Transplantation of primary cultured embryonic mesencephalic neural precursor cells for treating Parkinsonian rats

BACKGROUND： Choosing proper donor cells is one of keys in experimental and clinical studies on cell replacement therapy （CRT） for treating Parkinson disease （PD）. Embryonic mesencephalic precursor cells （MPCs） can stably differentiate into dopaminergic neuron after in vitro proliferated culture. As compared with embryonic stem cell and neural stem cell strains, cell composition of embryonic MPCs after primary culture is also the most close to that of embryonic mesencephalic ventral cell suspension without proliferated culture. Successful experience accumulated in the latter suggests that primary cultured embryonic MPCs might be the most potential donor cells in clinical application with CRT for treating PD so far. OBJECTIVE： To investigate the feasibility of primary cultured embryonic precursor cells cultured primarily as donor cells in CRT for treating PD in rats. DESIGN ： A randomized and controlled trial taking SD rats as experimental animals.SETTING： Department of Neurosurgery, Huashan Hospital Affiliated to Fudan University.MATERIALS： This experiment was carried out at the Institute of Neuroscience, Shanghai Institute for Biological Science, Chinese Academy of Sciences from July 2003 to June 2004. Totally 26 female SD rats, with body mass of 200 to 220 g, were provided by Shanghai Experimental Animal Center of Chinese Academy of Sciences. METHODS ： Stereotaxic injection of 6-hydroxydopamine into the medial forebrain bundle were perfored to develop PD model rat. Among 26 SD rats, 20 rats achieved a more than 5 turns/min in apomorphine induced rotation test, reaching the standard of PD model rats. Immunohistochemical detection was performed on 1 out of 20 model rats after execution, and the other 19 rats were randomly divided into control group （n=5）, sham transplantation group （n=5）and cell grafted group （n=9）. Primary cultured E12 MPC cell suspension （1.2×10^11 L^-1）were used as donor cells. 4μL primary cultured E12 MPC cell suspension prepared freshly was injected into the lesioned corpus striatum of rats in cell grafted group, and 4μL D-Hank＇s solution was injected in sham transplantation group in the same way. There was no injection in control group. Apomorphine-induced rotation rate of PD rats were recorded respectively in cell grafted group and sham transplantation group pre-operation （initial value） and at postoperative 2, 4, 6 and 16 weeks. Apomorphine-induced rotation rate of PD rats was recorded in control group at postoperative 2 months （initial value） and following 2,4,6 and 16 weeks. To determine TH antigen with immunohistological ABC method （DAB developing） at 6 months post-transplantation to investigate the differentiation and survival of donor cells in the host body.MAIN OUTCOME MEASURES： Apomorphine-induced rotation behavior before and after transplantation and the survival and differentiation of implanted cells in the host body at 6 months post-transplantation. RESULTS： Among 19 model rats, one rat died after transplantation respectively in the cell grafted group and sham transplantation group; finally 17 model rats entered the stage of result analysis. Relative apomorphine-induced rotation rate was significantly decreased in the cell grafted group as compared with that before transplantation , with significant difference （P 〈 0.01 .P 〈 0.05）;the mean value of relative apomorphine-induced rotation rate was significantly decreased at postoperative 16 weeks in cell grafted group as compared with that of corresponding relative rotation rate in control group , also with significant difference （P 〈 0.05）.Immunohistological results showed that donor cells could differentiate into large and multi-polar dopaminergic neurons in the host body. CONCLUSION ： Primary cultured embryonic MPCs can be used as the donor cells in CRT for treating PD.

Bis(7)-Tacrine, a Promising Anti-Alzheimer's Agent,Attenuates Glutamate-Induced Cell Injury in Primary Cultured Cerebrocortical Neurons of Rats

The effects of bis(7) tacrine, a novel dimeric acetylcholinesterase (AChE) inhibitor, on glutamate induced cell injury were investigated in primary cerebral cortical neurons of rats. Exposure of cultured neurons (12 days after plating) to 0.5 mmol/L glutamate for 30 min resulted in significant cell damage. Pretreatment with bis(7) tacrine (0.03 1.0 μmol/L) reduced the glutamate induced neurotoxicity in a concentration dependent manner and the maximal response was seen at 1 μmol/L with approximately 30% protection. A receptor binding assay showed that bis(7) tacrine can completely displace MK 801 binding to rat cortical membrane with an IC 50 of 0.57 μmol/L. These findings suggest that bis(7) tacrine can directly interact with N methyl D aspartate receptor channel complex, which may contribute to the inhibitor's protective effects against glutamate induced excitotoxicity. Thus, it is possible that anti glutamate/anti AChE synergism is responsible for potentially better Alzheimer's therapy of bis(7) tacrine relative to tacrine.

Manganese enhances the expression of the manganese superoxide dismutase in cultured primary chick embryonic myocardial cells

In the present study, the effect of manganese（Mn） on antioxidant status and the expression of the manganese superoxide dismutase（MnSOD） gene in cultured primary myocardial cells collected from the chick embryos was investigated. The hypothesis that Mn supplementation would enhance the expression of MnSOD in cultured primary myocardial cells of chick embryos was tested. Eggs collected from Mn-depleted Arbor Acres laying breeder hens were incubated for 10 days and then myocardial cells were isolated and cultivated for 8 days. The embryonic myocardial cells on day 6 were treated with Mn in the cell culture medium at different time points when the proportion of cells showing spontaneous contraction was over 95% after the 3-day primary culture. A completely randomized design involving a 3 Mn levels（0, 0.5 and 1.0 mmol L^（-1））×3 incubation time points（12, 24 and 48 h） factorial arrangement of treatments（n=6） was used in the current experiment. The results showed that MnSOD activity and m RNA expression level were induced by Mn and increased with incubation time, which supported the hypothesis that Mn would enhance the expression of the MnSOD gene, and thus might protect myocardial cells from oxidative stress during the chick embryonic development.

Effect of the gene silencing of phosphorus transporters on phosphorus absorption across primary cultured duodenal epithelial cell monolayers of chick embryos

The aim of the study was to investigate whether phosphorus(P) transporters, type IIb sodium-dependent phosphate cotransporter(NaP-IIb) and inorganic phosphate transporter 2(PiT2), were directly involved in P absorption across primary cultured duodenal epithelial cell monolayers of chick embryos. The siRNAs against NaP-IIb or PiT2 were designed, synthesized and transfected into primary cultured duodenal epithelial cells of chick embryos. Then, the inhibitory efficiency of siRNAs against NaP-IIb or PiT2 was analyzed, and the most efficacious siRNAs were selected to be used for subsequent P absorption experiments. Briefly, primary cultured duodenal epithelial cells of chick embryos were transfected with either NaP-IIb or PiT2 siRNAs and grown in confluent monolayers on transwell plates. The untransfected or transfected cell monolayers were then incubated in an uptake medium containing 0 or 0.25 mmol L^(–1) of P as KH_(2) PO_(4) to measure the P absorption across duodenal epithelial cell monolayers. The results showed that among the siRNAs designed, si-1372 and si-890 were demonstrated to be the most effective in inhibiting the NaPIIb and PiT2 expressions, respectively. Supplemental P increased(P=0.065) the protein abundance of PiT2 and enhanced(P<0.0001) P absorption in primary cultured duodenal epithelial cell of chick embryos. Furthermore, NaPIIb silencing decreased(P=0.07) P absorption across duodenal epithelial cell monolayers, while PiT2 silencing had no effect(P=0.345). It is concluded that the NaP-IIb, but not PiT2, might be directly involved in the P absorption of chick duodenal epithelial cells.

Protective Effects of Quercetin on Cadmium-induced Cytotoxicity in Primary Cultures of Rat Proximal Tubular Cells

Objective To investigate the protective effects of quercetin on cadmium-induced cytotoxicity in primary cultures of rat proximal tubular （rPT） cells. Methods Primary cultures of rPT cells undergoing exponential growth were incubated with 1.0 ug/mL quercetin and/or cadmium （2.5, 5.0 umol/L）, in a serum-free medium at 37℃ at different time intervals. Commercial kits were used and flow cytometric analyses were performed on rPT cell cultures to assay apoptosis and oxidative stress. Results Exposure of rPT cells to cadmium acetate （2.5, 5.0 umol/L） induced a decrease in cell viability, caused an increase in apoptotic rate and apoptotic morphological changes. Simultaneously, elevation of intracellular reactive oxygen species, malondialdehyde and calcium levels, depletion of mitochondrial membrane potential and intracellular glutathione, and inhibition of Na＋, K＋ -ATPase, Ca2＋ -ATPase, glutathione peroxidase （GSH-Px）, catalase （CAT）, and superoxide dismutase （SOD） activities were revealed during the cadmium exposure of rPT cells. However, simultaneous supplementation with 1 ug/mL quercetin protected rPT cells against cadmium-induced cytotoxicity through inhibiting apoptosis, attenuating lipid peroxidation, renewing mitochondrial function and elevating the intracellular antioxidants （non-enzymatic and enzymic） levels. Conclusion The present study has suggested that quercetin, as a widely distributed dietary antioxidant, contributes potentially to prevent cadmium-induced cytotoxicity in rPT cells.

Cytotoxicity of Modified Nonequilibrium Plasma with Chlorhexidine Digluconate on Primary Cultured Human Gingival Fibroblasts

The aim of this study was to investigate the cytotoxicity of modified nonequilibrium plasma with chlorhexidine digluconate（CHX） on human gingival fibroblasts（HGFs）, and to evaluate the biosecurity of modified nonequilibrium plasma with 2% CHX as a new method of root canal treatment. Tissue samples taken from human gingiva were primarily cultured and passaged. Cells from passages 3–7 were used. HGFs were treated by modified nonequilibrium plasma with 2% CHX for 0 min（control group）, 30 s, 1 min, 1.5 min, 3 min, 5 min, and 10 min, respectively, and then they were incubated for 0, 24, and 48 h. After that, cell counting kit-8（CCK-8） assay was applied to analyze the cytotoxicity of modified nonequilibrium plasma with 2% CHX on HGFs. There was no significant difference between the 0 h group treated with the modified nonequilibrium plasma for 1 min and the control group（P〉0.05）. However, there were significant differences between all the other treated groups and the control group（P〈0.05）. When treated for 1.5 min or shorter, the cell viability was obviously increased; while treated for 3 min or longer, it was obviously reduced. Moreover, when successively cultured for 0, 24, and 48 h, cell viability was decreased at first and then increased in the 3-min-treated and 5-min-treated groups. The modified nonequilibrium plasma with 2% CHX was of no influence on cell viability in 1.5 min treatment, and it could be safely used on root canal treatment.

Comparison of biological behavior of lacrimal gland adenoid cystic carcinoma with high-grade transformation cells

AIM:To evaluate the differences between human lacrimal gland adenoid cystic carcinoma with high-grade transformation(LACC-HGT)primar y cells cultured by high-grade transformation tissue and non-high-grade transformation(non-HGT)primary cells cultured by non-highgrade transformation tissue in proliferation,metastasis,drug susceptibility,and genes.METHODS:LACC-HGT primary cells were established by tissue block culture,and the 4^(th)to 10^(th)generation primary cells were selected as research objects.The cells were preliminarily identified by immunofluorescent staining.The differences between non-HGT and LACC-HGT primary cells in terms of proliferation,metastasis,and drug susceptibility were compared by cell counting kit-8(CCK-8)assay,wound healing,and drug sensitivity experiments.Differentially expressed genes were screened using mRNA array.Gene expression was analyzed using real-time quantitative polymerase chain reaction(RT-qPCR).RESULTS:LACC-HGT primary cells were successfully cultured by tissue block culture.Immunofluorescence staining results showed that cytokeratin(CK)and CK7 expression levels were positive in LACC-HGT primary cells.CCK-8 results showed that the proliferation ability of LACCHGT cells was significantly higher than that of non-HGT cells.Wound healing experiment showed that the migration ability of LACC-HGT cells was significantly higher than that of non-HGT cells.LACC-HGT cells were also less sensitive to cisplatin and paclitaxel than non-HGT cells.Compared with non-HGT cells,9566 differentially expressed genes were found in LACC-HGT primary cells,of which 5162 were upregulated and 4404 were down-regulated.The expression of N-acetylneuraminate pyruvate lyase(NPL),MARVEL domain containing 3(MARVELD3),syntabulin(SYBU),and allograft inflammatory factor 1(AIF1)was higher in LACCHGT cells than in non-HGT cells,whereas that of periostin(POSTN)was lower.CONCLUSION:LACC-HGT primary cells have faster proliferation,stronger migration ability,and poorer sensitivity to chemotherapy drugs than non-HGT primary cells.The expression of mRNAs in non-HGT and LACC-HGT primary cells are significantly different.These features are speculated to be the reasons why high-grade transformation tissues exhibit higher malignant degree and poorer prognosis than their counterparts.

Adherence of uropathogenic Escherichia coli to human primary epithelial cells of renal pelvis

Human primary epithelial cells of renal pelvis was established to investigate the adherence of uropathogenic Escherichia coli （UPEC） to this cell line, in which the primary cell culture was performed by using cultivation of the normal epithelium of renal pelvis in keratinocyte serum free medium （K-SFM） with epidermal growth factor （EGF） and bovine pituitary extract （BPE）. Both UPEC132 obtained from urine specimen of patients with pyelonephritis and the pilus-free representative strain E. coli K-12p678- 54 were used to study the adherence of these strains on human primary epithelial cells of renal pelvis. The UPEC adherence was performed with observation on the morphological changes of the adhered cells, while the adhesion rates and indices were calculated in different times of experiment. In addition, the virulence genes hly and cnfl of UPEC132 were detected by multiplex PCR assay. In this study, the human primary epithelial cells of renal pelvis was found to exhibit the character of the transitional epithelial cells. Compared with the control group, the adhesion rates and indices began to increase from 15 min of the experiment time and reached its peak in 120 min. The adhesion rate and index of UPEC132 to human primary epithelial cells of renal pelvis were 74.4% and 34.0 respectively. Many microscopic changes in the primary cells adhered with UPEC132 could be detected, such as rounding or irregularity in shape, unevenness in staining and the cytoplasmic and nuclear changes. It suggests that human primary epithelial cells of renal pelvis can be used for the experiment on UPEC adhesion, thus providing a basis for the further study on the pathogenesis of UPEC.

Effects of Aconitine on [Ca^(2+)] Oscillation in Cultured Myocytes of Neonatal Rats

In order to investigate the effects of aconitine on [Ca2＋] oscillation patterns in cultured myocytes of neonatal rats, fluorescent Ca2＋ indicator Fluo-4 NW and laser scanning confocal micro- scope （LSCM） were used to detect the real-time changes of [Ca2＋] oscillation patterns in the cultured myocytes before and after aconitine （1.0 μmol/L） incubation or antiarrhythmic peptide （AAP） and aconitine co-incubation. The results showed under control conditions, [Ca2＋] oscillations were irregu- lar but relatively stable, occasionally accompanied by small calcium sparks. After incubation of the cultures with aconitine, high frequency [Ca2＋] oscillations emerged in both nuclear and cytoplasmic regions, whereas typical calcium sparks disappeared and the average [Ca2＋] in the cytoplasm of the cardiomyocyte did not change significantly. In AAP-treated cultures, intracellular [Ca2＋] oscillation also changed, with periodic frequency, increased amplitudes and prolonged duration of calcium sparks. These patterns were not altered significantly by subsequent aconitine incubation. The basal value of [Ca2＋] in nuclear region was higher than that in the cytoplasmic region. In the presence or absence of drugs, the [Ca2＋] oscillated synchronously in both the nuclear and cytoplasmic regions of the same cardiomyocyte. It was concluded that although oscillating strenuously at high frequency, the average [Ca2＋] in the cytoplasm of cardiomyocyte did not change significantly after aconitine incuba- tion, compared to the controls. The observations indicate that aconitine induces the changes in [Ca2＋] oscillation frequency other than the Ca2＋ overload.

APOPTOTIC THRESHOLD OF ADRIAMYCIN AND CISPLATIN INHEPATOCELLULAR CARCINOMA

Objective: To investigate the apoptotic threshold of adriamycin (ADM) and cisplatin (CDDP) on hepatocellular carcinoma (HCC). Methods: Sensitivities of ADM and CDDP on HCC were studied by primary cell culture. Results: The apoptotic threshold of ADM and CDDP were 1.0 μg/ml and 1.5μg/ml respectively (its clinical dosage was 20 mg and 30 mg respectively). Conclusion: Understanding apoptotic threshold of anticancer drugs may reduce clinical dosages of anticancer drugs and reduce the incidence of multidrug resistance (MDR).

Stromal and Tumor Glioma-derived Cells with Similar Characteristics Have Differences in α-Smooth Muscle Actin Expression and Localization

Gliomas are solid brain tumors composed of tumor cells and recruited heterogenic stromal components.The study of the interactions between the perivascular niche and its surrounding cells is of great value in unraveling mechanisms of drug resistance in malignant gliomas.In this study,we isolated the stromal diploid cell population from oligodendroglioma and a mixed population of tumor aneuploid and stromal diploid cells from astrocytoma specimens.The stromal cells expressed neural stem/progenitor and mesenchymal markers showing the same discordant phenotype that is typical for glioma cells.Moreover,some of the stromal cells expressed CD133.For the first time,we demonstrated that this type of stromal cells had the typical myofibroblastic phenotype as theα-SMA+cells formedα-SMA fibers and exhibited the specific function to deposit extracellular matrix(ECM)proteins at least in vitro.Immunofluorescent analysis showed diffuse or focalα-SMA staining in the cytoplasm of the astrocytoma-derived,A172,T98G,and U251MG glioma cells.We could suggest thatα-SMA may be one of the main molecules,bearing protective functions.Possible mechanisms and consequences ofα-SMA disruptions in gliomas are discussed.

Reversing effect of exogenous WWOX gene expression on malignant phenotype of primary cultured lung carcinoma cells

Background Whether WW domain containing oxidoreductase （WWOX） gene is a tumor-suppressor is still controversial. Some researchers found that the transcription of the WWOX gene was lacking not only in tumor tissues but also in non-tumorous tissues and sometimes in normal tissues. Hence it is important to explore the role of the expression of the exogenous WWOXgene in the proliferation and apoptosis of primary cultured lung carcinoma cells. Methods Lipofection technique was used to determine primary cultured lung carcinoma cells containing the highly expressed exogenous WWOX gene and primary cultured cells with vectors as controls. An animal model of lung cancer was made by subcutaneous implantation of tumor cells into nude mice. RT-PCR, Western blotting, flow cytometry, and TUNEL were used to detect the transcription, expression of the exogenous gene and the effect of the expression of targeted genes on the proliferation and apoptosis of the primary cultured lung carcinoma cells. Results The growth, clone formation rate （CFR） （（5.33±1.53）%） of the primary lung cancer cells transfected with the WWOX gene, tumor size and weight were significantly lower than those of the non-transfected lung cancer cells （CFR： （14.33±1.53）%） and the primary tung cancer cells transfected with blank plasmids （CFR： （11.00±1.73）%, P 〈0.05）. The apoptosis level of primary lung cancer cells transfected with the WWOX gene （（40.72±5.20）%） was significantly higher than that of the non-transfected lung cancer cells （（2.76±0.02）%） and the primary lung cancer cells transfected with blank plasmids （（2.72±0.15）%, P 〈0.05）. Conclusion The expression of the exogenous WWOXgene can significantly inhibit the proliferation of lung cancer cells and induce their apoptosis, suggesting that the WWOX gene possesses tumor-suppressing effect.

Expression and Significance of LRIG1 Gene in Human Astrocytomas

Objective： LRIG1 gene is a newly found human gene that displays homologies to the Drosophila Kek-1 gene. Previous researches have shown that the LRIG1 gene almost expressed in all human tissues. However, its functions, particularly its functions in human tumors, are still unknown. The goals of the present study are to magnify the expression spectrum of the LRIG1 gene and determine their roles in the oncogenesis. Methods： A triphasic oligonucleotide probe was designed and used to detect the expression level of the LRIG1 gene in 16 astrocytomas and the corresponding tissues around the tumors by in situ hybridization. 11 primary astrocytoma cells were cultured. Among these, the expression level of the LRIG1 gene was checked by in situ hybridization and the expression of the Proliferating Cell Nuclear Antigen （PCNA） protein was detected by immunohistochemistry. Results： The expression of LRIG1 protein was detected in different degree in all the tumors and the surrounding tissues. Compared to the surrounding tissues, the expression of the tumors was lower. The decrease extends from the surrounding tissues to the tumors were correlation to the tumors＇ grades. The primary cultured cells also expressed LRIG1 to various extent and the expression of LRIG1 in the cultures was negatively correlated with the intensity of the PCNA staining. Conclusion： The LRIG1 protein may inhibit the growth of tumors of glial cells and the down-regulation of the LRIG1 gene may be involved in the development and progression of the tumor.

Curcumin pretreatment and post-treatment both improve the antioxidative ability of neurons with oxygen-glucose deprivation

Recent studies have shown that induced expression of endogenous antioxidative enzymes thr- ough activation of the antioxidant response element/nuclear factor erythroid 2-related factor 2 （Nrf2） pathway may be a neuroprotective strategy. In this study, rat cerebral cortical neurons cultured in vitro were pretreated with 10 ktM curcumin or post-treated with 5 pM curcumin, respectively before or after being subjected to oxygen-glucose deprivation and reoxygenation for 24 hours. Both pretreatment and post-treatment resulted in a significant decrease of cell injury as indicated by propidium iodide/Hoechst 33258 staining, a prominent increase of Nrf2 protein expression as indicated by western blot analysis, and a remarkable increase of protein expression and enzyme activity in whole cell lysates of thioredoxin before ischemia, after ischemia, and after reoxygenation. In addition, post-treatment with curcumin inhibited early DNA/RNA oxidation as indicated by immunocytochemistry and increased nuclear Nrf2 protein by inducing nuclear accumulation of Nrf2. These findings suggest that curcumin activates the expression of thi- oredoxin, an antioxidant protein in the Nrf2 pathway, and protects neurons from death caused by oxygen-glucose deprivation in an in vitro model of ischemia/reperfusion. We speculate that pharmacologic stimulation of antioxidant gene expression may be a promising approach to neu- roprotection after cerebral ischemia.

Three-dimensional perfused human in vitro model of nonalcoholic fatty liver disease

AIM To develop a human in vitro model of non-alcoholic fatty liver disease(NAFLD), utilising primary hepatocytes cultured in a three-dimensional(3D) perfused platform. METHODS Fat and lean culture media were developed to directly investigate the effects of fat loading on primary hepatocytes cultured in a 3D perfused culture system. Oil Red O staining was used to measure fat loading in the hepatocytes and the consumption of free fatty acids(FFA) from culture medium was monitored. Hepatic functions, gene expression profiles and adipokine release were compared for cells cultured in fat and lean conditions. To determine if fat loading in the system could be modulated hepatocytes were treated with known anti-steatotic compounds. RESULTS Hepatocytes cultured in fat medium were found to accumulate three times more fat than lean cells and fat uptake was continuous over a 14-d culture. Fat loading of hepatocytes did not cause any hepatotoxicity and significantly increased albumin production. Numerous adipokines were expressed by fatty cells and genes associated with NAFLD and liver disease were upregulated including: Insulin-like growth factorbinding protein 1, fatty acid-binding protein 3 and CYP7A1. The metabolic activity of hepatocytes cultured in fatty conditions was found to be impaired and the activities of CYP3A4 and CYP2C9 were significantlyreduced, similar to observations made in NAFLD patients. The utility of the model for drug screening was demonstrated by measuring the effects of known antisteatotic compounds. Hepatocytes, cultured under fatty conditions and treated with metformin, had a reduced cellular fat content compared to untreated controls and consumed less FFA from cell culture medium.CONCLUSION The 3D in vitro NAFLD model recapitulates many features of clinical NAFLD and is an ideal tool for analysing the efficacy of anti-steatotic compounds.

Effects of Single and Repeated Exposure to a 50-Hz 2-mT Electromagnetic Field on Primary Cultured Hippocampal Neurons

The prevalence of domestic and industrial electrical appliances has raised concerns about the health risk of extremely low-frequency magnetic fields（ELF-MFs）. At present, the effects of ELF-MFs on the central nervous system are still highly controversial, and few studies have investigated its effects on cultured neurons. Here, we evaluated the biological effects of different patterns of ELF-MF exposure on primary cultured hippocampal neurons in terms of viability, apoptosis, genomic instability,and oxidative stress. The results showed that repeated exposure to 50-Hz 2-mT ELF-MF for 8 h per day after different times in culture decreased the viability and increased the production of intracellular reactive oxidative species in hippocampal neurons. The mechanism was potentially related to the up-regulation of Nox2 expression.Moreover, none of the repeated exposure patterns had significant effects on DNA damage, apoptosis, or autophagy, which suggested that ELF-MF exposure has no severe biological consequences in cultured hippocampal neurons.

Different effects of isoflurane and sevoflurane on cytotoxicity in primary cortical neurons of rats

Background Isoflurane, a commonly used inhaled anesthetic, induces apoptosis in primary rat cortical neurons of rat in a concentration- and time-dependent manner by an unknown mechanism. We hypothesized that isoflurane induced apoptosis by causing abnormal calcium release from the endoplasmic reticulum （ER） via activation of inositol 1,4,5-trisphosphate （IP3） receptors. Sevoflurane has a reduced ability to disrupt intracellular calcium homeostasis and is a less potent cytotoxic agent. This study examined and compared the cytotoxic effects of isoflurane and sevoflurane on rat primary cortical neurons and their relationship with disruption of intracellular calcium homeostasis and production of reactive oxygen species （ROS）.Methods Primary rat cortical neurons were treated with the equivalent of 1 minimal alveolar concentration （MAC） of isoflurane and sevoflurane for 12 hours. MTT reduction and LDH release assays were performed to evaluate cell viability. Changes of calcium concentration in the cytosolic space, [Ca^2＋]c, and production of ROS were determined after exposing primary rat cortical neurons to isoflurane and sevoflurane. We also determined the effects of IP3 receptor antagonist xestospongin C on isoflurane-induced cytotoxicity and calcium release from the ER in primary rat cortical neurons.Results -Isoflurane at 1 MAC for 12 hours induced cytotoxicity in primary rat corticai neurons, which was also associated with a high and fast elevation of peak [Ca^2＋]c. Xestospongin C significantly ameliorated isoflurane cytotoxicity in primary cortical neurons, as well as inhibited the calcium release from the ER in primary cortical neurons. Isoflurane did not induce significant changes of ROS production in primary rat cortical neurons. Sevoflurane, at equivalent exposure to isoflurane, did not induce similar cytotoxicity or elevation of peak [Ca^2＋]c in primary rat cortical neurons.Conclusion These results suggested that isoflurane induced elevation in [Ca^2＋]c, partially via elevated activity of IP3 receptors, which rendered cells vulnerable to isoflurane neurotoxicity. ROS production was not involved in isoflurane-induced neurotoxicity. Sevoflurane, at an equivalent exposure to isoflurane, did not induce similar elevations of [Ca^2＋]c or neurotoxicity in primary cortical neurons of rat.

Establishment of a novel method for primary culture of normal human cervical keratinocytes

Background Cervical keratinocytes are recovered at a low numbers and frequently associated with contaminating human fibroblasts which rapidly overgrow the epithelial cells in culture with medium supplemented with 10% fetal bovine serum （FBS）. However, it is difficult to initiate keratinocyte cultures with serum-free keratinocyte growth medium alone because cell attachment can be poor. Therefore, the culture of these cells is extremely difficult. In this study, we described a modified culture medium and coated culture plastics for growing normal human cervical epithelial cells in vitro. Methods Normal cervical epithelial tissue pieces were obtained and digested with type I collagenase to dissociate the cells and a single cell suspension produced. The cells were cultured on plastic tissue culture substrate alone or substrate coated with collagen type I from rat tail, with modified keratinocyte serum-free medium （K-SFM） supplemented with 5% FBS. After attachment, the medium were replaced with K-SFM without FBS. The expression of basal keratins of the ectocervical epithelium, K5, K14 and K19 were assayed by immunofluorescence with monoclonal antibodies to identify the cell purity. Results Our results indicate that cells attached to the culture plastic more quickly in K-SFM supplemented with 5% FBS than in K-SFM alone, as well as to tissue culture plastic coated with collagen type I than plastic alone. The modified medium composed of K-SFM and 5% FBS combined with a specific tissue culture plastic coated with collagen type I from rat tail was the best method for culture of normal cervical epithelial cells. K5, K14 and K19 were assayed and keratinocyte purity was nearly 100%. Conclusion A novel, simple and effective method can be used to rapidly obtain highly purified keratinocytes from normal human cervical epithelium.

Effect of melatonin on the spatial and temporal changes of [Ca^(2+) ]i in single living cells of cortical neurons by laser scanning confocal microscopy

Objective To examine the effects of melatonin on the dynamic changes in the concentration of intracellular free Ca 2+ ([Ca 2+ ]i) in single intact cultured cortical neurons isolated from fetal rats, in order to explore the possible antiaging mechanisms of melatonin (MT) Methods Using the highly fluorescent Ca 2+ sensitive indicator Fluo 3/AM, cortical neurons cultured in a 35?mm Tissue Culture Dish were in incubated for 45?min at room temperature with 5?μmol/L Fluo 3/AM, resulting in proper intracellular dye concentration to provide adequate signal strength for detection and excellent Laser Scanning Confocal Microscopy (LSCM) imaging of [Ca 2+ ]i while not disturbing normal intracellular physiology The changes in fluorescent intensity were monitored by LSCM Results Bay K8644 (10 6 ?mol/L), KCl (20 ?mmol/L), sodium L glutamate (Glu, 50?μmol/L) caused a rapid increase of [Ca 2+ ]i in cortical neurons, and this increase could be significantly attenuated by 10 6 and 10 7 mol/L MT Conclusions MT could antagonize the extracellular Ca 2+ influx, reduce Ca 2+ overload, and have a protective effect on neurons This may be one of the important antiaging mechanisms of MT

Influence of moxa smoke on mitochondrial transmembrane potential and Bax/Bcl-2 in alveolar type Ⅱ epithelial A549 cells

Objective： To investigate the influence of moxibustion products on mitochondrial transmembrane potential （MTP） and mRNA expression of Bax/Bcl-2 in alveolar type Ⅱ epithelial A549 cells, and to further explore influence of moxibustion products on the oxidative damage of A549 cells. Methods： Smoke and particles generated by moxibustion were collected using the filter box for gas sampling. The moxa smoke extract （MSE） was diluted sequentially to the final concentrations of 0.05 mg/mL, 0.2 mg/mL, 0.2 mg/mL, 0.3 mg/mL and 0.4 mg/mL using the cell culture medium, and A549 cells were then intervened by the above MSE solution. Cell MTP was detected by JC-1 staining. Fluorescence quantitative polymerase chain reaction （PCR） was used to detect Bax/Bcl-2 mRNA expression of A549 cells. Results： Compared with cells in the normal control group, MTP was significantly decreased in cells of 0.3 mg/mL and 0.4 mg/mL MSE intervention groups （P〈0.01）; while MTP showed no significant changes in cells of 0.05 mg/mL, 0.1 mg/mL and 0.2 mg/mL MSE intervention groups （P〉0.05）; compared with cells in 0.05 mg/mL MSE intervention group, MTP was decreased significantly in cells of 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL and 0.4 mg/mL MSE intervention groups （P〈0.05）; compared with cells in 0.1 mg/mL MSE intervention group, MTP was decreased significantly in cells of 0.4 mg/mL MSE intervention group （P〈0.01）. Bax mRNA expression of cells in each concentration of MSE intervention group all showed no significant difference compared to that in the normal control group; Bcl-2 mRNA expression of cells was reduced with the increase of MSE intervention concentration. Wherein, Bcl-2 mRNA expressions of cells in 0.4 mg/mL and 0.3 mg/mL MSE intervention groups were significantly reduced compared with that of cells in the normal control group （P〈0.05）; Bcl-2 mRNA expression of cells in 0.4 mg/mL MSE intervention group was significantly reduced compared to that in 0.05 mg/mL MSE intervention group （P〈0.05）. Conclusion： Certain higher concentration of moxa smoke could reduce MTP and mRNA expression of the anti-apoptosis gene Bcl-2 in alveolar type Ⅱ epithelial A549 cells. Oxidative damage may be the important mechanism of apoptosis caused by the high concentration of moxa smoke solution, and further studies are necessary on the specific mechanisms.