The Study On Relationship Between Cellular Signal Transduction And Organophosphate Induced Delayed Neuropathy

ObjectivesThe experiment induces the model of organophosphate-induced delayed neuropathy(OPIDN) of hen by methamidophos and tricresyl phosphate, then observes the change of pathology and ultrastructure, measures the change of neuropathy target esterase(NTE) and other biochemistry indicators, detects the dynamic change trend of protein kinase C(PKC),protein kinase A(PKA),microtubulin-associated protein 2(MAP-2) andβ-actin in cerebral cellular signal transduction, illustrates the change of different signal protein content in methamidophos and tricresyl phosphate induced OPIDN, provides evidence to explore the mechanism of cellular signal transduction path in OPIDN.MethodsAfter one week adaptive feed, 44 healthy grown hens were randomly divided by weight into three groups: methamidophos group (n=20), tricresyl phosphate group (n=20) and control group (n=4). Methamidophos (10mg/kg) was administered by gavage for 14 days. Tricresyl phosphate (1ml/kg) was administered by gavage for 7 days in order to induce OPIDN. Control group received an equivalent volume of normal saline. The hen of three group are killed on the 7th day, 14th day, 21st day and 28th day after exposure, and the blood serum are taken. Cerebra, spinal cord, sciatic nerve and gastrocnemius are taken quickly. Tissue specimens were made in traditional processing techniques.Histological examination of these specimens were done by pathologists. The content of ALT, AST and ALP in blood serum was measured in kit, with Automatic Chemistry Analyzer measuring the content of CHE and Ca. NTE activity was measured with the classical method. Western blotting is used to determine the dynamic change trend of PKC,PKA,MAP-2 andβ-actin in brain.Results1.Establishing a model of OPIDN and observing the pathologic featuresWhen methamidophos (10mg/kg) was given at doses estimated to be greater than 5 times the LD50(unprotected by atropine), 60% of the hens in Methamidophos group could exhibit the clinical signs of OPIDN and the acute symptom is serious. However, they could still alive in acute stage. Tricresyl phosphate caused OPIDN in all hens at the 7th-10th days after exposure. The two groups'hens showed visible nervous lesion such as demyelination. Methamidophos group showed the axoplasm being moltening medullary sheath taking as air bubble and the damage becoming more severe at the 21st day, and ply being slack, fracture even crack. All hens in tricresyl phosphate group showed the same change in nerve system, which is according with clinical symptoms.2.Measuring the biochemistry indicators in serumThe ALT content of methamidophos group is significantly higher (P<0.05) than that of control group at the 7th day, 14th day and 21st day after exposure. The ALT content of tricresyl phosphate group is significantly higher (P<0.05) than that of control group at the 7th day and 14th day after exposure. The AST content of tricresyl phosphate group is significantly higher (P<0.05) than that of control group at the 7th to 14th day after exposure. The CHE content of two groups is significantly lower (P<0.05) than that of control group at the 7th day, 14th day ,21st and 28th day after exposure. The Ca content of methamidophos group is significantly higher (P<0.05) than that of control group at the 21st day and 28th day after exposure. The Ca content of tricresyl phosphate group is significantly lower (P<0.05) than that of control group at the 7th day after exposure.3.Measuring the activity of NTEThe activity of NTE of two groups is significantly lower(P<0.05) than that of control group at the 7th day, 14th day ,21st and 28th day after exposure.4.The changes in content of protein kinase,MAP-2andβ-actin in cerebraThe PKC content of methamidophos group decreased by 24%(P<0.05), 28%(P<0.05), 30%(P<0.05), 62%(P<0.01) at the 7th day, 14th day ,21st day, and 28th day respectively. The PKC content of tricresyl phosphate group decreased by 64%(P<0.01), 58%(P<0.01), 58%(P<0.01), 43%(P<0.05) at the 7th day, 14th day ,21st day, and 28th day after exposure respectively. No remarkeble difference was found in the PKA content between two groups and control group. Theβ-actin content in cerebra of methamidophos group increased by 30% (P<0.05) at the 14th day after exposure, and theβ-actin content in cerebra of tricresyl phosphate group decreseaed by 26%(P<0.01) at the 7th day after exposure compared to control group. The MAP-2 content in cerebra of methamidophos group is significantly higher (P<0.05) than that of control group at the 14th and 28th day after exposure, and the MAP-2 content in cerebra of tricresyl phosphate group significantly higher (P<0.01) than that of control group at the 7th day, 14th and 28th day after exposure.Conclusions1. Tricresyl phosphate could develop a hen model of OPIDN; without protective solute, the hen model of OPIDN developed by methamidophos has significantly improved. The two groups of hens showed visible nervous lesion such as demyelination.2. When OPIDN occurred and developed, the content of Ca significantly changed, that can offer an reference for future diagnose. The alterations of content of Ca indicate the relationship between Ca and OPIDN.3. To measure the inhibition of NTE with classical method, the NTE is obviously inhibited in the OPIDN, the detection had the limitations in stability of substrate.4. The content of PKA did not show a significant change, other nervous system should be study in future research. The content of PKC decreased during the process of OPIDN, which may be an candidate signal of OPIDN. Tricresyl phosphate and methamidophos resulted in significant alterations of MAP-2, PKC andβ-actin, among which the alterations of MAP-2 and PKC appeared to be more noticeable thanβ-actin. The significant alterations of content of MAP-2 in cerebra indicate the structure and function of microtubule are interfered.