Fetal Rat Hippocampal Neurons Research Articles

Ketamine exerts dual effects on the apoptosis of primary cultured hippocampal neurons from fetal rats in vitro.

Ketamine, a noncompetitive N-methyl D-aspartate (NMDA) receptor antagonist, is widely used in pediatric clinical practice. The neuroprotective and neurotoxic effects of ketamine on brain neurons during development remain controversial. The reason may be related to the different concentrations of ketamine used in practice and the small range of concentrations used in previous studies. In this study, cultured hippocampal neurons were treated with ketamine in a wide range of concentrations to comprehensively observe the effects of different concentrations of ketamine on neurons. We demonstrated that low concentrations of ketamine (10 μM, 100 μM and 1000 μM) promoted neuronal survival (p < 0.05) and reduced neuronal apoptosis (p < 0.05) compared with those of the control group. High concentrations of ketamine (2000 μM, 2500 μM and 3000 μM) reduced neuronal survival (p < 0.05) and promoted neuronal apoptosis (p < 0.05). The p38 MAPK inhibitor SB203580 reduced neuronal apoptosis induced by high concentrations of ketamine (2500 μM) (p < 0.05). Our findings indicate that ketamine exerts a dual effect on the apoptosis of primary cultured fetal rat hippocampal neurons in vitro and that the neurotoxic effects of ketamine are related to activation of the p38 MAPK signaling pathway.

The propofol-induced mitochondrial damage in fetal rat hippocampal neurons via the AMPK/P53 signaling pathway.

Propofol is a commonly used general anesthetic that may cause neuronal damage, especially in infants and young children. Mitochondria play an essential role in cellular metabolism and signal transduction. Propofol may cause neurotoxicity by inhibiting mitochondrial function, but the mechanism by this which occurs remains unclear. First, the primary rat hippocampal neurons were cultured for 7 days in vitro. The neurons were incubated with propofol at different times or different concentrations, and then the adenosine triphosphate (ATP), reactive oxygen species (ROS), mitochondrial membrane potential, and apoptosis-related proteins were analyzed. Based on the results of the 1st phase, the neurons were then incubated with propofol (100 µM) or corresponding reagents, including 5-aminoimidazole-4-carboxamide ribonucleotide, tenovin-1, and pifithrin-α. Subsequently, the ATP, ROS, mitochondrial membrane potential, phospho-adenosine 5'-monophosphate-activated protein kinase (p-AMPK), protein 53 (p53), and related apoptosis proteins were analyzed. Higher propofol concentrations or longer incubation times were associated with more pronounced decreases in ATP, B-cell lymphoma 2 (Bcl-2), and mitochondrial membrane potential, and more pronounced increases in ROS, BCL2-associated X (Bax), Cytochrome C (CytC), and cleaved caspase-9. Additionally, after incubation with propofol (100 µM), neuronal Bcl-2, p-AMPK, ATP, and mitochondrial membrane potential were downregulated, and ROS, p53, CytC, Bax, cleaved caspase-3, and cleaved caspase-9 were upregulated. AMPK activators or p53 inhibitors reversed the above-mentioned changes. Propofol (100 µM)-induced mitochondrial damage in fetal rat hippocampal neurons may be mediated by the AMPK/p53 signaling pathway. Propofol (100 µM) was shown to inhibit the activity of AMPK in neurons, upregulate the expression of p53, and then activate the mitochondrial-dependent apoptosis pathway, which may lead to neuronal apoptosis.

Hippocampal Neuronal Cultures to Detect and Study New Pathogenic Antibodies Involved in Autoimmune Encephalitis

Over the last 15 years, a new category of antibody-mediated diseases of the central nervous system (CNS) has been characterized and is now defined as "autoimmune encephalitis" (AE). There are currently 17 known AE syndromes, and all are associated with antibodies against the neuronal cell surface or synaptic proteins. The clinical syndromes are complex and vary according to the type of associated antibody. The best-known of these diseases is anti-N-methyl D-aspartate receptor (NMDAR) encephalitis, which is a prominent neuropsychiatric disorder associated with severe memory and behavioral impairments. The associated antibodies react with the GluN1 subunit of the NMDAR at the N-terminal domain. The approach most frequently used for the discovery and characterization of AE antibodies includes the culture of dissociated, fetal, rodent hippocampal neurons. During the process of antibody characterization, live neurons in culture are exposed to patients' serum or CSF, and the detection of reactivity indicates that the serum or CSF samples of the patient contain antibodies against neuronal surface antigens. Hippocampal cultures can also be used to determine whether the antibodies in patients are potentially pathogenic by examining if they cause structural or functional alterations of the neurons. The level of success of these studies depends on the quality of the cultures and on the protocols used to obtain and detect the reactivity of patient samples. This article provides an optimized protocol for primary cell culture of fetal rat hippocampal neurons combined with immunostaining to determine the presence of antibodies in the serum or CSF of patients. An example of how to examine the potential pathogenic effects of NMDAR antibodies using cultured neurons and calcium imaging is also presented.

Hippocampal Neuronal Cultures to Detect and Study New Pathogenic Antibodies Involved in Autoimmune Encephalitis.

Over the last 15 years, a new category of antibody-mediated diseases of the central nervous system (CNS) has been characterized and is now defined as "autoimmune encephalitis" (AE). There are currently 17 known AE syndromes, and all are associated with antibodies against the neuronal cell surface or synaptic proteins. The clinical syndromes are complex and vary according to the type of associated antibody. The best-known of these diseases is anti-N-methyl D-aspartate receptor (NMDAR) encephalitis, which is a prominent neuropsychiatric disorder associated with severe memory and behavioral impairments. The associated antibodies react with the GluN1 subunit of the NMDAR at the N-terminal domain. The approach most frequently used for the discovery and characterization of AE antibodies includes the culture of dissociated, fetal, rodent hippocampal neurons. During the process of antibody characterization, live neurons in culture are exposed to patients' serum or CSF, and the detection of reactivity indicates that the serum or CSF samples of the patient contain antibodies against neuronal surface antigens. Hippocampal cultures can also be used to determine whether the antibodies in patients are potentially pathogenic by examining if they cause structural or functional alterations of the neurons. The level of success of these studies depends on the quality of the cultures and on the protocols used to obtain and detect the reactivity of patient samples. This article provides an optimized protocol for primary cell culture of fetal rat hippocampal neurons combined with immunostaining to determine the presence of antibodies in the serum or CSF of patients. An example of how to examine the potential pathogenic effects of NMDAR antibodies using cultured neurons and calcium imaging is also presented.

Novel GPER Agonist, CITFA, Increases Neurite Growth in Rat Embryonic (E18) Hippocampal Neurons.

Numerous studies have reported neuroprotective and procognitive effects of estrogens. The estrogen 17β-estradiol (E2) activates both the classical nuclear estrogen receptors ERα and ERβ as well as the G protein-coupled estrogen receptor (GPER). The differential effects of targeting the classical estrogen receptors over GPER are not well-understood. A limited number of selective GPER compounds have been described. In this study, 10 novel compounds were synthesized and exhibited half-maximal effective concentration values greater than the known GPER agonist G-1 in calcium mobilization assays performed in nonadherent HL-60 cells. Of these compounds, 2-cyclohexyl-4-isopropyl-N-((5-(tetrahydro-2H-pyran-2-yl)furan-2-yl)methyl)aniline, referred to as CITFA, significantly increased axonal and dendritic growth in neurons extracted from embryonic day 18 (E18) fetal rat hippocampal neurons. Confirmation of the results was performed by treating E18 hippocampal neurons with known GPER-selective antagonist G-36 and challenging with either E2, G-1, or CITFA. Results from these studies revealed an indistinguishable difference in neurite outgrowth between the treatment and control groups, exhibiting that neurite outgrowth in response to G-1 and CITFA originates from GPER activation and can be abolished with pretreatment of an antagonist. Subsequent docking studies using a homology model of GPER showed unique docking poses between G-1 and CIFTA. While docking poses differed between the ligands, CIFTA exhibited more favorable distance, bond angle, and strain for hydrogen-bonding and hydrophobic interactions.

Effect of acupuncture serum on expression of microtubule associated protein-2 and nerve growth associated protein-43 in Mg2+-free-cultured hippocampal neurons of neonatal rats

To observe the effect of acupuncture serum on the expression of microtubule associated protein-2 (MAP-2) and nerve growth associated protein-43 (GAP-43) in cultured hippocampal neurons of convulsive rats. The acute convulsion model was induced by intraperitoneal injection of pentylenetetrazol in SD rats who were then randomized into model group and acupuncture group. Rats of the acupuncture group received manual acupuncture stimulation of "Baihui" (GV20) and "Dazhui" (GV14) for 30 min, once daily for 7 days. Then, the blood samples taken from the abdominal aorta of rats in the convulsion model and acupuncture groups were processed into serum samples, i.e. non-acupuncture serum and acupuncture se-rum. The primary-cultured hippocampal neurons of fetal rats were cultured for 10 days and then divided into normal extracellular fluid (normal) group, magnesium (Mg2+) free extracellular fluid group, acupuncture serum group and non-acupuncture serum group. At the 10th day, the neurons in the normal group were cultured continuously in extracellular fluid for 3 h, and then cultured in DMEM/F12(1∶1) medium (planting fluid); neurons in the Mg2+ free group were cultured in magnesium-free fluid medium to induce epileptic-like discharge; neurons in the acupuncture serum group were cultured in the mixed medium of planting fluid and 10% acupuncture serum; and neurons in the non-acupuncture serum were cultured in the mixed culture medium of planting fluid and non-acupuncture serum (10%). At last, these neurons in the above-mentioned groups were cultured in the magnesium-free extracellular fluid continuously for 2, 12 and 48 h, respectively, followed by detecting the expression levels of MAP-2 and GAP-43 proteins at the 3 time points by using immunofluorescence and Western blot, separately. The rate of MAP-2 positive cells and protein expression at 2, 12 and 48 h, and the rate of GAP-43 positive cells and protein expression at 12 and 48 h in the hippocampal neurons were significantly down-regulated in the Mg2+ free group in contrast to the normal group (P<0.05，P<0.01). Compared to the Mg2+ free group, the rates of MAP-2 and GAP-43 positive cells and protein expression at 2, 12 and 48 h were considerably up-regulated in the acupuncture serum group (P<0.05，P<0.01), but not in the non-acupuncture serum group (P>0.05). Acupuncture serum can significantly up-regulate the expression of MAP-2 and GAP-43 proteins in hip-pocampal neurons, which may play a positive role in improving synaptic plasticity and neuronal damage in convulsion rats.

Icariin Protects Hippocampal Neurons From Endoplasmic Reticulum Stress and NF-κB Mediated Apoptosis in Fetal Rat Hippocampal Neurons and Asthma Rats.

Icariin is a main component of the Chinese medicinal plant Epimedium brevicornu Maxim, exhibits potent activity against inflammatory diseases. Our previous data demonstrated the valid bioactivity of icariin on mitigating rodent asthma. Endoplasmic reticulum (ER) stress and nuclear factor-κB (NF-κB) pathway were involved in the pathogenesis of asthma. However, it remains poorly defined that whether icariin could inhibit ER stress and NF-κB mediated apoptosis in asthma and further influence the central neural system. Herein, we investigated the effects of icariin on primary cultured fetal rat hippocampal neurons and OVALPS-OVA induced asthma rat model. Asthma rat models were established by ovalbumin (OVA) and lipopolysaccharide (LPS) intraperitoneal injection and OVA inhalational challenge. Airway resistance was analyzed to evaluate lung function after last challenge and pathological changes were detected on lung tissues. Assessment of inflammatory cells counts in bronchoalveolar lavage fluids (BALF) were performed and ELISA was used to determine levels of interleukin (IL)-1β, tumor necrosis factor-α, IL-6, and interferon-γ in serum. Protein expression of BiP and IRE-1α, XBP-1s and phosphorylation-IκBα (p-IκBα), IκBα, and p65 as well as cytochrome c, caspase-3 (cleaved caspase-3), and caspase-9 (cleaved caspase-9) were tested by Western blot. We found that icariin could remarkably improve pulmonary function and reduce inflammatory cells in the lung, levels of inflammatory cytokines, and ER stress related proteins as well as NF-κB were prominently suppressed by icariin. Our results suggested that icariin had an inhibitory effect on airway inflammation and neuroprotective effect on ER stress and NF-κB mediated apoptosis in asthma rats and cultured fetal rat hippocampal neurons, which may provide new mechanistic insights into the asthma prevention and treatment of icariin.

Neuro-Protective Role of Metformin in Patients with Acute Stroke and Type 2 Diabetes Mellitus via AMPK/Mammalian Target of Rapamycin (mTOR) Signaling Pathway and Oxidative Stress.

BackgroundWe investigated the effects of metformin on neurological function and oxidative stress in patients with type 2 diabetes mellitus with acute stroke.Material/MethodsWe randomly assigned 80 acute stroke patients to 2 groups: the metformin combined group and the insulin group. Each group had 40 patients and all were treated with standard stroke treatment. The indexes of nervous functional score and oxidative stress were measured before and 2 weeks after treatment. The primary fetal rat hippocampal neurons were gradually matured after 7 days of culture, and divided into the control group (Con), the oxygen-glucose deprivation model group (Mod), and the metformin group (Met). In the Met group, 10 mmol/L metformin was added, and the Con group and the Mod group received equal volumes of cell culture fluid. Cell viability, cell apoptosis rate, and the expression of Bax, Bcl-2, AMPK, pAMPK and mTOR were detected; MDA content and SOD activity were also detected.ResultsBefore treatment, there was no difference in the metrical indexes between the 2 groups. After treatment, the treatment group was better than the control group in neurological function scores and multiple oxidative stress-related indicators. The experimental results of primary fetal rat hippocampal neuronal cells suggest that this mechanism of improvement is closely related to the AMPK/mTOR signaling pathway.ConclusionsMetformin can improve the neurological function and oxidative stress status of acute stroke patients with type 2 diabetes, and its mechanism may be related to the AMPK/mTOR signaling pathway and oxidative stress.

The tropical carrageenophyte Kappaphycus alvarezii extract promotes axodendritic maturation of hippocampal neurons in primary culture

Neurotrophic factors are essential to support neuronal development and to protect mature neurons from atrophy in neurodegenerative disorders. To investigate effects of the carrageenan-producing seaweed, Kappaphycus alvarezii, on the development and complexity of neuronal cytoarchitecture, ethanol extract of the seaweed was added to primary cultures of fetal rat hippocampal neurons. The neuronal morphology was observed under phase-contrast microscope and analyzed using ImageJ software. With an optimal concentration of 1 μg mL−1, the extract significantly increased axonal length, number of secondary axonal collateral branches, length of primary dendrites, and number of secondary dendritic branches by 58%, eightfold, 68%, and 2.6-fold, compared with control (p < 0.05). In Sholl’s analysis, the extract also significantly increased numbers of axodendritic intersections, branching points, and branching tips. It is assumed that several kinds of lipophilic compounds provide the neurotrophic activities to enhance neurites in a robust form. Thus, the K. alvarezii may be useful as a diet supplement or pharmaceuticals for people who are prone to neurological disorders.

Bupivacaine Indirectly Potentiates Glutamate-induced Intracellular Calcium Signaling in Rat Hippocampal Neurons by Impairing Mitochondrial Function in Cocultured Astrocytes.

Bupivacaine induces central neurotoxicity at lower blood concentrations than cardiovascular toxicity. However, central sensitivity to bupivacaine is poorly understood. The toxicity mechanism might be related to glutamate-induced excitotoxicity in hippocampal cells. The intracellular free Ca concentration ([Ca]i), mitochondrial membrane potential, and reactive oxygen species generation were measured by fluorescence and two-photon laser scanning microscopy in fetal rat hippocampal neurons and astrocytes. In astrocyte/neuron cocultures, 300 μM bupivacaine inhibited glutamate-induced increases in [Ca]i in astrocytes by 40% (P < 0.0001; n = 20) but significantly potentiated glutamate-induced increases in [Ca]i in neurons by 102% (P = 0.0007; n = 10). Ropivacaine produced concentration-dependent effects similar to bupivacaine (0.3 to 300 μM). Tetrodotoxin did not mimic bupivacaine's effects. In pure cell cultures, bupivacaine did not affect glutamate-induced increases in [Ca]i in neurons but did inhibit increased [Ca]i in astrocytes. Moreover, bupivacaine produced a 61% decrease in the mitochondrial membrane potential (n = 20) and a 130% increase in reactive oxygen species generation (n = 15) in astrocytes. Cyclosporin A treatment suppressed bupivacaine's effects on [Ca]i, mitochondrial membrane potential, and reactive oxygen species generation. When astrocyte/neuron cocultures were incubated with 500 μM dihydrokainic acid (a specific glutamate transporter-1 inhibitor), bupivacaine did not potentiate glutamate-induced increases in [Ca]i in neurons but still inhibited glutamate-induced increases in [Ca]i in astrocytes. In primary rat hippocampal astrocyte and neuron cocultures, clinically relevant concentrations of bupivacaine selectively impair astrocytic mitochondrial function, thereby suppressing glutamate uptake, which indirectly potentiates glutamate-induced increases in [Ca]i in neurons.

MicroRNA-132 protects hippocampal neurons against oxygen-glucose deprivation-induced apoptosis.

Hypoxic-ischemic brain injury (HIBI) results in death or long-term neurologic impairment in both adults and children. In this study, we investigated the effects of microRNA-132 (miR-132) dysregulation on oxygen-glucose deprivation (OGD)-induced apoptosis in fetal rat hippocampal neurons, in order to reveal the therapeutic potential of miR-132 on HIBI. MiR-132 dysregulation was induced prior to OGD exposure by transfection of primary fetal rat hippocampal neurons with miR-132 mimic or miR-132 inhibitor. The effects of miR-132 overexpression and suppression on OGD-stimulated hippocampal neurons were evaluated by detection of cell viability, apoptotic cells rate, and the expression of apoptosis-related proteins. Besides, TargetScan database and dual luciferase activity assay were used to seek a target gene of miR-132. As a result, miR-132 was highly expressed in hippocampal neurons following 2 h of OGD exposure. MiR-132 overexpression significantly increased OGD-diminished cell viability and reduced OGD-induced apoptosis at 12, 24, and 48 h post-OGD. MiR-132 overexpression significantly down-regulated the expressions of Bax, cytochrome c, and caspase-9, but up-regulated BCl-2. Caspase-3 activity was also significantly decreased by miR-132 overexpression. Furthermore, FOXO3 was a direct target of miR-132, and it was negatively regulated by miR-132. To conclude, our results provide evidence that miR-132 protects hippocampal neurons against OGD injury by inhibiting apoptosis.

The ethanol extract of the rhodophyte Kappaphycus alvarezii promotes neurite outgrowth in hippocampal neurons

Neurodegenerative disorders, which affect memory, cognition, and social functions, can be treated using neurotrophic agents to support neuronal development and protect mature neurons from atrophy. We screened 34 tropical seaweed species collected from Indonesian coastal areas for their neurite-outgrowth-promoting activity (NOPA) in fetal rat hippocampal neurons in vitro. Based on the number and total length of primary neurites, red seaweeds had greater NOPA than green and brown seaweeds. The red seaweed Kappaphycus alvarezii showed the highest NOPA. Addition of the ethanol extract to the culture (1 μg mL−1) significantly accelerated initial neuronal maturation from stage I to stage II (70 %; P < 0.05) within 24 h and increased the number of neurites that developed multipolar characteristics (48 %; P < 0.05). These results indicate that the aquaculturable carrageenan producer K. alvarezii might be a promising source of neurotrophic compounds to enhance memory and learning.

Effect of dexmedetomidine on expression of NGF in isolated hippocampal neurons of fetal rats incubated with propofol

Objective To evaluate the effect of dexmedetomidine on the expression of nerve growth factor(NGF)in isolated hippocampal neurons of fetal rats incubated with propofol. Methods Hippocampal neurons derived from the fetal rats of pregnant Sprague-Dawley rats at 5-13 days of gestation were primarily cultured for 7 days, and were inoculated in the culture plate at a density of 5×105 cells/ml.The neurons were randomly divided into 3 groups(n=15 each)using a random number table: control group(group C), propofol group(group P), and dexmedetomidine + propofol group(group DP). In group P, propofol with the final concentration of 100 μmol/L was added to the culture medium, and the cells were incubated for 3 h. In group DP, dexmedetomidine with the final concentration of 1 μmol/L was added to the culture medium, the cells were incubated for 30 min, and then propofol with the final concentration of 100 μmol/L was added to the culture medium, and the cells were incubated for 3 h. The viability of hippocampal neurons was assessed by CCK-8 assay.NGF mRNA expression was detected by real-time reverse transcriptase polymerase chain reaction.NGF protein expression was detected by Western blot. Results Compared with group C, the viability of hippocampal neurons was significantly decreased, and the expression of NGF protein and mRNA was down-regulated in group P(P<0.05). Compared with group P, the viability of hippocampal neurons was significantly increased, and the expression of NGF protein and mRNA was up-regulated in group DP(P<0.05). Conclusion Dexmedetomidine improve propofol-induced decrease in the viability of isolated hippocampal neurons of fetal rats through up-regulating the expression of NGF. Key words: Dexmedetomidine; Propofol; Hippocampus; Neurons; Nerve growth factor

Effects of dexmedetomidine on expression of phosphor-CREB in isolated hippocampal neurons of fetal rats

Objective To evaluate the effects of dexmedetomidine on the expression of phosphor-cAMP response element binding protein (p-CREB) in isoloated hippocampal neurons of fetal rats.Methods SpragueDawley rats on 16-18 days of gestation were sacrificed and the fetal rats were obtained.The hippocampi of fetal rats were isolated and hippocampal neurons were seeded in culture medium for 8 days.The cells were then divided into 4 groups (n =12 each) using a random number table:control group (group C),dexmedetomidine 0.001 μmol/L group (group D1),dexmedetomidine 0.010 μmol/L group (group D2),and dexmedetomidine 0.100μmol/L group (group D3).In D1.3 groups,dexmedetomidine with the final concentrations of 0.001 μmol/L,0.010 μmol/L,and 0.100 μmol/L was added to the culture medium,respectively,and then the cells were incubated for 3.5 h.The apoptosis in hippocampal neurons was detected by flow cytometry.The expression of p-CREB in hippocampal neurons was determined by RT-PCR and Western blot.Results Compared with group C,apoptosis rate was significantly decreased and the expression of p-CREB was up-regulated in D1.3 groups.Conclusion Dexmedetomidine inhibits apoptosis in isolated hippocampal neurons of fetal rats by up-regulating the expression of p-CREB. Key words: Dexmedetomidine; Cyclic AMP response element-binding protein; Fetus; Hippocampus; Neurons

Effect of different concentration of propofol on cell viability and apoptosis in hippocampal neurons of fetal rats

Objective To investigate the effect of different concentration of propofol on cell viability in hippocampal neurons of fetal rats in vitro.Methods Primary cultured hippocampal neurons were prepared from the fetuses of Sprague-Dawley rats after 17 d-18 d of gestation,and cultured for 7 d,immunocytochemical method was used to identify the cultured neurons.① The neurons were randomly divided into 12 groups (n=5):control group (group C) received no treatment,in group D 0.1％ dimethyl sulfoxide (DMSO) was added,in group P1-10 propofol was added at the final concentration of 0.01,0.1,0.5,1,5,10,50,100,150 μmol/L and 200 μmol/L respectively.The cells were selected after 24 h incubation for detection of the cell survival rate by cell viability[3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay].② The neurons were randomly divided into 7 groups(n=5):control group (group C) received no treatment,in group D 0.1％ DMSO was added,in group PⅠ,Ⅱ,Ⅲ,v,v propofol was added at the final concentration of 0.1,1,10,100 μmol/L and 200 μmol/L respectively.The cells were selected after 24 h incubation for detection of the neuronal apoptosis by immunofluorescence staining and flow cytometry.Results ① There was no significantly difference in the cell survival rate between group C and D (P＞0.05).Compared with group D,the cell survival rate was no significantly difference in group P1,2,5(P＞0.05),the cell survival rate was significantly increased in group P3(102.2±2.7)％,P4(102.9±2.7)％(P＜0.05) and that was significantly lower in group P6-10 in a concentration-dependent manner by (97.0±3.5)％,(90.1 ± 1.7)％,(79.8±0.9)％,(67.9± 1.1)％,(42.3±2.2)％ respectively (P＜0.05 or P＜0.01).② There was no significantly difference in the neuronal apoptosis rate between group C and D (P＞0.05).Compared with group D,the neuronal apoptosis rate was no significantly difference in group PⅠ (P＞0.05),the neuronal apoptosis rate was significantly decreased in group PⅡ (P＜0.05) and that was significantly higher in group PⅢ,Ⅳ,Ⅴ in a concentration-dependent manner by (8.2±0.8)％,(18.1±1.1)％,(29.1±2.1)％ respectively(P＜0.05 or P＜0.01).Conclusions The effect of propofol on cell viability and apoptosis in hippocampal neurons was bidirectional,propofol at the concentration of 1 μmol/L can inhibit the apoptosis and improve the survival rate of neurons,while decrease the cell viability through promoting the cell apoptosis in a concentration-dependent manner at the concentration of 10,100 μmol/L and 200 μmol/L. Key words: Propofol; Hippocampal neuron; Cell viability; Apoptosis

Effects of propofol on intracellular calcium ion concentration and NF-κB activity in hippocampal neurons of fetal rats in vitro

Objective To evaluate the effects of propofol on the intracellular calcium ion concentration （[Ca2＋ ]i） and nuclear factor kappa B （NF-κB） activity in hippocampal neurons of fetal rats in vitro .Methods Ten pregnant Sprague-Dawley rats at 16-18 days of gestation ,were sacrificed and the fetal rats were taken out from the abdominal cavity .The hippocampal neurons of the fetal rats were isolated and seeded in culture plates .After being cultured for 9 days ,the neurons were divided into 7 groups （ n=12 each ） using a random number table ：control group （C group） ,intralipid group （I group） and propofol 0.1 ,1 ,10 ,100 ,1 000 μmol/L groups （P1-5 groups） .In group I ,10% intralipid was added to the culture media until the final concentration reached 100μmol/L .In P1-5 groups ,propofol was added to the culture media until the final concentration reached 0.1 ,1 ,10 , 100 and 1 000μmol/L ,respectively .The cells were then incubated for 3 h .The [Ca2＋ ]i and cellular morphology of hippocampal neurons were examined by laser scanning confocal microscopy before incubation with propofol and within 10 min after the end of incubation with propofol .The expression of NF-κB protein in the nucleus was＆nbsp;detected at 7 days after the end of incubation with propofol by Western blot analysis to reflect NF-κB activity . Results Propofol increased [Ca2＋ ]i in P2-4 groups ,while decreased [Ca2＋ ]i in group P5 （ P0.05 ） .The structure of hippocampal neurons was normal in C ,I and P1 groups .The branchings of axons and dendrites in hippocampal neurons were significantly decreased in P 2-4 groups , while the structure of hippocampal neurons became fuzzy , the cell membrane was destroyed and the axons and dendrites were not seen in group P5 .Conclusion Propofol can produce neurotoxic effects on hippocampal neurons of fetal rats by changing the [Ca2＋ ]i and promoting NF-κB activation in vitro . Key words: Propofol; Fetus; Hippocampus; Neurons; Calcium; NF-kappa B

Effects of propofol on apoptosis in hippocampal neurons of fetal rats in vitro

Objective To evaluate the effects of propofol on apoptosis in the hippocampal neurons of fetal rats in vitro.Methods The isolated hippocampal neurons were seeded into 96-well plates or 24-well plates at a density of 5 × 104 cells/ml.The cells were randomly divided into 5 groups (n =18 each) using a random number table:control group (group C),in tralipid group (group Ⅰ) and propofol 1,10,100 μmol/L groups (P1-3 groups).In group Ⅰ,10％ intralipid was added to the culture media until the final concentration reached 100 μmol/L.In groups P1-3,propofol was added to the culture media until the final concentration reached 1,10 and 100 μmol/L,respectively,and the cells were then incubated for 3 h.The cell apoptosis was assessed by flow cytometry.The expression of Bcl-2 mRNA and caspase-3 mRNA was detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR).The expression of Bcl-2 and actived-caspase-3 protein was determined by Western blot analysis.The apoptosis rate was calculated.Results Compared with group C,the apoptosis rate was significantly increased,the expression of Bcl-2 mRNA and protein was down-regulated,and the expression of caspase-3 mRNA and actived-caspase-3 protein was up-regulated in P1-3 groups (P ＜ 0.05).There was no significant difference in the parameters mentioned above between group Ⅰ and group C (P ＞ 0.05).Conclusion Propofol induces apoptosis in isolated hippocampal neurons by inhibiting Bcl-2 expression and enhancing caspase-3 activity in fetal rats. Key words: Propofol ; Fetus ; Apoptosis ; Hippocampus

Ethanol modulates spontaneous calcium waves in axonal growth cones in vitro.

In developing neurons the frequency of long duration, spontaneous, transient calcium (Ca2+) elevations localized to the growth cone, is inversely related to the rate of axon elongation and increases several fold when axons pause. Here we report that these spontaneous Ca2+ transients with slow kinetics, called Ca2+ waves, are modulated by conditions of ethanol exposure that alter axonal growth dynamics. Using time-series fluorescence calcium imaging we found that acute treatment of fetal rat hippocampal neurons with 43 or 87 mM ethanol at an early stage of development in culture decreased the percent of axon growth cones showing at least one Ca2+ wave during 10 min of recording, from 18% in controls to 5% in cultures exposed to ethanol. Chronic exposure to 43 mM ethanol also reduced the incidence of Ca2+ waves to 8%, but exposure to 87 mM ethanol increased their incidence to 31%. Neither chronic nor acute ethanol affected the peak amplitude, time to peak or total duration of Ca2+ waves. In some experiments, we determined the temporal correlation between Ca2+ waves and growth and non-growth phases of axonal growth dynamics. As expected, waves were most prevalent in stationary or retracting growth cones in all treatment groups, except in cultures exposed chronically to 87 mM ethanol. Thus, the relationship between growth cone Ca2+ waves and axon growth dynamics is disrupted by ethanol.

Effect of melatonin on ketamine-induced apoptosis in hippocampal neurons of fetal rats

Objective To investigate the effect of melatonin on ketamine-induced apoptosis in hippocampal neurons of fetal rats.Methods Sixteen to eighteen day pregnant Sprague Dawley rats were anesthetized.The fetal rats were obtained under sterile condition and decapitated.The hippocampal neurons were isolated and primary cultured for 5 days.The primary cultured neurons were randomly divided into 5 groups (n =6 each):control group (group C),ketamine group (group K),and 1.0,2.5 and 5.0 mmol/L melatonin groups (groups M1-3 respectively).Ketamine with the final concentration of 1 000 μmol/L was added to the culture medium and the neurons were incubated for 3 h in group K.In groups M1-3,1.0,2.5 and 5.0 mmol/L melatonin were added to the culture medium,respectively,at 60 min before the addition of ketamine,and the neurons were incubated for 3 h.While the equal volume of normal saline was added instead in group C.The neuronal viability during the developmental phase was assessed by MTT assay.The mitochondrial membrane potential (Ψm) was measured by flow cytometry.The expression of cAMP response element binding protein phosphorylation (p-CREB (Ser133)),Bcl-2,Bax,and cytochrome C was detected by Western blot.Results Compared with group C,the neuronal viability and Ψm were significantly decreased,and the expression of p-CREB and Bcl-2 was down-regulated,while the expression of Bax and cytochrome C was up-regulated in group K (P ＜ 0.05).Compared with group K,Ψm was significantly increased in groups M2 and M3,and the neuronal viability was significantly increased,the expression of Bcl-2 was up-regulated,while the expression of Bax and cytochrome C was down-regulated in groups M1-3 (P ＜ 0.05).Conclusion Melatonin can protect the hippocampal neurons of fetal rats from apoptosis triggered by ketamine via regulating the expression of Bcl-2 and Bax,stabilizing Ψm,inhibiting the release of cytochrome C from mitoehondria,and preventing apoptosome formation. Key words: Melatonin; Ketamine; Membrane potential, mitochondrial; Hippocampus; Neurons; Apoptosis

Effects of propofol on mitochondrial membrane permeability during hypoxia/reoxygenation in rat hippocampal neurons

Objective To investigate the effects of propofol on mitochondrial membrane permeability during hypoxia/reoxygenation （H/R） in rat hippocampal neurons. Methods Primary cultured hippocampal neurons of fetal rats obtained from Wistar （17-18 days of gestation） were randomly divided into 3 groups： Ⅰ control group （group C）, Ⅱ H/R group and Ⅲ propofol ＋ H/R group. Neurons were cultured in the culture medium with combined oxygen glucose deprivation for 2 h followed by reoxygenation in group Ⅱ and Ⅲ. In group Ⅲ propofol was added to the culture medium with the final concentration of 20 μ mol/L before combined oxygen glucose deprivation.Neuronal viability was detected by MTT assay and mitochondrial membrane potential （MMP） with flow cytometry at 0, 4, 8, 12 and 24 h after reoxygenation （T1-5） and the permeability of cell membrane and mitochondrial membrane was monitored at T5 using laser confocal scanning microscope. Results The neuronal viability and MMP were significantly decreased （P 〈 0.05）, while the permeability of cell membrane and mitochondrial membrane was increased at T5 in group Ⅱ as compared to group Ⅰ . The neuronal viability at T1-4 and MMP at T1-5 were significantly increased （P 〈 0.05）, while the permeability of cell membrane and mitochondrial membrane was decreased at T5 in group Ⅲ compared to group Ⅱ . Conclusion Propofol can protect rat hippocampal neurons against H/R injury through increasing MMP, improving the cell and mitochondrial membrane permeability, and increasing the neuronal viability. Key words: Propofol; Cell hypoxia; Oxygen; Mitochondrial membranes; Permeability; Hippocampus; Neurons