腺相关病毒介导人骨形态发生蛋白7基因体外转染脂肪源性干细胞的研究

目的构建人骨形态发生蛋白7(hBMP7)基因重组腺相关病毒载体并观察其在脂肪源性干细胞(ADAS cells)中的表达,为骨组织工程探索新的基因治疗方法和细胞来源。方法以pcDNA1．1／Amp-hBMP7质粒为模板扩增,将回收的PCR产物片段克隆入pUC18载体,获得重组质粒pUC18-hBMP7。双酶切质粒pUC18-hBMP7、pSNAV,回收的两片段转化大肠杆菌DH5α感受态细胞,获得重组质粒PSNAV-hBMP7。用重组Ⅰ型单纯疱疹病毒HSV1-rc／△UL2感染BHK-21细胞,裂解细胞收获病毒液。采用“氯仿处理-PEG／NaCl沉淀-氯仿抽提”三个步骤分离浓缩和纯化,测定病毒滴度。培养大鼠ADAS cells,并检测细胞表面标记物。在体外分别转染rAAV2-hBMP7和rAAV2-GFP,流式细胞仪、Western-blot方法检测转染基因的表达。结果PCR、酶切鉴定以及测序分析表明,BMP7基因成功克隆入质粒pSNAV-hBMP7载体中,并在BHK21细胞中成功包装出具有感染活性的重组腺相关病毒载体rAAV2-hBMP7。早期对ADAS cells的转染效率可达90％, Western-Blot方法检测到第1周与第8周的转染组细胞均有蛋白水平的表达。结论成功构建了hBMP7基因重组腺相关病毒载体和培养出大鼠ADAS cells,rAAV2-hBMP7载体在体外可高效转染大鼠ADAS cells。     

重组人骨形成蛋白4基因腺相关病毒载体的构建

目的 构建重组人骨形成蛋白4（human bone morphogenetie protein4，hBMP-4）基因腺相关病毒载体。方法 根据hBMP4的基因序列设计引物，上游引入EcoRⅠ位点，下游引入Sal Ⅰ位点，以EX-A0242M01hBMP4为模板扩增目的基因。将hBMP-4基因克隆入pUC18载体中，获得重组质粒pUCl8hBMP-4。然后用EcoRⅠ与Sal Ⅰ酶切pUC18-hBMP-4及质粒pSNAV，回收酶切片段，T4DNA连接酶16C过夜，转化大肠杆菌DH5a感受肽细胞，筛选阳性克隆获得pSNAV-hBMP-4，EcoRⅠ／SalⅡ双酶切鉴定，并行全基因测序。转染BHK21细胞，G418筛选培养，获得抗药克隆细胞株。HsV1-rc／△UL2感染，包装此细胞株并收获病毒载体AAV-hBMP-4。行DNA点杂交法测定病毒滴度，SDS-PAGE分析判断病毒纯度。结果 PCR电泳及酶切鉴定表明，pSNAV-hBMP-4重组成功，基因测序显示装入pSNAV质粒中的hBMP-4基因正确；AAV-hBMP-4病毒的大致滴度为1．5×10^12μg／ml，分泌蛋白浓度为0．124mg／ml，病毒载体纯度在95％以上。结论 实验获得的病毒载体滴度高、感染性好，可以满足骨组织工程的需要。     

转化生长因子β1基因转染兔颞下颌关节滑膜间充质干细胞及其表达

目的 构建TGF-β1基因的重组腺相关病毒载体并将其转入兔颞下颌关节滑膜间充质干细胞(SMSCs)中,对转染后外源性TGF-β1 mRNA和蛋白的表达进行检测.方法 含TGF-β1全长cDNA的质粒pCMV6-XL4和质粒pAAV-MCS用EcoR Ⅰ+Xba Ⅰ进行双酶切后连接,转化大肠杆菌DHSα感受态细胞,获得重组质粒pAAV-MCS-TGF-β1.通过酶切和DNA测序鉴定重组质粒的正确性.采用磷酸钙共沉淀法,以重组质粒pAAV-MCS-TGF-β1和pAAV-RC、pHelper共转染AAV-293细胞,产生具有传染性的病毒颗粒;斑点杂交方法检测重组病毒的滴度,并转染体外培养的SMSCs.以逆转录一聚合酶链反应(RT-PCR)、Western blot分别检测目的 基因及蛋白的表达.结果 成功地构建TGF-β1基因腺相关病毒重组质粒,病毒滴度约为3.6×1013vp/ml,感染的SMSCs能稳定、高效地表达外源性目的 基因及蛋白.结论 重组腺相关病毒载体rAAV-TGF-β1能有效感染SMSCs并表达目的基因.     

改良法构建Ⅱ型重组腺相关病毒介导人TIMP1基因及鉴定

目的改良法构建携带金属蛋白酶抑制剂1（TIMP1）基因的Ⅱ型重组腺相关病毒（rAAV2）。方法采用PCR法从pDNR-LIB质粒中扩增人全长TIMP1基因，利用基因重组的方法将TIMP1全长cDNA插入通用型AAV2载体质粒pSNAV的多克隆位点，构建成pSNAV-TIMP1；用脂质体转染的方法将重组质粒转入BHK-21细胞中，G418细胞筛选得到转入重组质粒并能表达目的基因的细胞系BHK21／rAAV2-TIMP1；用具有rAAV2包装功能的重组Ⅰ型单纯疱疹病毒（rHSV1-rc／△UL2）感染BHK-21／rAAV2-TIMP1，纯化后得到rAAV2-TIMP1。结果用改良法成功构建rAAV2-TIMP1，病毒滴度达到1×10^12v.g．／ml。结论成功构建rAAV2-TIMP1，为其进一步应用于基因治疗的研究提供实验基础。     

人骨形成蛋白7基因重组2型腺相关病毒载体的构建及其在骨髓间充质干细胞中的表达

目的构建人骨形成蛋白7基因（hBMP7）重组腺相关病毒载体,并观察其在兔骨髓间充质干细胞中的表达。方法将骨形成蛋白7基因片段克隆入穿梭质粒pUC18获得重组质粒pUC18-hBMP7。KpnⅠ和鼠Ⅱ双酶切质粒pUC18-hBMP7／与pSNAV,用T4DNA连接酶连接分别回收的两片段后转化大肠杆菌DH5α感受态细胞,获得重组质粒PSNAV—hBMP7／,转染BHK-21细胞,筛选培养,用能表达Rap和Cap的重组Ⅰ型单纯疱疹病毒HSVl-rc／△UL2感染此细胞,裂解细胞收获病毒液。采用氯仿处理-PEG／NaCl沉淀-氯仿抽提法分离浓缩、纯化与测定病毒滴度。用rAAV2-hBMP7／和rAAV2-EGFP在体外分别转染兔骨髓间充质干细胞。流式细胞仪、RT-PCR和Western—blot方法检测兔骨髓间充质干细胞中hBMP-7基因的转录和表达。结果成功构建具有感染活性的重组腺相关病毒载体rAAV2-hBMP7／,病毒载体对兔骨髓间充质干细胞早期转染效率可达99．8％,hBMP7／在兔骨髓间充质干细胞中可得到转录和表达。结论成功构建了人骨形成蛋白7基因重组腺相关病毒载体,rAAV2-hBMP7／载体在体外可转染兔骨髓间充质干细胞,并获得较高的转染效率。     

靶向肝癌细胞的重组腺相关病毒载体的构建

目的　构建以甲胎蛋白增强子(AFP E)和白蛋白启动子(ALB P)联合转录调控序列(AFPenh+ALBprom, EP)为启动子的重组腺相关病毒基因治疗载体,用于肝细胞肝癌(HCC)的靶向基因治疗研究。方法　用PCR方法扩增EP基因片段,将EP基因片段顺义克隆至重组腺相关病毒载体系统(AAV Helper Free System)中表达质粒 pAAV IRES hrGFP的启动子位点,并替换其原有的巨细胞病毒(CMV)启动子,构建出以 EP为启动子的表达质粒 pAAV IRES hrGFP EP; 再将 pAAV IRES hrGFP EP与 AAV Helper Free System中的控制质粒pAAV RC和辅助质粒 pHelper用脂质体转染法共转染人胚肾细胞(293 细胞),生成以 EP 为启动子的、可用于HCC靶向基因治疗的重组腺相关病毒载体(rAAV EP)。结果　成功构建并包装出以 EP为启动子的重组腺相关病毒载体 rAAV2 EP,病毒滴度达1.2×105/ml。结论　构建的以腺相关病毒为载体、受 AFP E和 ALB P联合转录调控序列驱动的重组腺相关病毒载体 rAAV2 EP,可望能用于HCC靶向基因治疗研究,并为肝脏疾病的靶向基因治疗提供先进载体系统。     

重组腺相关病毒介导的血红素加氧酶1基因和绿色荧光蛋白基因在肝移植大鼠肝脏中的表达

目的 构建提纯重组腺相关病毒介导的血红素加氧酶1基因(HO-1)和绿色荧光蛋白基因(GFP),并探讨其在肝移植大鼠肝脏中的表达情况.方法 克隆大鼠HO-1,构建重组腺相关病毒-HO-1(rAAV-HO-1)载体,酶切鉴定并进行测序,氯化钙共沉淀法与辅助质粒Virus helper、AAV-cap-rep转染包装细胞,应用肝素层析柱法纯化浓缩病毒,实时荧光定量PCR测定病毒滴度.应用二套管法建立Wistar→Wistar大鼠原位肝移植模型.将提纯的重组腺相关病毒-GFP(rAAV-GFP)在供肝冷保存阶段经门静脉靶向转染并孵育2 h后行大鼠原位肝移植,分别于术后1、3个月处死大鼠取材,冰冻切片荧光显微镜下观察不同组织GFP的表达情况及转染效率.结果 rAAV-HO-1重组子经酶切电泳检测表明插入片段大小正确,测序结果与Genbank一致.rAAV-GFP靶向转染供肝1、3个月冰冻切片荧光显微镜下观察,移植肝GFP表达效率均＞80%,且在心、肺、脾、肾、肠等组织中未见报告基因的表达.结论 成功构建并提纯了携带HO-1、GFP基因的高滴度的腺相关病毒,验证了腺相关病毒介导的GFP在肝移植大鼠肝脏中稳定高效的表达.     

凋亡素重组腺相关病毒的构建及体外抑制膀胱癌效应

目的 探讨携带凋亡素基因的重组腺相关病毒构建方法,观察其体外抑制膀胱癌的效应.方法 构建重组质粒pAAV-VP3,经EcoRI/Sal Ⅰ双酶切鉴定和基因测序无误后,采用三质粒共转染法包装重组腺相关病毒rAAV-VP3.收获病毒后聚合酶链反应(PCR)扩增病毒液中的目的基因鉴定重组病毒,对其进行纯化后透射电镜观察病毒颗粒,并检测病毒滴度.按每个细胞5×105个载体基因组的剂量感染EJ细胞后,逆转录(RT) -PCR检测VP3基因在EJ细胞中的转录,Western blot法检测凋亡素蛋白的表达.透射电镜扫描观察感染重组病毒后肿瘤细胞的超微结构变化,流式细胞术(FCM)检测重组病毒对EJ细胞的影响.结果 转染72 h后重组腺相关病毒rAAV-VP3包装成功,滴度为5.1×1011个载体基因组/ml,电镜下可见病毒颗粒.感染EJ细胞后,可检测到VP3基因的转录和凋亡素蛋白表达,电镜观察到凋亡形态学改变,FCM检测S期细胞比例降低和G2/M期细胞比例增高,与对照组比较差异有统计学意义(P＜0.01).结论 重组腺相关病毒rAAV-VP3在体外能够发挥生物学活性,其介导的凋亡素表达抑制膀胱癌的体外效应为体内实验奠定了基础.     

hVEGF165及hBMP-7双基因共表达腺相关病毒载体的构建及鉴定

目的通过引入内部核糖体进入位点序列（internal ribosome entry site，IRES），构建带有hVEGF165及hBMP-7双基因的重组腺相关病毒载体（adeno—associated virus，AAV），并对其进行鉴定。方法以AAV腺相关病毒包装系统（helper—free system）为基础，将真核表达质粒pIRES中的IRES片段定向克隆至腺相关病毒骨架质粒pAAV-MCS中，形成含有IRES序列及上、下游多克隆位点的重组骨架质粒pAAV-MCSA—IRES—MCSB。PCR扩增hVEGF165和hBMP-7基因，先后亚克隆入重组腺相关病毒骨架质粒IRES序列上、下游的多克隆位点，获得重组质粒pAAV-hVEGF165—IRES—hBMP-7。将其和包装质粒pAAV-RC、辅助质粒pHelper三质粒共转染AAV-293细胞，包装重组腺相关病毒rAAV-hVEGF165-IRES—hBMP-7，同时包装含有绿色荧光蛋白（green fluorescent protein，GFP）标记的重组病毒rAAV-IRES—GFP作平行对照。荧光显微镜下监测病毒包装效率，收获重组病毒后感染AAV-HT1080细胞测定病毒滴度，并通过病毒基因组外源基因扩增鉴定重组病毒的包装是否成功。结果重组腺相关病毒骨架质粒pAAV-hVEGF-165-IRES—hBMP-7经双酶切鉴定正确。荧光显微镜下观察三质粒共转染AAV-293细胞72h后，病毒包装效率达95％～100％，收获的重组病毒具有较高浓度及活性，感染AAV-HT1080效率达90％，荧光计数法测定病毒感染滴度达5．5×10^11 vp／mL。提取重组病毒基因组成功扩增出外源目的基因hVEGF。及hBMP-7片段，重组病毒包装成功。结论成功构建带有hVEGF165及hBMP-7双基因的重组腺相关病毒rAAV-hVEGF165-IRES—hBMP-7，收获的病毒具有较高滴度，为今后利用腺相关病毒载体进行hVEGF。及hBMP-7双基因共表达影响骨修复的体外及体内研究提供实验基础。     

人PPFP基因的重组慢病毒载体的构建和表达

目的 构建含人PAX8/PPARγ/融合基因(PPFP)基因重组慢病毒载体并检测其表达性能.方法 从已构建好的含PPFP的质粒克降模板PEGFP-C-PPFP中,利用聚合酶链反应(PCR)方法钓取目的 基因PPFP,将该基因克隆到慢病毒载体表达质粒pGC-FU(含Flag基因)中,得到重组的pGC-FU-PPFP,通过PCR、酶切、测序和分析比对验证PPFP基因后,将pGC-FU-PPFP质粒和包装质粒pHelper1.0、pHelper2.0共同转染人胚胎肾上皮细胞株293T细胞,获得携带PPFP基因和Flag基因的重组慢病毒,收集并浓缩病毒上清液,测定重组病毒的滴度.通过Western blot鉴定PPFP-Flag融合蛋白在靶细胞内的表达进一步验证目的 基因在靶细胞中的表达.结果 经PCR扩增获得2591 bp的目的 基因片段,构建的重组质粒经PCR、酶切及测序和分析比对鉴定正确;该质粒与包装质粒共转染293T细胞获取的慢病毒滴度达3.5×10~7转导单位TU/ml;感染的293T细胞,Western blot结果显示条带大小为90 KDr,可判断目的 基因PPFP在293T细胞中表达.结论 成功构建PPFP基因慢病毒载体质粒pGC-FU-PPFP,并建立慢病毒过表达系统.     

Vasopressor hormone response following mesenteric traction during major abdominal surgery

Background : We investigated the vasopressor hormone response following mesenteric traction (MT) with hypotension due to prostacyclin (PGI₂) release in patients undergoing abdominal surgery with a combined general and epidural anesthesia. Methods : In a prospective, randomized, placebo-controlled study we administered 400 mg ibuprofen (i.v.) in 42 patients scheduled for abdominal surgery. General anesthesia was combined with epidural anesthesia (T4-L1). Before as well as 5, 15, 30, 45, and 90 min after MT we recorded plasma osmolality, hemodynamics and measured 6-keto-PGFlα (stabile metabolite of PGI₂), TXB₂ (stabile metabolite of thromboxane A₂) active renin, and arginine vasopressin (AVP) plasma concentrations by radioimmunoassay. Catecholamine levels were assessed by high-pressure liquid chromatography (HPLC) with electrochemical detection. Results : Following MT, arterial hypotension occurred along with a substantial PGI₂ release. This was completely abolished by ibuprofen administration. Although plasma levels of 6-keto-PGF_1α (1133 (708) vs. 60 (3) ng/L, median (median absolute deviation), P=0.0001, placebo vs. ibuprofen) remained significantly elevated, blood pressure was restored within 30 min after MT in the placebo group. At the same point in time plasma concentrations of TXB² (164 (87) vs. 58 (1) ng/L, P=0.0001), epinephrine (46 (33) vs. 14 (6) ng/L, P=0.001), AVP (41 ± (18) vs. 12 (7) ng/L, P=0.0004), and active renin (27 (12) vs. 12 (4) ng/L, P = 0.001) were significantly higher in placebo-treated patients. Conclusion : Under combined general and epidural anesthesia arterial hypotension following MT due to endogenous PGI₂ release is associated with enhanced release of AVP, active renin, epinephrine and thromboxane A₂, presumably contributing to hemodynamic stability within 30 min after MT.     

A Teaspoon Pump for Pumping Blood with High Hydraulic Efficiency and Low Hemolysis Potential

Abstract: Virtually all blood pumps contain some kind of rubbing, sliding, closely moving machinery surfaces that are exposed to the blood being pumped. These valves, internal bearings, magnetic bearing position sensors, and shaft seals cause most of the problems with blood pumps. The original teaspoon pump design prevented the rubbing, sliding machinery surfaces from contacting the blood. However, the hydraulic efficiency was low because the blood was able to "slip around" the rotating impeller so that the blood itself never rotated fast enough to develop adequate pressure. An improved teaspoon blood pump has been designed and tested and has shown acceptable hydraulic performance and low hemolysis potential. The new pump uses a nonrotating "swinging" hose as the pump impeller. The fluid enters the pump through the center of the swinging hose; therefore, there can be no fluid slip between the revolving blood and the revolving impeller. The new pump uses an impeller that is comparable to a flexible garden hose. If the free end of the hose were swung around in a circle like half of a jump rope, the fluid inside the hose would rotate and develop pressure even though the hose impeller itself did not "rotate"; therefore, no rotating shaft seal or internal bearings are required.     

A comparison of the inhibitory effects of four volatile anaesthetics on the metabolism of chlorzoxazone, a substrate for CYP2E1, in rabbits

Background: Halothane inhibits in vitro and in vivo activity of cytochrome P-450 (CYP) 2E1. There are several fluorinated volatile anaesthetics besides halothane, and most of them are defluorinated by CYP2E1. It is unclear whether other fluorinated anaesthetics inhibit the in vivo activity of CYP2E1.
Methods: We compared the inhibitory effects of therapeutic concentrations of four inhalational anaesthetics, halothane, enflurane, isoflurane, and sevoflurane, on chlorzoxazone metabolism in rabbits receiving artificial ventilation.
Results: All four inhalational anaesthetics decreased arterial blood pressure and increased plasma chlorzoxazone concentration. However, no significant differences in the plasma chlorzoxazone concentration were found between the four anaesthetics. The estimated chlorzoxazone clearance increased after beginning inhalation with all four agents, but no significant difference in clearance was noted between agents.
Conclusions: At therapeutic concentrations, the in vivo inhibitory effect on chlorzoxazone metabolism was similar for all four inhalational anaesthetics examined, even though their chemical characteristics and extent of hepatic metabolism differ considerably.     

Microspheres Based Detoxification System: A New Method in Convective Blood Purification

Abstract: A variety of protein-bound or hydrophobic substances, accumulating as a result of pathologic conditions such as exogenous or endogenous intoxications, are removed poorly by conventional detoxification methods because of low accessibility (hemodialysis), insufficient adsorption capabilities (hemosorption), low efficiency (peritoneal dialysis), or economic limitations (high-volume plasmapheresis). Combining advantages of existing methods with microspheric technology, a module-based system was designed. Major operating parameters of the latter can be modified to allow for adjustment to individual clinical situations. An extracorporeal blood circuit including a plasmafilter is combined with a secondary high-velocity plasma circuit driven by a centrifugal pump. Different microspheric adsorbers can be combined in one circuit or applied in sequence. Thus, a prolonged treatment can be tailored using specially designed selective adsorber materials. Comparing this system with existing methods (high-flux hemodialysis, molecular adsorbent recycling system), results from our in vitro studies and animal experiments demonstrate the superior efficiency of substance removal.     

Tissue perfusion in relation to cardiac output during continuous positive-pressure ventilation and administration of propranolol or verapamil

Background : Our objective was to determine whether administration of propranolol or verapamil modifies the hemodynamic adaptation to continuous positive-pressure ventilation (CPPV), in particular the regional distribution of cardiac output (CO).
Methods : General hemodynamics and regional blood flows assessed by microsphere technique (15 (μm) were recorded in 16 anesthetized pigs during spontaneous breathing (SB) and CPPV with 8 cm H₂O end-expiratory pressure (CPPV8) before and after intravenous administration of propranolol (0.3 mg · kg⁻¹ followed by 0.15 mg · kg⁻¹ · h⁻¹, n=8) or verapamil (0.1 mg · kg⁻¹ followed by 0.3 mg · kg⁻¹ · h⁻¹, n=8).
Results : CPPV8 depressed CO by 25% without shifts in its relative distribution with the exception of a noteworthy increase in adrenal perfusion. Propranolol increased arterial blood pressure, and due to a fall in heart rate, CO dropped by 25%. The kidneys and, to a lesser extent, the splanchic region and central nervous system received increased fractions of the remaining CO at the expense of skeletal muscle flow. Similar patterns were seen during SB and CPPV8 such that the combination of propranolol and CPPV8 depressed CO by 50%. The circulatory effects of verapamil were less evident but myocardial perfusion tended to increase.
Conclusions : The combination of propranolol or verapamil with CPPV does not result in any specific hemodynamic interaction in anesthetized pigs, except that the combined effect of propranolol and CPPV may severely reduce CO.     

Bariatric Surgery in Some "Central and East-European (former Eastern bloc)"Countries - Current Status and Prediction for the Next Millennium

Background: Obesity is increasing globallly, including in the formerly "Eastern Bloc" countries. Methods: A survey was made of obesity and bariatric surgery. Results: In the 8 East and Central European countries studied, with total population 300 million, roughly 43% of the population was overweight (BMI 25-30), 23% obese (BMI > 30), with about 15 million people morbidly obese (BMI > 40). From 0-10 morbidly obese individuals/100,000/year undergo bariatric surgery. Conclusion: Most countries were found to provide inadequate treatment for obesity.The majority of the morbidly obese are not treated effectively. However, health-care awareness of obesity and bariatric surgeons are slowly increasing.     

Volatile anaesthetics reduce adhesion of blood platelets under low-flow conditions in the coronary system of isolated guinea pig hearts

Background : Inhibitory effects of volatile anaesthetics on platelet aggregation have been demonstrated in several studies. However, the influence of volatile anaesthetics on intracoronary platelet adhesion has not been elucidated so far.
Methods : Isolated hearts of guinea pigs were perfused with buffer in the absence or presence of volatile anaesthetics (0.5 and 1 MAC) at constant coronary flow rates of 5 ml/min for 25 min, then 1 ml/min for 30 min and again 5 ml/min for 10 min. Before, during and after low-flow perfusion, a bolus of human platelets was applied into the coronary system. To simulate thrombogenic conditions, 0.3 U/ml human thrombin was infused during low-flow perfusion and reperfusion. The number of platelets sequestered to the endothelium was calculated from the difference between coronary in- and output of platelets. The myocardial production of lactate and consumption of pyruvate and coronary perfusion pressure were also determined.
Results : At a flow rate of 5 ml/min only about 3% of the applied platelets did not emerge from the coronary system, in any group. In contrast, 13.1±1.2% (mean±SEM) of infused platelets became adherent in low-flow perfusion in the control group without anaesthetic. The adherence was reduced with each 1 MAC isoflurane (to 6.2±1.2%), sevoflurane (to 4.4±0.9%) or halothane (to 3.2±1.5%) (each P <0.05 vs. control). Volatile anaesthetic, 0.5 MAC, did not inhibit platelet adhesion to a statistically significant extent in any case. Perfusion pressure and metabolic parameters were not statistically different between the control and the hearts exposed to anaesthetics.
Conclusion : Volatile anaesthetics in a concentration of 1 MAC can reduce the adhesion of platelets in the coronary system under reduced flow conditions. This action does not arise from vasodilation or inhibition of ischaemic stress.     

Synergistic interaction between the effects of propofol and midazolam with fentanyl on phrenic nerve activity in rabbits

Background: It has been shown that the depressive effects of both propofol and midazolam on consciousness are synergistic with opioids, but the nature of their interactions on other physiological systems, e. g. respiration, has not been fully investigated. The present study examined the effect of propofol and midazolam alone and in combination with fentanyl on phrenic nerve activity (PNA) and whether such interactions are additive or synergistic. Methods: PNA was recorded in 27 anaesthetised and artificially ventilated rabbits. In three groups, propofol, fentanyl and midazolam were administered intravenously in incremental doses to construct dose-response curves for the depressant effects of each one on PNA. In another two groups, the effect of pretreatment with either fentanyl 1 μg · kg^?1 i. v. or midazolam 0.05 mg · kg^?1 i. v. on the effects of propofol and fentanyl respectively on PNA were studied. Results: Propofol and fentanyl caused a dose-dependent depression of PNA with complete abolition at the highest total doses of 16 mg · kg^?1 i. v. and 32 μg · kg^?1 i. v., respectively. In contrast, midazolam in incremental doses to a total of 0.8 mg · kg^?1 reduced mean PNA by 63%, but approximately 12% of PNA remained at a total dose as high as 6.4 mg · kg^?1. The mean ED₅₀s, calculated from dose-response curves, were 5.4 mg · kg^?1, 3.9 μg · kg^?1 and 0.4 mg · kg^?1 for propofol, fentanyl and midazolam, respectively. Initial doses of either fentanyl 1 μg · kg^?1 i. v. or midazolam 0.05 mg · kg^?1 i. v. acted synergistically with subsequent doses of either propofol or fentanyl to abolish PNA at total doses of 8 mg · kg^?1 and 8 μg · kg^?1, respectively. Conclusion: Fentanyl has a synergistic interaction with both propofol and midazolam on PNA and hence potentially on respiration.     

Influence of dopexamine hydrochloride on haemodynamics and regulators of circulation in patients undergoing major abdominal surgery

Background: Catecholaminergic support is often used to improve haemodynamics in patients undergoing major abdominal surgery. Dopexamine is a synthetic vasoactive catecholamine with beneficial microcirculatory properties. Methods: The influence of perioperative administration of dopexamine on cardiorespiratory data and important regulators of macro- and microcirculation were studied in 30 patients undergoing Whipple pancreaticduodenectomy. The patients received randomized and blinded either 2 μg · kg^?1 · min^?1 of dopexamine (n=15) or placebo (n=15, control group). The infusion was started after induction of anaesthesia and continued until the morning of the first postoperative day. Endothelin-1 (ET-1), vasopressin, atrial natriuretic peptide (ANP), and catecholamine plasma levels were measured from arterial blood samples. Measurements were carried out after induction of anaesthesia, 2 h after onset of surgery, at the end of surgery, 2 h after surgery, and on the morning of the first postoperative day. Results: Cardiac index (CI) increased significantly in the dopexamine group (from 2.61±0.41 to 4.57±0.78 1 · min^?1 · m^?2) and remained elevated until the morning of the first postoperative day. Oxygen delivery index (DO₂I) and oxygen consumption index (VO₂I) were also significantly increased in the dopexamine group (DO₂I: from 416±91 to 717±110 ml/m² · m²; VO₂I: from 98±25 to 157±22 ml/m² · m²), being significantly higher than in the control group. pHi remained stable only in the dopexamine patients, indicating adequate splanchnic perfusion. Vasopressive regulators of circulation increased significantly only in the untreated control patients (vasopressin: from 4.37±1.1 to 35.9±12.1 pg/ml; ET-1: from 2.88±0.91 to 6.91±1.20 pg/ml). Conclusion: Patients undergoing major abdominal surgery may profit from prophylactic perioperative administration of dopexamine hydrochloride in the form of improved haemodynamics and oxygenation as well as beneficial influence on important regulators of organ blood flow.     

Systemic analgesia adapted to the children's condition

A concept of balanced analgesia using nonsteroidal anti-inflammatory drugs (NSAIDs), paracetamol (acetaminophen), opioids, and corticosteroids can also be used in patients with pre-existing illnesses. NSAIDs are the most effective treatment for acute pain of moderate intensity in children; however, these drugs should be avoided in patients at increased risk for serious side effects, e.g. patients with renal impairment, bleeding tendency, or extreme prematurity. NSAIDs can be given with minimal risks to the younger child with mild to moderate asthma, and, in these patients, the use of steroids can be encouraged; in addition to their antiemetic and analgesic action, a beneficial effect on asthma symptoms can be expected. In the non-intubated child with cerebral trauma, exaggerated sedation caused by opioids and increased bleeding tendency caused by NSAIDs must be avoided. In neonates and small infants, the oral administration of sucrose or glucose is helpful to minimize pain reaction during short uncomfortable interventions.