Propofol blocks apoptosis and Bcl-2 and Bax expression induced by hypoxia-reoxygenation in primary cultures of rat hippocampal astrocytes

BACKGROUND： Cerebral hippocampal astrocytes are more sensitive.to ischemic injury than neurons. Hypoxic-ischemic brain injury induces profound astrocyte apoptosis, and propofol may protect against astrocyte apoptosis. OBJECTIVE： To verify the protective effects of propofol against astrocyte apoptosis and to investigate anti-apoptotic Bcl-2 and pro-apoptotic Bax expression in primary cultures of rat hippocampal astrocytes exposed to hypoxia-reoxygenation for different periods of time following propofol treatment. DESIGN, TIME, AND SETTING： In vitro neural immunocytochemistry was performed at the Central Laboratory of Yunyang Medical College between September 2007 and March 2008.MATERIALS： A total of 30 Wistar rats, aged 1-3 days, wJth equal numbers of males and females, were included for isolation and culture of .hippocampal astrocytes. METHODS： Hippocampal astrocytes were purified and cultured for 3 weeks and treated with four culture conditions： 50 μL Hank＇s solution （normal control）; 0.2 mL/L Intralipid; 50 μL Hank＇s solution for 10 minutes followed by hypoxic incubation for 4 hours and normoxic incubation for 12, 24, 36, 48, 60 or 72 hours; propofol （250 μmol/L final） for 10 minutes followed by hypoxic incubation for 4 hours and normoxic incubation for 12, 24, 36, 48, 60 and 72 hours. MAIN OUTCOME MEASURES： （1） Morphologic changes in hippocampal astrocytes. （2） Levels of astrocyte apoptosis and Bcl-2 and Bax expression. RESULTS： Hypoxia and reoxygenation increased apoptosis over time, with Bcl-2 expression peaking at 24 hours and decreasing gradually （P 〈 0.01 ）; Bax expression peaked at 72 hours （P 〈 0.01）; the ratio of Bcl-2/Bax was 1.4, 0.8, and 0.6, respectively, at 24, 48 and 72 hours. Non-apoptotic astrocytes showed significant proliferation and swelling. Propofol treatment decreased apoptosis after hypoxia-reoxygenation （P 〈 0.01）, as well as Bct-2 and Bax expression （P 〈 0.05, P 〈 0.01）, with Bcl-2/Bax ratios of 1.6-1.8. Propofol treatmentalso blocked astrocyte proliferation and swelling. No apoptotic cells or Bcl-2/Bax expression was detected in astrocytes cultured in Hank＇s or Intralipid solution. CONCLUSION： Propofol protects astrocytes against injury caused by hypoxia and reoxygenation via a mechanism that involves maintaining high ratios of Bcl-2/Bax.

In vitro differentiation of human adipose-derived adult stromal cells into neuron-like cells in hippocampal astrocyte conditioned medium

BACKGROUND: At present, researches on differentiating from human adipose-derived adult stromal cells (hADASC) to neuron-like cells are focus on inducing by artificial-synthetic compound solution; however, hippocampal astrocyte conditioned medium (HCAM) can induce in vitro differentiation from hADASC to neuron-like cells is still unclear. OBJECTIVE: To observe whether HCAM can induce in vitro differentiation from hADASC to neuron-like cells. DESIGN: Randomized control study. SETTING: Department of Neurology, Taixing People's Hospital; Central Laboratory, North China Coal Medical College. MATERIALS: Donor of adipose tissue was donated by female volunteers suffering from caesarean section in the department of obstetrics & gynecology in our hospital and aged 20-35 years. Adipose tissue was collected from subcutaneous tissue of abdomen during the operation. In addition, 8 male newborn Wistar rats within 24 hours with average body mass of 20 g were provided by Animal Institute of Chinese Academy of Medical Sciences. Rabbit-anti-human Nestin polyclonal antibody, rabbit-anti-human glial fibriliary acidic protein (GFAP) polyclonal antibody, rabbit-anti-human neuro-specific enolase polyclonal antibody and mouse-anti-human microtubal associated protein 2 (MAP-2) polyclonal antibody were provided by Wuhan Boster Company. METHODS: The experiment was carried out in the Central Laboratory of North China Coal Medical College from October 2004 to June 2005. hADASC was cultured with HCAM and its growth and morphological changes were observed under inverted phase contrast microscope. Immunocytochemistry, immunofluorescence and Western blotting were used to evaluate the expressions of Nestin, which was a specific sign of nerve precursor, neuro-specific enolase and MAP-2, which was a specific sign of nerve cell, and GFAP, which was a specific sign of neuroglial cells. MAIN OUTCOME MEASURES: Nestin, which was a specific sign of nerve precursor, neuro-specific enolase and MAP-2, which was a specific sign of nerve cell, and GFAP, which was a specific sign of neuroglial cells. RESULTS: On the 3rd day of culture, partial hADASC started deformation from slender shuttle-shape cells to neuron-like cells. It suggested that cells stretched out apophysis, which were mainly double-pole or multiple-pole cells. Five days later, immunohistochemical detection suggested that expression of Nestin (10.5±0.037) was found out in cells; meanwhile, expressions of GFAP (38.4±0.052) and neuro-specific enolase (NSE) (15.7±0.023) were also found out in cells; however, expression of MAP-2 was not observed. Western blot indicated that, 5 days after effect of HCAM, Nestin was found out in hADASC; meanwhile, expressions of GFAP and neuro-specific enolase were also found out; however, expression of MAP-2 was not observed. CONCLUSION: HCAM can induce the differentiation from hADASC to neuron-like cells in vitro.

The effect of Modified Wen-Dan decoction on hippocampal astrocytes in depression model rats

Background:This study aimed to observe the effect of Modified Wen-Dan decoction on hippocampal astrocytes in depression model rats.Methods:A total of 48 rats were randomly divided into a control group,a model group,a Modified Wen-Dan decoction group(referred toas the traditional Chinese medicine group),and a fluoxetine control group(referred to as the Western medicine group).The control group was provided with a free diet and without any stimulation,while the remaining three groups were solitarily housed incombination with chronic unpredictable mild stress stimuli to replicate the depression rat model.The rats were subjected to the open-field and sucrose preference experiments for behavioral observation,and q-PCR was used to detect the mRNA of specific markers of hippocampal astrocytes,glial fibrillary acidic protein,and S100 calcium-binding proteinβ(S100β)in each group.Immunohistochemistry was used to observe the protein expression of glial fibrillary acidic protein and S100βin thehippocampus of rats of each group.Results:The behavioral results showed that,compared with the control group,the rats in the model group showed a decrease in the score of the open-field experiment and in the rate of sucrose preference(P<0.05).The score of the open-field experiment and the rate of sucrose preference was increased in the Chinese and Western medicine groups(P<0.05),compared to that of the model group.No significant difference was found between the Chinese and Western medicine groups(P>0.05).The q-PCR results showed that the mRNA and protein expression levels of GFAP and S100βwere downregulated in the model group compared to that of the control group(P<0.05).Compared with the model group,the mRNA expression levels of glial fibrillary acidic proteinand S100βwere upregulated in the traditional Chinese medicine group as well as in the Western medicine group(P<0.05).No significant difference between the Chinese and Western medicine groups was found.Conclusion:The mechanism of the antidepressant effect of Modified Wen-Dan decoction may be related to the improvement of hippocampal astrocyte dysfunction in rats.

Novel aspects of extracellular adenosine dynamics revealed by adenosine sensor cells

Adenosine modulates diverse physiological and pathological processes in the brain, including neuronal activities, blood flow, and inflammation. However, the mechanisms underlying the dynamics of extracellular adenosine are not fully understood. We have recently developed a novel biosensor, called an adenosine sensor cell, and we have characterized the neuronal and astrocytic pathways for elevating extracellular adenosine. In this review, the physiological implications and therapeutic potential of the pathways revealed by the adenosine sensor cells are discussed. We propose that the multiple pathways regulating extracellular adenosine allow for the diverse functions of this neuromodulator, and their malfunctions cause various neurological and psychiatric disorders.