Explore The Role Of Autophagy In Adult Hens Which Were Induced A Delayed Neurotoxicity

Objective:To build a delayed neurotoxicity (OPIDN) hen model induced by Tri-ortho-cresylphosphate, observe the dynamic of autophagy in TOCP-induced delayed neurotoxicity，Toreveal the relationship between autophagy and TOCP-induced OPIDN, to explore thepossible mechanisms of OPIDN， and to provid a theoretical basis for furtherunderstanding and revealing the mechanism of neurodegeneration.Method:The70chickens were randomly divided into2groups (the control group and theexperimental group). The experimental group （n=35）was once exposed to medical capsuleequipped with TOCP (750mg/kg) and the control group(n=35) was fed to the capsule fittedwith equal amount of vegetable oil. The hens were sacrificed at the1th、3th、5th、7th、10th、14th、21th exposured day; the spinal cord and the sciatic nerve was separated; theexpression of the autophagy related protein LC3was detected by Western blot; part offresh tissue was fixed and preparated to make paraffin slices and electron microscopy slices.The expression of the lysosomal membrane glycoprotein LAMP-1was detected byimmunohistochemistry.Results:1.General situation：after TOCP exposure, the hens appeared less activity, loss ofappetite.The activity and appetite were returned to original level at the third day.At the7thday,the OPIDN symptoms began to appear，the hen’s weight growth rate decreased whencompared with the control group at the14th and21th day(P <0.05).2.OPIDN clinical symptoms：in the7th day the experimental group hens appeared lessactivity, lower limb walked slightly uncoordinatly; in the10th day the hens appeared ataxic,like squatting, double lower limbs weakness, walking instability and the symptoms were progressively devoloped with time；The15th day, the experimented hens walked was veryuncoordinated, often fallen and gradually increased, the18th day, animals with severesymptoms appeared paralysis, was unable to stand and the symptoms lasted until the21thday. The control group hens were not observed any OPIDN symptoms.3.HE staining results:Compared with the control group,the spinal cord neuronsswollen markedly,part of the cells’ cytoplasm was light dyed and the nucleus wereambiguous;nucleus of the neurons appeared pyknosis and the cytoplasm hadvacuoles;Sciatic nerve fibers arranged sparse, appeared swelling, the colour become weakand the structure was disordered.4. Electron microscopy results: in the normal nerve tissue, the form of microglia andneurons were normal, the myelin was close, the lamellar was clear. The myelin in Sciaticnerve had onions tube-like change significantly and was blurred, multiple board layers wereloose, broken or even sputtering.5.With the extension of exposure time, the OPIDN symptoms were aggravated, usingWestern blot, immunohistochemical method to detect the expression of autophagy-relatedprotein LC3-Ⅱ in spinal cord, sciatic nerve, respectively. With the extension of the exposuretime, the protein expression increased, which indicated that OPIDN induced by TOCP maybe related with autophagy.6.To test the expression of LAMP-1in the spinal cord and sciatic nerve byimmunohistochemistry，protein expression was increased with the exposure time, and therewas a time-effect relationship.Conclusion:1.TOCP（Once exposure, TOCP750mg/kg）exposed in adult chickens of21days，theexperimental chickens appeared delayed neurotoxicity, the animals’ spinal cord, sciaticnerve showed signs of toxicity and the exposure time was longer, the poisoning was moreserious.2.TOCP had a effect on the expression of the autophagy-related protein LC3and thelysosome assoeiated memhrane protein1(LAMP-1) in spinal cord or the sciatic nerve. Theexpression level of LC3and LAMP-1were increased along with the exposed time of TOCP.The OPIDN induced by TOCP was probably relative with autophagy.