| Objective: To explore the mechanism of hippocampal plasticity-related gene1(PRG-1)involved in neonatal Repetitive noxious(RNS)stimuli-induced hyperalgesia and learning and memory impairment in rats through hippocampal synaptic changes,and to provide a new target for neonatal analgesia.Methods:1.Establishment of neonatal rat RNS model: In the first week of rat birth,at 0:00,6:00,12:00 and 18:00 every day,28 G blood collection was used to perform acupuncture on the four feet of the rat in turn to establish the rat RNS model to simulate repeated noxious stimuli to neonates.Rats in the control group were tactilely stimulated by cotton swabs at the same time and location.After each stimulation,the rats were placed in the previous rat cage to ensure that the separation time of each mother and child was less than 5 minutes to avoid the effects of prolonged mother and child separation.2.The rat model was evaluated by behavioral experiments: the body weight of the rats was monitored weekly after birth,and the effect of RNS modeling on the body weight of the rats was evaluated.The thermal pain threshold and mechanical pain threshold test were performed every two weeks to detect the pain sensitivity of the rats’ soles,and the Morris water maze test was performed at the 3th,6th and 9th weeks to evaluate the rat’s space learning and memory ability.In the 9th week,the inclined plate experiment was performed to evaluate the effects of RNS modeling and subsequent administration,virus injection and other operations on the muscle strength of rats.3.The expression of PRG-1 was detected by molecular biology experiments:Western blotting(WB),immunofluorescence,real-time quantitative-reverse -transcription polymerase chain reaction(qRT-PCR)experiments were used to detect the 3rd,6th and 9th weeks of PRG-1 expression in rats hippocampus.4.Changes of hippocampal dendritic spines were observed by morphological experiments: Golgi staining and Nissl staining were used to observe the changes of dendritic spines density and neuronal activity in rat hippocampal neurons at the 3 th and 9th weeks.5.Detecting the effects of PRG-1 on behavior and hippocampal synapses in RNS rats through PRG-1 intervention: Lentivirus was injected into the CA1 region of the bilateral hippocampus of rats at week 3 to interfere with PRG-1 expression,or at week 9 The PRG-1 agonist FTY720 was injected into the CA1 region of the bilateral hippocampus of the rats for three consecutive days,and then the changes of pain perception,learning and memory in the rats were observed by behavioral experiments,the expression of PRG-1 was observed by molecular biology experiments,and the Changes in hippocampal dendritic spine density were observed by Golgi staining.Results:1.The body weight results showed that there was no significant difference in body weight between RNS rats and CON rats in each period,indicating that RNS modeling in the neonatal period had no significant effect on the body weight of rats.At the ninth week,the inclined plane experiment showed that there was no significant difference in the maximum inclined plane inclination angle of the rats in each group on the inclined plate and kept still,indicating that the neonatal RNS modeling and subsequent administration,virus injection and other operations had no significant effect on the muscle strength of the rats.2.The test results of mechanical pain threshold and thermal pain threshold in rats showed that RNS rats had shorter paw withdrawal time with thermal pain and lower pressure on the needle tip during mechanical pain,suggesting that RNS modeling in the neonatal period induces persistent thermal pain sensation in rats from infancy to adulthood and mechanical hyperalgesia.The results of the water maze experiment showed that the total time and distance of the RNS rats to find the platform was longer,and the time spent exploring in the quadrant where the platform was located accounted for a smaller proportion,suggesting that the RNS modeling reduced the spatial learning and memory ability of the rats in each period.3.The results of WB experiment showed that the expression of PRG-1 in RNS rats increased at 3 weeks and decreased in adulthood;the results of immunofluorescence showed that the fluorescence intensity of PRG-1 in RNS rats was weaker in adulthood;the results of PCR experiments showed that the expression of PRG-1 in RNS rats was weaker PRG-1 mRNA levels were lower at 3 weeks,suggesting that RNS modeling increased PRG-1 expression in the rat hippocampus at3 weeks and decreased in adulthood.The results of Nissl staining showed that the Nissl staining in the CA1 and DG regions of rat hippocampal neurons became lighter,and the Golgi staining results showed that the density of dendritic spines in the hippocampus of RNS rats decreased,suggesting that the RNS model reduced rat hippocampal neuronal activity and dendritic spine density.4.The results of PRG-1 intervention showed that the PRG-1 overexpression or activation group prolonged thermal pain paw withdrawal time,and the mechanical pain paw withdrawal threshold increased,suggesting that PRG-1 overexpression and activation improved the hyperalgesia in RNS rats.In the PRG-1 overexpression and activation group,the time and distance to find the platform in the water maze were shortened,and the proportion of time spent exploring the platform in the quadrant was increased,suggesting that PRG-1 overexpression or activation improved the learning and memory impairment of RNS rats.The results of WB showed that the expression of PRG-1 in the hippocampus of the rats in the PRG-1 activation group was increased,and the results of PCR experiments showed that the content of PRG-1 mRNA in the hippocampus of the rats in the PRG-1 activation group was increased.The results of Golgi staining showed that the density of dendritic spines in the hippocampus of rats in the PRG-1 activation group increased.Conclusion:1.Postsynaptic membrane of hippocampal neurons PRG-1 improves synaptic plasticity by increasing the density of hippocampal dendritic spines,and relieves RNS-induced hyperalgesia and spatial learning and memory impairment-like behaviors in rats.2.The PRG-1 target drug FTY720 may become a new drug for pain treatment in neonatal rats. |
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