Effect of N-acetyl cysteine, rifampicin, and ozone on biofilm formation in pan-resistant Klebsiella pneumoniae: an experimental study

ABSTRACT BACKGROUND: To the best of our knowledge, this is the first study to evaluate the effectiveness of specific concentrations of antibiofilm agents, such as N-acetyl cysteine (NAC), rifampicin, and ozone, for the treatment of pan-resistant Klebsiella pneumoniae (PRKp). OBJECTIVES: We evaluated the effectiveness of antibiofilm agents, such as NAC, rifampicin, and ozone, on biofilm formation in PRKp at 2, 6, 24, and 72 h. DESIGN AND SETTING: This single-center experimental study was conducted on June 15, 2017, and July 15, 2018, at Istanbul Faculty of Medicine, Istanbul University, Turkey. METHODS: Biofilm formation and the efficacy of these agents on the biofilm layer were demonstrated using colony counting and laser-screened confocal microscopy. RESULTS: NAC at a final concentration of 2 μg/mL was administered to bacteria that formed biofilms (24 h), and no significant decrease was detected in the bacterial counts of all isolates (all P > 0.05). Rifampicin with a final concentration of 0.1 μg/mL was administered to bacteria that formed biofilm (24 h), and no significant decrease was detected in bacterial count (all P > 0.05). Notably, ozonated water of even 4.78 mg/L concentration for 72 h decreased the bacterial count by ≥ 2 log10. CONCLUSION: Different approaches are needed for treating PRKp isolates. We demonstrate that PRKp isolates can be successfully treated with higher concentrations of ozone.

Hypocrellin A-based photodynamic action induces apoptosis in A549 cells through ROS-mediated mitochondrial signaling pathway

Over recent decades, many studies have reported that hypocrellin A (HA) can eliminate cancer cells with proper irradiation in several cancer cell lines. However, the precise molecular mechanism underlying its anticancer effect has not been fully defined. HA-mediated cytotoxicity and apoptosis in human lung adenocarcinoma A549 cells were evaluated after photodynamic therapy (PDT). A temporal quantitative proteomics approach by isobaric tag for relative and absolute quantitation (iTRAQ) 2D liquid chromatography with tandem mass spectrometric (LC-MS/MS) was introduced to help clarify molecular cytotoxic mechanisms and identify candidate targets of HA-induced apoptotic cell death. Specific caspase inhibitors were used to further elucidate the molecular pathway underlying apoptosis in PDT-treated A549 cells. Finally, down-stream apoptosis-related protein was evaluated. Apoptosis induced by HA was associated with cell shrinkage, externalization of cell membrane phosphatidylserine, DNA fragmentation, and mitochondrial disruption, which were preceded by increased intracellular reactive oxygen species (ROS) generations. Further studies showed that PDT treatment with 0.08 µmol/L HA resulted in mitochondrial disruption, pronounced release of cytochrome , and activation of caspase-3, -9, and -7. Together, HA may be a possible therapeutic agent directed toward mitochondria and a promising photodynamic anticancer candidate for further evaluation.

Therapeutic efficiency of sodium hyaluronate eye drops on ocular surface disorders in patients with proliferative diabetic retinopathy after vitrectomy / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To observe the clinical therapeutic efficiency of 1g/L sodium hyaluronate eye drops on ocular surface disorders in patients with proliferative diabetic retinopathy(PDR)after 20G pars plana vitrectomy(PPV). <p>METHODS: Randomized single blind case control study was used. Patients with type 2 diabetes who were admitted to the department of ophthalmology, Xi'an No.4 Hospital for PDR and underwent 20G PPV surgery by the same surgeon were randomly divided into PDR control group(Group A)and sodium hyaluronate treatment group(Group B). Group B received continuous 1g/L sodium hyaluronate eye drops from 1d to 2mo after surgery. Before and 1wk, 1, 3mo after surgery, OSDI, SⅠt, BUT, fluorescein cornea staining(FL)and the tear film and corneal epithelial cell layer under corneal laser scanning confocal microscopy of the two groups were compared. <p>RESULTS:A total of 90 cases and 90 eyes were studied. In preoperative time, the two groups showed obvious dry eye syndrome, and compared with the control subjects, there were no significant difference existed between the two groups(<i>P</i>>0.05). In group A, ocular surface injury was further aggravated after surgery and failed to recover with time extension during the observation period.Some indexes of Group B improved at different observation time after surgery compared with those before surgery, and there were significant differences between group A and Group B after surgery(<i>P</i><0.05). <p>CONCLUSION: Applying 1g/L sodium hyaluronate eye drops could relief the uncomfortable feeling of ocular surface in patients with PDR after 20G vitrectomy and accelerate the recovery of ocular surface disorders.

Correlated Secondary Ion Mass Spectrometry-Laser Scanning Confocal Microscopy Imaging for Single Cell-Principles and Applications / 分析化学

Secondary ion mass spectrometry ( SIMS) as a powerful surface analysis technique has been widely applied in semiconductor industry and geology research. Recently, with the development of instrumental technology, SIMS is attracting more and more attention in life sciences. SIMS can provide surface MS spectra, 2D/3D chemical images and depth profiling of substances simultaneously. The minimal lateral resolution of 2D SIMS imaging is 80 to 100 nm, and the longitudinal resolution of 3D SIMS imaging is about 1-5 nm. However, due to lack of specific ions to render the structures of organelles, SIMS imaging for single cells still has great challenges. Optical microscopy, in particular laser scanning confocal microscopy ( LSCM) , has been emerged to be an indispensable technique for single cell imaging and can obtain high spatial 2D/3D imaging to visualize the structures of organelles. Thus, the combinational use of SIMS and LSCM, which takes advantages of SIMS for molecular imaging and LSCM for morphological imaging, has greatly extended the application of SIMS imaging and ensured its accuracy at single cells level, providing novel insights into better understanding of the biological events inside cells. In this review, we focus on the development and application of SIMS imaging and the correlated SIMS and LSCM imaging in the research of cell biology and drug discovery. We anticipate that the combinational use of SIMS and LSCM imaging has promising future in biomedicine and life sciences.

Effects of SNCG gene silencing on the cell cycle of endometrial carcinoma HEC-1A cells / 实用医学杂志

Objective To observe changes of cell cycle after stable silencing of SNCG gene in human endometrial carcinoma HEC-1A cells.Methods Flow cytometry was used to observe the changes of SNCG stable silencing HEC-1A cell growth cycle.The percentage of G2/M phase of HCE-1A cells after acridine orange staining was observed by laser scanning confocal microscopy.Results Flow cytometry results showed that the group of SNCG stable silencing in G1 and G2/M phase of the cells increased,but the percentage of cells in the S phase were decreased,the difference were both statistically significant (P ＜ 0.05).The results of cells after confocal microscopy observation of acridine orange staining,the group of SNCG stable silencing were observed in G2/M phase high percentage were statistically significant (P ＜ 0.05).Conclusion Two experimental methods commonly validated that the cell cycle of SNCG stable silencing on HEC-1A cells was arrested in G2/M phase,suggesting that SNCG gene is closely related to the cell growth cycle of endometrial carcinoma.

The changes of mitochondrial membrane potential detected by confocal microscopy in spermatocyte induced by bisphenol A / 中国医学装备

Objective:To detect changes in mitochondrial membrane potential after sperm mother cell damage induced by bisphenol A by laser scanning confocal microscopy(LSCM) and underline its potential action mechanism.Methods: The cultured spermatogenic cells were divided into 3 groups, respectively. Then 0, 10μmol/L, 100μmol/L of Bisphenol A(BPA) were added into the culture, after 3 hours culture, fluorescence probe JC-1 was used to lable the three groups. The fluorescence intensity of JC-1 in mitochondrial was then detected by LSCM. LSCM software was used to analyze the fluorescence intensity. Change of the mitochondrial membrane potential was represented by the change of fluorescence colors (relative proportion of red and green fluorescence is commonly used to measure mitochondrial depolarization ratio). Results:The ratio between the red and green fluorescence in the control group, low dose group and high dose group had significant difference. Intracellular mitochondrial membrane potential of Bisphenol A treatment group was lower than that of the control group, while mitochondrial membrane potential of high dose group was lower than that of the low dose group. Conclusion: Bisphenol A could damage spermatocytes, and as the dose increased, the more serious the injury. The method could real-time monitor with high sensitivity the change of intracellular mitochondrial membrane potential.

The effect of fluoride on the viability of rat ameloblast HAT-7 cells and Ca2 + concentration in the cells / 实用口腔医学杂志

Objective:To evaluate the effect of fluoride on the viability of rat ameloblast HAT-7 cells and calcium concentration in the cells.Methods:HAT-7 cells were exposed to NaF at 0,0.4,0.8,1.6,3.2 and 6.4 mmol/L for 24,48 and 72 h respectively. CCK-8 assay was performed to examine the cells proliferation;the apoptosis rate was determined by flow cytometry;Ca2 +concentration in the cells was detected by laser scanning confocal microscopy.Results:The cell proliferation was increased by NaF at 0.4 mmol/L and 0.8 mmol/L,whereas inhibited at 1.6 mmol/L and above.The effects were in a time-dependent manner.NaF increased apoptosis of the cells and increased Ca2 + concentration in the cells in a concentration-dependent manner.Conclusion:Fluoride at low doses promotes proliferation,at high doses inhibits proliferation of HAT-7 cells.NaF of 1.6 mmol/L or more induces apoptosis of HAT-7 cells and in-duce Ca2 + overloading in the cells.

Quantitative Assessment of Root Canal Roughness with Calcium-Based Hypochlorite Irrigants by 3D CLSM

Chemical solutions play important roles in endodontic treatment and promote ultrastructural changes in dentin surface. The aim of this study was to quantify root canal roughness at different concentrations of calcium hypochlorite (Ca(OCl)2) and sodium hypochlorite (NaOCl) by confocal laser scanning microscopy (CLSM). Fifty-two human mandibular premolars were sectioned and randomly organized into thirteen groups (n=8): saline (control); 1%, 2.5% and 5% NaOCl; 1%, 2.5% and 5% Ca(OCl)2; the hypochlorite groups were further divided into with or without EDTA. The chlorine concentrations of the different solutions were measured by iodine titration (%). The superficial roughness (Sa) was quantified by CLSM. Ca(OCl)2 presented substantial decrease in chlorine concentration that differed from the package indication, but without compromising the dentin ultrastructure changes. There were no significant differences in dentin roughness between Ca(OCl)2 or NaOCl at all studied concentrations. The combination with EDTA provided similar roughness values among the solutions (p>0.05). The 5% Ca(OCl)2 and NaOCl solutions significantly increased dentin roughness and did not differ from the EDTA association (p>0.05). Ca(OCl)2 promoted similar dentin roughness as the NaOCl at the same concentrations and combined with EDTA. It may be concluded that Ca(OCl)2 modified the root canal dentin roughness similarly to NaOCl, at the same concentrations and EDTA combinations used in this study. Ca(OCl)2 and NaOCl, both at 5%, significantly altered dentin roughness, overcoming EDTA association, thus Ca(OCl)2 concentrations ranging from 1% to 2.5% may be suitable solutions for root canal irrigation protocols.

Soluções químicas são fundamentais para o tratamento endodôntico; entretanto, promovem alterações ultraestruturais na superfície dentinária. O objetivo deste estudo foi quantificar a rugosidade da dentina radicular com diferentes concentrações de hipoclorito de cálcio (Ca(OCl)2) e hipoclorito de sódio (NaOCl) utilizando microscopia confocal à laser (CLSM). Foram utilizados 52 premolares humanos inferiores e aleatoriamente divididos em treze grupos (n=8): Soro fisiológico (controle); NaOCl a 1%, 2,5% and 5%; Ca(OCl)2 a 1%, 2,5% and 5%; os grupos de hipoclorito foram subdivididos pela associação ou não ao ácido etilenodiaminotetracético (EDTA). A concentração de cloro ativo foi avaliada para diferentes soluções utilizando titulação iodométrica (%). A rugosidade superficial (Sa) foi quantificada por CLSM. Ca(OCl)2 apresentou perda substancial de cloro ativo e que foi distinta da condição descrita pelo fabricante, sem entretanto comprometer as alterações no substrato dentinário. Não houve diferenças significantes na rugosidade dentinária produzida pelos Ca(OCl)2 e NaOCl em todas as concentrações estudadas e associação com EDTA. A associação ao EDTA produziu rugosidade semelhante entre as soluções (p>0.05). O Ca(OCl)2 e NaOCl na concentração de 5% aumentaram significativamente a rugosidade dentinária e não apresentaram diferenças dos valores obtidos com a associação de EDTA (p>0.05). O Ca(OCl)2 alterou a rugosidade da dentina radicular de forma semelhante ao NaOCl, nas concentrações e associações utilizadas neste estudo. Como a concentração de 5% de Ca(OCl)2 e NaOCl, apresentou maior rugosidade dentinária, independente da associação ao EDTA, pode-se concluir que Ca(OCl)2 nas concentrações de 1% e 2,5% pode ser considerado uma solução adequada para a irrigação de canais radiculares.

Effects ofβ1 adrenergic receptor autoantibodies on the micro-mechanics of a rat myocardial cell and the concentration of intracellular calcium / 中华微生物学和免疫学杂志

Objective To evaluate the effects of β1 adrenergic receptor (β1-AR) autoantibodies on the micro-mechanics of a single rat ventricular myocyte and the concentration of intracellular calcium for investigating the pathogenic mechanism of heart disease caused by β1-AR autoantibody on both cellular and molecular levels .Methods The micro-mechanics of an acutely isolated single myocardial cell from rat was detected by using the atomic force microscopy ( AFM) in combination with laser scanning confocal micro-scope (LSCM) before and after binding to β1-AR autoantibodies.The ventricular myocyte contraction and the intracellular calcium concentration were observed as well .Results The micro-mechanics of a single ventricular myocyte was increased from (44-51) nN to (76-82) nN after binding to β1-AR autoantibodies. Its contraction frequency was also increased from (0.17±0.04) Hz to (0.40±0.03) Hz (P<0.05).More-over, the intracellular calcium fluorescence intensity was increased significantly during contraction in com -parison with that before binding to β1-AR autoantibodies [ ( 102.1 ±12.3 ) % vs ( 154.3 ±16.7 ) %, P<0.01 ] .Conclusion β1-AR autoantibody could affect the contraction and the micro-mechanics of ventricu-lar myocytes and the intracellular calcium concentration in ventricular myocytes .

The diagnosis and monitor for fungal keratitis by detection of (1,3-) β-D-glucan in tears / 中华实验眼科杂志

Background The diagnosis and treatment of fungal keratitis are knotty.There is no quantitative method to identify the disease and judge the therapeutic effect of the antifungal agent.Studies have determined that serum (1,3-) β-D-glucan level can sensitively and specifically reflect the state of systemic mycotic-causing diseases.However,whether (1,3-) β-D-glucan level in tear can monitor and diagnose mycotic keratitis is unclear.Objective Purpose of this study was to investigate the change of tear (1,3-) β-D-glucan level following the administration of antifungal drug in fungal keratitis patients,and evaluate the diagnosis and monitor value of (1,3-) β-D-glucan in tears for fungal keratitis.Methods Sixty patients who were diagnosed as fungal keratitis by fungal culture were analyzed in Affiliated Hospital of Qingdao University Medical College from July 2010 to May 2012.The patients received the topical administration of antifungal drug for 28 days.Thirty healthy volunteers without eye disease served as normal controls.The tear of 50 μl was collected from each subject for the detection of (1,3)-β-D-glucan before the therapy,7,14,28 days after therapy and 7 days,14 days after the drugs were stopped,respectively.The dynamic changes of (1,3-) β-D-glucan levels in tears were evaluated and compared with the manifestation of the lesions under the laser scanning confocal microscope.The patients without hyphal by the laser scanning confocal microscopy and tear (1,3-)β-D-glucan level less than 20 ng/L were subsequently treated for another 7 days,and the following-up duration was 2 months.The informed consent was obtained before any medical examination was performed from each subject.Results (1,3-)β-D-glucan level in tears (Log value) was (6.37 ±0.48)ng/L in the patient group,and was significantly higher than (2.00±0.31) ng/L in the normal control group (t =2.89,P＜0.01).The lesion was smaller with the gradually clear border,and the number of mycelia was decreased under the laser scanning confocal microscope 7 days after treatment.(1,3-) β-D-glucan level in tears was gradually declined in a time-dependent manner after treatment.The (1,3)-β-D-glucan level in tears (Log) was (5.19 ± 0.42),(4.16 ± 0.33),(2.99 ±0.42),(2.91 ±0.39),(2.80±0.40) ng/L 7,14,28 days after treatment,and 7 days,14 days after the drugs were stopped,respectively,with a statistically significant difference in comparison with (6.37±0.48)ng/L before treatment (P＜0.01).(1,3)-β-D-gluean level in tears remained a lower level till the end of follow-up,and no recurrence of lesion was found in the patient group.Conclusions Detecting (1,3)-β-D-glucan level in tears is of good diagnosis and monitor value in the evaluation of fungal keratitis.

Laser scanning confocal microscopy findings of phlyctenular ophthalmia / 中华实验眼科杂志

Background Phlyctenular ophthalmia is a delayed hypersensitivity reaction to some microprotein and affected mainly by adolescent in high incidence.Objective This study was to investigate the microscopy findings of phlyctenular ophthalmia and evaluate the histological changes by laser scanning confocal microscope.Methods Twenty-nine eyes suffered from phlyctenular ophthalmia and twenty normal eyes were examined using laser scanning confocal microscope.The pictures were taken by a CCD camera.All the cases had initially chest X-ray,tuberculin test,bacterial and mycobacteria culture.Results Dendritic and inflammatory cells were increased and concentrated in conjunctiva,and epithelial cells were deformed and squamatizated.The capillaries engorged and the goblet cells were injured.The corneoscleral Vogt meshing of the phlyctenular keratitis was obscured and dendritic cells were intruded into the corneas.The corneal epithelium of phlyctenular keratitis was absent and the subepithelial nerve plexus were bended and fractured,and the dendritic and inflammatory cells were intruded.Scarring of corneal stroma was seen under the laser scanning confocal microscope.Conclusions Laser scanning confocal microscopy is valuable for basic research and clinical diagnosis of phlyctenular ophthalmia.

Exercise Training Could Reduce Age-Induced Microvascular Impairment Related to Its Anti-Oxidant Potential.

Objective: During aging, an ineffective perfusion of tissues/organs is a major risk factor for several diseases. Age-induced oxidative stress has been proposed to correlate with this age-related microvascular dysfunction including angiogenesis impairment. It has been demonstrated that exercise training could ameliorate oxidative damage, as well as, enhance angiogenesis in various organs. Therefore, the present study aims to investigate whether exercise training can prevent alterations of capillary vascularity in brain and bone during aging. Design and method: Male Wistar rats were divided into three groups: sedentary-young (aged 4-6 months), sedentary-aged (aged 20-22 months) and train-aged (aged 20-22 months). The exercise program included swimming training 5 days/week for 8 weeks. We directly observed microvasculature of brain and bone by using a laser scanning confocal microscopic system. The microvascular networks were visualized by fluorescein isothiocyanate labeled dextran and were analyzed for capillary vascularity by image analysis software. Blood was collected to determine the level of malondialdehyde, an indicator of oxidative stress. Results: In sedentary-aged group, the malondialdehyde level was significantly increased, whereas capillary vascularities in brain and bone were significantly decreased when compared to the sedentary-young group (P<0.05). In train-aged group, capillary vascularities in brain and bone were significantly higher, whereas the malondialdehyde level was significantly lower when compared to the sedentary-aged group (P<0.05). Besides, the result also showed a linear correlation between capillary vascularity and malondialdehyde level. Conclusion: The exercise training could attenuate age-induced suppression of capillary vascularity in brain and bone, closely related to exercise-ameliorated oxidative stress during aging.

Laser scanning confocal microscopy-assisted obtain of limbal tissue for the ex vivo culture of human limbal epithelial stem cells and identification / 中华实验眼科杂志

Background Human limbal allograft transplantation or limbal autograft transplantation are the primary approaches to the severe corneal-blindness,but their application in clinic were limited because of the defects of donor material.With the development of tissue engineering technology,transplantation of in vitro cultured limbal epithelial stem cells is being an advanced management.Objective The aim of this work was to expand human limbal epithelial stem cells ex vivo under the guidance of confocal microscope and to lay the foundation for fabricating ex vivo cultured cell sheets.Methods Ten eyes of ten patients were examined with the Heidelberg Retina Tomography Ⅲ Rostock Cornea Module(HRT3-RCM)to elucidate the structure of the human corneoscleral limbus and to correlate limbal epithelial dimensions.According to the analysis of the images of limbal epithelia,the limbal tissues provided by Eye Bank of Henan Eye Institute were cut into suitable explants.Then,this study was conducted to expand limbal epithelial stem cells ex vivo on denuded amniotic membrane.The phenotypes of primary cultured cells were evaluated by morphology and immunofluorescent staining with antibodies for limbal epithelial stem cell markers (p63,cytokeratinl9)and differentiation markers(keratin 3,involucrin).This experimental procedure was approved by the Ethic Committee of Henan Provincial People's Hospital.The written informed consent was obtained from subjects before initiation of any examination.Results The palisade morphology of human limbus was imaged clearly on the laser scanning in vivo confocal microscopy and many hyperreflective cells were observed in palisade basal cells.The cell-island phenomenon was seen in the basement membrane under the laser scanning in vivo confocal microscopy.The oblique sections of limbus showed many papilla-like epithelial columns below the superficial limbal epithelia.Throughout the experiment duration,the epithelial cells grew well with the migration rates from limbal tissue (68.62± 16.94)％ and the migration time(5.83 ±2.04)days,which depended on the tissue freshness.Compared with the second and forth batch of tissue,the migration rates of the third and sixth batch of tissues were significantly higher(P＜0.05),and the migration time was evidently longer in the forth and sixth batch of tissue compared with the first,second,third and fifth batch(P＜0.05).The positively expressing rates in the cultured corneal stem cells were 4.05％ and 36.52％ for p63,26.07％ and 40.55％ for CK19,57.88％ and 40.81％ for K3,64.66％ and 59.19％ for involucrin.Conclusion Human limbal epithelial stem cells can be successfully and purposefully obtained from the limbal tissue based on the guidance confocal miscroscope.The cultured corneal stem cells can grow well on the denuded amniotic membrane

Functional multineuron calcium imaging technology and its application prospect in neuropharmacology / 中国药理学通报

Functional multineuron calcium imaging(fMCI)is an optical recording technique to monitor neuron population action potentials in the spatiotemporal pattern by recording calcium signal changes in neurons.The review describes the technology of fMCI and its application prospect in neuropharmacology research.fMCI provides a kind of powerful tool to analyze various functions of brain and to research some central nervous system drug mechanism based on neural network.

Enhancing effect of negative electret on percutaneous drug absorption of meloxicam patch in SD rats / 第二军医大学学报

Objective: To determine meloxicam concentrations in both plasma and extract solution of tested-skin in SD rats and the fluorescence intensity, location of fluorescein sodium salt (NaFI) in striped rat skin within 4 h after transdermal administration, so as to investigate the enhancing effect of negative electret on percutaneous absorption of meloxicam and percutaneous absorption route of NaFI. Methods: Pharmaceutical method and grid-controlled constant corona charge technique were used to prepare electret meloxicam patch. High performance liquid chromatography (HPLC) method was employed to determine meloxicam concentration after transdermal administration. NaFI was used as probe to determine the localization and percutaneous absorption route of NaFI by using laser scanning confocal microscopy (LSCM). Results: (DNegative electret and its polypropylene(PP) electret meloxicam patch exhibited a good charge storage stability. (2)The results of HPLC demonstrated that electret had a remarkable enhancing effect on percutaneous absorption of meloxicam after application for 1-4 h. (3) LSCM further proved the enhancing effect of negative electret on percutaneous absorption of small molecules. We also found that the stratum corneum and the hair follicle areas were the two main pathways for the enhancing effect of the electret. Conclusion: Negative electret can be used as an enhancer for transdermal permeation of meloxicam.

Biocompatibility observation of titanium surfaces by laser scanning confocal microscope / 实用口腔医学杂志

Objective: To observe the biocompatibility of titanium implant surfaces using laser scanning confocal microscopy (LSCM). Methods; Titanium implant surfaces were prepared by 3 different methods. Osteoblasts were seeded onto the titanium surfaces. The cells were investigated by LSCM. Results: The sand-blasted titanium surfaces showed the best biocompatibility. Conclusion; The biocompatibility of titanium implant surfaces depends on the surface preparation method.

Expression and significance of cytochrome C and Bax in hippocampus of posttraumatic stress disorder rat / 解剖学报

Objective To observe the expression of cytochrome C (Cyt C)and apoptosis-related gene Bax in hippocampus of posttraumatic stress disorder(PTSD)rats in order to investigate their effects and relationships. Methods Sixty male Wistar rats were used in the present study. The single-prolonged stress(SPS)-method was carried out to set up the rat PTSD models. There were six groups:after SPS 1,4,7,14,28 days groups and control group. The expression of Cyt C and Bax proteins were detected with immunohistochemistry, double immunofluorescence, confocal laser scanning microscopy and Western blotting. Results The expression of Cyt C peaked at 4 days and maintained higher level at 7 days after SPS. At 7 days after SPS, strong immunoreactivity of Bax was noticed,At 14 days after SPS, the expression of Bax was down-regulated and then gradually decreased. Conclusion Bax is upregulated and promotes the releasing of Cyt C. And the releasing of Cyt C may be one of mechanisms of inducing apoptosis in the hippocampus of PTSD rats.

Observation of metastasis of colon adenocarcinoma Iovo cells in vitro under laser scanning confocal microscope / 国际生物医学工程杂志

Objective To explore the feasibility of applying laser scanning confocal microscopy to investigate the metastatic characteristic of tumor cells in vitro. Methods The confocal microscope combined with transwell assay was employed to observe the invasion and migration of the metastatic colon cancer ]ovo cells when stimulated by IL-8. Results The results show that the number of invasive lovo cells in the stimulated group was much higher than the unstimulated group (P<0.05). The migration way of lovo cells observed under the confocal microscopy was ameba like movement. These findings suggest that the metastatic ability of lovo cells stimulated by IL-8 was enhanced. Conclusion The metastatic characteristic of colon adenocarcinoma lovo cells is similar to the chemotactic movement of leukocytes.

The level of serum lipids,body-fat content of obesity rats,and the relationship between intracellular calciumand ventricular arrhythmia / 中国地方病学杂志

Objective To study the level of serum lipids and body-fat content of high-fat diet induced pbesity rats(DIO).explore the relationship between intracellular caleium and ventrieufar arrhythmia.Methods Sixty male Sprague-Dawley(SD)rats were divided into control group(15)and experiment group(45),high-fat diet was administrated for 12 weeks to established obesity model,15 rats were selected into obesity group according to their body weight gain.The standard 2-lead electrocardiograph was used to detect the incidence and scores of arrhythmia induced by barium chloride(BaCl2,0.1 mg/kg)for 1 hour on every 8 rats from different groups respectively.Body-fat content.the level of serum total cholesterol(TC),triglyceride(TG),low density lipopmtein cholesterol (LDL-C) and high density lipopmtein cholesterol (HDL-C)were measured.The epididymal(EP),retroperitoneal (RE) and mesenteric(ME)white adipose pads was measured to obtain the body fat content.Single ventricular myrocytes of rats were isolated by enzymatic dissociation.The confocal laser scanning microscope was used to record basic intraceUular calcium level([Ca2+]i).Results The body-fat content in obesity group[(7.71±0.74)%] was significantly higher than control group[(4.69±0.37)%](t＝3.650,P＜0.05).The level of serum TC,TGand LDL-C were significantly higher(t＝3.801,2.778,3.536.P＜0.05) in obesity group[(1.26±0.04),(0.58±0.10),(0.51±0.04)mmol/L]than those in control group[(0.92±0.08),(0.29+0.03),(0.31±0.04) mmol/L].The level of serum HDL-C wa8 decreased gradually from control group[(0.53±0.05)mmol/L] toobesity group[(0.52±0.02)mmol/L],but there waft no significant difference between them(t＝0.186,P＞0.05).The incidence of arrhythmia induced by BaCl2(0.1 mg/kg)in obesity group was significantly higher than controlgroup(X2＝5.333,P＜0.05),and the scores of arrhythmla was increased in obesity group(2.5±0.6)too.The fluorescence intensity standing for[Ca2+]i was increased significantly(t＝2.409,P＜0.05)from obesity group(247.96±20.03)to control group (174.25±23.13).Conclusion As the free cytosolic calcium begin to accumulate,the arrhythmia morbidity is increased in obesity rats.

Changes of Cytosolic Ca2+ under Metabolic Inhibition in Isolated Rat Ventricular Myocytes

To characterize cytosolic Ca2+ fluctuations under metabolic inhibition, rat ventricular myocytes were exposed to 200microM 2, 4-dinitrophenol (DNP), and mitochondrial Ca2+, mitochondrial membrane potential (delta psi m), and cytosolic Ca2+ were measured, using Rhod-2 AM, TMRE, and Fluo-4 AM fluorescent dyes, respectively, by Laser Scanning Confocal Microscopy (LSCM). Furthermore, the role of sarcolemmal Na+/Ca2+ exchange (NCX) in cytosolic Ca2+ efflux was studied in KB-R7943 and Na+-free normal Tyrode's solution (143 mM LiCl ). When DNP was applied to cells loaded with Fluo-4 AM, Fluo-4 AM fluorescence intensity initially increased by 70+/-10% within 70+/-10 s, and later by 400+/-200% at 850+/-46 s. Fluorescence intensity of both Rhod-2 AM and TMRE were initially decreased by DNP, coincident with the initial increase of Fluo-4 AM fluorescence intensity. When sarcoplasmic reticulum (SR) Ca2+ was depleted by 1microM thapsigargin plus 10microM ryanodine, the initial increase of Fluo-4 AM fluorescence intensity was unaffected, however, the subsequent progressive increase was abolished. KB-R7943 delayed both the first and the second phases of cytosolic Ca2+ overload, while Na+-free solution accelerated the second. The above results suggest that: 1) the initial rise in cytosolic Ca2+ under DNP results from mitochondrial depolarization; 2) the secondary increase is caused by progressive Ca2+ release from SR; 3) NCX plays an important role in transient cytosolic Ca2+ shifts under metabolic inhibition with DNP.