| Objectiven-hexane a solvent widely used in many industrial processes such as the preparation of fabrics, adhesives, lacquers and other coatings, etc. n-Hexane is metabolized oxidatively to a number of compounds, including 2,5-hexanedione (HD), which is thought to be the ultimate neurotoxic agent. Continued exposure to hexane or HD produces loss of sensorial and motor function in arms and legs, which is the result of the nerve damage characterized as a central-peripheral distal axonopathy. The polyneuropathy was initially reported in shoe-makers who were exposed to n-hexane for months in Italy in 1957. More and more neuropathy patients induced by occupational contact to HD have been reported in our country recently.The corresponding pathological alterations observed in the axonopathy include the accumulation of neurolfi laments (NFs) proximal to the nodes of Ranvier, a Wallerian-type degeneration consisting of accumulation of mitochondria, vesicles, microtubules and dense bodies distal to the axonal swellings, retraction of myelin from Ranvier nodes and segmental demyelination, and distal axon atrophy. The mechanisms of HD-induced axonopathy aren't clear now, and the pathological alterations indicate that NF and MT are involved in the axonapathy. It is well known that NF subunit proteins are decreased substantially in both of peripheral nerves and central nervous regions from HD-exposed rats, and the evidence suggests that the decrements in axonal NF contents are partly responsible for HD-induced axon atrophy. However, the effect of HD on NF in different forms such as cytoskeletal proteins, monopolymer, oligopolymer, etc. has been studied infew papers. As regards to MT and tubulin, the reports related to HD neuropathty exhibit that P -tubulin contents in sciatic nerve and spinal cord were altered. The alteration and contribution of MFs or actin in HD axonopathy haven't been reported. With regards to possible mechanisms to declined protein contents, the studies on the dorsal root ganglia from moderately affected rats intoxicated with HD at 175, 250 and 400 mg/kg, have shown small but significant reductions (approximately 20%) in mean NF subunit mRNA levels, while P - tubulin mRNA expression keeps unaffected. In short, although previous studies have demonstrated decreases in NF contents of HD-intoxicated rats, the contributions of NFs, MFs, MTs and the mechanisms of how such changes in NF proteins might occur in HD axonopathy haven't been demonstrated in detail.In the present investigation,the animal model of HD-induced axonopathy in rat was established, and the relative levels of NF-L, NF-M, NF-H, a -tubulin, P -tubulin, and P -actin in the tissues of sciatic nerves, spinal cords and cerebrum were determined to investigate the mechanisms of the axonopathy. In addition, the levels of gene expression of NF-L, NF-M, and NF-H subunit, a -tubulin, P -tubulin, and P -actin mRNA, was quantified using reverse transcription-polymerase chain reaction (RT-PCR) to investigate the mechanisms from which the alterations might occur. The contents of cytoskelatal proteins in the rat serum were determined to investigate the possibility that was used as the effect biomarker to evaluate the HD neurotoxicity and nervous injury. MethodsHD was administered by intraperitoneal injection to randomly assigned groups of adult male Wistar rats to establish animal model of axonopathy, according to the following daily dosing schedules: 200 or 400 mg/kg/d for continuous 6 weeks (five times per week).The excised nervous tissues of the sciatic nerves, spinal cords and cerebrum were homogenized and then centrifuged at 28, 000 Xg for 30min. The relative levels of NF-L, NF-M, NF-H, a -tubulin, P -tubulin, and P -actinin the supernatant and pellet of central and peripheral nervous tissue homogenates were determined by immunoblotting.The tissues of sciatic nerves, spinal cords and cerebrum were excised, and the total mRNA was isolated from flash frozen tissues using Trizol reagent. The levels of gene expression of NF-L, NF-M, and NF-H subunit, a -tubulin, 3 -tubulin, and ï¿¡ -actin mRNA, was quantified using RT-PCR.The peripheral bloods were sampled, and the serum was separated routinely. The contents of cytoskelatal proteins in the rat serum were investigated by immunoblotting. Results1 .The establishment of the animal modelHD intoxication was associated with evident neurological deficits in rats. After 6 weeks of HD treatment (five times per week) at 200 or 400mg/kg dose-rate, the rats were at completely different levels of neurological deficits; i.e., slight neurotoxicity (obvious movement abnormalities characterized by dropped hocks and tail dragging) or moderate-severe neurotoxicity (dragging hindlimbs and complete absence of rearing), respectively. In addition, neurobehavioral abnormalities were presented including increased hindpaw-lick latency, tail-flick latency and hindpaw-withdrawal latency to pain, and increased hindlimb landing foot splay.2. The alterations in cytoskeletal proteins of nervous tissues In the sciatic nerves, HD intoxication at 200 or 400mg/kg was associated with significant decreases in the pellet fractions, with 67% and 47% (PO.01) relatively of the control group, while supernatant NF-L could not be assayed. For NF-M, the levels were declined to 12% or 5%(P<0.01) in the pellet, with 24% and 6%(P<0.01) in the supernatant respectively. For NF-H, the levels were dropped to 62% and 56%(P<0.01) in the pellets, with 65% and 29%(P<0.01) in the supernatants separately. For ï¿¡ -actin, the levels were dropped to 98% or 93%(P>0.05) in the pellets, with 63%(P<0.05)and 51%(P<0.01) in the supernatants individually respectively. Fora -tubulin, the levels were dropped to 109% or 87%(P>0.05) in the pellets, with 70%(P<0.05) and 58%(P<0.01) in the supernatant respectively. For P-tubulin, the levelswere regulated to 88% or 92%(P>0.05) in the pellets, with 62%(P<0.05) and 22%(P<0.01) in the supernatant respectively.In the spinal cord, the levels of NF-L in 200 or 400mg/kg group were declined to 65% and 58%(P<0.01) significantly of the control group in the pellet fractions, with 65% and 21% in the supernatant relatively. For NF-M, the levels were declined to 68% and 51%(P<0.01) in the pellet, with 74% and -24%(P<0.01) in the supernatant respectively. For NF-H, the levels were dropped to 71% and 60%(P<0.01) in the pellets, with 77%(P<0.05) and 48%(P<0.01) in the supernatants separately. For3-actin, the levels were increased to 110% and 116%(P>0.05) in the pellets, with 99% and 76%(P>0.05) in the supernatants individually. Fora -tubulin, the levels were dropped to 88% or kept unaffected with 100%(P>0.05)in the pellets, while they were declined to 59%(P<0.01) at 200mg/kg and increased to 126%(P<0.01) at 400mg/kg in the supernatant respectively. For P -tubulin, the levels were regulated to 97% or 105%(P>0.05) in the pellets, while they were declined to 59%(P<0.01) at 200mg/kg and increased to 156%(P<0.01) at 400mg/kg in the supernatant respectively.In the cerebrum, HD intoxication at 200 or 400mg/kg was associated with significant decreases in the pellet fractions, with 57% and 49% (PO.01) relatively of the control group, while the supernatant NF-L could not be assayed. For NF-M, the levels were declined to 66% or 33% (PO.01) in the pellets, while the supernatant NF-M could not be also assayed. For NF-H, the levels were dropped to 73% and 68%(P<0.01) in the pellets, with 65% and 51%(P<0.01) in the supernatants separately. ForP-actin, the levels were regulated to 102% or 88%(P>0.05) in the pellets, with 87% and 105%(P>0.05) in the supernatants individually. Fora -tubulin, the levels were increased to 104% or 106%(P>0.05)in the pellets, with 87%(P<0.05) and 114%(P<0.01) in the supernatant respectively. ForP -tubulin, the levels were regulated to 91% or 100%(P>0.05) in the pellets, with 107% and 101%(P>0.05) in the supernatant respectively.3. The mRNA expression of cytoskeletal proteinsIn sciatic nerves, the expression of NF-L mRNA was increased significantly, and they were elevated to 126% and 152% separately at 200 and 400mg/kg dosages.' The levels of NF-H mRNA kept unaffected, with the relative contents of 100% and 95%(P>0.05) respectively. NF-M mRNA was not amplified. The expression of P -actin mRNA also kept steady, with relative contents of 95% and 102%(P>0.05). The levels of a -tubulin mRNA were decreased to 97% and 94%(P>0.05), while the levels of P -tubulin mRNA were increased significantly to 119%(P>0.05) and 143%(P<0.01) of the control.In the spinal cord, the expression of NF-L mRNA at 200 and 400mg/kg dosage was decreased significantly to 54% and 51%(P <0.01) relatively. The levels of NF-M mRNA were decreased to 86% and 85% respectively, not significantly compared with the control (P>0.05). The expression of NF-H mRNA at 200 mg/kg dosage was decreased to 90%(P>0.05) relatively, and 85%(P<0.05) at 400 mg/kg dosage. The levels of P -actin mRNA was decreased to 88% and 80% respectively, not significantly compared with the control(P>0.05). The levels of a -tubulin mRNA kept unaffected, with relative contents of 96% and 97% respectively. The expression of P -tubulin mRNA at 200 and 400mg/kg dosage was decreased to 87% and 80%(P<0.01 Relatively, not significantly different compared with the control(P>0.05).In the cerebrum, the expression of NF-L mRNA at 200 and 400mg/kg dosage was decreased significantly to 41% and 34%(P<0.01) relatively. The levels of NF-M mRNA were decreased to 99% and 90% respectively, not significantly compared with the control (P>0.05). The expression of NF-H mRNA at 200 mg/kg dosage was decreased to 99%(P>0.05) relatively, and 85%(P<0.05) at 400 mg/kg dosage. The levels of P -actin mRNA was decreased to 92% and 85% respectively, not significantly different compared with the control(P>0.05). The levels of a -tubulin mRNA at 200 mg/kg dosage was decreased to 94(P>0.05) relatively, and 79%(P<0.05) at 400 mg/kg dosage. The expression of P -tubulin mRNA at 200 mg/kg dosage was decreased to 86%(P>0.05) relatively, and 78%(P<0.05) at 400 mg/kg dosage.4.The alterations in cytoskeletal proteins in the serum The protein levels of NF-L,NF-M,NF-H, 3 -actin, a -tubulin and 0 -tubulin in the serum of 200mg/kg dosage showed small increase, with relative contents of 105% to 121% of the control group(P>0.05). The contents of NF-L and P -actin exhibited significant reductions by 39% and 49% (P<0.05 or PO.01) respectively in the serum from rats treated with 400mg/kg dosage. The levels of a -tubulin protein were increased to 120% (PO.05), while the contents of NF-M, NF-H, 0 -actin, and ï¿¡ -tubulin exhibited no significant alterations, with the relative contents of 86% to 121 % (P>0.05) of the control. Conclusion1 .HD intoxication was associated with significant declines in NF contents in rat sciatic nerves, spinal cord, and cerebrum, and declines in the levels of |3 -actin in the sciatic nerves. For a -tubulin and ï¿¡ -tubulin, there were significant decreases in sciatic nerves following exposure to HD, while significant declines for 200 mg/kg and obvious increase for 400 mg/kg in spinal cord.2.The decreases in cytoskeletal proteins of sciatic-tibial nerve tended to be greater than those in the spinal cord and the cerebrum. Among three NF subunits, the trend for the declines in NF-M was greater than those in NF-L and NF-H.3.HD might not only induce alterations in protein contents, but also interfere with the kinetics of the polymer exchange of NF and MT.4.HD induced increases in the gene expression of NF-L and 3 -tubulin in the rat sciatic nerves. In addition, HD-intoxication was associated with reductions in expression of NF-L and NF-H mRNA of the spinal cord and the cerebrum, and decreases in the expression of a -tubulin and 3 -tubulin mRNA in the cerebrum.5.The alterations in NF-L, 3 -actin and a -tubulin of the rat serum might be used as the effect biomarker to evaluate the HD neurotoxicity and nervous injury.6.The cytoskeletal proteins including NF, MF, MT were involved in the peripheral axonopathy induced by HD. |
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