| Part ⅠObjective:Quantitatively study the age-related changes of neuronal nuclear volume and diameter in the hippocampal formation and its main functional subregions of rats from postnatal 15 days to 36 months,in order to determine if there is atrophy of neuronal nuclei in the aging rats hippocampal formation and its main functional subregions.Methods:5 animals were randomly sampled from same cohort of normal male Sprague-Dawley rats at 15-day-old,3-month-old,12-month-old,24-month-old and 36-month-old,respectively.After perfusion fixation,the brain was taken out,and the left or right cerebral hemisphere was selected in turn,then completely stripped off the hippocampal formation,serial sections(section thickness 40 μm)were cut after embedding in a resin of glycol methacrylate,and 3-4 sections were systematically sampled and stained with periodic acid,Schiff’s reagent and hematoxylin.The nuclear sizes of hippocampal neurons were estimated with stereological methods:first,the nuclei were sampled in a uniform random way using the optical disector;then,a Feret diameter of the largest nuclear profile at its focal plane(optical section)was measured as the nuclear diameter,and the nuclear volume was measured at the clearest focal plane of the nucleolus with the nucleator.Results:1.The mean volume of neuronal nuclei in hippocampal formation was the largest at 15 days of age,which was 642±27μm3,and decreased from 15 days of age.Compared with the mean volume of 15 days old,3 months,12 months,24 months,and 36 months of age are significantly reduced by 16%,36%,29%and 39%,respectively,(P<0.05).There was significant statistical difference between 12,24,and 36 months of age group(P<0.05),compared with the group of 3-month-old,respectively.but there was no significant statistical difference between the group of 12,24 and 36 months of age(P>0.05).The smallest mean volume of neuronal nuclei was 392±24μm3 at the age of 36 months.The mean diameter of neuronal nuclei in hippocampal formation was the largest at 15 days of age,which was 9.93±0.12μm,and decreased from 15 days of age.Compared with the mean diameter of 15 days old,3 months,12 months,24 months,and 36 months of age are reduced by 2.8%(P>0.05),11.1%(P<0.05),7.0%(P<0.05)and 12.5%(P<0.05),respectively.Compared with the mean diameter of 3 months,12 months,24 months,and 36 months of age are reduced by 8.2%(P<0.05),4.3%(P>0.05)and 9.9%(P<0.05),respectively.2.The mean volume of neuronal nuclei in the CA was the largest at 15 days of age,which was 744±31μm3,it remained unchanged from 15 days to 3 months of age and decreased from 3 months of age.Compared with the mean volume of 15 days and 3 months of age,the neuronal nuclei mean volume of 12,24 and 36 months of age decreased significantly by about 26%,17%and 26%,respectively(P<0.05).There was no significant statistical difference in the volume of neuronal nuclei between 12 months and 36 months of age,and the smallest mean volume was 538±38μm3 at 12 months of age.The mean diameter of neuronal nuclei increased slightly from 15 days old,reached the maximum of 10.81±0.07μm at 3 months of age,and decreased from 3 months of age.Compared with 3 months of age,the average diameter of neuronal nuclei of 12 months,24 months and 36 months of age decreased by 8.1%(P<0.05),3.7%(P>0.05)and 9.1%(P<0.05),respectively.3.The mean volume of neuronal nuclei in the DG was the largest at 15 days of age,which was 417±19μm,and decreased from 15 months of age.Compared with the mean volume of 15 days of age,the neuronal nuclei mean volume of 3,12,24 and 36 months of age decreased significantly by about 20%,41%,35%and 46%,respectively(P<0.05).There was no significant statistical difference in mean volume between 12,24,36 months of age(P<0.05).The smallest mean volume was 227±11μm3 at the age of 36 months.The average diameter of neuronal nuclei was the largest at 15 days of age,which was 8.63±0.14μm.and decreased from 15 days of age.Compared with the mean diameter of 15 days old,the neuronal nuclei mean diameter of 3,12,24 and 36 months of age decreased by 3.4%(P>0.05),11.2%(P<0.05),9.3%(P<0.05)and 13.4%(P<0.05)respectively.Compared with the mean diameter of 3 months old,the average neuronal nuclei diameter of 12-month-old,24-month-old and 36-month-old decreased by 8.0%(P<0.05),6.1%(P<0.05)and 10.3%(P<0.05),respectively.4.The mean volumes of neuronal nuclei in the Sub was the largest at 15 days of age,which was 690±59μm3.There was no significant statistical difference in mean volume between 5 age-groups(P>0.05).The mean diameter of neuronal nuclei was the largest at 15 days old,which was 10.16±0.24μm.There was no significant difference in mean diameter of neuronal nuclei between 3-month-old,12-month-old,24-month-old,36-month-old and 15-day-old(P>0.05).5.The mean diameters of the neuronal nuclei in hippocampal formation and its main functional subregions was basically consistent with the change trend of the mean volume of neuronal nuclei.Part ⅡObjective:To determine whether the changes of nuclear size of hippocampal neurons in rats of different ages are related to different perfusion methods(15 days and 3 months age group with infiltration fixation,12,24and 36 months age group with perfusion fixation).We quantitatively study the changes of nuclear volume and diameter of rat hippocampal formation neurons after perfusion fixation and infiltration fixation.The purpose of this study was to illustrate whether perfusion fixation and infiltration fixation will affect the nuclear size of rat hippocampal neurons.Methods:12 normal male 3-month-old Sprague-Dawley rats with a body weight 201.8±4.4 g were randomly divided into two groups:the perfusion fixation group(n=6)and the infiltration fixation group(n=6).In one group,the brain was taken out after cardiac perfusion and fixation,and in the other group,the brain was directly removed without perfusion and stored in fixation liquid,and then the hippocampal formation was completely stripped off.Each hippocampal formation was first sliced into 6 pieces perpendicular to the C-shaped hippocampal long axis and with approximately equal thickness,and 3 pieces were randomly selected at equal distance.After that,3 pieces of hippocampal tissue were embedded in a tissue block with methacrylic resin,and each tissue block was cut into a section(section thickness 40 μm)and stained with periodic acid,Schiff’s reagent and hematoxylin.The nuclear volume of CA pyramidal layer neurons and DG granular layer neurons was estimated by stereological method:firstly,the nuclei of pyramidal layer or granular layer neurons were randomly selected by optical disector,and then the nuclear Feret diameters of pyramidal or granular layer neurons were measured in the focus plane(optical section)of their largest profile.finally,the volume of nuclear was measured by nucleator at the clearest focal plane of their nucleolus.Results:1.The mean volume of neuronal nuclei of CA pyramidal layer in perfusion fixation group and infiltration fixation group was 735±50μm3 and 767±38μm3,respectively,and there was no significant difference between the two groups(P>0.05).The mean volume of neuronal nuclei of DG granular layer in perfusion fixation group and infiltration fixation group was 374±20μm3 and 422±11μm3,respectively,and there was no significant difference between the two groups(P>0.05).2.The mean diameter of neuronal nuclei of CA pyramidal layer in perfusion fixation group and infiltration fixation group was 10.73±00.3μm and 10.93±0.39μm,respectively,and there was no significant difference between the two groups(P>0.05).The mean diameter of neuronal nuclei of DG granular layer in perfusion fixation group and infiltration fixation group was8.74±0.23μm and 9.1±0.15μm,respectively,and there was no significant difference between the two groups(P>0.05).Conclusions:1.Our study found that the nuclear mean volume of neurons in hippocampal formation,CA and DG significantly decreased with age,but there was no significant change in the Subiculum(P>0.05).The mean volume of neuronal nucleus in the DG exhibited significantly decreased at 3 months old,while that in the CA significantly decreased at 12 months old,then mean volume of neuronal nucleus in the CA and DG increased slightly at 24 months old,eventually down the lowest at 36 months old,indicating that the nuclear mean volume of neurons in some specific regions of hippocampal formation atrophied with the increase of age.Moreover,the age of nuclear atrophy of neurons in different functional subregions is also different,and this regional difference may be due to the difference of neuroplasticity in the effects of different hippocampal subregions on the internal and external environment.In addition,the mean volume of neuronal nucleus in the CA and DG increased slightly at 24 months of age,which may suggest that some functions(such as cellular excitability)of hippocampal neurons increased to a certain extent from 12 months to 24 months of age.There was no significant change in the mean volume of neuronal nucleus in hippocampal formation、CA and DG between 12 and 36 months of age,indicating that:In general,there was no age-related change in the nuclear size of neurons throughout the hippocampus from middle age to old age,suggesting that age-related decline in learning and memory,if present,may be unrelated to or not necessarily associated with overall decline in neuronal function in the hippocampus.This does not rule out the possibility,though,that some of the neurons may change in form or function.2.The mean diameter of the nucleus of hippocampal neurons is positively correlated with the mean volume of the nucleus,suggesting that the diameter of the neuronal nucleus can reflect the nuclear volume.3.These two fixation methods of perfusion and infiltration had no significant effect on the nuclear size of hippocampal neurons in rats.However,it is worth noting that the nuclear size of hippocampal neurons after perfusion fixation was smaller than that after infiltration fixation.although the difference was not statistically significant. |
