Age-related Changes In The Volume Of Hippocampal Formation And Its Major Subdivisions And The Number Of Neurons In Them In Rats

Part ⅠObjective:To explore the age-related changes in the volume of hippocampal formation and its major subdivisions using stereological methods.The purpose of this study is to provide important morphological quantitative data for the volume changes in hippocampal formation and its major subdivisions during the development,maturation and aging of rats.Methods:5-6 animals were randomly sampled from a same cohort of normal male Sprague-Dawley rats at 15 days,3,12,24 and 36 months of age,respectively.After perfusion fixation,the brain was taken out,and the left or right hemispheres were selected in turn to remove the complete hippocampal formation.Each hippocampal formation was weighed,and its density was measured by immersing it in different concentrations of ethanol or sucrose(each corresponds to a known density),and the total volume of hippocampal formation(V)was obtained by weight divided by density.The hippocampal formation was embedded in the methacrylate resin perpendicular to the short axis of it,and then was cut into 40μm-thick continuous sections.1/6 or 1/9 of the sections were sampled in a systematic(equally spaced)manner and stained with periodic acid-schiff reagent(PAS)and hematoxylin.The complete histological micrograph of each hippocampal formation section was taken with 1.25-fold objective of Olympus BX51 light microscope.And then stereological measurement(point counting)of hippocampal formation was performed to obtain the volume fraction of each functional subdivisions of the hippocampal formation(Vv),using Photoshop software.And the volume of each major subdivision was obtained by multipling the Vv by the total volume of the hippocampal formation.Results:1.From 15 days to 3 months of age,the total volume of hippocampal formation increased significantly by 208%,and the volume of Ammon’s horn(CA)and dentate gyrus(DG)in the hippocampal formation increased significantly by 202%and 277%,respectively.The volume of molecular layer,pyramidal layer and polymorph layer of the CA increased by 203%,181%and 213%,respectively;and the volume of molecular layer,particle layer and polymorph layer of the DG increased by 344%,209%and 194%,respectively.2.From 3 to 36 months of age,there was no significant change in the volume of hippocampal formation and its main subdivisions(P>0.05).Part ⅡObjective:To unbiasedly estimate the total numbers and their age-related changes of neurons in the hippocampus and its major subdivisions using the unbiased stereological method—fractionator.The purpose of this study is to provide important morphological quantitative data for the changes in the number of neurons in the hippocampal structure and its major subdivisions during development,maturation and aging of rats.Methods:The experimental animals,materials,embedding,sectioning and staining were the same as the Part I.1/4 or 1/5 sections were selected in a systematic random manner,and vision fields were randomly selected in the delineated hippocampal formation subdivions by using the visiopharm stereology equipment.Each vision field was superimposed with a counting frame(30 ×23 μm2).The number of nucleoli of neurons in the 10 μm thickness of section was counted from 3 μm under the upper surface of the section according to the optical disector counting rule.The total number of neurons in the hippocampal formation and its major subdivisions was estimated according to the fractionator.Results:1.There were(42.38±3.62)×105 neurons in the hippocampal formation of 15 days old rats.There was no significant change from 15 days to 36 months of age(P>0.05).2.The number of neurons in the CA was largest at 15 days of age,which was(18.13±1.18)× 105.The number of neurons in the CA decreased gradually from 15 days to 36 months,but there was no significant difference between 15 days,3 months,12 months and 24 months of age(P>0.05).At 36 months of age,the number of the CA neurons was(12.19±1.28)×105,which was significantly reduced by 33%,28%and 27%compared with 15 days,3 months and 12 months(P<0.05),and 22%less than that of 24 months(P>0.05).3.The number of neurons in the pyramidal layer of the CA was(15.83±1.50)× 105 at 15 days,decreased gradually from 15 days to 36 months,and was(10.79 ±1.15)×105 at 36 months,but there was no significant difference among the age groups(P>0.05).4.The number of neurons in the DG and granular layer of the DG were(16.57±2.71)×105 and(16.0±2.69)×105 at 15 days,respectively,which gradually increased from 15 days to 36 months of age,and were(23.36±2.49)×105 and(22.79±2.34)×105 at 36 months,respectively.But there was no significant difference among the age groups(P>0.05).Conclusions:1.In this study,for the first time,five important age groups(15 days,3 months,12 months,24 months,36 months of age)were sampled from a same cohort of SD rats;and the hippocampal formation was separated and embedded in resin along its short axis;the age-related changes of the volume of hippocampal formation and its major subdivisions and their numbers of neurons were quantitatively studied by stereology.The methods and results would provide important reference for future research.2.From 15 days to 3 months old,the total volume of hippocampal formation increased significantly,and the total number of neurons in the hippocampal formation did not change significantly;the total volume of the CA and DG also increased significantly;the number of neurons in the CA decreased,and the number of neurons in the DG increased,but the differences were not statistically significant.This suggested that the increases of the volumes of hippocampal formation and its major subdivisions were not accompanied by significant changes of the number of neurons in the development stage of rats.3.From 3 months to 36 months of age(late senility),there were no significant age-related changes in the total volume of hippocampal formation and the total number of neurons in the hippocampal formation of rats;there was no significant change in the volume of the CA;the number of neurons in the CA decreased gradually,but only decreased significantly at 36 months of age;there was no significant change in the volume of the DG,and the number of neurons in the DG increased gradually,but the difference was not statistically significant.4.Our results showed that the number of neurons in the CA decreased gradually from 15 days of age,but did not decrease significantly until the late senility,which might be closely related to learning and memory decline associated with aging.The number of neurons in the DG increased gradually from 15 days to the late senility,which might be related to neurogenesis in rodents DG in a lifetime.