Direct Current Stimulation in Cell Culture Systems and Brain Slices-New Approaches for Mechanistic Evaluation of Neuronal Plasticity and Neuromodulation: State of the Art.

Non-invasive direct current stimulation (DCS) of the human brain induces neuronal plasticity and alters plasticity-related cognition and behavior. Numerous basic animal research studies focusing on molecular and cellular targets of DCS have been published. In vivo, ex vivo, and in vitro models enhanced knowledge about mechanistic foundations of DCS effects. Our review identified 451 papers using a PRISMA-based search strategy. Only a minority of these papers used cell culture or brain slice experiments with DCS paradigms comparable to those applied in humans. Most of the studies were performed in brain slices (9 papers), whereas cell culture experiments (2 papers) were only rarely conducted. These ex vivo and in vitro approaches underline the importance of cell and electric field orientation, cell morphology, cell location within populations, stimulation duration (acute, prolonged, chronic), and molecular changes, such as Ca2+-dependent intracellular signaling pathways, for the effects of DC stimulation. The reviewed studies help to clarify and confirm basic mechanisms of this intervention. However, the potential of in vitro studies has not been fully exploited and a more systematic combination of rodent models, ex vivo, and cellular approaches might provide a better insight into the neurophysiological changes caused by tDCS.

Ozone-Based Eye Drops Activity on Ocular Epithelial Cells and Potential Pathogens Infecting the Front of the Eye.

Confirmation of the biological effectiveness of new ophthalmic preparations introduced in the market is an important element in maintaining the safety of using this type of medications. This study aimed to investigate the activity of Ozodrop® on human corneal and conjunctival epithelial cells, as well as its antibacterial and antifungal activity. Cytotoxicity analyses of ocular surface epithelial cells were performed in vitro by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) and Neutral Red uptake assays. The level of nitric oxide released by the cells was assessed by the Griess method. The reduction of the DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical by the tested formulation was analyzed. Microbiological tests were also performed. It was found that the Ozodrop® preparation exhibited biological activity, but was less active than the reference antibiotics and the anti-yeast agent. The cytotoxic activity of the Ozodrop® formulation was dependent on the time of cell exposure to it. No toxic effect was observed in the short-term, for up to 3 h. It appeared after 24 h of exposure of the cells to the preparation. The drops showed antioxidant activity in the specified concentration range. They also stimulated the release of nitric oxide, mainly by corneal epithelial cells. The Ozodrop® formulation exhibits biological activity that can be considered useful in the treatment of infections in the front part of the eye.

Synthesis, Structure and Antiproliferative Activity of New pyrazolo[4,3- e]triazolo[4,5-b][1,2,4]triazine Derivatives.

BACKGROUND: Triazoles and their fused derivatives are an important class of compounds that exhibit interesting biological properties, such as antiasthmatic, antimicrobial, antifungal, analgesic, antiallergic, antiinflammatory, herbicidal, plant growth regulative activity, and anti-HIV-1 activities. Moreover, anticancer activity of 1,2,4-triazole containing derivatives has been documented. Due to the fact a convenient approach toward polycyclic frameworks containing fused 1,2,4-triazoles was described. OBJECTIVE: The objective of this article is the synthesis of new pyrazolo[4,3-e]triazolo[4,5- b][1,2,4]triazine derivatives with potential antiproliferative activity. METHODS: Cancer cell proliferation was analysed by means of MTT assay after 96 h treatment. IC50 was calculated using computerized linear regression analysis of quantal log doseprobit functions, according to the method of Litchfield and Wilcoxon. X-ray data were collected on the Bruker SMART APEX II CCD diffractometer; The structure was solved by direct methods using SHELXS-2013 and refined by full-matrix least-squares with SHELXL-2014/7. All calculations were performed using WINGX version 2014.1 package. RESULTS: The series of pyrazolo[4,3-e]triazolo[4,5-b][1,2,4]triazine derivatives were synthesized. MTT assay revealed that the compounds inhibited cancer cells growth at concentrations below 10 µM. The tested compounds showed higher antiproliferative activity than popular cytostatics cisplatin (lung carcinoma) and 5-fluorouracil (colon adenocarcinoma). X-ray examinations showed that final products in the crystalline phase have a linear form. CONCLUSION: In the paper we have reported the synthesis and spectroscopic analysis of new condensed tricyclic derivatives of the pyrazolo[4,3-e]triazolo[4,5-b][1,2,4]triazine. MTT analysis revealed concentration-dependent decrease in lung A549 and colon LS180 cancer cells proliferation. In order to explain the molecular mechanisms involved in anticancer activity of pyrazolo[4,3- e]triazolo[4,5-b][1,2,4]triazine derivatives, our research will be continued.

hASC and DFAT, Multipotent Stem Cells for Regenerative Medicine: A Comparison of Their Potential Differentiation In Vitro.

Adipose tissue comprises both adipose and non-adipose cells such as mesenchymal stem cells. These cells show a surface antigenic profile similar to that of bone-marrow-derived MSC. The cells derived from the dedifferentiation of mature adipocytes (DFAT) are another cell population with characteristics of stemness. The aim of this study is to provide evidence of the stemness, proliferation, and differentiation of human adipose stem cells (hASC) and DFAT obtained from human subcutaneous AT and evaluate their potential use in regenerative medicine. Cell populations were studied by histochemical and molecular biology techniques. Both hASC and DFAT were positive for MSC markers. Their proliferative capacity was similar and both populations were able to differentiate into osteogenic, chondrogenic, and adipogenic lineages. DFAT were able to accumulate lipids and their lipoprotein lipase and adiponectin gene expression were high. Alkaline phosphatase and RUNX2 gene expression were greater in hASC than in DFAT at 14 days but became similar after three weeks. Both cell populations were able to differentiate into chondrocytes, showing positive staining with Alcian Blue and gene expression of SOX9 and ACAN. In conclusion, both hASC and DFAT populations derived from AT have a high differentiation capacity and thus may have applications in regenerative medicine.

Mesenchymal stem and progenitor cells of rats' bone marrow under chronic action of ionizing radiation. / MEZENKhIMAL'NI STOVBUROVI KLITYNY TA KLITYNY POPEREDNYKY KISTKOVOGO MOZKU ShchURIV V UMOVAKh KhRONIChNOÏ DIÏ IONIZUIuChOÏ RADIATsIÏ.

Under the influence of ionizing radiation on hematopoietic system, the level of its injury is determined not only by the radiosensitivity of hematopoietic stem cells, but also by radiation induced changes in microenvironment func tioning, in particular, mesenchymal stem cells as its components. OBJECTIVE: to define functioning characteristics of mesenchymal stem and progenitor cells of rats' bone marrow under prolonged action of ionizing radiation as a result of 90Sr incorporation. MATERIALS AND METHODS: We applied the model of Wistar rats' internal irradiation with 90Sr radionuclide and per formed the in vitro cultivation of their bone marrow mesenchymal cells. Colony forming efficiency in the in vitro cell culture was determined, as well as the possibility of these cells to form feeder layers and to support rat bone mar row hematopoietic cells in the culture of diffusion chambers in vitro. RESULTS AND CONCLUSIONS: We established that chronic action of incorporated 90Sr radionuclide induced considerable decrease in proliferative activity of mesenchymal stem cells comparing to control, as well as the inhibition of the capability to prolonged support of hematopoietic processes in vitro by their feeder layers.Thus, bone marrow mesenchymal stem cells and their closest progeny - progenitor cells were characterized by rather high radiosensitivity under the influence of ionizing radiation, which was revealed in considerable decline of their functional activity in cell culture in vitro comparing to control indices as a result of irradiation.

Aptamer-polymer functionalized silicon nanosubstrates for enhanced recovered circulating tumor cell viability and in vitro chemosensitivity testing.

Selection of the optimal chemotherapy regimen for an individual cancer patient is challenging. The existing chemosensitivity tests are costly, time-consuming, and not amenable to wide utilization within a clinic. This limitation might be addressed by the recently proposed use of circulating tumor cells (CTCs), which provide an opportunity to noninvasively monitor response to therapy. Over the past few decades, various techniques were developed to capture and recover CTCs, but these techniques were often limited by a capture and recovery performance tradeoff between high viability and high efficiency. In this work, we used anti-epithelial cell adhesion molecule coated aptamer-poly (N-isopropylacrylamide) functionalized silicon nanowire substrates to capture and release epithelial cell adhesion molecule-positive CTCs at 32°C and 4°C, respectively. Then, we applied the nuclease to digest the aptamer to release the captured CTCs (near or at the end of the polymer brush), which cannot be released by heating/cooling process. High viability and purity CTCs could be achieved by decreasing the heating/cooling cycles and enzymatic treatment rounds. Furthermore, the time-saving process is helpful to maintain the morphology and enhance vitality of the recovered CTCs and is beneficial to the subsequent cell culture in vitro. We validated the feasibility of chemosensitivity testing based on the recovered HCC827 cells using an adenosine triphosphate-tumor chemosensitivity assay, and the results suggested that our method can determine which agent and what concentration have the best chemosensitivity for the culturing recovered CTCs. So, the novel method capable of a highly effective capture and recovery of high viability CTCs will pave the way for chemosensitivity testing.

Expression of glutathione transferases in corneal cell lines, corneal tissues and a human cornea construct.

Glutathione transferase (GST) expression and activity were examined in a three-dimensional human cornea construct and were compared to those of excised animal corneas. The objective of this study was to characterize phase II enzyme expression in the cornea construct with respect to its utility as an alternative to animal cornea models. The expression of the GSTO1-1 and GSTP1-1 enzymes was investigated using immunofluorescence staining and western blotting. The level of total glutathione transferase activity was determined using 1-chloro-2,4- dinitrobenzene as the substrate. Furthermore, the levels of GSTO1-1 and GSTP1-1 activity were examined using S-(4-nitrophenacyl)glutathione and ethacrynic acid, respectively, as the specific substrates. The expression and activity levels of these enzymes were examined in the epithelium, stroma and endothelium, the three main cellular layers of the cornea. In summary, the investigated enzymes were detected at both the protein and functional levels in the cornea construct and the excised animal corneas. However, the enzymatic activity levels of the human cornea construct were lower than those of the animal corneas.

Effects of low-dose T-2 toxin on cultured cartilage cells and molecular mechanism / 中华地方病学杂志

Objective Through observing the effect of low-dose T-2 toxin on chondrocyte,to study the molecular mechanism of cartilage damage.Methods The primary chondrocytes were isolated from articular cartilage of d 1-2 Wistar neonate rats through enzymatic digestion.Different doses (0.005,0.010,0.100 μg/L) of T-2 toxin were added after 24 h in vitro culture.The survival rate of chondrocytes was detected with Trypan blue staining.Echylosis (matrix metalloproteinase,MMP1) was analyzed by immunohistochemistry.The damage of articular chondrocyte was observed by transmission electron microscope.Results ①Cell morphology of in vitro cultured chondrocyte:the newly isolated chondrocytes were spherical.After 24 hours,the adherent cells gradually began to stretch the triangle or polygon;the nucleus was large and round;the cell was clear and transparent,containing secretory granules.②Cell proliferation:T-2 toxin had a significant inhibitory effect on chondrocyte proliferation,the higher the concentration of T-2 toxin,the significant the inhibitory effects [0.000 μg/L (control) group:3.45 × 108/L,0.005 μg/L T-2 toxin group:3.45 × 108/L,0.010 μg/L T-2 toxin group:2.06 × 108/L,x2 =9.554,P ＜ 0.05].③Immunohistochemical observation:dysplasia,nucleus condensation and membrane rupture were observed in T-2 toxin treated group,brown staining was observed in all groups at varying degrees.The deepest staining was in 0.005 μg/L T-2 toxin group,with the strongest secretion of MMP1;with increasing doses of the toxin,the damage to cartilage cells was severe,MMP1 secretion was less,staining was weak,and the weakest staining was in the 0.100 μg/L T-toxin group.④Under transmission electron microscopy:in control group,cytoplasm was rich in rough endoplasmic reticulum,nuclear membrane and cell membrane were clear;in 0.005 μg/L T-2 toxin group,the cell nucleus showed pyknosis,organelles were decreased in cytoplasm;in 0.100 μg/L T-2 toxin group,the microvilli was dropped out of cartilage surface,nuclear changes were obvious,and mitochondria was myeloid degeneration;rough endoplasmic reticulum was degranulation and expansion into cystiform,chondrocytes were apoptosis occasionally,the cell nucleus showed pyknosis,and the formation of high-density plaque.Conclusion Low dose of T-2 toxin could damage the primary cultured articular chondrocyte in vitro.The results have showed that there are damaged cytostasis,chondrocyte degeneration,necrosis and apoptosis.

Effect of hydroxycamptothecin combined with etoposide on apoptosis of human Tenon capsule fibroblast and its mechanism / 中华实验眼科杂志

Background Previous study showed that both hydroxycamptothecin (HCPT) and etoposide (VP-16) can induce the apoptosis of human Tenon capsule fibroblasts (HTFs).However,whether the combination of HCPT with VP-16 enhance the efficacy of drugs is unknown.Olbjeetive This study was to investigate the synergistic effect and its mechanism of HCPT combined with VP-16 on apoptosis of HTFs.Methods Human Tenon capsule tissue was obtained from the eye bank of Jiangsu Province People's Hospital.HTFs were cultured in vitro using explant method and identified by immunofluorescence with vimentin.The fourth generation of cells were incubated in 96-well plate,and different concentrations of HCPT (1,5,10,50,100 mg/L),VP-16 (0.6,2.5,5.0,25.0,50.0 mg/L) and HCPT+VP-16 (2:1,final concentrations 0.80,3.75,7.50,37.50,75.00 mg/L) were added for 24 hours.The inhibiting rate of drugs to HTFs growth was detected using CCK-8 kit.The HTFs were divided into blank control group,HCPT (50 ng/L) treated group,VP-16 (25 mg/L) treated group and HCPT+ VP-16 (37.5 mg/L) treated group,and the apoptosis rates of HTFs in various groups were assayed by flow cytometry.The expressions of caspase-3,cleaved caspase-3,bax,bcl-2,JNK,p-JNK,Akt,p-Akt in the cells were detected by Western blot assay.Results Cultured cells grew well with the polygon shape and positive response for vimentin.The inhibiting rate was elevated with the increase of drug dosage 24 hours after addition of drugs (HCPT:F=41.34,P=0.00 ; VP-16:F =62.60,P =0.00 ; HCPT+VP-16:F =46.77,P =0.00).The half maximal inhibitory concentrations (IC50) of HCPT,VP-16,HCPT+VP-16 were 80.99,27.93,19.81 mg/L,respectively,and the combined index (CI) of HCPT with VP-16 was 0.399,showing a stronger synergistic action.The apoptotic rates of HTFs were (4.87±0.78) ％,(11.20± 1.94)％,(12.67±1.51)％ and (19.77±2.01)％ in the blank control group,HCPT treated group,VP-16 treated group and HCPT+VP-16 treated group,respectively,with a significant difference among them (F=18.23,P ＜ 0.01),and the apoptotic rate was significantly raised in the HCPT + VP-16 treated group,HCPT treated group and VP-16 treated group compared with the blank control group (q'=15.67,16.32,26.88,all at P＜0.01).Compared with the blank control group,the grey scale values of cleaved caspase-3,bax,p-JNK in the cells of HCPT+VP-16 treated group,HCPT treated group and VP-16 treated group were significantly increased (all at P＜0.01),and those in the HCPT+VP-16 treated group significant ascent in comparison with the HCPT treated group and VP-16 treated group (all at P＜0.01).However,the changes of caspase-3,JNK and Akt expression were insignificant.The grey scale values of bcl-2 and p-Akt in the HTFs of the HCPT,VP-16 and HCPT+VP-16 treated groups were significantly lower than those of the blank control group,with a dominant reducing in the HCPT+VP-16 treated group (all at P＜0.01).Conclusions HCPT and VP-16 induce the apoptosis of HTFs in vitro at a dose-dependent manner.The combination of HCPT with VP-16 has a stronger synergistic efficacy.The up-regulation of p-JNK and bax as well as the down-regulation of p-Akt and bcl-2 in HTFs are involved in the coaction of HCPT and VP-16.

The impact of the bone marrow-derived mesenchymal stem cell on the microenvironment of the human limbal stem cell / 中华实验眼科杂志

Background When limbal stem cell deficiency (LSCD) occurs,not only the limbal stem cells (LSCs) were damaged,but also the LSCs matrix microenvironment was under destruction.The treatment of LSCD include both replenishing of stem cells and restoration of microenvironment.So far,the method to improve the microenvironment of LSCD still exist limitation and urgently need to establish more appropriate microenvironment for the LSCs growth in vitro.Objective This study was to investigate whether the human bone marrow-derived mesenchymal stem cells (BMSCs) can be used as the ideal cells to repair limbal microenvironment and its possible mechanism of repairing limbal microenvironment during the human LSCs amplification in vitro as feeder cells.Methods BMSCs were cultured and passaged in vitro,and flow cytometry was used to assay the expressions of CD45,CD71,CD90,CD105 and HLA-DR and directionally induced BMSCs to the osteoblasts and adipocytes.BMSCs were treated using mitomycin C (MMC) to use as the feeder cells.LSCs were separately co-cultured with BMSCs,Swiss-3T3 feeder cells and free-feeder cells,and colony-forming efficiency (CFE) of the LSCs was compared among different co-cultured groups.LSCs were then cultured sequentially and identified by flow cytometry.Expression of cytokines in BMSCs was confirmed by reverse transcriptional polymerase chain reaction (RT-PCR).Results Cultured BMSCs showed a good homogeneity,with a lot of expressions of interstitial cell markers such as CD71,CD90,CD105 and less expressions of hematopoietic cell markers including CD45 and HLA-DR.After separately cocultured with feeder cells for 12 days,the CFE of the LSCs co-cultured with BMSCs,Swiss-3T3 and no feeder cells was 3.67％ ±0.58％,4.30％ ± 1.54％ and 0.20％ ±0.10％,showing a statistical significant difference among the three groups(F =15.420,P =0.040).There was no statistically significant difference in the C FE of the LSCs between the BMSCs feeder group and the Swiss-3T3 feeder cells group(P =0.456),between the BMSCs feeder group and the free-feeder cells group or the Swiss-3T3 co-culture group and the free-feeder cells group (P =0.005,0.002).LSCs presented with a positive response for ABCG2 antigen in the co-cultured with BMSCs group.Basic fibroblast growth factor(bFGF),stem cell factor (SCF) and N-cadherin(N-cad) were positively expressed in the BMSCs as feeder cells.Conclusions Human BMSCs-derived feeder cells can improve the growth of the stromal microenvironment of the LSCs and enhance their proliferation ability.Human BMSCs are suitable for engineering of epithelial sheets.

Biological characteristics of in vitro trabecular cells from primary open-angle glaucomatous eye / 中华实验眼科杂志

Background Primary open angle glaucoma (POAG) is a major blindness-causing disease,characterized by elevated intraocular pressure due to an insufficient outflow of aqueous humor. The trabecular meshwork lining the aqueous outflow pathway modulates the aqueous outflow facility. To study the biological characteristics of the trabecular meshwork cells has important significance. Objective This study was to culture the trabecular cells from primary open-angle glaucomatous eye (POAG) and study the biologic characteristics of passaged cells. Methods The deep scleral tissue with trabecular meshwork was obtained during the trabeculectomy from 8 eyes with POAG. The trabecular cells were primarily cultured and passaged in vitro. The generation 3 cells were identified by immunochemistry with the laminin (LM), fibronectin (FN) and neuron specific endolase (NSE)monoclonal antibodies. The ultrastructure was examined to observe the biological characteristics of the cells under the transmission electronic microscope. The experimental results were compared among POAG group, normal control group and blank control group. Results The primarily cultured POAG trabecular cells migrated from the edge of tissue mass about 10 days. The cells of generation 3 presented the logarithmic phase in the first 4 days and fused in the 7th day. FN,LM and NSE were positively expressed in the generated cells in POAG group and normal control group rather than blank control group. The MOD values of the generation 3 cells for FN in POAG group and normal control group were 0. 35 ± 0.06 and 0. 26 ± 0. 01, and those for LM were 0. 34 ± 0. 03 and 0. 25 ± 0. 02 respectively, showing statistically significant difference between these two groups ( FN: t = 14. 446, P＜0.001; LM: t = 9. 346, P＜0. 001 ). The microvilli, cytolysosome and phagocytic vesicle were obviously decreased in the trabcular cells of POAG group compared with normal control group under the transmission electron microscope. Conclusion The trabecular meshwork cells from POAG can be successfully cultured and passaged in vitro. It provides a cytology basis for further glaucoma research.

Effect of 5-Azacytidine on In-Vitro Proliferation and Myogenic Differentiation of Rat Bone Marrow Mesenchymal Stem Cells / 广州中医药大学学报

0.05),but 5-Aza at the concentration of 15?mol/L inhibited the growth of MSCs(P

In-vitro Culture of Astrocytes from Cerebral Cortex of Neonatal Rats / 广州中医药大学学报

Objective To establish a method for the in-vitro culture of astrocytes from cerebral cortex of neonatal rats.Methods Astrocytes were isolated from cerebral cortex of the neonatal SD rats aged 24 hours,and then were cultured in the high-glucose DMEM medium with 10% fetal bovine serum added.The morphological features of astrocytes were examined under inverted microscope.The growth of the third generation of astrocytes was observed with counting plate method and methyl thiazolyl tetrazolium (MTT) assay.Results The astrocytes from cerebral cortex of neonatal rats grew well,the astrocytes showed typical morphological features,and no other kinds of cells was found.Conclusion A simple and practical in-vitro culture method for rats astrocytes is established.