in both CS_2-treated groups.
Conclusions
1 The intoxication of HD and CS_2 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 CS_2 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 CS_2 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 CS_2 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 CS_2.
8 The alteration of cytoskeleton induced by HD and CS_2 was involved in the development of HD or CS2- induced neuropathy.
引文
1 Couri D, Milks M. Toxicity and metabolism of the neurotoxic hexacarbons n-hexane, 2-hexanone, and 2, 5-hexanedione. Annu Rev Pharmacol Toxicol. 1982; 22: 145-66.
2 Spencer PS, Bischoff MC, Schaumburg HH. On the specific molecular configuration of neurotoxic aliphatic hexacarbon compounds causing central-peripheral distal axonopathy. Toxicol Appl Pharmacol. 1978; 44: 17-28.
3 Fedtke N and Bolt HM. The relevance of 4, 5-dihydroxy-2-hexanone in the excretion kinetics of n-hexane metabolites in rat and man. Arch. Toxicol. 1987; 61: 131-137.
4 WHO working group, n-Hexane Source. Environmental Health Criteria. 1991; 122: 133.
5 Green EC, Hunter A. Toxicity of carbon disulfide in developing rats: LD50 and effects on the hepatic mixed-function oxidase enzyme system. Toxicology and applied pharmacology. 1983; 78: 130-138.
6 Stetkiewicz J and Wronska-Nofer T. Updating of hygiene standards for carbon disulfide based on health risk assessment. Int J Occup Med Environ Health. 1998; 11(2): 129-143.
7 Beauchamp RO, Bus JS, Popp JA, Boreiko C J, and Goldberg L. A critical review of the literature on carbon disulfide toxicity. Crit Rev Toxicol. 1983; 11: 169-278.
8 林斯星，陈嘉斌，吴子俊．一起严重的正己烷职业中毒事故报告．中国工业医学杂志，1997；10：172-173．
9 杨玉珍．14例正己烷中毒患者的观察及护理．中国职业医学．1995；2(2)：63．
10 ATSDR (Agency for Toxic Substances and Disease Registry). Toxicological profile for n-hexane. 1999.
11 何凤生．中华职业医学．北京：人民卫生出版社，1999，62．
12 Cavanagh JB. The significance of the "dying back" process in experimental and human neurological disease. Int Rev Exp Pathol. 1964; 3: 219-267.
13 Spencer PS, schaumburg HH. central peripheral distal axonopathy: The pathology of dying-back polyneuropathies: progress in neuropathology. Vol 3. New york: Grune & Stratton, 1976, 253-295.
14 Sills RC, Harry GJ, Morgan DL, Valentine WM, Graham DG. Carbon disulfide neurotoxicity in rats: V. Morphology of axonal swelling in the muscular branch of the posterior tibial nerve and spinal cord. Neurotoxieology. 1998; 19:117-127.
15 Colombi A, Maroni M, Picchi O, Rota E, Castano P, Foa V. Carbon disulfide neuropathy in rats. A morphological and ultrastructural study of degeneration and regeneration. Clinical Toxicology. 1981; 18(12): 1463-1474.
16 Schaumburg HH, Spencer PS. Degeneration in central and peripheral nervous systems produced by pure n-bexane: an experimental study. Brain. 1976, 99(2): 183-192.
17 LoPachin RM, Ross JF, Reid ML, Das S, Mansukhani S, Lehning EJ. Neurological evaluation of toxic axonopathies in rats: acrylamide and 2,5-hexanedione. Neurotoxicology. 2002; 23:95-110.
18 Spencer PS, Sabri MI, Schaumburg HH, Moore CL. Does a defect of energy metabolism in the nerve fiber underlie axonal degeneration in polyneuropathies? Ann. Neurol. 1979; 5: 501-507.
19 Sabri MI, Moore CL, Spencer PS. Studies on the biochemical basis of distal axonopathies--I. Inhibition of glycolysis by neurotoxic bexacarbon compounds. J Neurochem. 1979; 32(3): 683-689.
20 Monaco S, Autilio-Gambetti L, Zabel D, Gambetti P. Giant axonal neuropathy: acceleration of neurofilament transport in optic axons. Proc. Natl Acad. Sci. USA. 1985; 82: 920-924.
21 Monaco S, Jacob J, Jenich H, Patton A, Autilio-Gambetti L, Gambetti P. Axonal transport of neurofilament is accelerated in peripheral nerve during 2,5-hexanedione intoxication. Brain Res. 1989; 491: 328-334.
22 Monaco S, Wongmongkolrit T, Shearson CM, Patton A, Schaetzle B, Autilio-Gambetti L, Gambetti P, Sayre LM. Giant axonopathy characterized by intermediate location of axonal enlargements and acceleration of neurofilament transport. Brain Res. 1990; 519: 73-81.
23 McKenna MJ and DiStefano V. Carbon disulfide. Ⅱ. A proposed mechanism for the action of carbon disulfide on dopamine beta-hydroxylase. J Pharmacol Exp Ther. 1977; 202(2): 253-266.
24 Teisinger J. New advances in the toxicology of carbon disulfide. Am Ind Hyg Assoc J. 1974; 35(2): 55-61.
25 Genter MB, Szakal-Quin G, Anderson CW, Anthony DC, Graham DG. Evidence that pyrrole formation is a pathogenetic step in gamma-diketone neuropathy. Toxicol Appl Pharmacol. 1987; 87(2): 351-362.
26 Genter St Clair MB, Amarnath V, Moody MA, Anthony DC, Anderson CW, Graham DG. Pyrrole oxidation and protein cross-linking as necessary steps in the development of gamma-diketone neuropathy. Chem Res Toxicol. 1988; 1(3): 179-185.
27 Sayre LM, Shearson CM, Wongmongkolrit T, Medori R, Gambetti P. Structural basis of gamma-diketone neurotoxicity: non-neurotoxicity of 3, 3-dimethyl-2, 5-hexanedione, a gamma-diketone incapable of pyrrole formation. Toxicol Appl Pharmacol. 1986; 84(1): 36-44.
28 Valentine WM, Amamath V, Graham DG., Morgan DL, and Sills RC. CS2-mediated cross-linking of erythrocyte spectrin and neurofilament protein: dose response and temporal relationship to the formation of axonal swellings. Toxicol Appl Pharmacol. 1997; 142(1): 95-105.
29 Valentine WM, Graham DG, and Anthony DC. Covalent cross-linking of erythrocyte spectrin by carbon disulfide in vivo. Toxicol Appl Pharmacol. 1993; 121(1): 71-77.
30 Hirai T, Mizutani M, Kimura T, Ochiai K, Umemura T, Itakura C. Neurotoxic effects of 2, 5-hexanedione on normal and neurofilament-deficient quail. Toxicol Pathol. 1999; 27(3): 348-353.
31 Stone JD, Peterson AP, Eyer J, Oblak TG, Sickles DW. Neurofilaments Are Nonessential to the Pathogenesis of Toxicant-Induced Axonal Degeneration J Neurosci. 2001; 21(7): 2278-2287.
32 Sickles DW, Pearson JK, Beall A, Testino A. Toxic axonal degeneration occurs independent of neurofilament accumulation. J Neurosci Res. 1994; 39(3): 347-354.
33 何凤生，薛启蒙．神经系统中毒及代谢性疾病．人民军医出版社，2002，75-77．
34 Hemandez-Viadel M, Montoliu C, Monfort P, Canales J J, Erceg S, Rowan M, Ceccatelli S, Felipo V. Chronic exposure to 2, 5-hexanedione impairs the glutamate-nitric oxide-cyclic GMP pathway in cerebellar neurons in culture and in rat brain in vivo. Neurochem Int. 2003; 42(7): 525-533.
35 Prieto-Castello MJ, Hernandez-Viadel ML, Cardona A, Marhuenda D, Felipo V. Activation of soluble guanylate cyclase by nitric oxide is increased in lymphocytes from both rats chronically exposed to 2, 5-hexanedione and workers chronically exposed to n-hexane. Toxicology. 2007; 229(1-2): 73-78.
36 Lehning EJ, Dyer KS, Jortner BS, LoPachin RM. Axonal atrophy is a specific component of 2,5-hexanedione peripheral neuropathy. Toxicol. Appl. Pharmacol. 1995; 135: 58-66.
37 Lehning EJ, Jortner BS, Fox JH, Arezzo JC, Kitano T, LoPachin RM. gamma-diketone peripheral neuropathy. I. Quality morphometric analyses ofaxonal atrophy and swelling. Toxicol. Appl. Pharmacol. 2000; 165: 127-140.
38 LoPachin RM, He D, Reid ML, Opanashuk LA. 2, 5-Hexanedione-induced changes in monomeric neurofilament protein content of rat spinal cord fractions. Toxicol. Appl. Pharmacol. 2004; 198, 61-73.
39 LoPachin RM, He D, Reid ML. 2,5-Hexanedione-induced changes in the neurofilament subtmit pools of rat peripheral nerve. Neurotoxicology, 2005, 26: 229-240.
40 高艳华，梁友信，傅慰祖．CS_2对大鼠神经微丝及红细胞膜结构的影响．中国工业医学杂志，1998；11(5)：267-270．
1 LoPachin RM, Ross JF, Reid ML, Das S, Mansukhani S, Lehning EJ. Neurological evaluation of toxic axonopathies in rats: acrylamide and 2, 5-hexanedione. Neurotoxicology 2002; 23: 95-110.
2 Moser VC, Phillips PM, Morgan DL, et al. Carbon disulfide neurotoxicity in rats: Ⅶ. Behavioral evaluations using a functional observational battery. Neurotoxicology, 1998; 19 (1): 147-157.
3 Schaumburg HH, Spencer PS. Degeneration in central and peripheral nervous systems produced by pure n-hexane: an experimental study. Brain, 1976; 99(2): 183-192.
4 Spencer P. Neuropathic potential of n-hexane in the presence of other hexane isomers[M]. Washington, DC: American Petroleumlnstitute (API Medical Research Publication, 1982: 30.
5 Spencer PS, Bischoff MC, Schaumburg HH. On the specific molecular configuration of neurotoxic aliphatic hexacarbon compounds causing central-peripheral distal axonopathy. Toxicol Appl Pharmacol, 1978; 44 (1): 17-28.
6 Shell L, Rozum M, Jortner BS, Ehrich M. Neurotoxicity of acrylalnide and 2, 5-hexanedione in rats evaluated using a functional observational battery and pathological examination. Neurotoxicol Teratol, 1992; 14(4): 273-283.
7 Gerber GJ, O'Shaughnessy D. Comparison of the behavioral effects of neurotoxie and systemically toxic agents: how discriminatory are behavioral tests of neurotoxicity? Neurobehav Toxieol Teratol, 1986; 8(6): 703-710.
8 邵蓓，何金彩，倪佩琦，等．慢性正己烷中毒的肌电图表现．临床脑电学杂志，1999；8： 87．
9 吴小炼，朱松华．75例正己烷中毒性周围神经病临床分析．临床神经病学杂志，2002； 15(3)：179-180．
10 杨文英，安榕榕，林文珍．68例慢性正己烷中毒的肌电图神经传导速度分析，临床神经电生理学杂志，2003；12(2)：95-96．
11 Bleecker ML. Parkinsonism: a clinical marker of exposure to neurotoxins. Neurotoxieol Teratol. 1988; 10(5): 475-478.
12 何凤生，薛启蒙．神经系统中毒及代谢性疾病，人民军医出版社，2002．75-77．
13 Johnson BL, Boyd J, Burg JR, etal. Effects on the peripheral nervous system of workers' exposure to carbon disulfide. Neurotoxicology. 1983; 4(1): 53-66.
14 Vanhoome MH, Ceulemans L, De Bacquer DA, etal. An epidemiologie study of the effects of carbon disulfide on the peripheral nerves. Int J Oceup Environ Health, 1995; 1(4): 295-302.
15 Seppalainen AM, Linnoila I. Electrophysiological studies on rabbits in long-term exposure to carbon disulfide. Scand J Work Environ Health. 1975; 1(3): 178-183.
16 Rebert CS, Beeker E. Effects of inhaled carbon disulfide on sensory-evoked potentials of Long-Evans rats. Neurobehav Toxicol Teratol. 1986; 8(5): 533-541.
1 Lee, M. K., Cleveland, D. W. Neuronal intermediate filaments. Annu Rev Neurosci, 1996; 19: 187-217.
2 Lee, M. K., Xu, Z., Wong, P. C., Cleveland, D. W., 1993. Neurofilaments are obligate heteropolymers in vivo. J Cell Biol, 1993; 122, 1337-1350.
3 Fadic, R., Vergara, J., Aivarez, J. Microtubules and caliber of central and peripheral processes of sensory axons. J. Comp. Neurol. 1985; 236, 258-264
4 Pannese, E., Ledda, M., Arcidiacono, G., Rigamonti, L., Procacci, P. Density and distribution of microtubules in the axons of the lizard dorsal root. J Submicrosc Cytol, 1984; 13: 169-181.
5 Lehning, E. J., Dyer, K. S., Jortner, B. S., LoPachin, R. M. Axonal atrophy is a specific component of 2, 5-hexanedione peripheral neuropathy. Toxicol. Appl. Pharmacol. 1995; 135: 58-66.
6 Lehning E J, Jortner BS, Fox JH, Arezzo JC, KitanoT, LoPachin RM. gamma-diketone peripheral neuropathy. I. Quality morphometric analyses ofaxonal atrophy and swelling. Toxicol. Appl. Pharmacol. 2000; 165: 127-140.
7 Chiu FC, Opanashuk LA, He DK, Lehning EJ, and LoPachin RM. gamma-diketone peripheral neuropathy. Ⅱ. Neurofilament subunit content. Toxicol Appl Pharmacol, 2000; 165(2): 141-147.
8 Karlsson JE, Rosengren LE, Haglid KG. Quantitative and qualitative alterations of neuronal and glial intermediate filaments in rat nervous system after exposure to 2,5-hexanedione. J Neurochem, 1991; 57(4): 437-444.
9 Nagano M, Yamamoto H, Harada K, Miyamoto E, Futatsuka M. Comparative study of modification and degradation of neurofilament proteins in rats subchronically treated with allyl chloride, acrylamide, or 2, 5-hexanedione. Environ Res, 1993; 63(2): 229-240.
10 Monaco S, Autilio-Gambetti L, Lasek R J, Katz M J, Gambetti P. Experimental increase of neurofilament transport rate: decreases in neurofilament number and in axon diameter. Neuropathol Exp Neurol, 1989; 48(1): 23-32.
11 Monaco S, Autilio-Gambetti L, Zabel D, Gambetti P. Giant axonal neuropathy: acceleration of neurofilament transport in optic axons. Proc Natl Acad Sci USA, 1985; 82(3): 920-924.
12 Pyle SJ, Amarnath V, Graham DG, Anthony DC. Decreased levels of the high molecular weight subunit of neurofilaments and accelerated neurofilament transport during the recovery phase of 2,5-hexanedione exposure. Cell Motil Cytoskeleton, 1993; 26(2): 133-143.
13 LoPachin RM, He D, Reid ML, Opanashuk LA. 2, 5-Hexanedione-induced changes in the monomeric neurofilament protein content of rat spinal cord fractions. Toxicol Appl Pharmacol, 2004; 198(1): 61-73.
14 Lopachin RM, He D, Reid ML. 2, 5-hexanedione-induced changes in the neurofilament subunit pools of rat peripheral nerve. Neurotoxicol, 2005; 26(2): 229-240.
15 Hall ES, Hall S J, Boekelheide K. Sertoli cells isolated from adult 2, 5-hexanedione-exposed rats exhibit atypical morphology and actin distribution. Toxicol Appl Pharmacol, 1992; 117(1): 9-18.
16 Hall ES, Eveleth J, Boekelheide K. 2, 5-Hexanedione exposure alters the rat Sertoli cell cytoskeleton. Ⅱ. Intermediate filaments and actin. Toxicol Appl Pharmacol, 1991; 111(3): 443-453.
17 Valentine WM, Amarnath V, Graham DG, Morgan DL, and Sills RC. CS2-mediated cross-linking of erythrocyte spectrin and neurofilament protein: dose response and temporal relationship to the formation of axonal swellings. Toxicol Appl Pharmacol, 1997; 142(1): 95-105.
18 Valentine WM, Graham DG, and Anthony DC. Covalent cross-linking of erythrocyte spectrin by carbon disulfide in vivo. Toxicol Appl Pharmacol. 1993; 121 (1): 71-77.
19 高艳华，梁友信，傅慰祖．二硫化碳对大鼠神经微丝及红细胞膜结构的影响．中国工业医学杂志，1998；11(5)：267-270．
20 Elhanany E. Identification of endogenously phosphorylated KSP sites in rat neurofilament protenin. J Neurochem, 1992; 58(4): 1975-1984.
21 Elhanany E, Jaffe H, Link WT, et al. Identification of endogenously phosphorylated KSP sites in the high-molecular-weight rat neurofilament protenin. J Neurochem, 1994; 63(6): 2324-2335.
22 米瑞发，范明，周长满．神经丝磷酸化及其生理病理作用．神经解剖学杂志，1998：14(2)： 199-202．
23 Geisler N, Weber K. Self-assembly in Vitro Of the 68,000 molecular weight component of the mammalian neurofilament triplet proteins into intermediate-sized filaments. J Mol Biol. 1981, 151(3): 565-571.
24 Friede RL, Samorajski T. Axon caliber related to neurofilaments and microtubules in sciatic nerve fibers of rats and mice. Anat Rec. 1970; 167(4):379-87.
25 Peters A, Vaughn JE. Microtubules and filaments in the axons and astrocytes of early postnatal rat optic nerves. J Cell Biol. 1967; 32(1): 113-9.
26 Hirokawa N.Axonal transport and the cytoskeleton. Curr Opin Neurobiol. 1993; 3(5): 724-731.
27 Black MM, Keyser P, Sobel E.Interval between the synthesis and assembly of cytoskeletal proteins in cultured neurons. J Neurosci. 1986; 6(4): 1004-1012.
28 Hoffman PN, Lasek RJ. The slow component of axonal transport. Identification of major structural polypeptides of the axon and their generality among mammalian neurons. J Cell Biol. 1975; 66(2):351-66.
29 Yabe JT, Chan WK, Chylinski TM, Lee S, Pimenta AF, and Shea TB. The predominant form in which neurofilament subunits undergo axonal transport varies during axonal initiation, elongation, and maturation. Cell Motil Cytoskeleton. 2001; 48(1): 61-83.
30 Elder GA, Friedrich VL Jr, Margita A, Lazzarini RA.Age-related atrophy of motor axons in mice deficient in the mid-sized neurofilament subunit. J Cell Biol, 1999; 146(1):181-192.
31 Julien JP, Mushynski WE. Neurofilaments in health and disease. Prog Nucleic Acid Res Mol Biol. 1998; 61(1): 1-23.
32 Lariviere RC, Julien JP. Functions of intermediate filaments in neuronal development and disease.J Neurobiol. 2004; 58(1):131-148.
33 LoPachin RM, Ross JF, Reid ML, Das S, Mansukhani S, Lehning EJ. Neurological evaluation of toxic axonopathies in rats: acrylamide and 2,5-hexanedione. Neurotoxicology. 2002; 23, 95-110.
34 Ladefoged O, Roswall K, Larsen JJ. Acetone potentiation and influence on the reversibility of 2,5-hexanedione-induced neurotoxicity studied with behavioural and morphometric methods in rats. Pharmacol Toxicol. 1994;74(4-5):294-9
35 Rebert, C., and Sorenson. 1983. Concentration-related effects of n-hexane on evoked responses from brain and peripheral nerve of the rat. Neurobehav Toxicol Teratol, 5: 69-76.
36 Pryor, G.T., Dickinson, J., Howd, R.A., and Rebert, C.S. 1983. Neurobehavioural effects of subchronic exposure of weanling rats to toluene or hexane. Neurobehav Toxicol Teratol, 5: 47-52.
37 Hirokawa, N.,and Takeda, S. 1998. Gene targeting studies begin to reveal the function of neurofilament proteins. J Cell Biol. 143(1): 1-4.
38 Zhu Q, Couillard-Despres S, and Julien JP. Delayed maturation of regenerating myelinated axons in mice lacking neurofilaments. Exp. Neurol. 1997; 148: 299-316.
39 Elder GA, Friedrich VL, Bosco P, Kang C, Gourov A, Tu PH, Lee VM, and Lazzarini RA. Absence of the mid-sized neurofilament subunit decreases axonal calibers, levels of light neurofilament (NF-L), and neurofilament content. J Cell Bio1.1998; 141(3): 727-739.
40 Rao MV, Houseweart MK, Williamson TL, Crawford TO, Folmer J, and Cleveland DW. Neurofilament-dependent radial growth of motor axons and axonal organization of neurofilaments does not require the neurofilament heavy subunit (NF-H) or its phosphorylation. J Cell Bio1.1998; 143(1): 171-181.
41 Zhu Q, Lindenbaum M, Levavasseur F, Jacomy H, and Julien JP. Disruption of the NF-H gene increases axonal microtubule content and velocity of neurofilament transport: relief of axonopathy resulting from the toxin beta, beta'-iminodipropionitrile. J Cell Biol. 1998; 143(1): 183-193.
42 Pachter JS, Liem RK. The differential appearance of neurofilament triplet polypeptides in the developing rat optic nerve. Dev Biol. 1984; 103, 200-210.
43 Tohyama T, Lee, VM, Rorke, LB, Trojanowski JQ. Molecular milestones that signal axonal maturation and the commitment of human spinal cord precursor cells to the neuronal or glial phenotype in development. J Comp Neurol. 1991; 310, 285-299.
44 Uchida A, Tashiro T, Komiya Y, Yorifuji H, Kishimoto T, Hisanaga S. Morphological and biochemical changes of neurofilaments in aged rat sciatic nerve axons. J Neurochem. 2004; 88, 735-745.
45 Trojanowski JQ, Wa!kenstein N, Lee VM. Expression of neurofilament subunits in neurons of the central and peripheral nervous system: an immunohistochemical study with monoclonal antibodies. J Neurosci. 1986; 6: 50-60.
46 Shaw G, Weber K. Differential expression of neurofilament triplet proteins in brain development. Nature. 1982; 298(5871): 277-279.
47 Ching GY, Liem RK. Assembly of type IV neuronal intermediate filaments in nonneuronal cells in the absence of preexisting cytoplasmic intermediate filaments. J Cell Biol. 1993; 122(6): 1323-1335.
48 Mishra DP, Pal R, Shaha C. Changes in cytosolic Ca2+ levels regulate Bcl-xS and Bcl-xL expression in spermatogenic cells during apoptotic death. J Biol Chem. 2006; 281, 2133-2143.
49 张艳淑，刘强，刘清君，段化伟，何风生，郑玉新．2，5-己二酮对运动神经元钙稳态的影响．中华劳动卫生职业病杂志，2006；24(5)：270-272．
50 Fujiwara I, Takahashi S, Tadakuma H, Funatsu T, Ishiwata S. Microscopic analysis of polymerization dynamics with individual actin filaments. Nat Cell Biol, 2002, 4: 666-673.
51 Pollard TD. Rate constants for the reactions of ATP-and ADP-actin with the ends of actin filaments. J Cell Biol, 1986, 103: 2747-2754.
52 Ma L, Rohatgi R, Kirschner MW. The Arp2/3 complex mediates actin polymerization induced by the small GTP-binding protein Cdc42. Proc Natl Acad Sci U S A. 1998, 95 (26):15362-15367.
53 Cooper JA. Effects of cytochalasin and phalloidin on actin. J. Cell Biol, 1987, 105: 1473-1478.
54 Sampath P, Pollard TD. Effects of cytochalasin, phalloidin and pH on the elongation of actin filaments. Biochem, 1991, 30: 1973-1980.
55 Lankford KL, Letourneau PC.Evidence that calcium may control neurite outgrowth by regulating the stability of actin filaments. J Cell Biol. 1989; 109(3): 1229-1243.
56 Kiselar JG, Janmey PA, Almo SC, Chance MR. Visualizing the Ca2+-dependent activation of gelsolin by using synchrotron footprinting. Proc Natl Acad Sci U S A. 2003; 100(7): 3942-3947.
57 Kinosian HJ, Newman J, Lincoln B, Selden LA, Gershman LC, Estes JE. Ca2+ regulation of gelsolin activity: binding and severing of F-actin. Biophys J. 1998; 75(6): 3101-3109.
58 Perrone Capano C, Pernas-Alonso R, di Porzio U. Neurofilament homeostasis and motoneurone degeneration. Bioessays. 2001; 23:24-33.
59 Rao MV, Campbell J, Yuan A, Kumar A, Gotow T, Uchiyama Y, Nixon RA. The neurofilament middle molecular mass subunit carboxyl-terminal tail domains is essential for the radial growth and cytoskeletal architecture of axons but not for regulating neurofilament transport rate. J Cell Biol, 2003; 163(5): 1021-1031.
60 Boekelheide K. 2,5-Hexanedione alters microtubule assembly. Ⅰ. enhanced tubulin polymerization. Toxicol Appl Pharmacol. 1987, 88(3): 370-382.
61 Boekelheide K.2,5-Hexanedione alters microtubule assembly. Ⅱ. Enhanced polymerization of crosslinked tubulin. Toxicol Appl Pharmacol, 1987, 88(3): 383-396.
62 Liuzzi FJ, Bufton SM, Vinik AI. Streptozotocin-induced diabetes mellitus causes changes in primary sensory neuronal cytoskeletal mRNA levels that mimic those caused by axotomy. Exp Neurol, 1998; 154: 381-388.
63 Tetzlaff W, Leonard C, Krekoski CA, Parhad IM, Bisby MA. Reductions in motoneuronal neurofilament synthesis by successive axotomies: a possible explanation for the conditioning lesion effect on axon regeneration. Exp Neurol, 1996; 139(1): 95-106.
1 Opanashuk LA, He DK, Lehning EJ, and Lopachin RM. Diketone peripheral Neuropathy Ⅲ. Neurofilament gene expression. Neurotoxicology, 2001; 22: 215-220.
2 Kittttr S, Hob J, Endo H, Tourtellotte W, Weeks BS, Markesbery W, Adler W. Cytoskeletal neurofilament gene expression in brain tissue from Alzheimer's disease patients. I. Decrease in NF-L and NF-M message. J Geriatr Psychiatry Neurol. 1994, 7(3): 153-158.
3 Mohiuddin L, Femyhough P, Tomlinson DR. Reduced levels of mRNA encoding endoskeletal and growth-associated proteins in sensory ganglia in experimental diabetes. Diabetes. 1995; 44(1): 25-30.
4 Lariviere RC, Julien JP. Functions of intermediate filaments in neuronal development and disease. J Neurobiol. 2004; 58(1): 131-148.
5 Lewis SA, Cowan NJ. Genetics, evolution, and expression of the 68, 000-mol-wt neurofilament protein: isolation of a cloned eDNA probe. J Cell Biol. 1985; 100(3): 843-850.
6 Beaudet L, Charron G, Julien JP. Origin of the two mRNA species for the human neurofilament light gene. Biochem Cell Biol. 1992; 70(5): 279-284.
7 Parhad IM, Swedberg EA, Hoar DI, Krekoski CA, Clark AW. Neurofilament gene expression following beta, beta'-iminodipropionitrile (IDPN) intoxication. Brain Res. 1988; 464(4): 293-301.
8 Beaudet L, Cote F, Houle D, Julien JP. Different posttranscriptional controls for the human neurofilament light and heavy genes in transgenic mice. Brain Res Mol Brain Res. 1993; 18(1-2): 23-31.
9 Sills RC, Harry GJ, Morgan DL, Valentine WM, Graham DG. Carbon disulfide neurotoxicity in rats: V. Morphology of axonal swelling in the muscular branch of the posterior tibial nerve and spinal cord. Neurotoxicology. 1998; 19(1): 117-127.
10 Monteiro M J, Hoffman PN, Gearhart JD, Cleveland DW. Expression of NF-L in both neuronal and nonneuronal cells of transgenic mice: increased neurofilament density in axons without affecting caliber. J Cell Biol. 1990; 111 (4): 1543-1557.
11 Tu PH, Elder G, Lazzarini RA, Nelson D, Trojanowski JQ, Lee VM. Overexpression of the human NFM subunit in transgenic mice modifies the level of endogenous NFL and the phosphorylation state ofNFH subunits. J Cell Biol. 1995; 129(6): 1629-1640.
12 Ching GY, Liem RK. Assembly of type IV neuronal intermediate filaments in nonneuronal cells in the absence of preexisting cytoplasmic intermediate filaments. J Cell Biol. 1993; 122(6): 1323-1335
13 Lee MK, Xu Z, Wong PC, Cleveland DW. Neurofilaments are obligate heteropolymers in vivo. J Cell Biol. 1993; 122(6): 1337-1350.
14 Lemischka 1R, Farmer S, Racaniello VR, Sharp PA. Nucleotide sequence and evolution of a mammalian alpha-tubulin messenger RNA. J Mol Biol. 1981; 151 (1): 101-120.
15 Lemischka I, Sharp PA. The sequences of an expressed rat alpha-tubulin gene and a pseudogene with an inserted repetitive element. Nature. 1982; 300(5890): 330-335.
16 Liu NK, Xu XM. beta-Tubulin Is a More Suitable Internal Control than beta-Actin in Western Blot Analysis of Spinal Cord Tissues after Traumatic Injury. J Neurotrauma. 2006; 23(12): 1794-1801.
17 何彬，范明，甘思德．神经再生过程中脊髓运动神经元α管蛋白基因的表达．科学 通报，1993；9(1)：129-131．
18 米瑞发，甘思德，范明．神经再生过程中脊髓运动神经元β、γ肌动蛋白基因表达的变化．科学通报，1994；39(6)：542-544．
19 Mi RF, Shi XQ, Fan M, et al. Expression of neurofilament gene in spinal motomeurons during neural regeneeration. Chin Sci Bull, 1999; 44(2): 225-229.
1 Perrin BJ, Huttenlocher A. Caipain. Int J Biochem Cell Biol. 2000; 34, 722-725.
2 Suzuki K, Sorimachi H, Yoshizawa T, Kinbara K, Ishiura S. Calpain: novel family members, activation, and physiologic function. Biol Chem Hoppe Seyler. 1995; 376: 523-529.
3 Ray SK, Banik NL. Calpain and its involvement in the pathophysiology of CNS injuries and diseases: therapeutic potential of calpain inhibitors for prevention of neurodegeneration. Curr. Drug Target CNS Neurol. Disord.2003; 2: 173-189.
4 Guo-Ross S, Yang E, Bondy SC. Elevation of cerebral proteases after systemic administration of aluminum. Neurochem. Int. 1998; 33: 277-282.
5 Gupta RP, Abou-Donia MB. Alterations in the neutral proteinase activities of central and peripheral nervous systems of acrylamide-, carbon disulfide-, or 2,5-hexanedione-treated rats. Mol Chem Neuropathol, 1996; 29(1): 53-66.
6 Nagano M, Yamamoto H, Harada K, Miyamoto E, Futatsuka M. Comparative study of modification and degradation of neurofilament proteins in rats subchronicaUy treated with allyl chloride, acrylamide, or 2,5-hexanedione. Environ Res, 1993; 63(2): 229-240.
7 Tanii H, Hayashi M, Hashimoto K. Neurofilament degradation in the nervous system of rats intoxicated with acrylamide, related compounds or 2,5-hexanedione. Arch Toxicol, 1988; 62 (1): 70-75.
8 Karlsson JE, Rosengren LE, Haglid KG. Quantitative and qualitative alterations of neuronal and glial intermediate filaments in rat nervous system after exposure to 2,5-hexanedione. J Neurochem, 1991, 57(4): 437-444.
9 Banik NL, Hogan, EL, Powers JM, Whetstine LJ. Degradation of cytoskeletal proteins in spinal cord injury. Neurochem. Res. 1982; 7: 1465-1475.
10 Banik NL, Matzelle DC, Gantt-Wilford G, Osborne A, Hogan EL. Increased calpain content and progressive degradation of neurofilament protein in spinal cord injury. Brain Res. 1997; 752: 301-306.
11 Ray SK, Hogan EL, Banik NL. Calpain in the pathophysiology of spinal cord injury: neuroprotection with calpain inhibitors. Brain Res. Rev. 2003; 42:169-185.
12 Chakrabarti AK, Yoshida Y, Powers JM, Singh I, Hogan EL and Banik NL. Calcium-activated neutral proteinases in rat brain and subcellular fractions. J. Neurosci. Res. 1988; 20: 351-358.
13 Banay-Schwartz, M., Deguzman, T., Palkovits, M. and Lajtha, A. Calpain activity in adult and aged human brain regions. Neurochem. Res. 1994; 19, 563-567.
14 Ray SK, Shields DC, Saido TC, Matzelle DC, Wilford GG, Hogan EL, Banik NL. Calpain activity and translational expression increased in spinal cord injury. Brain Res. 1999; 816(2): 375-380.
15 Nixon RA, Quackenbusch R, Vitto A.Multiple calcium-activated neutral proteinases (CANP) in mouse retinal ganglion cell neurons: specificities for endogenous neuronal substrates and comparison to purified brain CANE J. Neurosci. 1986; 6:1252-1263.
16 Lapadula DM, Suwita E, and Abou-Donia MB. Cross-linking of neurofilament proteins of rat spinal cord in vivo after administration of 2,5-hexanedione, J. Neurochem. 1986; 46: 1843-1850.
17 Heijink E, Scholten SW, Bolhuis PA, de Wolff FA. Effects of 2,5-hexanedione on calpain mediated degradation of human neurofilaments in vitro. Chem. Biol. Interact. 2000; 129(3):231-247.
18 Rajgopal Y, Vemuri MC. Calpain activation and alpha-spectrin cleavage in rat brain by ethanol. Neurosci Lett. 2002; 321 (3): 187-191.
19 Takeuchi KH, Saito K-I, and Nixon RA. Immunoassay and activity of calcium-activated neutral proteinase (mCANP): distribution in soluble and membrane-associated fractions in human and mouse brain. J Neurochem, 1992; 58: 1526-1532.
20 Mishra DP, Pal R, Shaha C. Changes in cytosolic Ca2+ levels regulate Bcl-xS and Bcl-xL expression in spermatogenic cells during apoptotic death. J Biol Chem. 2006; 281(4): 2133-2143.
21 Lapadula DM, Suwita E, and Abou-Donia MB. Evidence for multiple mechanisms responsible for 2,5-hexanedione-induced neuropathy. Brain Res. 1988; 458:123-131.
22 Watson DF, Fittro KP, Hoffman PN, and Griffin JW. Phosphorylation-related immunoreactivity and the rate of transport of neurofilaments in chronic 2,5-hexanedione intoxication. Brain Res. 1991; 539: 103-109.
23 LoPachin RM, He D, Reid ML, Opanashuk LA. 2,5-Hexanedione-induced changes in the monomeric neurofilament protein content of rat spinal cord fractions. Toxicol Appl Pharmacol, 2004; 198(1): 61-73.
24 Lopachin RM, He D, Reid ML. 2,5-hexanedione-induced changes in the neurofilament subunit pools of rat peripheral nerve. Neurotoxicol, 2005; 26(2): 229-240.
25 Gou JP, Leterrier JF.Possible involvement of ubiquitination in neurofilament degradation. Biochem. Biophys. Res. Commun. 1995; 217(2):529-538.
26 Ehlers MD. Deconstructing the axon: Wallerian degeneration and the ubiquitin-proteasome system. Trends Neurosci. 2004; 27(1): 3-6.
27 Hernandez, F., Diaz-Hernandez, M., Avila, J., Lucas, J. J. 2004. Testing the ubiquitin-proteasome hypothesis of neurodegeneration in vivo. Trends Neurosci. 27(2): 66-69.
28 Zhai Q, Wang J, Kim A, Liu Q, Watts R, Hoopfer E, Mitchison T, Luo L, He Z. Involvement of the ubiquitin-proteasome system in the early stages of wallerian degeneration. Neuron. 2003, 39(2):217-225.
2 Spencer PS, Bischoff MC, Schaumburg HH. On the specific molecular configuration of neurotoxic aliphatic hexacarbon compounds causing central-peripheral distal axonopathy. Toxicol Appl Pharmacol. 1978; 44: 17-28.
3 Fedtke N and Bolt HM. The relevance of 4, 5-dihydroxy-2-hexanone in the excretion kinetics of n-hexane metabolites in rat and man. Arch. Toxicol. 1987; 61: 131-137.
4 WHO working group, n-Hexane Source. Environmental Health Criteria. 1991; 122: 133.
5 Green EC, Hunter A. Toxicity of carbon disulfide in developing rats: LD50 and effects on the hepatic mixed-function oxidase enzyme system. Toxicology and applied pharmacology. 1983; 78: 130-138.
6 Stetkiewicz J and Wronska-Nofer T. Updating of hygiene standards for carbon disulfide based on health risk assessment. Int J Occup Med Environ Health. 1998; 11(2): 129-143.
7 Beauchamp RO, Bus JS, Popp JA, Boreiko C J, and Goldberg L. A critical review of the literature on carbon disulfide toxicity. Crit Rev Toxicol. 1983; 11: 169-278.
8 林斯星，陈嘉斌，吴子俊．一起严重的正己烷职业中毒事故报告．中国工业医学杂志，1997；10：172-173．
9 杨玉珍．14例正己烷中毒患者的观察及护理．中国职业医学．1995；2(2)：63．
10 ATSDR (Agency for Toxic Substances and Disease Registry). Toxicological profile for n-hexane. 1999.
11 何凤生．中华职业医学．北京：人民卫生出版社，1999，62．
12 Cavanagh JB. The significance of the "dying back" process in experimental and human neurological disease. Int Rev Exp Pathol. 1964; 3: 219-267.
13 Spencer PS, schaumburg HH. central peripheral distal axonopathy: The pathology of dying-back polyneuropathies: progress in neuropathology. Vol 3. New york: Grune & Stratton, 1976, 253-295.
14 Sills RC, Harry GJ, Morgan DL, Valentine WM, Graham DG. Carbon disulfide neurotoxicity in rats: V. Morphology of axonal swelling in the muscular branch of the posterior tibial nerve and spinal cord. Neurotoxieology. 1998; 19:117-127.
15 Colombi A, Maroni M, Picchi O, Rota E, Castano P, Foa V. Carbon disulfide neuropathy in rats. A morphological and ultrastructural study of degeneration and regeneration. Clinical Toxicology. 1981; 18(12): 1463-1474.
16 Schaumburg HH, Spencer PS. Degeneration in central and peripheral nervous systems produced by pure n-bexane: an experimental study. Brain. 1976, 99(2): 183-192.
17 LoPachin RM, Ross JF, Reid ML, Das S, Mansukhani S, Lehning EJ. Neurological evaluation of toxic axonopathies in rats: acrylamide and 2,5-hexanedione. Neurotoxicology. 2002; 23:95-110.
18 Spencer PS, Sabri MI, Schaumburg HH, Moore CL. Does a defect of energy metabolism in the nerve fiber underlie axonal degeneration in polyneuropathies? Ann. Neurol. 1979; 5: 501-507.
19 Sabri MI, Moore CL, Spencer PS. Studies on the biochemical basis of distal axonopathies--I. Inhibition of glycolysis by neurotoxic bexacarbon compounds. J Neurochem. 1979; 32(3): 683-689.
20 Monaco S, Autilio-Gambetti L, Zabel D, Gambetti P. Giant axonal neuropathy: acceleration of neurofilament transport in optic axons. Proc. Natl Acad. Sci. USA. 1985; 82: 920-924.
21 Monaco S, Jacob J, Jenich H, Patton A, Autilio-Gambetti L, Gambetti P. Axonal transport of neurofilament is accelerated in peripheral nerve during 2,5-hexanedione intoxication. Brain Res. 1989; 491: 328-334.
22 Monaco S, Wongmongkolrit T, Shearson CM, Patton A, Schaetzle B, Autilio-Gambetti L, Gambetti P, Sayre LM. Giant axonopathy characterized by intermediate location of axonal enlargements and acceleration of neurofilament transport. Brain Res. 1990; 519: 73-81.
23 McKenna MJ and DiStefano V. Carbon disulfide. Ⅱ. A proposed mechanism for the action of carbon disulfide on dopamine beta-hydroxylase. J Pharmacol Exp Ther. 1977; 202(2): 253-266.
24 Teisinger J. New advances in the toxicology of carbon disulfide. Am Ind Hyg Assoc J. 1974; 35(2): 55-61.
25 Genter MB, Szakal-Quin G, Anderson CW, Anthony DC, Graham DG. Evidence that pyrrole formation is a pathogenetic step in gamma-diketone neuropathy. Toxicol Appl Pharmacol. 1987; 87(2): 351-362.
26 Genter St Clair MB, Amarnath V, Moody MA, Anthony DC, Anderson CW, Graham DG. Pyrrole oxidation and protein cross-linking as necessary steps in the development of gamma-diketone neuropathy. Chem Res Toxicol. 1988; 1(3): 179-185.
27 Sayre LM, Shearson CM, Wongmongkolrit T, Medori R, Gambetti P. Structural basis of gamma-diketone neurotoxicity: non-neurotoxicity of 3, 3-dimethyl-2, 5-hexanedione, a gamma-diketone incapable of pyrrole formation. Toxicol Appl Pharmacol. 1986; 84(1): 36-44.
28 Valentine WM, Amamath V, Graham DG., Morgan DL, and Sills RC. CS2-mediated cross-linking of erythrocyte spectrin and neurofilament protein: dose response and temporal relationship to the formation of axonal swellings. Toxicol Appl Pharmacol. 1997; 142(1): 95-105.
29 Valentine WM, Graham DG, and Anthony DC. Covalent cross-linking of erythrocyte spectrin by carbon disulfide in vivo. Toxicol Appl Pharmacol. 1993; 121(1): 71-77.
30 Hirai T, Mizutani M, Kimura T, Ochiai K, Umemura T, Itakura C. Neurotoxic effects of 2, 5-hexanedione on normal and neurofilament-deficient quail. Toxicol Pathol. 1999; 27(3): 348-353.
31 Stone JD, Peterson AP, Eyer J, Oblak TG, Sickles DW. Neurofilaments Are Nonessential to the Pathogenesis of Toxicant-Induced Axonal Degeneration J Neurosci. 2001; 21(7): 2278-2287.
32 Sickles DW, Pearson JK, Beall A, Testino A. Toxic axonal degeneration occurs independent of neurofilament accumulation. J Neurosci Res. 1994; 39(3): 347-354.
33 何凤生，薛启蒙．神经系统中毒及代谢性疾病．人民军医出版社，2002，75-77．
34 Hemandez-Viadel M, Montoliu C, Monfort P, Canales J J, Erceg S, Rowan M, Ceccatelli S, Felipo V. Chronic exposure to 2, 5-hexanedione impairs the glutamate-nitric oxide-cyclic GMP pathway in cerebellar neurons in culture and in rat brain in vivo. Neurochem Int. 2003; 42(7): 525-533.
35 Prieto-Castello MJ, Hernandez-Viadel ML, Cardona A, Marhuenda D, Felipo V. Activation of soluble guanylate cyclase by nitric oxide is increased in lymphocytes from both rats chronically exposed to 2, 5-hexanedione and workers chronically exposed to n-hexane. Toxicology. 2007; 229(1-2): 73-78.
36 Lehning EJ, Dyer KS, Jortner BS, LoPachin RM. Axonal atrophy is a specific component of 2,5-hexanedione peripheral neuropathy. Toxicol. Appl. Pharmacol. 1995; 135: 58-66.
37 Lehning EJ, Jortner BS, Fox JH, Arezzo JC, Kitano T, LoPachin RM. gamma-diketone peripheral neuropathy. I. Quality morphometric analyses ofaxonal atrophy and swelling. Toxicol. Appl. Pharmacol. 2000; 165: 127-140.
38 LoPachin RM, He D, Reid ML, Opanashuk LA. 2, 5-Hexanedione-induced changes in monomeric neurofilament protein content of rat spinal cord fractions. Toxicol. Appl. Pharmacol. 2004; 198, 61-73.
39 LoPachin RM, He D, Reid ML. 2,5-Hexanedione-induced changes in the neurofilament subtmit pools of rat peripheral nerve. Neurotoxicology, 2005, 26: 229-240.
40 高艳华，梁友信，傅慰祖．CS_2对大鼠神经微丝及红细胞膜结构的影响．中国工业医学杂志，1998；11(5)：267-270．
1 LoPachin RM, Ross JF, Reid ML, Das S, Mansukhani S, Lehning EJ. Neurological evaluation of toxic axonopathies in rats: acrylamide and 2, 5-hexanedione. Neurotoxicology 2002; 23: 95-110.
2 Moser VC, Phillips PM, Morgan DL, et al. Carbon disulfide neurotoxicity in rats: Ⅶ. Behavioral evaluations using a functional observational battery. Neurotoxicology, 1998; 19 (1): 147-157.
3 Schaumburg HH, Spencer PS. Degeneration in central and peripheral nervous systems produced by pure n-hexane: an experimental study. Brain, 1976; 99(2): 183-192.
4 Spencer P. Neuropathic potential of n-hexane in the presence of other hexane isomers[M]. Washington, DC: American Petroleumlnstitute (API Medical Research Publication, 1982: 30.
5 Spencer PS, Bischoff MC, Schaumburg HH. On the specific molecular configuration of neurotoxic aliphatic hexacarbon compounds causing central-peripheral distal axonopathy. Toxicol Appl Pharmacol, 1978; 44 (1): 17-28.
6 Shell L, Rozum M, Jortner BS, Ehrich M. Neurotoxicity of acrylalnide and 2, 5-hexanedione in rats evaluated using a functional observational battery and pathological examination. Neurotoxicol Teratol, 1992; 14(4): 273-283.
7 Gerber GJ, O'Shaughnessy D. Comparison of the behavioral effects of neurotoxie and systemically toxic agents: how discriminatory are behavioral tests of neurotoxicity? Neurobehav Toxieol Teratol, 1986; 8(6): 703-710.
8 邵蓓，何金彩，倪佩琦，等．慢性正己烷中毒的肌电图表现．临床脑电学杂志，1999；8： 87．
9 吴小炼，朱松华．75例正己烷中毒性周围神经病临床分析．临床神经病学杂志，2002； 15(3)：179-180．
10 杨文英，安榕榕，林文珍．68例慢性正己烷中毒的肌电图神经传导速度分析，临床神经电生理学杂志，2003；12(2)：95-96．
11 Bleecker ML. Parkinsonism: a clinical marker of exposure to neurotoxins. Neurotoxieol Teratol. 1988; 10(5): 475-478.
12 何凤生，薛启蒙．神经系统中毒及代谢性疾病，人民军医出版社，2002．75-77．
13 Johnson BL, Boyd J, Burg JR, etal. Effects on the peripheral nervous system of workers' exposure to carbon disulfide. Neurotoxicology. 1983; 4(1): 53-66.
14 Vanhoome MH, Ceulemans L, De Bacquer DA, etal. An epidemiologie study of the effects of carbon disulfide on the peripheral nerves. Int J Oceup Environ Health, 1995; 1(4): 295-302.
15 Seppalainen AM, Linnoila I. Electrophysiological studies on rabbits in long-term exposure to carbon disulfide. Scand J Work Environ Health. 1975; 1(3): 178-183.
16 Rebert CS, Beeker E. Effects of inhaled carbon disulfide on sensory-evoked potentials of Long-Evans rats. Neurobehav Toxicol Teratol. 1986; 8(5): 533-541.
1 Lee, M. K., Cleveland, D. W. Neuronal intermediate filaments. Annu Rev Neurosci, 1996; 19: 187-217.
2 Lee, M. K., Xu, Z., Wong, P. C., Cleveland, D. W., 1993. Neurofilaments are obligate heteropolymers in vivo. J Cell Biol, 1993; 122, 1337-1350.
3 Fadic, R., Vergara, J., Aivarez, J. Microtubules and caliber of central and peripheral processes of sensory axons. J. Comp. Neurol. 1985; 236, 258-264
4 Pannese, E., Ledda, M., Arcidiacono, G., Rigamonti, L., Procacci, P. Density and distribution of microtubules in the axons of the lizard dorsal root. J Submicrosc Cytol, 1984; 13: 169-181.
5 Lehning, E. J., Dyer, K. S., Jortner, B. S., LoPachin, R. M. Axonal atrophy is a specific component of 2, 5-hexanedione peripheral neuropathy. Toxicol. Appl. Pharmacol. 1995; 135: 58-66.
6 Lehning E J, Jortner BS, Fox JH, Arezzo JC, KitanoT, LoPachin RM. gamma-diketone peripheral neuropathy. I. Quality morphometric analyses ofaxonal atrophy and swelling. Toxicol. Appl. Pharmacol. 2000; 165: 127-140.
7 Chiu FC, Opanashuk LA, He DK, Lehning EJ, and LoPachin RM. gamma-diketone peripheral neuropathy. Ⅱ. Neurofilament subunit content. Toxicol Appl Pharmacol, 2000; 165(2): 141-147.
8 Karlsson JE, Rosengren LE, Haglid KG. Quantitative and qualitative alterations of neuronal and glial intermediate filaments in rat nervous system after exposure to 2,5-hexanedione. J Neurochem, 1991; 57(4): 437-444.
9 Nagano M, Yamamoto H, Harada K, Miyamoto E, Futatsuka M. Comparative study of modification and degradation of neurofilament proteins in rats subchronically treated with allyl chloride, acrylamide, or 2, 5-hexanedione. Environ Res, 1993; 63(2): 229-240.
10 Monaco S, Autilio-Gambetti L, Lasek R J, Katz M J, Gambetti P. Experimental increase of neurofilament transport rate: decreases in neurofilament number and in axon diameter. Neuropathol Exp Neurol, 1989; 48(1): 23-32.
11 Monaco S, Autilio-Gambetti L, Zabel D, Gambetti P. Giant axonal neuropathy: acceleration of neurofilament transport in optic axons. Proc Natl Acad Sci USA, 1985; 82(3): 920-924.
12 Pyle SJ, Amarnath V, Graham DG, Anthony DC. Decreased levels of the high molecular weight subunit of neurofilaments and accelerated neurofilament transport during the recovery phase of 2,5-hexanedione exposure. Cell Motil Cytoskeleton, 1993; 26(2): 133-143.
13 LoPachin RM, He D, Reid ML, Opanashuk LA. 2, 5-Hexanedione-induced changes in the monomeric neurofilament protein content of rat spinal cord fractions. Toxicol Appl Pharmacol, 2004; 198(1): 61-73.
14 Lopachin RM, He D, Reid ML. 2, 5-hexanedione-induced changes in the neurofilament subunit pools of rat peripheral nerve. Neurotoxicol, 2005; 26(2): 229-240.
15 Hall ES, Hall S J, Boekelheide K. Sertoli cells isolated from adult 2, 5-hexanedione-exposed rats exhibit atypical morphology and actin distribution. Toxicol Appl Pharmacol, 1992; 117(1): 9-18.
16 Hall ES, Eveleth J, Boekelheide K. 2, 5-Hexanedione exposure alters the rat Sertoli cell cytoskeleton. Ⅱ. Intermediate filaments and actin. Toxicol Appl Pharmacol, 1991; 111(3): 443-453.
17 Valentine WM, Amarnath V, Graham DG, Morgan DL, and Sills RC. CS2-mediated cross-linking of erythrocyte spectrin and neurofilament protein: dose response and temporal relationship to the formation of axonal swellings. Toxicol Appl Pharmacol, 1997; 142(1): 95-105.
18 Valentine WM, Graham DG, and Anthony DC. Covalent cross-linking of erythrocyte spectrin by carbon disulfide in vivo. Toxicol Appl Pharmacol. 1993; 121 (1): 71-77.
19 高艳华，梁友信，傅慰祖．二硫化碳对大鼠神经微丝及红细胞膜结构的影响．中国工业医学杂志，1998；11(5)：267-270．
20 Elhanany E. Identification of endogenously phosphorylated KSP sites in rat neurofilament protenin. J Neurochem, 1992; 58(4): 1975-1984.
21 Elhanany E, Jaffe H, Link WT, et al. Identification of endogenously phosphorylated KSP sites in the high-molecular-weight rat neurofilament protenin. J Neurochem, 1994; 63(6): 2324-2335.
22 米瑞发，范明，周长满．神经丝磷酸化及其生理病理作用．神经解剖学杂志，1998：14(2)： 199-202．
23 Geisler N, Weber K. Self-assembly in Vitro Of the 68,000 molecular weight component of the mammalian neurofilament triplet proteins into intermediate-sized filaments. J Mol Biol. 1981, 151(3): 565-571.
24 Friede RL, Samorajski T. Axon caliber related to neurofilaments and microtubules in sciatic nerve fibers of rats and mice. Anat Rec. 1970; 167(4):379-87.
25 Peters A, Vaughn JE. Microtubules and filaments in the axons and astrocytes of early postnatal rat optic nerves. J Cell Biol. 1967; 32(1): 113-9.
26 Hirokawa N.Axonal transport and the cytoskeleton. Curr Opin Neurobiol. 1993; 3(5): 724-731.
27 Black MM, Keyser P, Sobel E.Interval between the synthesis and assembly of cytoskeletal proteins in cultured neurons. J Neurosci. 1986; 6(4): 1004-1012.
28 Hoffman PN, Lasek RJ. The slow component of axonal transport. Identification of major structural polypeptides of the axon and their generality among mammalian neurons. J Cell Biol. 1975; 66(2):351-66.
29 Yabe JT, Chan WK, Chylinski TM, Lee S, Pimenta AF, and Shea TB. The predominant form in which neurofilament subunits undergo axonal transport varies during axonal initiation, elongation, and maturation. Cell Motil Cytoskeleton. 2001; 48(1): 61-83.
30 Elder GA, Friedrich VL Jr, Margita A, Lazzarini RA.Age-related atrophy of motor axons in mice deficient in the mid-sized neurofilament subunit. J Cell Biol, 1999; 146(1):181-192.
31 Julien JP, Mushynski WE. Neurofilaments in health and disease. Prog Nucleic Acid Res Mol Biol. 1998; 61(1): 1-23.
32 Lariviere RC, Julien JP. Functions of intermediate filaments in neuronal development and disease.J Neurobiol. 2004; 58(1):131-148.
33 LoPachin RM, Ross JF, Reid ML, Das S, Mansukhani S, Lehning EJ. Neurological evaluation of toxic axonopathies in rats: acrylamide and 2,5-hexanedione. Neurotoxicology. 2002; 23, 95-110.
34 Ladefoged O, Roswall K, Larsen JJ. Acetone potentiation and influence on the reversibility of 2,5-hexanedione-induced neurotoxicity studied with behavioural and morphometric methods in rats. Pharmacol Toxicol. 1994;74(4-5):294-9
35 Rebert, C., and Sorenson. 1983. Concentration-related effects of n-hexane on evoked responses from brain and peripheral nerve of the rat. Neurobehav Toxicol Teratol, 5: 69-76.
36 Pryor, G.T., Dickinson, J., Howd, R.A., and Rebert, C.S. 1983. Neurobehavioural effects of subchronic exposure of weanling rats to toluene or hexane. Neurobehav Toxicol Teratol, 5: 47-52.
37 Hirokawa, N.,and Takeda, S. 1998. Gene targeting studies begin to reveal the function of neurofilament proteins. J Cell Biol. 143(1): 1-4.
38 Zhu Q, Couillard-Despres S, and Julien JP. Delayed maturation of regenerating myelinated axons in mice lacking neurofilaments. Exp. Neurol. 1997; 148: 299-316.
39 Elder GA, Friedrich VL, Bosco P, Kang C, Gourov A, Tu PH, Lee VM, and Lazzarini RA. Absence of the mid-sized neurofilament subunit decreases axonal calibers, levels of light neurofilament (NF-L), and neurofilament content. J Cell Bio1.1998; 141(3): 727-739.
40 Rao MV, Houseweart MK, Williamson TL, Crawford TO, Folmer J, and Cleveland DW. Neurofilament-dependent radial growth of motor axons and axonal organization of neurofilaments does not require the neurofilament heavy subunit (NF-H) or its phosphorylation. J Cell Bio1.1998; 143(1): 171-181.
41 Zhu Q, Lindenbaum M, Levavasseur F, Jacomy H, and Julien JP. Disruption of the NF-H gene increases axonal microtubule content and velocity of neurofilament transport: relief of axonopathy resulting from the toxin beta, beta'-iminodipropionitrile. J Cell Biol. 1998; 143(1): 183-193.
42 Pachter JS, Liem RK. The differential appearance of neurofilament triplet polypeptides in the developing rat optic nerve. Dev Biol. 1984; 103, 200-210.
43 Tohyama T, Lee, VM, Rorke, LB, Trojanowski JQ. Molecular milestones that signal axonal maturation and the commitment of human spinal cord precursor cells to the neuronal or glial phenotype in development. J Comp Neurol. 1991; 310, 285-299.
44 Uchida A, Tashiro T, Komiya Y, Yorifuji H, Kishimoto T, Hisanaga S. Morphological and biochemical changes of neurofilaments in aged rat sciatic nerve axons. J Neurochem. 2004; 88, 735-745.
45 Trojanowski JQ, Wa!kenstein N, Lee VM. Expression of neurofilament subunits in neurons of the central and peripheral nervous system: an immunohistochemical study with monoclonal antibodies. J Neurosci. 1986; 6: 50-60.
46 Shaw G, Weber K. Differential expression of neurofilament triplet proteins in brain development. Nature. 1982; 298(5871): 277-279.
47 Ching GY, Liem RK. Assembly of type IV neuronal intermediate filaments in nonneuronal cells in the absence of preexisting cytoplasmic intermediate filaments. J Cell Biol. 1993; 122(6): 1323-1335.
48 Mishra DP, Pal R, Shaha C. Changes in cytosolic Ca2+ levels regulate Bcl-xS and Bcl-xL expression in spermatogenic cells during apoptotic death. J Biol Chem. 2006; 281, 2133-2143.
49 张艳淑，刘强，刘清君，段化伟，何风生，郑玉新．2，5-己二酮对运动神经元钙稳态的影响．中华劳动卫生职业病杂志，2006；24(5)：270-272．
50 Fujiwara I, Takahashi S, Tadakuma H, Funatsu T, Ishiwata S. Microscopic analysis of polymerization dynamics with individual actin filaments. Nat Cell Biol, 2002, 4: 666-673.
51 Pollard TD. Rate constants for the reactions of ATP-and ADP-actin with the ends of actin filaments. J Cell Biol, 1986, 103: 2747-2754.
52 Ma L, Rohatgi R, Kirschner MW. The Arp2/3 complex mediates actin polymerization induced by the small GTP-binding protein Cdc42. Proc Natl Acad Sci U S A. 1998, 95 (26):15362-15367.
53 Cooper JA. Effects of cytochalasin and phalloidin on actin. J. Cell Biol, 1987, 105: 1473-1478.
54 Sampath P, Pollard TD. Effects of cytochalasin, phalloidin and pH on the elongation of actin filaments. Biochem, 1991, 30: 1973-1980.
55 Lankford KL, Letourneau PC.Evidence that calcium may control neurite outgrowth by regulating the stability of actin filaments. J Cell Biol. 1989; 109(3): 1229-1243.
56 Kiselar JG, Janmey PA, Almo SC, Chance MR. Visualizing the Ca2+-dependent activation of gelsolin by using synchrotron footprinting. Proc Natl Acad Sci U S A. 2003; 100(7): 3942-3947.
57 Kinosian HJ, Newman J, Lincoln B, Selden LA, Gershman LC, Estes JE. Ca2+ regulation of gelsolin activity: binding and severing of F-actin. Biophys J. 1998; 75(6): 3101-3109.
58 Perrone Capano C, Pernas-Alonso R, di Porzio U. Neurofilament homeostasis and motoneurone degeneration. Bioessays. 2001; 23:24-33.
59 Rao MV, Campbell J, Yuan A, Kumar A, Gotow T, Uchiyama Y, Nixon RA. The neurofilament middle molecular mass subunit carboxyl-terminal tail domains is essential for the radial growth and cytoskeletal architecture of axons but not for regulating neurofilament transport rate. J Cell Biol, 2003; 163(5): 1021-1031.
60 Boekelheide K. 2,5-Hexanedione alters microtubule assembly. Ⅰ. enhanced tubulin polymerization. Toxicol Appl Pharmacol. 1987, 88(3): 370-382.
61 Boekelheide K.2,5-Hexanedione alters microtubule assembly. Ⅱ. Enhanced polymerization of crosslinked tubulin. Toxicol Appl Pharmacol, 1987, 88(3): 383-396.
62 Liuzzi FJ, Bufton SM, Vinik AI. Streptozotocin-induced diabetes mellitus causes changes in primary sensory neuronal cytoskeletal mRNA levels that mimic those caused by axotomy. Exp Neurol, 1998; 154: 381-388.
63 Tetzlaff W, Leonard C, Krekoski CA, Parhad IM, Bisby MA. Reductions in motoneuronal neurofilament synthesis by successive axotomies: a possible explanation for the conditioning lesion effect on axon regeneration. Exp Neurol, 1996; 139(1): 95-106.
1 Opanashuk LA, He DK, Lehning EJ, and Lopachin RM. Diketone peripheral Neuropathy Ⅲ. Neurofilament gene expression. Neurotoxicology, 2001; 22: 215-220.
2 Kittttr S, Hob J, Endo H, Tourtellotte W, Weeks BS, Markesbery W, Adler W. Cytoskeletal neurofilament gene expression in brain tissue from Alzheimer's disease patients. I. Decrease in NF-L and NF-M message. J Geriatr Psychiatry Neurol. 1994, 7(3): 153-158.
3 Mohiuddin L, Femyhough P, Tomlinson DR. Reduced levels of mRNA encoding endoskeletal and growth-associated proteins in sensory ganglia in experimental diabetes. Diabetes. 1995; 44(1): 25-30.
4 Lariviere RC, Julien JP. Functions of intermediate filaments in neuronal development and disease. J Neurobiol. 2004; 58(1): 131-148.
5 Lewis SA, Cowan NJ. Genetics, evolution, and expression of the 68, 000-mol-wt neurofilament protein: isolation of a cloned eDNA probe. J Cell Biol. 1985; 100(3): 843-850.
6 Beaudet L, Charron G, Julien JP. Origin of the two mRNA species for the human neurofilament light gene. Biochem Cell Biol. 1992; 70(5): 279-284.
7 Parhad IM, Swedberg EA, Hoar DI, Krekoski CA, Clark AW. Neurofilament gene expression following beta, beta'-iminodipropionitrile (IDPN) intoxication. Brain Res. 1988; 464(4): 293-301.
8 Beaudet L, Cote F, Houle D, Julien JP. Different posttranscriptional controls for the human neurofilament light and heavy genes in transgenic mice. Brain Res Mol Brain Res. 1993; 18(1-2): 23-31.
9 Sills RC, Harry GJ, Morgan DL, Valentine WM, Graham DG. Carbon disulfide neurotoxicity in rats: V. Morphology of axonal swelling in the muscular branch of the posterior tibial nerve and spinal cord. Neurotoxicology. 1998; 19(1): 117-127.
10 Monteiro M J, Hoffman PN, Gearhart JD, Cleveland DW. Expression of NF-L in both neuronal and nonneuronal cells of transgenic mice: increased neurofilament density in axons without affecting caliber. J Cell Biol. 1990; 111 (4): 1543-1557.
11 Tu PH, Elder G, Lazzarini RA, Nelson D, Trojanowski JQ, Lee VM. Overexpression of the human NFM subunit in transgenic mice modifies the level of endogenous NFL and the phosphorylation state ofNFH subunits. J Cell Biol. 1995; 129(6): 1629-1640.
12 Ching GY, Liem RK. Assembly of type IV neuronal intermediate filaments in nonneuronal cells in the absence of preexisting cytoplasmic intermediate filaments. J Cell Biol. 1993; 122(6): 1323-1335
13 Lee MK, Xu Z, Wong PC, Cleveland DW. Neurofilaments are obligate heteropolymers in vivo. J Cell Biol. 1993; 122(6): 1337-1350.
14 Lemischka 1R, Farmer S, Racaniello VR, Sharp PA. Nucleotide sequence and evolution of a mammalian alpha-tubulin messenger RNA. J Mol Biol. 1981; 151 (1): 101-120.
15 Lemischka I, Sharp PA. The sequences of an expressed rat alpha-tubulin gene and a pseudogene with an inserted repetitive element. Nature. 1982; 300(5890): 330-335.
16 Liu NK, Xu XM. beta-Tubulin Is a More Suitable Internal Control than beta-Actin in Western Blot Analysis of Spinal Cord Tissues after Traumatic Injury. J Neurotrauma. 2006; 23(12): 1794-1801.
17 何彬，范明，甘思德．神经再生过程中脊髓运动神经元α管蛋白基因的表达．科学 通报，1993；9(1)：129-131．
18 米瑞发，甘思德，范明．神经再生过程中脊髓运动神经元β、γ肌动蛋白基因表达的变化．科学通报，1994；39(6)：542-544．
19 Mi RF, Shi XQ, Fan M, et al. Expression of neurofilament gene in spinal motomeurons during neural regeneeration. Chin Sci Bull, 1999; 44(2): 225-229.
1 Perrin BJ, Huttenlocher A. Caipain. Int J Biochem Cell Biol. 2000; 34, 722-725.
2 Suzuki K, Sorimachi H, Yoshizawa T, Kinbara K, Ishiura S. Calpain: novel family members, activation, and physiologic function. Biol Chem Hoppe Seyler. 1995; 376: 523-529.
3 Ray SK, Banik NL. Calpain and its involvement in the pathophysiology of CNS injuries and diseases: therapeutic potential of calpain inhibitors for prevention of neurodegeneration. Curr. Drug Target CNS Neurol. Disord.2003; 2: 173-189.
4 Guo-Ross S, Yang E, Bondy SC. Elevation of cerebral proteases after systemic administration of aluminum. Neurochem. Int. 1998; 33: 277-282.
5 Gupta RP, Abou-Donia MB. Alterations in the neutral proteinase activities of central and peripheral nervous systems of acrylamide-, carbon disulfide-, or 2,5-hexanedione-treated rats. Mol Chem Neuropathol, 1996; 29(1): 53-66.
6 Nagano M, Yamamoto H, Harada K, Miyamoto E, Futatsuka M. Comparative study of modification and degradation of neurofilament proteins in rats subchronicaUy treated with allyl chloride, acrylamide, or 2,5-hexanedione. Environ Res, 1993; 63(2): 229-240.
7 Tanii H, Hayashi M, Hashimoto K. Neurofilament degradation in the nervous system of rats intoxicated with acrylamide, related compounds or 2,5-hexanedione. Arch Toxicol, 1988; 62 (1): 70-75.
8 Karlsson JE, Rosengren LE, Haglid KG. Quantitative and qualitative alterations of neuronal and glial intermediate filaments in rat nervous system after exposure to 2,5-hexanedione. J Neurochem, 1991, 57(4): 437-444.
9 Banik NL, Hogan, EL, Powers JM, Whetstine LJ. Degradation of cytoskeletal proteins in spinal cord injury. Neurochem. Res. 1982; 7: 1465-1475.
10 Banik NL, Matzelle DC, Gantt-Wilford G, Osborne A, Hogan EL. Increased calpain content and progressive degradation of neurofilament protein in spinal cord injury. Brain Res. 1997; 752: 301-306.
11 Ray SK, Hogan EL, Banik NL. Calpain in the pathophysiology of spinal cord injury: neuroprotection with calpain inhibitors. Brain Res. Rev. 2003; 42:169-185.
12 Chakrabarti AK, Yoshida Y, Powers JM, Singh I, Hogan EL and Banik NL. Calcium-activated neutral proteinases in rat brain and subcellular fractions. J. Neurosci. Res. 1988; 20: 351-358.
13 Banay-Schwartz, M., Deguzman, T., Palkovits, M. and Lajtha, A. Calpain activity in adult and aged human brain regions. Neurochem. Res. 1994; 19, 563-567.
14 Ray SK, Shields DC, Saido TC, Matzelle DC, Wilford GG, Hogan EL, Banik NL. Calpain activity and translational expression increased in spinal cord injury. Brain Res. 1999; 816(2): 375-380.
15 Nixon RA, Quackenbusch R, Vitto A.Multiple calcium-activated neutral proteinases (CANP) in mouse retinal ganglion cell neurons: specificities for endogenous neuronal substrates and comparison to purified brain CANE J. Neurosci. 1986; 6:1252-1263.
16 Lapadula DM, Suwita E, and Abou-Donia MB. Cross-linking of neurofilament proteins of rat spinal cord in vivo after administration of 2,5-hexanedione, J. Neurochem. 1986; 46: 1843-1850.
17 Heijink E, Scholten SW, Bolhuis PA, de Wolff FA. Effects of 2,5-hexanedione on calpain mediated degradation of human neurofilaments in vitro. Chem. Biol. Interact. 2000; 129(3):231-247.
18 Rajgopal Y, Vemuri MC. Calpain activation and alpha-spectrin cleavage in rat brain by ethanol. Neurosci Lett. 2002; 321 (3): 187-191.
19 Takeuchi KH, Saito K-I, and Nixon RA. Immunoassay and activity of calcium-activated neutral proteinase (mCANP): distribution in soluble and membrane-associated fractions in human and mouse brain. J Neurochem, 1992; 58: 1526-1532.
20 Mishra DP, Pal R, Shaha C. Changes in cytosolic Ca2+ levels regulate Bcl-xS and Bcl-xL expression in spermatogenic cells during apoptotic death. J Biol Chem. 2006; 281(4): 2133-2143.
21 Lapadula DM, Suwita E, and Abou-Donia MB. Evidence for multiple mechanisms responsible for 2,5-hexanedione-induced neuropathy. Brain Res. 1988; 458:123-131.
22 Watson DF, Fittro KP, Hoffman PN, and Griffin JW. Phosphorylation-related immunoreactivity and the rate of transport of neurofilaments in chronic 2,5-hexanedione intoxication. Brain Res. 1991; 539: 103-109.
23 LoPachin RM, He D, Reid ML, Opanashuk LA. 2,5-Hexanedione-induced changes in the monomeric neurofilament protein content of rat spinal cord fractions. Toxicol Appl Pharmacol, 2004; 198(1): 61-73.
24 Lopachin RM, He D, Reid ML. 2,5-hexanedione-induced changes in the neurofilament subunit pools of rat peripheral nerve. Neurotoxicol, 2005; 26(2): 229-240.
25 Gou JP, Leterrier JF.Possible involvement of ubiquitination in neurofilament degradation. Biochem. Biophys. Res. Commun. 1995; 217(2):529-538.
26 Ehlers MD. Deconstructing the axon: Wallerian degeneration and the ubiquitin-proteasome system. Trends Neurosci. 2004; 27(1): 3-6.
27 Hernandez, F., Diaz-Hernandez, M., Avila, J., Lucas, J. J. 2004. Testing the ubiquitin-proteasome hypothesis of neurodegeneration in vivo. Trends Neurosci. 27(2): 66-69.
28 Zhai Q, Wang J, Kim A, Liu Q, Watts R, Hoopfer E, Mitchison T, Luo L, He Z. Involvement of the ubiquitin-proteasome system in the early stages of wallerian degeneration. Neuron. 2003, 39(2):217-225.