| Objective:In this study,LV-rno-miR-375-3p was injected into the left lateral ventricle and then the hypoxic-ischemia brain damage(HIBD)model was established.We here study the effects of 375-3p on long-term learning and memory ability and circadian rhythm in neonatal rats to know the effects of microRNA(LV-mo-miR-375-3p)on long-term behavior and rhythm of hypoxic-ischemic factors in mammals.Provide a reference for clinical long-term intervention.Methods:1.In the first part,The newborn SD rats within 24 hours after birth were randomly divided according to the random distribution principle.They were divided into miR-control sham,miR-control HIBD,miR-375-3psham group and miR-375-3pHIBD group.After anesthesia with ether,a precise left lateral ventricle injection was performed by using a stereotactic locator through a micro-injector.Then,when 7 days old,the HIBD model and the sham operation group were made.Here,the newborn rat HIBD model was made by the Rice-Vannucci method.Clearly separate the left neck artery and disconnect it after ligation.The rats were placed in an anoxic silo which is full of mixed gases of 8%O2 and 92%N2 for 3 hours,then returned them to the cage of the mother.On the 10th day,live imaging techniques were used to understand viral transfection status.2.we Start behavioral testing after the rats were fed to 3 weeks of age.At this time,the rats are relatively active.After 6 days of swimming training with the Morris water maze,the positioning navigation experiment and the space exploration experiment were completed to test the difference of spatial learning and memory ability between the miR-control sham,miR-control HIBD,miR-375-3psham group and miR-375-3p HIBD group.In the wheel running test,all rats were kept in a single cage and were free to move in the cage.Each group is in LD(12:12)mode:turn on at 08:00,turn off after 20:00 for one week and then change to DD(completely dark)mode for 2 weeks.Check the running wheel interface connection every day.Analysis the differences of circadian rhythm changes in the four groups of rats under exclusion of light.Motor function and anxiety in the four groups of rats were tested by open field experiment.Using the Y-maze test,the residence time and the number of shuttles in each arm of the four groups of rats were tested to understand the difference in learning and memory between the four groups.3.After the SD rats have completed the behavioral test,that is,at the 14th week of age,the rat's brain was dissected.The appearance of the brain tissue was observed after hypoxia and the pineal gland was extracted(related to the rhythm)to analysis fluorescence expression differences by TaqMan RT-qPCR and flow cytometry.Results:1.Lentiviral-coated LV-rno-miR-375-3p and control virus were injected into the lateral ventricle for 10 days.Through the Living imager equipment,it was clear that the normal SD rats had no fluorescence imaging,while the LV-rno-miR-375-3p and the control virus group SD rats showed significant fluorescence,Confirming successful in situ viral injection2.The result of running wheel is completed by the software clocklab3.Data analysis software is done by Math Works.There was no statistically significant difference between the miR-control HIBD group and the miR-375-3pHIBD group(p>0.05).However,there was a difference in the onset time of the rats and the onset time difference between the two group was more than half an hour.It was found to be statistically significant between the two groups(P<0.05).It is indicated that miR-375-3p has no obvious effect on the exercise cycle of rats after hypoxia-ischemia,but it has a great influence on the exercise phase of rats.3.After the rats were trained by swimming intensive training,the average escape latency gradually decreased.In the space exploration experiment,there was a statistically significant difference between the miR-375-3p HIBD and the miR-control HIBD group crossing the platform(P<0.05).There was also a statistically significant difference between miR-375-3p HIBD and miR-375-3p sham(P<0.05),but there was no significant difference in the quadrants of the groups(P>0.05).The space exploration experiment indicated that the lateral ventricle was injected with lentivirus,and under the overexpression of miR-375-3p,the spatial learning and memory ability decreased.4.1n the open field experiment,between the miR-375-3p HIBD group and the miR-control HIBD group,the activity distance in the open field was statistically different among the two groups(P<0.05).The activity distance of the miR-control HIBD group was statistically significant(P<0.05).The data of the two groups at the speed of motion showed statistical significance(P<0.05).5.There was no significant difference in the retention time of the target arm of the Y maze(P>0.05),and the number of times between the two groups was not statistically significant(P>0.05).6.Through the collection and preparation of cell suspensions of pineal(related to rhythm)specimens,it was found that RT-qPCR and flow cytometry techniques further verified the molecular level of SD rats injected with LV-mo-miR-375-3p.Both volume and fluorescence imaging were significantly higher than the normal control group.It indicates that the virus is successfully injected,and the infection has a long-lasting ability,which also indicates the reliability of the previous behavioral experiment results.However,the inadequacy of the experiment is that the fluorescence detection of the empty virus group has not been carried out.Conclusion:After making the HIBD model by overexpressing miR-375-3p,we can find that miR-375-3p can alter the abnormality of long-term exercise,learning and memory ability and circadian rhythm in neonatal rats with hypoxia-ischemia.Previous studies have reported that HIBD can cause learning and memory defects in neonatal rats.After overexpression of miR-375-3p,it can be aggravated by this phenomenon,and the learning and memory function is further reduced.In terms of circadian rhythm,the effect on the day and night cycle is not obvious,but the activity start time can be significantly changed.In the open field experiment,the activity speed,the activity distance and the central area residence time were all changed,which was more anxious than other rats.The emotional impact is more obvious,and it is related to the huge damage caused by the neonatal hypoxic-ischemic brain damage,and the neurological abnormalities such as mental retardation,cerebral palsy,anxiety and the like are similar.When routinely raised to 14 weeks of age,we improved RT-qPCR and flow cytometry,and we found that miR-375-3p can continue to express for a long time. |
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