Experimental Research On Development And Senescence Of C57/BL6 Mouse Hippocampal Formation

ObjectiveTo investigate the morphological changes, the cell proliferation and apoptosis during the development and Senescence of C57/BL6 mouse hippocampal formation.MethodsSerial sections and stereological methods were used to quantitatively analyze the different areas of developing and senescent mouse hippocampal formation in molecular layers, pyramidal layers (granular layers) and polymorphic layers on E10,12,14,16,18,20 day and P1,3,5,7,14,21,28 day and P2,3,6,12,15,18 month. Immunohistochemistry, immunofluorescence, transmission electron microscope and western blotting were used to qualitatively and quantitatively analyze the cell proliferation,apoptosis and apoptosis-related protein bcl-2, bax and caspase-3 expression in the hippocampal formation at different time points.Results1. Volume changes of development and aging of C57/BL6 mouse hippocampal formation.1.1 Results of light microscopy observation. Hippocampal formation primordium first appeared at embryonic day 12(E12).A"C"outline can be seen in pyramidal layer of CA,and the extra arm's blanket of DG's granular layer formed at E18.After birth,CA developed and maturated gradually.The blanket of DG's granular layer formed at postnatal day 7(P7d).At P21d,the inner arm's thickness of DG's granular layer was equal to the extra's,and the subgranular layer presented until 15 month(15M).1.2 Stereology analysis resultsThe volume of hippocampal formation,CA,DG,CA's located layers and DG's presented layers increased slowly before P7,quickly during P7 to P14 and slowly again after P14,but all came stable after 3M.2. Cell proliferation and apoptosis during development and aging of C57/BL6 mouse hippocampal formation.2.1 Expression of DCXA large number of immunoreactive cells can be seen in hippocampal primordium at E12d. At E18d immunoreactive cells differed at various layers of the hippocampus.During P1～P14d,positive cells were located at the exterior of pyramidal layer as a compact zone. It was the widest at P1d but disappeared at P14d. During P1～P7d,positive cells were differed at every layer of DG.At P14d immunoreactive cells were mainly located at the 1/2 of the granular layers. During P28d～18M positive cells were mainly differed at the subgranular layers. During P1d～P7d Vv of positive cells in CA pyramidal layer decreased. During P1d～P7d Vv of DG granular layer increased,but it decreased during P14d～18M. P1d～P3d V of CA pyramidal layer increased but decreased at P7d. V of DG granular layer increased during P1d～P14d,but decreased during P21d～18M.The Vv of positive cells in CA pyramidal layer decreased in CA3 areas but increased in CA1 at P1d. At P7d it decreased in all areas. The Vv was larger in CA3 than in CA1 during E18d～E20d,but smaller from P1d to P7d. The results of Western Blotting shown that:The expression of DCX rised form E18d to P3d,but decreased during P3d～18M.2.2 Results of Hoechst33258 stainingE12d ~ E16d apoptotic cells increased gradually. At E18d apoptotic cells dispersed, mainly in the polymorphic layer and molecular layer of CA and DG. P1 ~ P7d apoptotic cells gradually decreased. P14d ~ 2M a few of apoptotic cells were mainly distributed in the dentate gyrus and a little in the hippocampal fissure and hippocampal alveus. 3M ~ 18M only in the dentate gyrus granule cell layers, seen sub-scattered.P1d～P14d the apoptosis rate of polymorphic layer and molecular layer in CA and DG decreased especially at P14d. P21d～2M only a little scattered. At 3M there were no distribution. Principal cell layers of CA and DG had the highest apoptosis rate at P1d, and then gradually decreased.After P14d there were no distribution in the pyramidal layer, a little scattered in the granular layer even after 3M.2.3 Results of half thin and ultra-thin slices observationsThe number of proliferation cells decreased gradually from embryonic day to postnatal day in mouse hippocampal formation.There was little or no apoptotic cells at E12d, maximum at P1d but decreased at P7d in mouse hippocampal formation.3. Studies on the apoptosis related gene in the development and aging of C57/BL6 mouse hippocampal formation.3.1 Expression of Bcl-2 and BaxAt E16d a few positive cells scattered in hippocampal formation.At E18d there were more immunoreactive cells in the pyramidal layer and the hilus of DG,and it was intensive in CA3 and sparse in CA1. P1～P7d two kinds of positive cells were all gradually increased. P14～P21d two kinds of positive cells were all gradually decreased in CA. During this period Bcl-2 positive cells gradually decreased and mainly in subgranule layer at P21d in DG.But Bax positive cells continue to increase at P14d,decreased at P21d,and came to be stable after P28d. E18 d～P21d ,AO of Bcl-2 and Bax positive cells in every area first increasesd and then decreased, at P7d was maximum,and stabilized after P28d. Bcl-2/Bax of each area suddenly decreased at P1d,and there is no difference in other time points.Stereology and Western Blotting results was similar to upward.3.2 Expression of Caspase-3There was little expression in hippocampal formation primordium on E12d.On E16d positive cells increased, mainly located in the hippocampal plate and the marginal layer. E18d~P3d there were more positive cells than E16d, distributed in every layer,esbecialy in the CA pyramidal layer and DG granular layer. P5~P14d positive cells gradually decreased .During P21d ~ 18M only a few positive cells can be seen in various districts.The results of Western Blotting shown that:On E18~P3d the expression of Caspase-3 increased, reduced on P5～P21 d and stabilized on P21d.Conclusions1.C57/BL6 mouse hippocampal formation are formed at E12 and become mature at 3M basicly.The volumes of aging mouse hippocampal formation have no obvious changes.2. The proliferation and apoptosis of hippocampal formation's primary cell layer of C57/BL6 mouse gradually increased at embryonic day. At postnatal day it decreased, but remain at a relatively high level and at the same level.A week latter they were all at a low level,but the proliferation was in comparative advantage.After P14d the were both rare or none.3 Bcl-2, Bax and Caspase-3 all played an important role in the cell apoptosis during early development of C57/BL6 mouse hippocampal formation.