| Acrylamide(Acrylamide,ACR)is a common chemical raw materials,widely used in mining,paper industry,textile industry,sewage treatment,soil regulation and scientific experiments a variety of fields etc.In general polyacrylamide has no toxicity,but animal experiment has confirmed that ACR monomer has neurotoxicity,reproductive toxicity,genetic toxicity and carcinogenicity.It has been found it has neurotoxicity in the human body,mainly show as muscle weakness,ataxia,hypersensitivity and so on are a series of clinical symptoms.For a long time,research on diseases of the central nervous system are mainly concentrated in the part of gray matter.According to the studies in recent years,lesions of white matter are closely associated with many diseases of the central nervous system.As it is less part of the blood vessels distribution in white matter,white matter is more vulnerable to damage than the part of gray matter.Studies have shown that children who’s cerebral white matter was injury,can increase 15 times of incidence with cerebral palsy.Feeding period of pregnancy is women’s special physiological period,due to the lower resistance and more sensitive to all kinds of harmful factors.It may be related to the change of hormone level,and the incidence rate of cerebral demyelination is higher in 3months after delivery.In recent years,with the development of perinatal technology,the newborn survival rates are increase,but the child in diseases of the nervous system is also a trend of increase.Infants and young children are a critical period of growth and development,due to the function of each organhas not yet developed,it is more vulnerable to a variety of factors and increasing risk of a variety of infantile encephalopathy.so far,there has not been related research reports on the toxic effects of ACR on brain white matter and sheath of lactational maternal and infants at home and abroad.Focus on the influence of ACR on myelin sheath of the two kinds of vulnerable groups,is help to guarantee the health of both maternal and child,and improve the quality of birth.Therefore we proposed to establish the model of maternal exposure SD rats,to study the effect of ACR on the myelin sheath of mothers and young rats by immune histochemical methods,western immunoblot method,luxol fast blue and shoot electron-microscopy.The myelin is an important part of neuronal development which formed by the oligodendrocytes protrusions twines around the neuronal axons,this study is to further testing the influence of the formation of young rat’s neurons and synapse after ACR infected.Method:1.The establishment of animal model: 40 female and 16 males 6 weeks SPF SD rats,including eight spare females,are weight 180-200 g.Adaptability raised after 6 weeks,females cohabited with males in 1:1 ratio,daily morning check the seminal plugs,as finding the seminal plugs,confirm the mating success,and was defined as gestational day 0.Pregnant rats were randomly divided into four groups,each group consists of 8 females,from the 15 days began to gavage,acrylamide dose of 4.5 mg/kg,9 mg/kg,18 mg/kg,and the control group given ddH2 O,continuously administrated until the 14 days after the young rat birth(on the same day delivery marked for the day after the birth of the "0" day).Assessed the body weight of each young rat in the 3 days after born and randomly remained only 10,respectively got the tissues on the 7 and 14 days after birth and each of 5 only.2.The toxic effects of ACR on mouse myelin was examined: behavior detection of the female rats;Nissl staining to detect changes of rats cortex Nissl body;HE staining to detect the changing structure when detecting the expression of lipids by LFB in the corpus callosum of mother rats;Immune histochemical method and western immunoblot method to detect the expression of GFAP,PLP and MBP protein of the corpus callosum of mother rats.3.The effect of ACR on myelination in offspring rats was examined: behavior detection of young rats;LFB staining was used to detect the expression of lipid in the white matter;Immune histochemical method and immunofluorescence method to detect the expression of internal capsule and the corpus callosum myelin associated protein PLP,MBP of the P7,P14 young rat;Western immunoblot method of quantitative analysis to detect theexpression of PLP,MBP protein in P7,P14 young rats’ brain;Transmission electron microscope to observed the structure of myelin sheath in the brain frontal cortex white matter of P14 young rats.4.The effects of ACR on the development of neurons and synapses in offspring rats were examined: immune histochemical method to detect the expression of DCX,GAP-43,GFAP in P14 young rat hippocampal;Western immunoblot method to detect the expression of DCX,SYP and GFAP in the young rats’ brain;Transmission electron microscope to observe the structure of the rat frontal cortex synapses.Result:1.ACR impact on mothers:compared with the control group and low dose group,gait score in medium and high dose group was obviously increased after ACR infected(P<0.01).Compared with control group,high dose group of female postpartum weight decreased obviously(P<0.05).Nissl staining showed that the cortex Nissl body is fewer and smaller volume in the high dose group of rats;HE staining showed that the corpus callosum was smaller volume in high dose group than that in control group,and the internal structure was osteoporosis.The LFB dyeing lighter in each dose group exposuring to ACR.Immune histochemical method and western immunoblot method detection results show that the expression of GFAP was relatively less in low,middle and high dose groups than in control group;While compared with the control group,the expression of middle and high dose groups brains sheath protein PLP and MBP were decreased.2.ACR influence of myelination in young rat brain:Compared with control group,the survival rate of young rats in high dose group was significantly decreased(P<0.05).Compared with control group,body weight of P0,P7,P14 young rat in high dose group were significantly lower(P<0.05).The LFB staining was bright blue in the control group while the staining became shallow or disappeared after ACR exposure.According to the results of the immunohistochemical detection and western immunoblot method,the expression of sheath protein PLP and MBP in P0,P7,P14 each dose group of young rat brains was decreased after ACR infected(P<0.05).Transmission electron microscopy showed that the number of myelin distribution a in P14 high dose group of young rat,the arrangement of the myelin sheath was disordered,and there was a loose separation between the layers,and even the formation of vesicles.3.ACR influence of neurons and synapses of P14 young rats: according to the results of immune histochemical method,the positive expression of DCX,GAP-43,GFAP in the hippocampal dentate gyrus of P14 young rat is less than in the control group(P<0.05).According to the results of western immunoblot method,compared with control group,the positive expression of DCX,SYP,GFAP in young rat brain of high dose group were significantly decreased(P<0.05).Transmission electron microscopy showed that in the control group,there was more synaptic in the brain frontal cortex,the dyeing was depth andthe cell structure was clear,as in the high dose group,was less synapses,the dyeing was shallow and cell structure was not clear.Conclusion:1.Mother rats and offsprings showed neurotoxicity after maternal exposure to ACR.2.ACR may affect the structure of the corpus callosum of mother rats by reduceing the expression of lipids,PLP and MBP.3.ACR may be delay myelination on the brain of offspring rats by reducing the expression of lipids,PLP and MBP of sphingomyelin.4.ACR may be interference the growth of neurons and synapses by reducing the expression of DCX,SYP,GAP-43 and GFAP in the brain of offspring rats. |
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