| Objective:Establish neurobehavioral test combination in mice. After DiBP subchronic oral exposure in mice, learning and memory function were evaluated by the established neurobehavioral test combination and the the cell signaling mechanisms were explored. The results can provide us with important insights into the molecular mechanisms of cognitive dysfunction induced by DiBP,and at the same time to provide the scientific basis for risk assessment.Method:1 Establish neurobehavioral of test combination1.1 sensory-motor function of test combination1.1.1 Experimental DesignMale C57BL/6 mice were divided into five groups,15 in each. The mouse in group 1 served as control, and received water. The animals in group 2,3,4 and 5 were given acrylamide by gavages (5,10,20,30mg/kg bw 5 days/week)last for 8 weeks. During the first 3 weeks Neurobehavioral indexes was determined per week. The trial lasted 8 weeks, Neurobehavioral tests were determined again.1.1.2 methods of sensory and motor testingNeurobehavioral tests included hotplate, rotarod, grip strength, motor activity, measurements of hind limb landing foot splay.1.2 Learning-memory function of test combination1.2.1 Animal selection and exposure mode216 male KunMing mice were Accommodating to the animal house for 3 days, then dividing into 6 groups according to their weights. That is, one control group, four experimental groups (50mg/kg bw; 250mg/kg bw;500mg/kg bw;1000mg/kg bw) and Positive control group.The mice were fed with the corn oil in control group, and the other groups were fed with the related dose of diisobutyl phthalate mixture by gavages last for 8 weeks. The mice of positive control group were given intraperitoneal injection of Scopolamine at the 5mg/kg bw dosage before Neurobehavioral tests.1.2.2 Learning-memory Behavioral analysisThe effects on learning and memory behavior, such as locomotor activity in the open field (OPF), learning ability in the passive avoidance response (PA) and active avoidance(AA), spatial learning ability in the Morris water maze (MWM) were examined at the beginning and the end of the experiment.2 Effects of DiBP on learning and memory function in mice and the cell signaling mechanisms involved2.1 Animal selection and exposure mode120 male KunMing mice were Accommodating to the animal house for 3 days, then dividing into 5 groups according to their weights. That is, one control group, four experimental groups (50mg/kg bw; 250mg/kg bw;500mg/kg bw;1000mg/kg bw).The mice were fed with the corn oil in control group, and the other groups were fed with the related dose of diisobutyl phthalate mixture by gavages last for 8 weeks.2.2 Learning-memory Behavioral analysisThe effects on learning and memory behavior, such as locomotor activity in the open field (OPF), learning ability in the passive avoidance response (PA) and active avoidance(AA), spatial learning ability in the Morris water maze (MWM) were examined at the end of the experiment.2.3 Determination of DiBP in brain tissueAt the end of experimental time,six mice were randomly selected to detect the DiBP in brain tissue by HPLC. Determine whether it can through blood-brain barrier.2.4 Hippocampal pathology2.4.1 Optical microscope sample preparation Mice were anesthetized with 1% sodium pentobarbital(i.p) and the brain were fixed by heart perfusion with cold saline until the effluent become clear, followed by 4% paraformaldehyde in PBS. After fully fixed, rapid separate the brain and hippocampus on the ice.the brains were post-fixed in the same fixative overnight for preparing sections,then dehydrated,paraffin embedding and slice up.2.4.2 Hematoxylin-Eosin stainingThe paraffin sections were stained with HE,and morphology of the hippocampus were observed under light microscope.2.4.3 Observation of Nissl bodiesThe paraffin sections were used for Nissl staining. The numbers of Nissl-positive cell in hippocampal pyramidal cell layer(CAl and CA3) and the dentate gyrus(DG) were counted under light microscope. Total cell count was averaged from at least 3 sections per animal.2.4.4 observed with transmission electron microscopeAt the end of experimental time,six mice were randomly selected to detect hippocampal ultrastructural alterations on electron microscopy.2.5 Synapse counts in the neuropilEach specimen in the electron microscope magnification to 15,000 times, randomly selected 10 neuropil and counting the number of synapses.2.6 Apoptosis rate of hippocampal nerve cellsThe apoptosis rate of hippocampal nerve cells was measured by Flow cytometry (FCM).2.7 Oxidation status of miceAt the end of experimental time, mice were killed, and then choosed the tissues to test. The biomarkers, such as SOD, GSH-PX, MDA,8-OHdG in livers and brains tissue and comet assay in blood.2.8 Detection of amino acid neurotransmitters in brain tissueThe content of amino acid neurotransmitters was determined by LC/MS.2.9 Detection of cAMP in hippocampal tissueThe content of cAMP was determined by euzymelinked immunosorbent assay(ELISA).2.10 The plasticity-related proteins in hippocampus including phospho-N-methyl-D-aspartate receptor 1(p-NMDAR1),postsynaptic density-95(PSD95),phosphor- cAMP response element binding (p-CREB),protein kinase A Catalytic subunit (p-PKA C),Calcium/calmodulin kinaseâ…¡(CaMKâ…¡) and TrkB receptor were measure by western blotting.2.11 The mRNA expressions including CREB,BDNF,CaMKâ…¡,c-fos and c-jun were checked by real time reverse transcription-polymerase chain reaction(RT-PCR).Result:1 Establish neurobehavioral of test combination1.1 sensory-motor function of test combination1.1.1 There was no mice died in the experiment and no ACR-related adverse effect on the body weight.1.1.2 Before the experiment, sensory and motor ability of each group has no significant difference. After the exposure had ceased, Nociception, Grip Strength, Rotating Rod and Hindlimb Foot Splay were affected at all.1.2 Learning and memory function of test combination1.2.1.There was no mice died in the experiment and no effect on the body weight.1.2.2 Before the experiment,learning and memory ability of each group has no significant difference. Positive control group appeared to show that the time in center was longer than that of solvent control (P< 0.05);In the morris water maze test, latency to find a hidden platform was significantly increased in the positive control group (P< 0.01). In the Passive Avoidance(PA), the exposed animals of positive control group showed learning impairment as compared to unexposed mice(P<0.05).1.2.3 After the DiBP exposure had ceased, total locomotor activity in OPF was not affected at all, but the 1000mg/kg bw group appeared to show that the time in center was longer than that of solvent control (P< 0.05);In the morris water maze test, latency to find a hidden platform was significantly increased in the 500mg/kg bw group and 1000mg/kg bw group (P< 0.01). In the Passive Avoidance(PA) and Active Avoidance(AA) test, the exposed animals of 500mg/kg and 1000mg/kg bw group showed learning impairment as compared to unexposed mice(P<0.05).2 Effects of DiBP on learning and memory function in mice and the cell signaling mechanisms involved2.1 There was no mice died in the experiment and no effect on the body weight.2.2 DiBP was detected in500mg/kg bw and 1000mg/kg bw group and dose-effect relationship was observed. At the same time DiBP wasn't measured in control, 50mg/kg bw and 250mg/kg bw group.2.3 After the exposure had ceased, Spatial Reference Memory, Passive Avoidance ability and Active Avoidance ability were affected by DiBP.2.4 The results of HE stain and Nissle stain displayed the morphology in hippocampus of control group was normal,and that ofl OOOmg/kg bw group was minimal abnormal, the number of hippocampal neurons,which in CA1, CA3 and DG region, are less than that of control group(P<0.05). lost. While the other dose groups are also normal and number of Nissl bodies, compared with the control group no significant difference.2.5 On transmission electron microscopy neuronal ultrastructural alterations, such as damage of nuclear membrane,mitochondria, rough surfaced endoplasmic reticulum(RER) and polyribosome,were found in DiBP-exposed hippocampal neurons compared with controls. In addition, the number of nerve synapses shows decreased in the 500mg/kg bw group and 1000mg/kg bw group compared with the control group (P< 0.01),which was observed under the transmission electron microscope.2.6 The apoptosis rate of hippocampal nerve cells increased remarkably in 1000mg/kg bw group(P<0.05).2.7The activities of SOD and GSH-PX in experimental groups significantly decreased (P<0.05), and the experimental groups (50mg/kg bw group and 250mg/kg bw group VS control group) contrasting to the control group, the MDA content increases significantly (P<0.05), and the 8-OHdG content (500mg/kg bw group and 1000mg/kg bw group VS control group) increase significantly (P<0.05). The comet assay show that the length of DNA migration (the experimental mice groups VS the control group) varies significantly (P<0.05). 2.8 GAB A content of brain in 1000mg/kg bw group was significantly less than control group(P<0.05).2.9 The cAMP content of brain in 1000mg/kg bw group was significantly less than control group(P<0.05).2.10 DiBP down-regulates the expressions of synapse plasticity-related proteins in hippocampus,Such as expressions of PSD95,NMDAR1,p-CAMKâ…¡,p-PKA C and p-CREB decreased in experimental groups.2.11 The relative level of CREB,BDNF,CaMKâ…¡,c-fos and c-jun mRNA has been down-regulated through cAMP/PKA-CREB signaling pathway in experimental groups.Conclusion:1. hotplate test, rotarod test, grip strength test, measurements of hind limb landing foot splay test and motor activity test can be seen as a screening test combination for Sensory-Motor function. Open field, Morris water maze, passive avoidance test and activety avoidance test can be seen as a screening test combination for Learning-Memory function.2. Mice subchronic exposure to DiBP, learning and memory ability can be impaired. DiBP can go through blood-brain barrier into the brain tissue after oral intake.3. DiBP affected cAMP-PKA signaling pathway of hippocampus in mice, then decreased the p-CREB protein expression levels, inhibited the formation of LTP and impaired the learning and memory function. This may be the important molecular mechanism of learning and memory dysfunction caused by DiBP exposure.4. DiBP could reduce the antioxidant defense system function of mouse and lead to DNA damage.
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