Effect of a photosensitization reaction performed during the first 3 min after exposure of rat myocardial cells to talaporfin sodium in vitro

To better understand the mechanism of photodynamic cardiac ablation, we studied the effects of a photosensitization reaction (PR) performed during the first 3 min after rat myocardial cells were exposed to talaporfin sodium. A 3-mm-square microelectrode array with 64 electrodes was used to continuously measure the action potentials of the myocardial cells. A 30 μg/mL talaporfin sodium solution, a chlorine photosensitizer, was used, along with a 663-nm red diode laser (86 mW/cm² for up to 600 s). The first trough of the measured action potential waveform corresponding to Na⁺ dynamics decreased exponentially with increasing PR duration. The average decay time of the exponential function from PR onset was 20.1 s. Marked morphological changes in the myocardial cells was observed after the PR. These results indicated that the behavior of the action potential waveform measured by the microelectrode array might be used as a less invasive method to evaluate the electrophysiological effects of a PR on myocardial cells.     

Myocardial necrosis depth prediction during extracellular photosensitization reaction of talaporfin sodium by defined index using fluorescence measurement

An application of photodynamic therapy for myocardial ablation, which would induce myocardial electrical conduction block, is proposed. For the proposed application, an extracellular photosensitization reaction (PR) is performed while photosensitizer is distributed in myocardial interstitial space by employing a short drug-light interval. Because the myocardial necrosis depth must be accurately controlled to prevent surrounding tissue injury during the myocardial ablation procedure, the necrosis depth during PR needs to be predicted. The purpose of this study is to investigate the availability of predicting PR-induced myocardial necrosis depth (d _nec) using a defined fluorescence-fall amount (FA), which is the calculated result of fluorescence intensity decrease from the start of the PR multiplied by irradiation duration and corresponds to photosensitizer consumption amount under an assumption that the photosensitizer consumption rate is faster than the photosensitizer supply rate. The correlation between FA and d _nec was experimentally investigated in vivo using an open-chested canine heart model with 2.5 and 5.0 mg/kg of talaporfin sodium at an irradiance of 5–20 W/cm² for 5–20 s. The fluorescence measurement was performed at a wavelength of 710 nm during the PR to derive FA. One week after the PR, a uniform necrosis depth was measured histopathologically as d _mnec. A logarithmic correlation between d _mnec and FA was confirmed with R ²?=?0.69–0.80 and a d _mnec range of 0.2–7.1 mm. The defined FA might be useful for predicting d _nec for the extracellular PR in myocardium when using talaporfin sodium.     

Development of a practical animal model of photodynamic therapy using a high concentration of extracellular talaporfin sodium in interstitial fluid: influence of albumin animal species on myocardial cell photocytotoxicity in vitro

Photodynamic reaction-induced photocytotoxicity using talaporfin sodium is inhibited by serum proteins binding to talaporfin sodium. The serum albumin binding site for talaporfin sodium differs among animal species. To identify a practical animal therapeutic model, we studied the ability of human, canine, bovine, and porcine albumin to influence talaporfin sodium-induced photocytotoxicity in rat myocardial cells in vitro. Human, canine, bovine, and porcine serum albumins were used. The ratio of talaporfin sodium binding, which is strongly associated with photocytotoxicity, was measured by ultrafiltration with an albumin concentration of 0.5–20 mg/ml and 20 μg/ml talaporfin sodium to mimic interstitial fluid. Rat myocardial cell lethality was measured by the WST assay 2 h after samples were exposed to a radiant exposure of 20 J/cm² by a red diode laser (Optical Fuel?, Sony, Tokyo, Japan) with a wavelength of 663 nm. The binding ratio dependence on albumin concentration differed among the animal species. Bovine albumin exhibited the largest difference from human albumin, with a maximum difference of 31% at 2 mg/ml albumin. The cell lethality characteristic was similar between human and canine albumin. The cell lethality dependence on albumin was not in the same order as the binding ratio. Cell lethality was lowest for human albumin with higher albumin concentrations between 5 and 20 mg/ml. There were no significant differences in cell lethality between bovine and porcine albumin and between human and canine albumin. We suggest that the canine model may be a useful animal therapeutic model for evaluating photodynamic therapy using a high concentration of the photosensitizer in the extracellular space.     

Killing tumor cells: the effect of photodynamic therapy using mono-L-aspartyl chlorine and NS-398

BACKGROUND: Photodynamic therapy (PDT) is a useful treatment for malignant tumors. PDT involves the administration of a photosensitive drug that is selected by neoplastic tissues and their vasculature. One such photosensitizer is mono-l-aspartyl chlorine e6 (NPe6). Recent evidence suggests that the presence of the cyclooxygenase-2 (COX-2) inhibitor NS-398 may potentiate the effect of photosensitizing agents. This study was designed to determine if the addition of NS-398 to NPe6-induced PDT in single or fractionated dosing would result in greater tumor kill. METHODS: Colon-38 tumor was subcutaneously implanted into both flanks of mice and allowed to grow to 0.5 to 1.0 cm. Mice were randomly allocated to 5 groups: (1) single dose of NPe6; (2) fractionated dose of NPe6; (3) NS-398 only; (4) single dose of NPe6 + NS-398; and (5) fractionated dose of NPe6 + NS-398. The left flank was shielded from exposure to irradiation. Tumor size was measured before initiation of PDT and at the time of sacrifice. RESULTS: The initial tumor weights of both flanks were not significantly different between all groups. Tumor weights at the time of death after PDT using NPe6 were significantly less than their paired tumors in the untreated flanks (P <0.0001). Tumor weights in the treated flanks were significantly less in the group receiving the fractionated dosing of NPe6 as compared to the single dose of NPe6 (P = 0.0037). NS-398 plus the single dose of NPe6 significantly decreased tumor weight in the PDT-treated flank (P = 0.035) at a level equivalent to that observed with fractionated dosing of the photosensitizer in the absence of NS-398. NS-398 did not significantly further decrease tumor weight in the group that received the fractionated dose of NPe6. CONCLUSIONS: Fractionated dosing of NPe6 demonstrated the best tumor kill. However, NS-398 did not potentiate the effect of PDT using fractionated dosing of NPe6. While PDT using the single NPe6 dose significantly decreased tumor weight, the addition of NS-398 potentiated the killing effect.     

阿仑膦酸钠对假体周围界膜组织分泌IL-6影响的实验研究

目的观察阿仑膦酸钠对假体周围界膜组织分泌IL-6的影响。方法无菌条件下从股骨分离界膜组织,并将其放入RPMI培养液中培养,取12孔培养板3块,分空白对照组、低浓度固邦组(10%)、高浓度固邦组(20%),培养72h,取上清液,酶联法测定IL-6。结果与对照组相比,低浓度和高浓度固邦组都能显著抑制界膜组织分泌IL-6(P<0.01)。结论阿仑膦酸钠能显著抑制假体周围界膜组织分泌IL-6。     

Effect of gossypol on the concentration of sodium and potassium in the rat epididymis

The effects of administration of gossypol acetic acid (7.5 mg/kg daily for 4 weeks) on the concentration of Na+ and K+ in the rat epididymis was assessed. Epididymal fluid samples, collected by micropuncture, from the caput, corpus, proximal cauda and distal cauda epididymis from gossypol-treated and control animals were analysed for Na+ and K+ concentrations. Gossypol-treated males failed to impregnate healthy females, presumably because their sperm were immotile. In gossypol-treated rats, Na+ levels decreased significantly (P less than 0.01) in the caput, corpus, proximal and distal cauda epididymis. In contrast, the K+ concentration was increased significantly (P less than 0.05) only in the caput and corpus epididymis. This altered electrolyte milieu may be responsible, to some extent, for immotility and hence infertility.     

复合头孢曲松钠和BMP的壳聚糖抗菌成骨生物陶瓷的制备实验研究

目的 使用壳聚糖和磷酸钙骨水泥(CPC)复合头孢曲松钠和BMP,制备出一种具有成骨和抗感染作用的植骨材料.方法 使用不同成分和比例的壳聚糖固化液将磷酸钙骨水泥、头孢曲松钠和BMP共混制备抗菌成骨生物陶瓷,绘制头孢曲松钠的紫外吸光度-浓度标准直线,测定生物陶瓷中头孢曲松钠体外释放浓度.微生物法测定生物陶瓷体外抑菌效果.生物陶瓷埋于大鼠腿部肌袋研究其成骨性能.将生物陶瓷填充大鼠污染骨缺损模型,研究其抑菌和成骨作用.结果 生物陶瓷的优化制备方案为0.1 g磷酸钙骨水泥复合10.4 mg头孢曲松钠和0.5 mg BMP与2.4 ml同化液C混合后在60℃、100%湿度下固化24 h.该生物陶瓷体外释放稳定,微生物测定结果表明头孢曲松钠在体外释放持续1周高于金黄色葡萄球菌最小抑菌浓度,达到了局部抗菌效果,组织切片显示8周后埋于大鼠腿部肌袋的生物陶瓷周围有骨组织产生,污染骨缺损实验中白细胞计数和组织切片显示实验组较对照组炎症反应轻微,成骨显著.结论 复合头孢曲松钠、壳聚糖和BMP的抗菌成骨生物陶瓷有望成为治疗污染性骨缺损的理想材料.     

在体外乳鼠心肌细胞缺氧的损伤及利多卡因的保护作用

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基质金属蛋白酶1组织抑制剂短发夹式RNA对肾小球系膜细胞凋亡及细胞外基质的影响

目的 构建靶向基质金属蛋白酶1组织抑制剂(TIMP-1)的短发夹RNA(shRNA)表达载体，并观察其对大鼠肾小球系膜细胞的凋亡和细胞外基质分泌的影响。方法 构建携带绿色荧光蛋白(EGFP)基因的RNA干扰真核表达载体pEGFP-1/TIMP-1 shRNA， 利用该载体介导两个TIMP-1 shRNA，分别为TIMP-1 shRNA1和TIMP-1 shRNA2。用荧光显微镜观察EGFP的表达。采用 RT-PCR和Western 印迹测定宿主细胞TIMP-1在基因和蛋白水平的沉默效果。用 ELISA检测细胞外基质成份的表达。以流式细胞仪检测细胞的凋亡情况。 结果 (1)测序证实成功构建了针对TIMP-1的shRNA表达载体pEGFP-1/TIMP-1 shRNA；(2)荧光显微镜下，转染组细胞内均见绿色荧光；未转染组未见绿色荧光；(3)基因和蛋白水平的检测显示，与阴性对照组相比，TIMP-1 shRNA1组的TIMP-1明显受抑制(P < 0.05)，TIMP-1 shRNA2组抑制效果不明显；(4)ELISA结果显示， 正常对照组FN、Ⅳ型胶原蛋白和 LN的表达不随时间的延长而变化；未转染组和阴性对照组在高糖诱导下， FN、Ⅳ型胶原蛋白和 LN的表达随时间的延长而增加；48、72 h的shRNA1组、抗体组和反义组对 FN、Ⅳ型胶原蛋白、LN表达明显低于阴性对照组，以shRNA1组最明显；(5)流式细胞仪结果显示，高糖诱导的各组细胞凋亡率随时间的延长而升高。在同一时间点shRNA1组细胞的凋亡率最高，反义组细胞的凋亡率次之，48 h和72 h的shRNA1组细胞的凋亡率与其他各组比较， 差异有统计学意义(P < 0.05)。 结论 pEGFP-1介导的TIMP-1 shRNA1能更加有效的抑制大鼠肾小球系膜细胞中TIMP-1的表达，促进肾小球系膜细胞的凋亡并使细胞外基质的成份表达下降。     

Effects of melatonin on diclofenac sodium treated rat kidney: a stereological and histopathological study

Abstract

Objective: In this study, we aimed to investigate the effect of diclofenac sodium (DS) and melatonin (MEL) on kidney of the prenatally administered rats. Materials and methods: Pregnant rats were divided into the control, physiological saline, DS, and DS?+?MEL groups. All injections were given beginning from the 5th day after mating to the 15th day of the pregnancy. Physical dissector and Cavalieri principle were used to estimate the numerical density and total number of glomeruli and the volumetric parameters of kidney, respectively. Results: Our stereological results indicated that DS application during the pregnancy lead to decrease in the mean volume, numerical density, and total number of the glomeruli (p?<?0.05). In addition, we determined that usage of the MEL with the DS caused increases in the mean volume, numerical density, and total number of the glomeruli (p?<?0.05). So, there was no significant difference in terms of the any parameter between the CONT and DS?+?MEL groups (p?>?0.05). Light microscopic investigation showed congestion in blood vessels and shrinkage of the Bowman's space in the DS group. Moreover, there was degeneration in nephrons including glomerulosclerosis and tubular defects, and an increase in the connective tissue in the kidneys of the DS-treated group. However, usage of the MEL with the DS caused preventing of these pathological alterations in the kidney. Discussion: We suggested that DS might lead to adverse effects in the kidneys of the rats that are prenatally subjected to this drug. Fortunately, these adverse effects can be prevented by the melatonin supplementation.     

戊聚硫钠对慢性非细菌性前列腺炎大鼠动物模型治疗的研究

目的:探讨戊聚硫钠(PPS)对慢性非细菌性前列腺炎(CNP)大鼠的治疗作用。方法:选取6月龄健康成年雄性SD大鼠80只,体重315~450 g,去势后用苯甲酸雌二醇0.25 mg/(kg.d)皮下注射,每日1次,连续注射30 d建模。成功建立CNP大鼠动物模型后,随机等分为安慰剂组和PPS治疗组,每组40只,分别用PPS、生理盐水灌胃治疗,连续8周后,处死动物,前列腺组织,HE染色,观察其病理变化。结果:建模后各组大鼠前列腺出现不同程度的慢性炎症改变和炎症细胞的浸润。PPS治疗组大鼠前列腺炎症较治疗前明显好转,而安慰剂组炎症未见好转。结论:PPS对大鼠CNP有一定治疗作用,其作用机制可能与PPS可修复大鼠前列腺受损的GAG层,抑制炎症的发生相关。     

阿仑膦酸钠治疗男性骨质疏松症临床研究

目的 探讨阿仑膦酸钠对男性骨质疏松症患者骨密度、血生化及骨标志物的影响.方法 选择2012年1月～2013年1月在我科门诊及住院50岁以上男性骨质疏松患者共169例,每人每天服用元素钙600 mg,活性维生素D0.25 μg作为基础补充剂,每周服用阿仑膦酸钠70 mg,共治疗12个月.观察骨密度、骨标志物等指标,骨密度测定采用双能X线吸收法,骨标志物测定采用酶联免疫吸附法.结果 研究结果显示,治疗1年后,L2、L3、L2～4、Neck、Ward's三角骨密度分别为0.791±0.150 g/cm2、0.817±0.149 g/cm2、0.827±0.154 g/cm2、0.875±0.153 g/cm2、0.703±0.138 g/cm2、0.522±0.133 g/cm2,均较治疗前0.772±0.144 g/cm2、0.800±0.156 g/cm2、0.861-±0.168 g/cm2、0.685±0.109 g/cm2、0.490 ±0.121 g/cm2有明显提高,差异具有统计学意义(P＜0.05),其他部位骨密度无明显差异(P＞0.05);治疗后血清CTX、BGP、BAP为0.20±0.11 ng/ml、7.73±4.11 ng/ml、14.57±7.20 ng/ml,较治疗前0.32±0.23 ng/ml、11.39±5.6 ng/ml、16.17±8.81 ng/ml显著降低,差异具有统计学意义(P＜0.05).结论 阿仑膦酸钠能有效降低破骨细胞活性,抑制骨破坏,显著提高骨量,对老年男性骨质疏松疗效显著.     

Death of rat sympathetic ganglion cells in vitro caused by neurite transection: effect of extracellular calcium

Calcium entry into neurons secondary to excitotoxic insults is believed to cause neuronal death after trauma and ischemia, but the role of calcium influx in neuronal death after neurite transection independent of excitotoxicity has not been clearly defined. This study assesses the effect of variations in extracellular calcium concentration ([Ca2+]e) from 50 nM to 5 mM on cell death, in 14-day-old cultures of dissociated sympathetic neurons from the superior cervical ganglia of newborn rats. The neurites were transected with a custom-made injury device, and cell death was assessed with propidium iodide and fluorescence microscopy. We found that neurite transection caused a significant increase (p < 0.05) in cell death at all [Ca2+]e studies, but there was no significant difference in mortality at the various [Ca2+]e. Cell death significantly increased between 2 and 24 h postinjury at all three [Ca2+]e. Cell death increased with decreasing distance between the cell body and the transection site, and there was a significant decrease in mortality at distances greater than 0.66 mm, irrespective of the [Ca2+]e. These results suggest that influx of extracellular calcium is not responsible for posttransection cell death, suggesting that calcium release from internal stores or calcium-independent cell death mechanisms are triggered by neurite transection.     

Therapeutic effects of sodium butyrate on glioma cells in vitro and in the rat C6 glioma model

OBJECTIVE: Preliminary in vitro studies have indicated that sodium butyrate inhibits the proliferation of cultured glioma cells and induces cellular differentiation, making it potentially useful as a therapeutic agent for patients with glioblastoma multiforme. The purpose of this study was to expand on the preliminary research by investigating the effects of sodium butyrate on multiple cell lines, explanted cells from glioblastoma tumor specimens, and in vivo in the rat C6 glioma brain tumor model. METHODS: Four malignant glioma cell lines (A-172, T98G, U118MG, and C6) and two primary cell cultures derived from human glioblastoma tumor specimens were treated with 2 mmol/L sodium butyrate for up to 72 hours. Sodium butyrate-induced effects on cell morphology, proliferation, cell cycle distribution, migration, glial fibrillary acidic protein staining, and S100beta protein content were determined. For in vivo studies, a total of 64 male Wistar-Furth rats underwent operations to implant C6 glioma cells stereotactically or were used as controls. The rats were treated with escalating doses of sodium butyrate by microinfusion with Alzet minipumps (Durect Corp., Cupertino, CA). RESULTS: Sodium butyrate treatment in vitro produced changes in morphology and glial fibrillary acidic protein expression indicative of cellular differentiation. In cell lines and explanted cells, sodium butyrate consistently inhibited glioblastoma cell proliferation (to 51 +/- 6% that of controls) and migration (to 46 +/- 17%). Intratumoral infusion of 40 mmol/L sodium butyrate prolonged the survival of Wistar-Furth rats with intracerebral C6 tumors (P = 0.013) without detectable toxicity. CONCLUSION: These data support further consideration of direct interstitial infusion of sodium butyrate in a Phase I clinical study for patients with recurrent glioblastoma multiforme.     

硫酸软骨素对牛磺胆酸钠诱导的大鼠离体胰腺细胞骨架损伤的影响

为探讨硫酸软骨素（CS）对牛磺胆酸钠诱导的大鼠离体胰腺腺泡细胞氧化应激损伤及细胞骨架蛋白F actin结构的影响。笔者将雄性Wistar大鼠36只，经分离提纯获取胰腺腺泡细胞悬液，牛磺胆酸钠处理离体胰腺细胞后，随机分3组即细胞模型（细胞骨架损伤）组，CS处理组和对照组。各组分别于30min，1h，3h进行MDA， GSH， SOD和ATP含量测定。离心细胞涂片后，以罗丹明-法罗丁F actin染色，在共聚焦激光显微镜下观察F actin结构的变化；用流式细胞术检测细胞的F actin蛋白含量。结果示,模型组GSH，SOD和ATP明显下降（P<0.05），MDA明显升高(P<0.05)。CS组GSH，SOD，ATP下降幅度小于模型组，差异有显著性（P<0.05）；MDA升高幅度小于模型组，差异有显著性（P<0.05）。模型组细胞骨架蛋白F actin解聚并弥漫分布于胞浆内，其蛋白含量持续下降（P<0.05）；CS组F actin结构较稳定，其蛋白水平明显高于模型组（P<0.05）。提示：牛磺胆酸钠诱导的离体胰腺细胞早期已存在内源性抗氧化物质的显著下降，脂质过氧化增加和ATP耗竭加重了细胞损伤。CS可通过减轻氧化应激损伤维持ATP含量，缓解F actin的降解，稳定细胞骨架结构从而减轻细胞损伤。     

Low-power laser biostimulation enhances nerve repair after end-to-side neurorrhaphy: a double-blind randomized study in the rat median nerve model

Previous studies have shown that low-power laser biostimulation (lasertherapy) promotes posttraumatic nerve regeneration. The objective of the present study was to investigate the effects of postoperative lasertherapy on nerve regeneration after end-to-side neurorrhaphy, an innovative technique for peripheral nerve repair. After complete transection, the left median nerve was repaired by end-to-side neurorrhaphy on the ulnar donor nerve. The animals were then divided into four groups: one placebo group, and three laser-treated groups that received lasertherapy three times a week for 3 weeks starting from postoperative day 1. Three different types of laser emission were used: continuous (808 nm), pulsed (905 nm), and a combination of the two. Functional testing was carried out every 2 weeks after surgery by means of the grasping test. At the time of withdrawal 16 weeks postoperatively, muscle mass recovery was assessed by weighing the muscles innervated by the median nerve. Finally, the repaired nerves were withdrawn, embedded in resin and analyzed by light and electron microscopy. Results showed that laser biostimulation induces: (1) a statistically significant faster recovery of the lesioned function; (2) a statistically significant faster recovery of muscle mass; (3) a statistically significant faster myelination of the regenerated nerve fibers. From comparison of the three different types of laser emissions, it turned out that the best functional outcome was obtained by means of pulsed-continuous-combined laser biostimulation. Taken together, the results of the present study confirm previous experimental data on the effectiveness of lasertherapy for the promotion of peripheral nerve regeneration and suggest that early postoperative lasertherapy should be considered as a very promising physiotherapeutic tool for rehabilitation after end-to-side neurorrhaphy.