Preliminary Study On Mice Hippocampal Neurogenesis And Microenviromental Changes During Ageing

Objective: Neural Stem Cells (NSCs) is a cell population in the brainof adult mammals having capacity of self-renew and multi-directionaldifferentiation. After asymmetric division, progenitor differentiate fromNSCs migrate to functional areas gradually and undergoing plastic changescontinuously, establish synaptic connections with other neurons and finallygain matured neuron function. This process is called adult neurogenesis.Neural stem cells play an important role in maintaining of brain functionand structure by participating in neurogenesis under modulation ofmicroenvironment. There are two majior neurogenesis regions in themammalian nervous system： subventricular zone (SVZ)and dentategyrus(DG) of hippocampus. It has been widely accepted that the cellpopulation of dentate gyrus is closely related to advanced features of thebrain, such as cognition, memory, etc. It is reported that usually adulthippocampal NSCs keep in a quiescent state, but in the whole life the cellswill leave the G0phase several times to asymmetric division and will differentiate into neurons, oligodendrocytes or astrocytes.After that,theycome back to the quiescent state. Number of hippocampal neurons reducedin the aging period, which is closely related to the decline in memory andcognitive function. Benedetta Artegiani made four hypothesis about thereason of reduction of hippocampal neurogenesis during aging:1. A largenumber of neural stem cells apoptosis.2. The ratio of neural stem cells in aquiescent state raised;3.The proportion of neural stem cells thatdifferentiated into glial cells increased;4. A large number of newbornneurons apoptosis rapidly.Although the neurogenesis in adult hippocampus is decreased paralledto age, there is few report about the very detail of disorder of hippocampalhappened in aging, and the change in microenviroment is still unknown. Inour study proliferation of SGZ cell, number of neural stem cells, number ofNBs decreased. From these results we can get some idea about theneurogenesis in adult hippocampus. At the same time oxidative damage andantioxidant capacity, levels of neurotrophic factors, the expression ofmatrix metalloproteinase-9and SA-β-Gal were checked to see the changein hippocampus microinviroment. Expression of age-related gene p19Arf,p21Cip/Waf1mRNA in hippocampus is also checked. The purpose of thestudy is to understand changes of hippocampal neurogenesis andneurogenesis of microinviroment during aging. Methods1.27Male C57BL/6mice at the age of2-3,6-8, and18-20months weredesigned for three different age groups, named group A, group B and groupC.2. Mice were deeply anesthetized and perfused.The brains were removedand imbedded with paraffin. Serial coronal sections were obtained on avibratome to immunohistochemical stain by SP method and then thenumber of nestin+cell, DCX+cell and PCNA+cell in SVZ were counted.3. Mice were deeply anesthetized and perfused. The brains were removedand imbedded with OCT. Serial coronal sections were obtained on avibratome to carry on MMP-9immunofluorescence staining and SA-β-galstaining. Mice were decapitated and the brains were separated. Thehippocampus were separated, weighed, homogenized and centrifuged.Theclear supernatant were separated to detect content of GSH and activity ofGSH-PX by colorimetry, to determination of malondialdehyde level basedon its reaction with thiobarbituric acid (TBA).4. Mice were decapitated and the brains were separated.The hippocampuswere separated, weighed, homogenized and centrifuged. The supernatantwere kept to detection of BDNF, GDNF levels by ELISA.5. Mice were decapitated and the brains were separated. The hippocampuswere separated. The total RNA were extracted from hippocampus to detectmRNA of p19Arf、p21Cip1/Waf1by RT-PCR. Results1. The indexs of hippocampal neurogenesis：1) The number of NSCs （nestin-positive cells）in SGZ were younggroup (61.33±23.02), middle-aged group (35±11.53), old group(19.33±8.33);2) The number of active-proliferating cell (PCNA-positive cells) inSGZ were young group (56.67±9.29), middle-aged group (19±6.22), oldgroup (4.33±2.08); the number of NBs (DCX-positive cells) were younggroup(82.33±8.50), middle-aged group (30±3),old group(12.33±1.52).2.The indexs of hippocampal neurogenesis microenvironment：1）The indexs of antioxidant capacity and oxidative damage: thecontents of GSH in the hippocampus were young group (9.77±3.125)mg/gprot, middle-aged group (4.94±1.59) mg/gprot, old group (3.94±1.11)mg/gprot；the activity of GSH-PX were young group (124.09±23.34)active unit， middle-aged group (106.48±14.24) active unit, old group（60.621±14.71）active unit; the content of MDA in the hippocampus wereyoung group (8.3±0.8) nmol/mg, middle-aged group (9.7±1.57) nmol/mg,old group (12.8±1.9) nmol/mg.2）The degree of hippocampal tissue aging：expression of SA-β-gal inhippocampus of senescent mouse significant increased. The positiveexpression of SA-β-gal were only found in CA while no expression in DG. 3) The levels of neurotrophic factors of hippocampal: The levels ofBDNF were young group (107.42±18.55) pg/ml, middle-aged group(89.04±15.7) pg/ml, old group (58.45±15.29) pg/ml; the levels of GDNFwere young group (384.56±59.12) pg/ml, middle-aged group(297.59±14.69) pg/ml, old group (203.33±34.83) pg/ml.4）The degree of hippocampal tissue matrix degradation：expressionof MMP-9of in hippocampus of senescent mouse significant increased.The expression of MMP-9were only found in CA while no expression inDG.3. The expression of mRNA of p19Arf, p21Cip1/Waf1of senescent miceincreased compared with young mice.Conclusions1. In SGZ of senescent mice, the number of NSCs, the number ofactive-proliferating cells, the number of NBs significant decreasedcompared with young mouse. The result suggetted mice hippocampalneurogenesis decreased during aging.2. The antioxidant capacity and neurotrophic factor levels inhippocampus of senescent mice were significantly decreased.Oxidativedamage and matrix degradation in hippocampus of senescent miceincreased. It suggested that these changes in hippocampusmicroenvironment may be related to the neurogenesis reduction of senescent mice.3. Expression of age-related genetic p19Arf, p21Cip1/Waf1mRNAincreased, suggests that signal pathway p19Arf-Mdm2-p53-p21Cip1/Waf1maybe associated with the decrease of hippocampal neurogenesis in aging.