The Compare Study Of The Effects Induced By Hydrogen Peroxide, Amyloid β(1-42) And Tau Protein On Two Primary Astrocyte Cultures

Aging is an inevitable event when we enjoy increased longevity. Many reports pointed out that the decreased numbers of neurons and the increased numbers and volumes of glia may be the main basic changes of brain aging. Aging is associated with a dramatic increase for the risk of many aging related neurodegenerative disorders, such as Alzheimer's disease (AD). Currently, it was confirmed that amyloid β and tau protein, the major components of senile plaques and neurofibrillary tangles, respectively, had been considered as central mediators of the pathogenesis of Alzheimer's disease.Glia greatly outnumber other cells in our central nervous system, especially astrocytes comprised 25% percent of all the brain cells. Astrocytes can widely participate in the activities in our brain, such as the maintenance and regulations of the extracellular environment; provision of nutrients; energy substrates and neurotransmitter precursors; free radical scavenging; guidance of neuronal migration during development; and immune/ inflammatory functions. To our knowledge, astrocytes, especially young or immature astrocytes can strongly support neurons. During aging, astrocytes have the decreased capabilities to do so. In particular, aged astrocytes could not provide the necessary cell surface molecules, extracellular matrix molecules and neurotrophic factors.Commonly, the loss of neurons was regarded as the main cause of the memory deficiency in aging related diseases, so the study of the neurons was always the focus in aging and aging related neurodenegerative disorders. Glia fibrillary acid protein (GFAP) is the major intermediate filament of astrocytes in adult nervous system, vimentin as another intermediate filament has the low level of expression. The hallmark of reactive gliosis in CNS ischemia, trauma or in neurodegeneration is characteristic hypertrophy of cellular processes of astrocytes and upregulation of GFAP and vimentin, accompanying alterations in expression of many proteins.The hypertrophy of astrocytes plays a protective role against the development and progression of brain injury, however sometimes the hypertrophy is lethal to astrocytes. Theeffects of the astrocytic hypertrophy during normal aging were often neglected because the astrocytic activation was secondary to neuron loss. Finch (2003) recently had shown that astrocytes were already activated without the presence of obvious, diagnosable pathology during aging. Our current data also indicated that significant age related increases in the numbers of astrocytes and the expressions of GFAP in the hippocampus of aged SAM mice. As well, the expression levels of GFAP in aged SAMP8 mice were significantly greater than those in matched aged SAMR1 mice.Currently, it was established that there were wide intercommunications between astrocytes and neurons. Activated neurons can release many neurotransmitters to stimulate astrocytes to take up Ca²⁺, which plays an important role in activation of astrocytes. Activated astrocytes also can express many surface molecules; release a lot of neurotrophic factors and cytokinases. Such active substances can reinforce the feed loop of astrocytes onto neurons to modulate the release of the neurotransmitters or directly to inhibit or activate the postsynaptic neuron activities. The modulation of neural functions was inevitably influenced by the astrocytic hypertrophy during aging.Normally, the β-amyloid precursor protein and tau protein were mainly expressed in neurons, and could also be present at low level in astrocytes. However, Miyazono et al (1993) had confirmed that there were many tau immunopositive astrocytes in the brain of AD and other neurodegenerative diseases. As mentioned above, the metabolic turnover of GFAP was increased in aged glia. Accumulations of tau, although not neurofibrillary tangles (NFT), have also been found in astrocytes and oligodendrocytes of aged baboons. The expressed β-amyloid precursor protein (APP) and tau protein in aged glia might affect astrocytic functions. Up to date, there were limited reports about these effects induced by APP and tau protein in primary cultured astrocytes.Senescence-accelerated mouse (SAM) strains were originated from the ancestral AKR/J strains as established by Takeda in 1981, and were seen as a murine model for accelerated aging. At present, there are at least 13 lines of SAM: nine senescence-prone strains and four senescence-resistant strains. The senescence-accelerated-prone mice (SAMP) exhibit accelerated aging with a shortened life span, increased amyloidosis, mitochondrial dysfunction, as well as learning and memory deficits. Senescence-accelerated-resistant mice (SAMR) exhibit normal aging features. Therefore, the SAM mice are good models to study astrocytes during aging.Until now, the studies about β amyloid in aged astrocytes were very limited, and also few research works about tau protein in in vitro astrocytes were reported. In our present study, two strains of primary cultured astrocytes were developed separately from the cortex of SAM P8 and R1 neonatal mice (1-3 days) to model the astrocytes during aging. In the following experiments, the cell samples prepared were treated separately by different concentrations of hydrogen peroxide, Amyliod β1-42 and tau protein, and the effects in two lines of astrocytes were investigated and compared.Part â…  The establishment of two astrocyte stains and the cell proliferation assayAims: to establish the two astrocyte strains from the cortex of SAM P8 and R1 neonatal mice, and to investigate their proliferative capabilities. Methods: (1)the primary astrocytes were prepared from the cortex of SAM P8 and R1 neonatal mice (1-3 days) by dissection and tissue culture methods. Then the acquired cell suspensions were plated into sterile flasks, and cultured under 37°C 5% CO2 environment. During the culture period, the cells were observed and photographed to compare the proliferation of the two astrocytes. At 7-8 days after in vitro subculture, the cells plated into flasks were purified by shaking at 200 rpm 37℃ for 8 hours to dislodge the contaminated cells, such as microglia and other cells, then the astrocytes were cultured for another week before subsequent experiments; (2)the purity of two astrocytes were assayed by immunocytochemical assay using the antibody of astrocytic marker protein GFAP;(3) Cell proliferation was also tested by 3-(4,5-dimethylthiazol-2-yl)-2,2-diphenyltetrazolium bromide (MTT) reduction assay. Results: (1) Under light microscopy, the cells plated at 3 days after in vitro subculture began to extend their side processes, but no concentric reparatitions; after 5 days after in vitro subculture, the proliferating centers were observed and surrounded by flat and polygonal astrocytes; at 7 days after in vitro subculture, the astroglial populations began to increase strongly with concentric repartitions; at about 14-16 days in vitro (DIV), the astroglial culture was definitely confluent; after 4 passages of subculture, some astrocytes lost their abilities to proliferate and adopted an "epithelioid" morphology. (2)the cells plated onto 11 mm round coverslips pretreated by poly-dl-ornithine were stained by GFAP immunocytochemistry and photographed. About 95 percent of total astrocytes were GFAP positive although some cells were stained dim, and astrocytes exhibit stellate, unipolar or polygonal morphology. (3)At 72 hours after subculture, the cells strongly proliferated and entered into exponential phrase. After 7 days after subculture, the cells become confluent and started to stop dividing. Compared the proliferation of astrocytes originated from SAM P8 at 72h after subculture, the SAM R1 derived astrocytes had a higher proliferative ability. Conclusions: Through primary cultures obtained from the cortex of SAM P8 and R1 neonatal mice (1-3 day), we had established highly enriched populations of astrocytes that were about 95% purity as judged by immunophenotypical expression of GFAP, they all had strongly proliferative function.Part â…¡ The effects induced by hydrogen peroxide on two strain astrocytesAims: to investigate and compare the effects induced by different concentrations of hydrogen peroxide (H₂O₂) (0,100,200,400μM) in two SAM (P8 and R1) derived astrocytes.Methods: two group astrocytes were treated by different concentrations of H₂O₂ (0, 100, 200, 400μM) for one or four hours, scanning electron microscopy(SEM) was used to observe the astrocytic morphologies; MTT assays were performed to test the reduction ability; cell death was assay by propidium iodide (PI) under fluorescence microscope; cell apoptosis was assayed by in situ terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling(TUNEL) assay; Cell necrosis was assayed by lactate dehydrogenase(LDH) immunocytochemical assay; the expressions of GFAP, superoxide dismutase (SOD), Caspase-3 and B-cell lymphoma 2-associated protein (Bax) were detected by Western blot. Results: after treatments by different concentrations of H₂O₂ (0, 100, 200, 400 μM),our SEM results illustrated that the cultures of astrocytes from both R1 and P8 strains were usually star shaped with numerous short cilia on the cell surfaces. Long projections also appeared from all around the culture cells, making contacts with other culture cells. These cells usually had club endings. Exposure of R1 strain culture astrocytes from one to four hours of H₂O₂ at a dosage of 100μM showed little damage effect. The astrocytes maintained an abundant amount of cilia and side projections and with increasing dosage (4 hours of 200μM), roughly 30% of the cilia were lost and side projections became short. At even higher dosage (4 hours of 400μM), most cilia were lost although some side projections were still evident. A similar situation occurred in the P8 strain cultured astrocytes. With increasing dosage, the cilia became less and side projections decreased and shortened. The only difference was that the loss of cilia was more vigorous in the P8 cells than in the R1 cells, with cilia decreasing in number rapidly at a dosage of 200μM for 4 hours. When the cells were fractured, in both the control untreated R1 and P8 cells, thin fibers (microtubules) with small globules were observed internally. In both R1 and P8 astrocytes, after 4 hours treatment by low dosage (100μM), the microtubules and globules were still present. Treatment of a high dosage (400μM) of H₂O₂, within a shorter period of an hour resulted in an increase of globular material, while a high total dosage (200μM in 4 hours) eliminated globular material and caused coagulation and thickening of microtubules. (2) For the MTT assays, the percentage of MTT reduction decreased significantly (p<0.05) with increasing H₂O₂ concentrations as compared to that of the controls (cultures not exposed to H₂O₂) derived from both P8 and R1 strains. Further, the MTT reduction of the P8 control was 81.4±7.4% when compared to the R1 control as 100%, and the difference was statistically significant (p<0.05). (3) Overall cell death in astrocytes derived from both P8 and R1 mice was assessed by the PI assay. Results showed cell death percentage increased with increasing H₂O₂ concentrations. With no addition of H₂O₂, the cell death in R1 was 11±2.53% and P8 was 10.6±2.22%. As compared to the cell death percentage of the above control (zero H₂O₂ concentration), the increases at 200μM and 400μM of H₂O₂ treatment were statistically significant (p<0.05). At 200μM of H₂O₂, the difference between P8 samples (18.1±1.78%) and R1 samples (27.5±2.61%) was statistically significant (p<0.05). (4) The extent of apoptosis in the astrocyte culture was visualized by TUNEL in situ hydridization. The percentage of astrocytes stained positive for TUNEL increased withincreasing H₂O₂ concentrations in cultures derived from both P8 and R1 strains, and the increases were statistically significant (p<0.05) as compared to the controls (cultures not exposed to H₂O₂). (5) The percentage of cell stained positive with LDH increased with increasing H₂O₂ concentrations in cultures derived from both P8 and R1 strains. The increases were statistically significant (p<0.05) from controls (cultures not exposed to H₂O₂) when the P8 and R1 cultures were treated with 400 μM of H₂O₂. The differences between P8 samples and R1 samples were statistically significant (p<0.05) only when treated with 400 μM of H₂O₂. (6) When treated with increased concentrations of H₂O₂, the decreases in GFAP levels were statistically significant (p<0.05) as compared to the controls (cultures not exposed to H₂O₂). In addition, there were large and statistically significant (p<0.05) differences in GFAP levels between P8 and R1 samples treated with each of the H₂O₂ concentrations as well as between controls; SOD levels shown by Western blot increased with H₂O₂ concentrations in cultures derived from both P8 and R1 strains. Again, only when treated with a H₂O₂ concentration of 400 μM, were the increases in SOD levels statistically significant (p<0.05) as compared to the controls (cultures not exposed to H₂O₂). Likewise, the difference between P8 samples and R1 samples was statistically significant (p<0.05) when treated with 400 μM of H₂O₂; in both P8 and R1 samples, Caspase-3 levels increased significantly (p<0.05) when treated with 400 μM of H₂O₂ as compared with the controls (cultures not exposed to H₂O₂). In addition, the differences between P8 samples and R1 samples was statistically significant (p<0.05) at this concentration of H₂O₂; Bax expression level were increased in all groups treated by H₂O₂, especially in R1 samples. There were no statistical significant differences in P8 samples as compare with the controls (cultures not exposed to H₂O₂). The difference between P8 samples (1.122±0.224) and the corresponding R1 samples (1.525±0.182) was statistically significant (p<0.05) when treated with 400 μM of H₂O₂. Conclusions: A mild but statistically significant difference was observed in the numbers of cell death between R1 and P8 cells after H₂O₂ treatment. Cellular changes were equivalent in both strains after injury, including loss of cilia and side projections. High total dose of H₂O₂ treatment (e.g. 400μM for only one hour) caused increased cellular synthesis, while high total dose of H₂O₂ treatment (e.g. 200μM for four hours) downregulated in intracellular synthesis and caused coagulation of microtubules. Our results showed that the oxidative stress had similar effects in both strains of astrocytes: decreases in MTT recution and GFAP levels and increases in cell death by PI staining, TUNEL, LDH staining and the expression levels of SOD, Caspase-3 and bax. At a H₂O₂ concentration of 400 μM, the differences of the above parameters between P8 cultures and R1 cultures were statistically significant (p<0.05). This strongly suggested that astrocytes derived from P8 and R1 strains reacted to oxidative stress with similar mechanisms and consequences. However, the mechanisms were not able to compensate for the oxidative stress in the P8 strain at a H₂O₂ concentration of 400 μM remains elusive. Different age astrocytes may play an alternative role in detoxification of toxicants, and may exert an important function in CNS aging and aging related neurodenegerative disorders.Part â…¢ The effects Induced by Aβ(1-42) and Tau protein on two strain astrocytesAims: to investigate and compare the different effects induce by Aβ(1-42) and tau protein on two strain astrocytes(SAM P8 and R1). Methods: two strain astrocytes (SAM P8 and R1) prepared as above were separately treated by Aβ (1-42)(1μM or 5μM), tau protein (100nM) and mixed solutions [Aβ(1-42) 1μM or 5μM + tau protein 100nM] or DMEM/F-12 medium without fetal bovine serum (FBS) (control group) for 24 hours, then the following experiments were performed. Using immunocytochemical assay, the expression level of protein kinase c(cPKC), hexokinase, LDH, Amyloid β precursor protein (APP), N-methyl-D-aspartate (NMDA) receptor and GFAP were investigated; using Western blot method the expression levels of cPKC, GFAP, LDH, Caspase-3, NMDA receptor, hexokinase, APP and S100β protein were separately detected. The coexpression of GFAP and tubulin in astrocytes treated by Aβ (1-42) and tau protein were analysed by confocal laser scanning microscopy (CLSM). Results: (1) Compared with the corresponding R1 astrocytes, the In vitro P8 astrocytes had low expression levels of cPKC. By the treatment of single tau protein or combined treatment of Aβand tau protein for 24h, the expressions of cPKC in P8 astrocytes could be significantly increased; there were significant differences between the treated groups and the control groups. Compared with the expression of cPKC in R1 astrocytes, tau protein (100nM) could significantly enhance the expressions of cPKC in P8 astrocytes. Aβ solely enhanced the the expressions of cPKC in R1 astrocytes, but there were no obvious effects by combined treatments of Apand tau protein. (2) compared with the corresponding R1 astrocytes (including the control group), In vitro P8 astrocytes had lower expression levels of hexokinase. Aβ or tau could solely significantly enhance the expression levels in both R1 and P8 astrocytes. When treated at the dosage of Aβ5μM, the expression of HXK in P8 astrocytes could be significantly decreased compared with that in R1 astrocytes. The combined treatments of Aβand tau protein could significantly enhance the expressions of HXK in P8 astrocytes, and decreased the expressions in R1 astrocytes, especially by the treatment of Aβ(5μM)and tau protein (100nM) .(3) Compared with the corresponding R1 astocytes, in vitro P8 astrocytes had lower expression level of LDH (p<0.05). Compared with the expression of each control group, there were significantly increased expressions of LDH in two astrocytes (R1 and P8) with 24 hour's sole or combined treatment by Aβ and tau protein(p<0.05).(4) Both in vitro astrocytes could express APP. Compared with the corresponding R1 astrocytes, in vitro P8 astrocytes had lower expression levels of APP. Especially the combined treatment of Aβ5μM and tau (100nM) could significantly decrease the expressions of APP in both astrocytes(p<0.05).(5) The expressions of NMDA receptors were obviously detected in both SAM P8 and R1 samples. Compared with the correspondingR1 astrocytes, P8 cells had the lower expression of NMDA receptors. By the sole or combined treatments of Aβ (1-42) and tau protein, the expressions of NMDA receptors in P8 astrocytes could be enhanced, particularly by tau or the combined treatment of tau and Aβ. In R1 astrocytes sole Aβ could enhance the expression of NMDA receptors, but tau or the combined treatment of tau and Aβ could lower the expressions. (6) Compared with the corresponding R1 astrocytes, P8 astrocytes in vitro had lower expression of Caspase-3. The sole or combined treatments of Aβ (1-42) and tau could enhance the expressions of Caspase-3, especially by treatment of tau or the combined treatments of tau and Aβ (1-42). Compared with the expressions in the corresponding R1 astrocytes, there were significantly decreased expression levels of Caspase-3 by treatment of sole tau or by combined treatments of tau and Aβ (1-42). (7) Compared with the expressions of S100β in the corresponding R1 astrocytes, in vitro P8 astrocytes could express lower levels of S100β without or with the treatments of Aβ(1-42) and/or tau protein. Compared with the expression level in the control group, the expressions of S100β in R1 astrocytes could be significantly enhanced by sole treatment of Aβ(1-42) or tau protein. In P8 astrocytes the expression levels of S100β could be decreased by sole treatment of Aβ(1-42) or tau protein, but can be significantly increased by combined treatments of Aβ(1-42) and tau protein(p<0.05).(8) The expression levels of GFAP could be significantly increased in both two astrocyte samples with addition of Aβ or tau or mixed solutions. Compared with the expressions in the corresponding controls, the expressions of GFAP could be significantly enhanced by combined treatments of Aβ and tau in both two astrocytes. Compared with the expressions in the corresponding R1 astrocyes, P8 astrocytes had the lower levels of GFAP after treatment of tau protein or combined treatments of tau and Aβ. Conclusions: In the present studies, we first investigated the effects of sole Aβ or tau protein or mixed solutions in two primary astrocyte cultures originated from senescence accelerated mice (SAM P8 and R1). There were similar but mild different consequences in the expressions of cPKC, HXK, LDH, APP, NMDA receptors, Caspase-3, S100β and GFAP between P8 samples and the corresponding R1 sampels. These data provided clues to study the functional changes of astrocytes during normal aging and aging related disorders.