硫化氢后处理对大鼠缺氧-复氧心肌细胞的保护作用

目的探讨硫化氢(H2S)后处理对缺氧-复氧损伤成年大鼠心肌细胞的影响,评价其心肌保护作用及其可能的机制。方法分离成年SD大鼠心肌细胞,随机均分为四组:正常组(N组)、缺氧-复氧组(HR组)、缺氧后处理组(IPTC组)和H2S后处理组(S组),在激光扫描共聚焦显微镜下检测F-肌动蛋白/G-肌动蛋白荧光强度及Western blot技术检测各组心肌细胞p38MAPK磷酸化(p-p38MAPK)水平;再将上述四组又分为加或不加细胞松弛素D(CyD)两个亚组,激光扫描共聚焦显微镜下检测细胞内Ca2+、pH值荧光强度。结果 HR组、IPTC组和S组F/G-肌动蛋白明显高于N组(P0.05);IPTC组和S组F/G-肌动蛋白明显高于HR组(P0.05),且S组明显高于IPTC组(P0.05)。与无CyD处理比较,CyD处理时四组Ca2+荧光强度明显升高(P0.05);N组与HR组pH荧光强度升高,IPTC组与S组pH荧光强度降低。无CyD处理时N组、IPTC组和S组Ca2+荧光强度明显低于、pH值荧光强度明显高于HR组(P0.05);CyD处理后HR组、IPTC组和S组Ca2+荧光强度明显高于,IPTC组和S组pH值荧光强度明显低于N组(P0.05)。心肌细胞内HR组p-p38MAPK水平明显高于N组、IPTC组和S组(P0.05)。结论硫化氢后处理可以促进F-肌动蛋白的重塑,稳定缺血缺氧成年大鼠心肌细胞内环境;硫化氢后处理可降低缺血缺氧成年大鼠心肌细胞p38MAPK磷酸化水平。     

骨髓c-Kit+Lin-细胞移植改善肝纤维化的实验研究

目的:探讨骨髓c-Kit+Lin-(CD117)细胞在体外扩增后移植治疗小鼠肝纤维化的效果。方法:采用免疫磁珠分选法分离小鼠骨髓CD117细胞,将细胞在添加了肝细胞生长因子(HGF),干细胞因子(SCF),FLt-3配基(FL),促血小板生成素(TPO),白血病抑制因子(LIF)的无血清培养基中培养扩增;制备同种小鼠的酒精性肝纤维化模型,成模后的小鼠分别尾静脉注射体外扩增的CD117细胞(移植组)或等体积缓冲液(模型组),同时以正常小鼠为对照(正常组),30 d后观察各组小鼠的肝脏病理改变以及肝功能与肝纤维化指标。结果:添加上述细胞因子的无血清培养基能有效扩增CD117细胞;正常组小鼠肝脏无病理改变,模型组呈明显的肝细胞变性和纤维化改变,移植组较模型组的病变程度明显减轻;与正常组比较,模型组和移植组均有不同程度的血清谷丙转氨酶(ALT),谷草转氨酶(AST),透明质酸(HA),层黏连蛋白(LN)升高和清蛋白(ALB)降低,但移植组上述指标改变较模型组明显减轻,差异均有统计学意义(均P<0.05)。结论:合适剂量的HGF+SCF+FL+TPO+LIF细胞因子组合能有效扩增CD117细胞;CD117细胞移植对小鼠肝纤维化有治疗作用。     

Phalloidin对钙离子载体A23187诱导的人精子顶体反应的作用

目的:探讨F-肌动蛋白在人精子顶体反应(AR)过程中的重要作用及可能机制。方法:将每例标本分为A、B、C、D、E 5组,共30例。以链球菌溶血素O(SLO)、Phalloid in、钙离子载体A23187分别处理各实验组人精子,其中A:A23187 3μmol/L;B:Phalloid in 40μmol/L+A23187 3μmol/L;C:SLO 0.5 U/m l+A23187 3μmol/L;D:SLO0.5 U/m l+Phalloid in 40μmol/L+A23187 3μmol/L;E:不添加任何试剂。分别采用罗丹明标记山豌豆凝集素(Rhodam ine-PSA,10μg/m l)荧光染色检测其AR,计算各组AR发生率(AR%)。结果:在5个实验组中,除A组与B组比较差异无显著性(P>0.05)外,其余各组两两比较差异均具有显著的统计学意义(P<0.01)。结论:Phalloid in对质膜完整的人精子AR发生率无影响。而在添加SLO的人精子模型中,Phalloid in较对照组能显著降低人精子AR发生率,提示F-肌动蛋白的聚合作用对人精子AR的发生具有重要意义,并且主要发生在精子顶体的内部。     

JMJD3在顺铂致急性肾损伤小鼠肾纤维化中的作用

目的:评价含十字形结构域蛋白3(JMJD3)在顺铂致急性肾损伤小鼠肾纤维化中的作用。方法:健康C57BL/6雄性小鼠48只,8~10周龄,体重20~30 g,采用随机数字表法分为4组( n＝12)：对照组(CON组)、对照+ JMJD3抑制剂组(CON-A组)、顺铂组(CIS组)和顺铂+ JMJD3抑制剂组...     

mTOR/HIF-1α信号通路在布-加综合征肝纤维化中的作用机制研究

目的:探讨mTOR/HIF-1α信号通路在布-加综合征(B-CS)肝纤维化中的作用机制。方法:雄性C57小鼠20只按随机数字表法分为假手术(Sham)组、假手术+雷帕霉素(Sham+Ra)组、B-CS组、B-CS+雷帕霉素(B-CS+Ra)组,每组均5只。于肝后段下腔静脉(IVC)部分结扎构建B-CS小鼠模型;Sham...     

氯离子转运蛋白NKCC1在老年小鼠术后认知功能障碍中的作用

目的 探讨氯离子转运蛋白钠离子钾离子氯共转运体1(NKCCl)在老年小鼠术后认知功能障碍(POCD)中的作用。方法 选择老年雄性C57BL/6小鼠40只,18～20月龄,体重28～32 g。将小鼠随机分为四组：对照+生理盐水组(N组),对照+布美他尼(NKCC1抑制剂)组(B组),POCD模型+生理盐水组(M组)和POCD模型+布美他尼组(MB组),每组10只。采用异氟醚麻醉和剖腹探查术建立POCD动物模型,于术后3～7 d行旷场与条件恐惧实验评估认知功能;行为学结束后采用Western blot法检测海马组织NKCC1、钾离子氯离子共转运体2(KCC2)、脑源性神经营养因子(BDNF)及突触后密度蛋白95(PSD95)的蛋白含量,免疫组织荧光检测海马组织CA1、CA3、DG区NKCC1免疫荧光强度。结果 与N组比较,M组场景僵直时间明显缩短(P<0.05),海马NKCC1蛋白含量明显升高(P<0.05),BDNF、PSD95蛋白含量明显降低(P<0.05),海马组织CA1、CA3、DG区NKCC1免疫荧光强度明显增强(P<0.05)。N组和B组各指标差异均无统...     

CXC趋化因子受体6在小鼠心脏移植排斥反应中的表达及意义

目的 研究CXC趋化因子受体6(CXCR6)在同种异体小鼠心脏移植中的表达及CXC趋化因子配体16(CXCL16)与CXCR6相互作用对移植物存活时间的影响.方法 以野生型Balb/c小鼠(H-2d)为供者(同种移植组),或以野生型C57BL/6小鼠(H-2b)为供者(同系移植组),以野生型C57BL/6小鼠为受者分别行小鼠腹腔异位心脏移植.测定同系和同种移植组小鼠移植心脏CXCR6mRNA的表达,并测定受者脾脏CD8+T淋巴细胞CXCR6的表达.另制作小鼠同种异位心脏移植模型(Balb/c小鼠为供者,C57BL/6小鼠为受者),将其分为实验组和对照组,实验组受者移植当天至发生排斥反应时腹腔注射抗CXCL16抗体,对照组受者同期注射对照抗体.记录两组移植心脏存活时间.进行CD8+T淋巴细胞的细胞毒试验,即用Balb/c小鼠脾细胞免疫C57BL/6小鼠后,获取C57BL/6小鼠脾脏CD8+T淋巴细胞,将Balb/c小鼠脾细胞与C57BL/6小鼠CD8+T淋巴细胞混合培养,分别加入抗CXCL16抗体、小鼠IgG(对照抗体)和抗CD40L抗体.结果 同种移植组移植心脏中CXCR6 mRNA的表达以及脾脏CD8+T淋巴细胞上CXCR6的表达均高于同系移植组和正常对照组.抗CXCL16抗体对CD8+T淋巴细胞的细胞毒活性无影响.与对照组相比较,实验组小鼠移植心脏存活时间并未明显延长.结论 小鼠心脏移植排斥反应中CD8+T淋巴细胞CXCR6的表达上升,阻断CXCL16/CXCR6相互作用并不能延长移植心脏的存活时间.     

CXC趋化因子受体6在小鼠心脏移植排斥反应中的表达及意义

目的 研究CXC趋化因子受体6(CXCR6)在同种异体小鼠心脏移植中的表达及CXC趋化因子配体16(CXCL16)与CXCR6相互作用对移植物存活时间的影响.方法 以野生型Balb/c小鼠(H-2d)为供者(同种移植组),或以野生型C57BL/6小鼠(H-2b)为供者(同系移植组),以野生型C57BL/6小鼠为受者分别行小鼠腹腔异位心脏移植.测定同系和同种移植组小鼠移植心脏CXCR6mRNA的表达,并测定受者脾脏CD8+T淋巴细胞CXCR6的表达.另制作小鼠同种异位心脏移植模型(Balb/c小鼠为供者,C57BL/6小鼠为受者),将其分为实验组和对照组,实验组受者移植当天至发生排斥反应时腹腔注射抗CXCL16抗体,对照组受者同期注射对照抗体.记录两组移植心脏存活时间.进行CD8+T淋巴细胞的细胞毒试验,即用Balb/c小鼠脾细胞免疫C57BL/6小鼠后,获取C57BL/6小鼠脾脏CD8+T淋巴细胞,将Balb/c小鼠脾细胞与C57BL/6小鼠CD8+T淋巴细胞混合培养,分别加入抗CXCL16抗体、小鼠IgG(对照抗体)和抗CD40L抗体.结果 同种移植组移植心脏中CXCR6 mRNA的表达以及脾脏CD8+T淋巴细胞上CXCR6的表达均高于同系移植组和正常对照组.抗CXCL16抗体对CD8+T淋巴细胞的细胞毒活性无影响.与对照组相比较,实验组小鼠移植心脏存活时间并未明显延长.结论 小鼠心脏移植排斥反应中CD8+T淋巴细胞CXCR6的表达上升,阻断CXCL16/CXCR6相互作用并不能延长移植心脏的存活时间.     

红景天苷对低氧环境下大鼠阴茎海绵体平滑肌细胞α-肌动蛋白和骨桥蛋白表达的影响

目的:探讨红景天苷对低氧SD大鼠阴茎海绵体平滑肌细胞(CCSMC)α-肌动蛋白、骨桥蛋白(OPN)表达的影响。方法:体外培养SD大鼠CCSMC,免疫组化法鉴定细胞;设正常对照组(21%O2浓度)、低氧组(1%O2浓度)、低氧+红景天苷1 mg/L组、低氧+红景天苷3 mg/L组、低氧+红景天苷5 mg/L组、低氧+前列腺素E1(PGE1)0.4μg/L组,分别培养48 h;RT-PCR法分别测定各组α-肌动蛋白、OPN的相对表达量。结果:体外培养的CCSMC生长良好,抗平滑肌α-肌动蛋白单克隆抗体免疫组化染色阳性;与正常对照组比较,低氧组细胞的α-肌动蛋白表达量下降、OPN表达量升高(P<0.01);与低氧组比较,红景天苷5 mg/L组α-肌动蛋白表达量升高、OPN表达量降低(P<0.01),与PGE1作用相当(P﹥0.05)。结论:低氧可引起SD大鼠CCSMC收缩型标志物α-肌动蛋白表达降低,合成型标志物OPN表达升高,推测低氧可能引起CCSMC由收缩型向合成型转化。红景天苷能抑制低氧引起的CCSMCα-肌动蛋白表达降低、OPN表达升高,浓度为5 mg/L时与PGE1作用几乎相当。     

CXC趋化因子受体6在小鼠心脏移植排斥反应中的表达及意义

目的 研究CXC趋化因子受体6(CXCR6)在同种异体小鼠心脏移植中的表达及CXC趋化因子配体16(CXCL16)与CXCR6相互作用对移植物存活时间的影响.方法 以野生型Balb/c小鼠(H-2d)为供者(同种移植组),或以野生型C57BL/6小鼠(H-2b)为供者(同系移植组),以野生型C57BL/6小鼠为受者分别行小鼠腹腔异位心脏移植.测定同系和同种移植组小鼠移植心脏CXCR6mRNA的表达,并测定受者脾脏CD8+T淋巴细胞CXCR6的表达.另制作小鼠同种异位心脏移植模型(Balb/c小鼠为供者,C57BL/6小鼠为受者),将其分为实验组和对照组,实验组受者移植当天至发生排斥反应时腹腔注射抗CXCL16抗体,对照组受者同期注射对照抗体.记录两组移植心脏存活时间.进行CD8+T淋巴细胞的细胞毒试验,即用Balb/c小鼠脾细胞免疫C57BL/6小鼠后,获取C57BL/6小鼠脾脏CD8+T淋巴细胞,将Balb/c小鼠脾细胞与C57BL/6小鼠CD8+T淋巴细胞混合培养,分别加入抗CXCL16抗体、小鼠IgG(对照抗体)和抗CD40L抗体.结果 同种移植组移植心脏中CXCR6 mRNA的表达以及脾脏CD8+T淋巴细胞上CXCR6的表达均高于同系移植组和正常对照组.抗CXCL16抗体对CD8+T淋巴细胞的细胞毒活性无影响.与对照组相比较,实验组小鼠移植心脏存活时间并未明显延长.结论 小鼠心脏移植排斥反应中CD8+T淋巴细胞CXCR6的表达上升,阻断CXCL16/CXCR6相互作用并不能延长移植心脏的存活时间.     

丰富环境对新生小鼠暴露于七氟醚所致微清蛋白中间神经元表型缺失及认知功能损伤的影响

目的观察微清蛋白(parvalbumin,PV)在七氟醚麻醉致小鼠认知功能损害中的表达变化,并研究丰富环境是否能改善此种变化。方法 6日龄新生雄性C57BL/6小鼠144只随机均分为四组(n=36):对照+标准环境组(CS组)、对照+丰富环境组(CE组)、七氟醚+标准环境组(SS组)及七氟醚+丰富环境组(SE组)。SS及SE组分别在出生后第6、7、8天每天接受1次2h的3%七氟醚麻醉+30%氧气;CS及CE组在相应日龄吸入30%氧气。从P8到P90,CE及SE组小鼠每天接受2h丰富环境饲养,CS及SS组则在标准环境下饲养。小鼠于P42行旷场实验,于P42-43和P89-90分别行场景条件性恐惧实验。并于P9、P14、P42、P60及P90取小鼠皮层及海马组织,采用Western blot检测小鼠额叶皮层及海马中微清蛋白的表达。结果旷场实验测试中,四组探索路程和中央格停留时间差异无统计学意义。场景性恐惧实验测试中,SS组P43僵直反应时间百分比明显低于CS组(P0.05),但在P90差异无统计学意义;在P9和P14,SS组前额皮层及海马中PV表达明显低于CS组(P0.01),但在P60、P90恢复正常;在P42,SE组前额皮层及海马中PV表达明显高于SS组(P0.05);线性回归分析表明:小鼠前额皮层中PV表达量与环境诱导僵直时间成显著正相关(r=0.670 7,P=0.000 1),小鼠海马中PV表达量与环境诱导僵直时间成显著正相关(r=0.509 6,P=0.001 9)。结论七氟醚暴露(P6-P8)可致小鼠认知功能损伤伴随前额皮层和海马PV表达降低,丰富环境可减轻此种损伤。     

成年小鼠七氟醚麻醉手术后海马突触结合蛋白-Ⅰ表达的变化

目的 评价突触结合蛋白-Ⅰ(synaptotagmin-Ⅰ,syt-Ⅰ)在七氟醚麻醉的成年小鼠海马中的表达变化. 方法 成年雄性C57BL/6小鼠120只,16周龄,体重(21.2±2.2)g,采用随机数字表法分为4组(每组30只):吸氧+生理盐水+戊巴比妥钠组(C组)、吸氧+生理盐水+腹腔探查术+戊巴比妥钠组(S组)、七氟醚麻醉+腹腔探查术+生理盐水组(SS组)和七氟醚+腹腔探查术+地塞米松组(SD组).麻醉前1h,SD组腹腔注射地塞米松(2 mg/kg),余3组腹腔注射等量生理盐水.C组、S组1.5％戊巴比妥钠腹腔注射麻醉,SS组、SD组3.4％～3.6％七氟醚麻醉,S组、SS组、SD组行腹腔探查术.麻醉手术结束24 h后,水迷宫训练后测定各组的潜伏期、穿越平台次数、目标象限游泳时间.水迷宫测试结束后2h进行条件恐惧训练,条件恐惧训练结束24 h后进行场景恐惧和条件恐惧测试.行为学测试结束后取小鼠海马组织,Western blot检测syt-Ⅰ、IL-1β、S100A8含量,RT-PCR检测S100A8、Toll样受体4(toll-like receptor 4,TLR4)、IL-6、IL-1β、TNF-α,mRNA含量. 结果 与C组比较,S组和SS组第3、4、5天潜伏期延长、穿越平台次数减少、目标象限游泳时间缩短、场景恐惧时间下降(P均＜0.05);与SS组比较,SD组潜伏期缩短、穿越平台次数增加、目标象限游泳时间增加、场景恐惧时间增加(P均＜0.05).4组条件恐惧僵直反应差异无统计学意义(P＞0.05).与C组比较:S组和SS组IL-1β和S100A8蛋白及其mRNA表达增加,TNF-α mRNA表达增加(P＜0.05);SS组syt-Ⅰ及TLR4 mRNA含量降低,IL-1β和S100A8蛋白及其mRNA表达增加(P＜0.05). 结论 syt-Ⅰ表达下降参与了七氟醚麻醉手术后成年小鼠认知功能障碍.     

IL-17A对脂多糖致老年大鼠早期中枢炎症和场景性恐惧实验的作用

目的探讨IL-17A对脂多糖(LPS)致老年大鼠早期中枢炎症和恐惧实验的影响。方法雄性SD大鼠70只,18月龄,首先取30只大鼠随机均分为五组:腹腔注射生理盐水(生理盐水组,A组)、腹腔注射LPS 500μg/kg 6h组(B组)、12h组(C组)、24h组(D组)、48h组(E组)。检测LPS注射后各组大鼠海马IL-17A的表达。随后,将剩余40只大鼠随机均分为四组:空白对照组(O组)、IL-17A抗体组(P组)、LPS腹腔注射组(Q组)、IL-17A抗体+LPS腹腔注射组(R组)。P组和R组大鼠侧脑室给予IL-17A抗体3μl(200μg/μl),O组和Q组给予同体积生理盐水;30min后,Q组和R组大鼠腹腔注射LPS(500μg/kg),O组和P组给予同体积生理盐水,24h后各组行场景性恐惧实验,记录四组大鼠的僵直时间,检测海马TNF-α和IL-6水平及CA1区Iba1阳性细胞的表达。结果 B、C和D组大鼠海马中IL-17A的表达明显高于A组(P0.01),E与A组IL-17A的表达差异无统计学意义;Q组和R组大鼠僵直反应时间明显短于O组(P0.05或P0.01),R组大鼠僵直反应时间明显长于Q组(P0.01);Q组和R组大鼠海马TNF-α和IL-6的水平明显高于O组(P0.01),R组大鼠海马TNF-α和IL-6水平明显低于Q组(P0.01);Q组和R组大鼠海马CA1区Iba1阳性细胞数目明显多于O组,R组大鼠海马CA1区Iba1阳性细胞数目明显少于Q组(P0.05)。结论 IL-17A参与LPS引起的老年大鼠早期中枢炎症因子TNF-α和IL-6的表达、小胶质细胞的活化以及场景性恐惧实验的僵直时间改变。     

The effect of three inhaled anesthetics in mice harboring mutations in the GluR6 (kainate) receptor gene

Combinations of GluR5-GluR7, KA1, and KA2 subunits form kainate receptors, a subtype of excitatory ionotropic glutamate receptors. Isoflurane enhances the action of kainate receptors comprising GluR6 subunits expressed in oocytes. To test whether alterations of the GluR6 subunit gene affect the actions of inhaled anesthetics in vivo, we measured the minimum alveolar concentration of desflurane, isoflurane, and halothane in mice lacking the kainate receptor subunit GluR6 (GluR6 knockout mice) and mice with a dominant negative glutamine/arginine (Q/R) editing mutation in membrane domain 2 of the GluR6 receptor (GluR6 editing mutants), which increases the calcium permeability of kainate receptors containing GluR6Q. We also measured the capacity of isoflurane to interfere with Pavlovian fear conditioning to a tone and to context. Absence of the GluR6 subunit did not change the minimum alveolar concentration of isoflurane, desflurane, or halothane. Possibly, kainate receptors assembled from the remaining kainate receptor subunits compensate for the absent subunits and thereby produce a normal minimum alveolar concentration. A Q/R mutation that dominantly affects kainate receptors containing the GluR6 subunit in mice increased isoflurane minimum alveolar concentration (by 12%; P < 0.01), decreased desflurane minimum alveolar concentration (by 18%; P < 0.001), and did not change halothane minimum alveolar concentration (P = 0.25). These data may indicate that kainate receptors containing GluR6Q subunits differently modulate, directly or indirectly, the mechanism by which inhaled anesthetics cause immobility. The mutations of GluR6 that were studied did not affect the capacity of isoflurane to interfere with fear conditioning.     

单次短期七氟醚暴露联合母婴分离对新生大鼠远期认知功能的影响

目的 探讨单次短期七氟醚暴露联合母婴分离对新生大鼠发育期大脑远期认知功能的影响及可能机制。方法 选择出生后第6天的雄性SD新生大鼠64只。采用随机数字法将新生大鼠分为五组：对照组(C组,n=13)、七氟醚组(S组,n=13)、母婴分离组(M组,n=12)、七氟醚+母婴分离组(SM组,n=13)和布美他尼组(SMB组,n=13)。C组自由饮水饮食。S组于出生后第6天暴露于2.1%七氟醚1 h。M组于出生后第10天母婴分离3 h。SM组于出生后第6天暴露于2.1%七氟醚1 h,并于出生后第10天母婴分离3 h。SMB组于出生后第6天七氟醚暴露前腹腔注射Na⁺-K⁺-2Cl^-同向转运蛋白(NKCC1)抑制剂布美他尼1.82 mg/kg,后暴露于2.1%七氟醚1 h,于出生后第10天母婴分离3 h。于大鼠出生后第44天行旷场实验,记录运动总距离和穿越中央格次数。出生后第54或55天行新物体识别实验,记录大鼠对新物体和旧物体的探索时间,计算辨别指数。出生后第84～86天行场景性条件性恐惧实验,记录场景性恐惧测试阶段和条件性恐惧测试...     

Determination of the EC50 amnesic concentration of etomidate and its diffusion profile in brain tissue: implications for in vitro studies

BACKGROUND: Etomidate is a widely used general anesthetic that has become a useful tool to investigate mechanisms of anesthetic action in vivo and in brain slices. However, the free aqueous concentration of etomidate that corresponds to amnesia in vivo and the diffusion profile of etomidate in brain slices are not known. METHODS: The authors assessed the effect of intraperitoneally injected etomidate on contextual fear conditioning in mice. Etomidate concentrations in brain tissue were obtained by high-performance liquid chromatography. Uptake studies in 400-microm-thick brain slices were used to calculate the diffusion and partition coefficients of etomidate. A diffusion model was used to calculate the expected concentration profile within a brain slice as a function of time and depth. The predicted rate of drug equilibration was compared with the onset of electrophysiologic effects on inhibitory circuit function in recordings from hippocampal brain slices. RESULTS: Etomidate impaired contextual fear conditioning with an ED50 dose of 11.0+/-0.1 mg after intraperitoneal injection, which corresponded to an EC50 brain concentration of 208+/-9 ng/g. The brain:artificial cerebrospinal fluid partition coefficient was 3.35, yielding an EC50,amnesia aqueous concentration of 0.25 microm. The diffusion coefficient was approximately 0.2x10 cm/s. The development of etomidate action in hippocampal brain slices was compatible with the concentration profile predicted by this diffusion coefficient. CONCLUSIONS: The free aqueous concentration of etomidate corresponding to amnesia, as defined by impaired contextual fear conditioning in mice, is 0.25 microM. Diffusion of etomidate into brain slices requires approximately an hour to reach 80% equilibration at a typical recording depth of 100 microm. This information will be useful in designing and interpreting in vitro studies using etomidate.     

Organization of cytoskeletal F-actin, G-actin, and gelsolin in the adhesion structures in cultured osteoclast.

Immunofluorescence using Gc protein (group-specific component or vitamin D binding protein [DBP]) as a marker of G-actin showed that nonfilamentous, monomeric G-actin is a component of the podosomes of osteoclasts cultured on glass plates or bone slices. Typical individual podosomes of the well-spread cells on glass plates were rosette in form. When viewed from the basolateral surface, the core portion of the dotlike podosomes was associated with packed F-actin filaments surrounded by G-actin organized in a ringlike structure. The podosomes, when viewed perpendicular to the substrate, showed a conical shape as a bundle of short F-actin core and a ring of G-actin. With cell spreading on glass plates, the clustering of the podosomes formed a continuous belt of tightly packed podosomes as an adhesion structure at the paramarginal area. In addition, these structures were seen on the ventral cell surface. Similar changes in cell shape were seen in the osteoclasts when they were plated on bone slices. With the loss of dotlike podosomes, a continuous band of F-actin was formed around the resorption lacunae. It became evident then that F- and G-actin dissociated from each other in the podosomes. The staining patterns of G-actin varied from a discrete dot to a diffuse one. Toward the nonresorption phase, the osteoclasts lost their continuous F-actin band but dotlike podosomes appeared in the leading and the trailing edges. In such a cell undergoing translational movements, G-actin was located diffusely in the cytoplasm behind the lamellipodia and along some segments of the leading edge. Cytochalasin B treatment caused cells to disorganize the actin cytoskeletal architecture, which indicated the disassembling of F-actin into G-actin in podosomes and disappearance of actin-ring of cultured osteoclasts. Staining with polyclonal actin antibody or monoclonal beta-actin was overlapped with the distribution pattern of G- and F-actin. Gelsolin was detected in the region of the adhesion area corresponding to the podosome. The observation that F-actin, G-actin, and gelsolin were detected in the osteoclastic adhesion structures suggests that the podosomes may represent sites where a rapid polymerization/depolymerization of actin occurs. These dynamic changes in cytoskeletal organization and reorganization of G-actin may reflect changes in the functional polarization of the osteoclast during the bone resorption cycle and suggest the important role of G-actin in the regulation of osteoclast adhesion.     

Determination of the EC50 Amnesic Concentration of Etomidate and Its Diffusion Profile in Brain Tissue: Implications for In Vitro Studies

Background: Etomidate is a widely used general anesthetic that has become a useful tool to investigate mechanisms of anesthetic action in vivo and in brain slices. However, the free aqueous concentration of etomidate that corresponds to amnesia in vivo and the diffusion profile of etomidate in brain slices are not known.

Methods: The authors assessed the effect of intraperitoneally injected etomidate on contextual fear conditioning in mice. Etomidate concentrations in brain tissue were obtained by high-performance liquid chromatography. Uptake studies in 400-[mu]m-thick brain slices were used to calculate the diffusion and partition coefficients of etomidate. A diffusion model was used to calculate the expected concentration profile within a brain slice as a function of time and depth. The predicted rate of drug equilibration was compared with the onset of electrophysiologic effects on inhibitory circuit function in recordings from hippocampal brain slices.

Results: Etomidate impaired contextual fear conditioning with an ED50 dose of 11.0 +/- 0.1 mg after intraperitoneal injection, which corresponded to an EC50 brain concentration of 208 +/- 9 ng/g. The brain:artificial cerebrospinal fluid partition coefficient was 3.35, yielding an EC50,amnesia aqueous concentration of 0.25 [mu]m. The diffusion coefficient was approximately 0.2 x 10-6 cm2/s. The development of etomidate action in hippocampal brain slices was compatible with the concentration profile predicted by this diffusion coefficient.     

Effects of lipopolysaccharide on actin reorganization and actin pools in endothelial cells

Objective: To investigate the dose and time dependent effects of lipopolysaccharide ( LPS ) on cytoskeletal F-acitn and G-actin reorganizations by visualizing their distribution and measuring their contents in human umbilical vein endothelial cell line ECV-304. Methods: F-actin was labeled with rhodamine-phalloidin and G-actln with deoxyribonuclease I (DNase I)conjugated with fluorescein isothiocyanate ( FITC ). Contents of cytoskeletal proteins were obtained by flow cytometry. Results: F-actin was mainly distributed peripherally in endothelial ceils under normal conditions. LPS stimulation caused the formation of stress fibers and fdopodia. G-actin was normally seen in perinuclear and nuclear areas in control ECV-304 cells. Under LPS stimulation, G-actin dots appeared in the cytoplasmic region. The actin disorganization was accompanied by the time-and dose-dependent decrease in F-actin pool and increase in G-actin pool. Conclusions: LPS can induce characteisic morphological alterations of actin cytoskeleton and formation of intercellular gap in endothefial cells, accompanied by changes in F-actin and G-actin pools.