Establishment Of Amouse Model Of Fourth Ventricle Injury And Analysis Of Fourth Ventricle Cell Group

There are many and complex cell types in mammalian nervous system,and heterogeneity exists among cells.The nervous system is the dominant functional regulatory system in the body.Only by thoroughly understanding the cell types and molecular mechanisms of the nervous system,can we better study the functions of the nervous system and the occurrence of nervous system related diseases.The emergence of single-cell transcriptome provides convenience for the study of intercellular heterogeneity,which mainly obtains the information of individual cells in an unbiased way,so that our study of tissue is no longer to obtain the information of mixed cell populations.The previous work of our laboratory found that there were resting stem cells in the fourth ventricle of adult mice,and after the fourth ventricle was damaged,the stem cells were activated.Therefore,we carried out two aspects of research on the fourth ventricle.The results are as follows:(1)Objective to construct a mouse model of hemorrhagic brain injury(ICH).The autogenous blood from the tail of the mice was injected into the brain of the mice5.88-6.5mm behind the anterior fontanelle point and 0.5-0.55 mm to the right with the stereotactic apparatus.The second injection was adopted.The autologous blood was injected twice at the rate of 2 μl/min during the injection of 3.4-3.5mm,and stayed for5 minutes,and then 20 ul was injected at the speed of 2 μl/min at 3.75-3.95 mm.The results showed that the injected blood was scattered around the fourth ventricle,which indicated that the stereotactic parameters were accurate and ICH model mice were successfully established.(2)ICH model mice were selected to take samples at 6h and 24 h after injury.For neurobehavioral assessment of the 27 ICH model mice,longa 5-point scale and modified Garcia JH score were used at 6 h of injury.The mice with Longa 5 score more than 1 and the modified Garcia JH score less than 13 were used in neurological rating and subsequent experiments.A total of nine unqualified injured mice were excluded.Combined with the data of neurobehavioral assessment(6h-7d)and TUNEL cell apoptosis detection(6h-72h)in brain slices of three model mice,it was found that the repair system of ICH model mice began to operate 12 hours after injury,reached a rapid repair device after 24 hours of injury,slowed down after 48 hours of injury,and gradually returned to normal state,which can be used as a basis for studying the repair mechanism of injury in the fourth ventricle.(3)Based on the single nuclear transcriptome data of 5 samples(control group,injured side and uninjured side after injury for 6 hours,injured side and uninjured side after injury for 24 hours),the control group samples were clustered into 28 clusters by unsupervised dimensionality reduction clustering,18 clusters for injured side after injury for 6 hours,and 21 clusters for uninjured side after injury for 6 hours.There were 20 clusters on the injured side 24 hours after injury and 23 clusters on the uninjured side 6 hours after injury.(4)Based on the clustering results of the control group,the cell atlas of the area around the fourth ventricle was constructed by automatic annotation,marker gene alignment and GO / KEGG enrichment analysis.The neurons included typeⅠA spiral ganglion neurons(15.97%),type ⅠC spiral ganglion neurons(5.01%)and type Ⅱspiral ganglion neurons(11.82%),typical interneurons(13%),GABAergic interneurons(chandelier like cells,0.56%),and trans laminar interneurons(Martinotti’s cells,3.35%),GABAergic neurons(2.77%),Purkinje cells(1.32%).Endothelial cells(0.6%).Glial cells include astrocytes(1.76%),radial astrocytes(Bergman glia cells,2.53%),mature oligodendrocytes(9.28%),oligodendrocyte precursor cells(1.95%),microglia,ependymal cells(0.23%),choroid plexus epithelial cells(0.74%)and olfactory ensheathing glia(0.63%).Neural stem cells include neuroblasts(0.95%)and quiescent neural stem cells(9.18%).(5)By neuronal subpopulation analysis,it was found that one of the granule neuron subpopulations may be early developmental cells;Among the three type IA spiral ganglion neuron subpopulations,there are Ecel1 specific(1A-sgn2)and Ccbe1specific(1A-sgn3)subpopulations;Among the three type IC spiral ganglion neuron subpopulations,there are Cpne4 and Ebf2 specific(1C-sgn1)as well as Fam107 b specific(1C-sgn3)subpopulations;Three subsets of type II spiral ganglion neurons expressed genes specifically,Rpl7a-ps3,Gm10036 and Rpl39 l,which was expressed only in 2-sgn 1,ntrk3 which was highly expressed in 2-sgn 2 and expressed only in this subset,and COL27A1,which was expressed only in 2-sgn 3;There are two specific subsets of interneurons: Nfia,St18,Nfib and Satb1 specific subsets(Inter-1),Gulp1,Foxo1,Sema3 c and Casz1 specific subsets(Inter-2);Of the three purkinje cell subpopulations,there are an Fgf7 specific subpopulation(purk 1),Eln specific subpopulation(purk 2),and Rmst,Plce1,and Cacna1 e specific subpopulations(purk 3).(6)By glial subpopulation analysis,two subpopulations of oligodendrocytes,with Mirg,Hs3st4 specific expression subset(oligo 1);Among the two astrocyte subpopulations,there are Slc6a5 specific(Astro 1),Baiap3,Col25a1,Gm2824,and Sst specific subpopulations(Astro 2);Of the three bergmann glial subpopulations,with Plch1,Chst9,and Adamts16 specificity(Berg 1)and 0610040j01 Rik specificity(Berg 2).(7)Through the integrated analysis of 2 samples injured for 6 hours,the results showed that the injured mice were seriously injured at 6 hours after injury,and both sides of the fourth ventricle were significantly affected.Through the integrated analysis of 2 samples injured for 24 hours,it is preliminarily considered that the cells on the uninjured side of the injured mice recover quickly after 24 hours of injury,thus new cells are quickly produced to repair the injury.Through the integration analysis of the injured side samples of the experimental group and the control group,the results showed that the cells around the fourth ventricle of mice may have a protective mechanism after the fourth ventricle tissue was damaged,and new cells could be produced quickly to fill the missing cells.In summary,this paper completes the mouse fourth ventricle injury model and the construction of cell atlas around the fourth ventricle in mice,which will provide the basis for the study of the function of the fourth ventricle and related diseases.