应用脉冲电磁场刺激人骨髓间充质干细胞体外培养的生物学特性的研究

目的　应用脉冲电磁场 (PEMFs)刺激体外分离培养的人骨髓间充质干细胞 (hMSCs) ,对其生物学特性进行研究。方法　PEMFs (1 2Hz、 1 1mT、 8h/d)刺激hMSCs体外培养 ,计算细胞的倍增时间 ,观察细胞超微结构 ,放免法测定骨钙素的分泌情况和钙化结节VonKossa染色。结果　脉冲电磁场作用后的细胞增殖能力提高 ,细胞形态发生变化 ,透射电镜观察提示细胞成熟 ,VonKossa钙染色阳性 ,骨钙素的分泌活性增高。结论　hMSCs经合理的PEMFs体外刺激培养后 ,不仅能够大量扩增细胞 ,同时能够向成骨细胞转化 ,具有一定的成骨活性 ,是一种较为理想的骨种子细胞来源     

应用脉冲电磁场刺激人骨髓间充质干细胞体外培养的生物学特性的研究

目的应用脉冲电磁场(PEMFs)刺激体外分离培养的人骨髓间充质干细胞(hMSCs)，对其生物学特性进行研究。方法PEMFs(12Hz、1．1mT、8h／d)刺激hMSCs体外培养，计算细胞的倍增时间，观察细胞超微结构，放免法测定骨钙素的分泌情况和钙化结节Von Kossa染色。结果脉冲电磁场作用后的细胞增殖能力提高，细胞形态发生变化，透射电镜观察提示细胞成熟，Von Kossa钙染色阳性，骨钙素的分泌活性增高。结论hMSCs经合理的PEMFs体外刺激培养后，不仅能够大量扩增细胞，同时能够向成骨细胞转化，具有一定的成骨活性，是一种较为理想的骨种子细胞来源。     

骨髓间充质干细胞与骨组织共培养模型的建立

目的:建立骨髓间充质干细胞(BMSCs)与骨组织共培养模型,模拟体内成骨环境,以全面研究骨髓间充质干细胞及细胞支架复合物的生物性状.方法:通过插入式培养皿和培养板来构建细胞一组织共培养的模型,将新鲜兔骨碎粒及骨块与骨髓间充质干细胞共培养,观察共培养条件下细胞的形态学变化、ALP活性及矿化能力,免疫组化检测Ⅰ型胶原、骨钙素.结果:构建出骨髓间充质干细胞与骨组织共培养的模型.共培养的间充质干细胞同期ALP活性高于普通培养对照组,其Ⅰ型胶原、骨钙素免疫组化阳性,对照组Ⅰ型胶原免疫组化弱阳性、骨钙素免疫组化阴性.结论:体外构建的共培养模型部分模拟了体内成骨环境;经过共培养的细胞呈现成骨细胞的表型.     

低氧环境下共培养的骨膜细胞和髓核细胞骨向分化能力的研究

[目的]探讨低氧环境对体外共培养的骨膜细胞、髓核细胞骨向分化能力的影响。[方法]采用组织块法分离兔骨膜细胞,胰酶、胶原酶消化法获取髓核细胞,传至3代进行共培养实验,实验分为2组:正常氧组(20%O2)、低氧组(5%O2),共培养后用CCK-8检测细胞增殖情况,用AKP试剂盒、BCA试剂盒检测碱性磷酸酶活性,RT-PCR检测骨钙素、Ⅰ型胶原、RUNX2以及HIF-1a mRNA的表达,免疫组化试剂盒检测Ι型胶原的表达,茜素红染色检测钙盐沉积或钙结节。[结果]骨膜细胞和髓核细胞在体外成功分离和培养,共培养后保持了较高的增殖率,经过成骨诱导培养后成功诱导为成骨细胞,细胞增殖曲线为"S"型,两组在1、3、5、7、9 d的光吸收值(OD)比较差异有统计学意义(P0.05);低氧组的骨钙素、Ⅰ型胶原、RUNX2以及HIF-1a mRNA表达水平高于常氧组(P0.05),碱性磷酸酶(ALP)活性高于常氧组(P0.05),细胞成骨染色(茜素红染色、免疫组化)结果显示低氧组较常氧组表达增多。[结论]低氧条件下体外共培养的骨膜细胞和髓核细胞经成骨诱导后向成骨细胞分化的能力更强。     

成骨诱导的兔骨髓基质干细胞成骨活性的表达及维持

目的观察成骨诱导的兔骨髓基质干细胞(BMSCs)体内、外环境下成骨活性的表达及维持。方法观察BMSCs在体外成骨诱导培养条件下的成骨分化特性;构建兔BMSCs与活骨组织共培养模型模拟体内“成骨环境”,将成骨诱导的MSCs置于共培养及普通传代培养条件下进行传代培养,观察经成骨诱导的BMSCs在体外及模拟体内的培养条件下细胞的表型维持情况。结果药物成骨诱导培养的BMSCs,其ALP活性及骨钙素均显著高于普通培养组(P<0.05);经过诱导培养的BMSCs,其Ⅰ型胶原、骨钙素免疫组化阳性。RT-PCR法半定量测定Ⅰ型胶原mRNA,成骨诱导培养的Ⅰ型胶原mRNA表达量明显高于普通传代培养对照组。药物成骨诱导后的细胞在体外普通传代培养传5代后,细胞碱性磷酸酶(ALP)活性、骨钙素水平及Ⅰ型胶原表达稳定维持在较高水平,保持其成骨细胞的表型;在共培养条件下,ALP活性、骨钙素水平Ⅰ型胶原表达保持在高水平,且ALP活性、骨钙素水平在大部分时间点均高于普通传代培养。结论药物成骨诱导培养呈现促BMSCs向成骨方向转化的特点,能使ALP、骨钙素及Ⅰ型胶原表达短期内达到高水平;经成骨诱导的BMSCs在体外或模拟的体内传代培养条件下,均能维持成骨表型,保持成骨活力。     

脱细胞软骨基质来源的多孔支架复合山羊髓核细胞体内初步构建组织工程髓核的实验研究

[目的]探讨脱细胞软骨基质多孔支架复合PKH26标记的山羊髓核细胞体内异位构建组织工程髓核的可行性.[方法]制备脱细胞软骨基质来源的多孔支架,扫描电镜(scanning electron microscope,SEM)观察、天狼星红染色、HE染色观察、MTT毒性检测;分离山羊髓核细胞,通过倒置显微镜观察、番红O染色、Ⅱ型胶原免疫组化染色进行鉴定;将PKH26标记的山羊髓核细胞接种支架上,体外培养3d后进行LIVE/DEAD活性染色,将细胞支架复合物置入裸鼠皮下,培养6周,病理切片,荧光显微镜下观察,进行番红O、Ⅰ、Ⅱ型胶原免疫组化染色.[结果]扫描电镜观察支架孔隙相连通且分布均匀,天狼星红染色支架呈黄绿相间色,HE支架淡染,MTT检测细胞增殖曲线无统计学差异(P＞0.05);P1代髓核细胞呈软骨样细胞形态,番红O染色、Ⅱ型胶原免疫组化染色均阳性,PKH26标记后的细胞呈红色荧光;体外LIVE/DEAD染色细胞呈绿色荧光,体内培养6周后,带红色荧光的细胞填满支架孔隙,番红O、Ⅱ型胶原免疫组化染色阳性,Ⅰ型胶原免疫组化染色弱阳性.[结论]以脱细胞软骨基质多孔支架复合山羊髓核细胞在体内能够形成组织工程髓核样组织.     

经骨向诱导的骨髓间充质干细胞在脱钙骨上生长行为的实验研究

目的:建立成骨细胞-脱钙骨支架复合物,观察其诱导成骨能力,从而探寻组织工程化骨的体外构建方法.方法:运用细胞沉淀法将大鼠BMSCs诱导形成的成骨细胞接种于脱钙骨支架,通过扫描电镜(SEM)、免疫荧光观察脱钙骨表面细胞生长情况,通过上清液碱性磷酸酶(ALP)活性及Ⅰ型前胶原羧端肽(PICP)、骨钙素检测观察成骨细胞活性.结果:扫描电镜示成骨细胞在脱钙骨表面生长良好,上清液ALP、PICP及骨钙素表达均明显高于对照组(P＜0.05),并随时间的增长而显著增加(P＜0.05).结论:采用细胞沉淀法成功将成骨细胞接种于脱钙骨支架,成骨细胞在支架材料中增殖旺盛,细胞活性状态良好,具有良好的骨形成能力,脱钙骨支架可作为骨组织工程中载体支架的较优选择.     

人间充质干细胞在骨组织工程中的应用

目的从数量与功能两方面探讨人间充质干细胞(humanmesenchymalstemcells,hMSCs)作为骨组织工程种子细胞的可行性。方法髂骨穿刺,梯度离心分离获得hMSCs,体外扩增培养;对第3代细胞用地塞米松、β-甘油磷酸、维生素C等成骨诱导培养,检测细胞碱性磷酸酶、骨钙素的表达和钙结节形成情况;诱导细胞与可吸收性复合支架材料体外构建复合体,植入裸小鼠皮下,对照组仅植入支架材料,术后3周取材检测骨钙素、Ⅰ型胶原表达。结果4ml骨髓含3.2×107～6.0×107个单个核细胞,培养3周收获hMSCs2.5×107～4.3×107;成骨诱导后,表达碱性磷酸酶、骨钙素阳性细胞数>50%,对照组<10%;诱导细胞与材料复合后植入体内仍然高表达骨钙素、Ⅰ型胶原。结论hMSCs能够满足体外构建组织工程化骨,对种子细胞数量和功能的要求。     

不同年龄人骨髓间充质干细胞体外增殖及成骨分化的研究

目的 :观测年龄对人骨髓间充质干细胞 (humanmesenchymalstemcells ,hMSCs)体外增殖、成骨分化的影响。方法 :使用密度梯度离心法分离不同年龄人骨髓MSCs进行培养 ,保留贴壁细胞传代 ,观察细胞生长情况 ,检测其增殖活性、碱性磷酸酶活性 (ALP)、诱导后骨钙素定量测定。结果 :低龄hMSCs较高龄hMSCs体外生长快、MTT、ALP及骨钙素浓度高。结论 :hMSCs的增殖和成骨分化的能力和活性随着年龄的增加而降低。     

体外负压培养对hBMSCs成骨活性影响的初步研究

[目的]探讨体外负压培养对hBMSCs成骨活性的影响.[方法]取第3代hBMSCs分为实验组和对照组,实验组进行间歇性负压培养,设置压力为17 kPa,30 min/次,4次/d,干预2周;对照组于普通CO,培养箱中常规培养.倒置显微镜下观察细胞形态,MTT法和流式细胞仪检测细胞增殖和凋亡,检测ALP活性,茜素红染色观察钙节结形成,免疫组织化学检测Ⅰ型胶原和VEGF的表达.[结果]诱导2周,实验组细胞增殖能力下降,S期细胞百分比为(5.14±1.56)%,较对照组(13.45±3.51)%约下降62.4%,细胞凋亡增加.实验组ALP活性为(15.68±1.97)mU/mg,对照组为(6.344±1.21)mU/mg,两组比较差异有统计学意义(P<0.05);与对照组比较,实验组钙结节形成增多,VEGF表达较对照组显著提高;实验组Ⅰ型胶原表达呈阳性,对照组呈阴性反应.[结论]负压能抑制hBMSCs增殖,促进细胞凋亡,但可以提高细胞成骨活性.     

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.     

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.     

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.     

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.     

Is the recovery profile of mivacurium independent of the rate of decay of its plasma concentration in patients with normal plasma cholinesterase activity?

Background: The duration of action of muscle relaxants is poorly correlated to the rate of decay of their plasma concentration. The plasma concentration of mivacurium may rapidly decrease below its active concentration because of the extensive hydrolysis of mivacurium. By inflating a tourniquet on one upper limb for 3 min after the administration of atracurium, mivacurium or vecuronium, we studied the influence of the initial decline of their plasma concentration on their effect. Methods: In 50 patients anaesthetised with thiopental, isoflurane and fentanyl, the effect of bolus doses of 0.15 or 0.25 mg . kg^?1 mivacurium (MIV 15, MIV 25), 0.3 or 0.5 mg . kg^?1 atracurium (ATR 30, ATR 50) and 0.06 or 0.1 mg . kg^?1 vecuronium (VEC 06, VEC 10) were measured on both arms (evoked response of the adductor pollicis to train-of-four stimulation every 12 s), a tourniquet being applied on one arm just before and during 3 min after the muscle relaxant bolus. Results: Tourniquet inflation of 3 min almost abolished the neuromuscular effect of mivacurium. In the vecuronium groups and in the ATR 50 group, tourniquet inflation did not modify the maximum degree of depression of the twitch response. Also, the duration of action of vecuronium was unaffected by the tourniquet. In the ATR 30 group, times to return of the twitch response to 25% (duration 25%) and 75% (duration 75%) of control response were significantly shorter in the cuffed arm, 23 min vs 27 min, and 41 min vs 45 min, respectively. In the ATR 50 group, only duration 25% was significantly shorter in the cuffed arm (41 min vs 45 min). Conclusion: The results suggest that the rate of decline of the plasma concentration of mivacurium is so rapid, that a very low and almost clinically ineffective concentration is present as soon as 3 min after its administration. The results also indicate that the recovery from a mivacurium-induced neuromuscular blockade is not influenced by the rate of decay of its plasma concentration in patients with genotypically normal plasma cholinesterase.     

Membranes in Artificial Organs

Abstract: Membrane processes play a pivotal and enabling role in modern replacement therapy for acute and chronic organ failure and in the management of immunologic diseases. In fact, virtually all contemporary extracorporeal blood purification methods employ membrane devices, and the next generation of artificial organs and tissue engineering therapies are almost certain to be similarly grounded in membrane technology. In this short essay, we comment on the similarities and differences among synthetic membranes and their natural counterparts and also provide a critical overview of the demographics and technology of hemodialysis, hemofiltration, apheresis, oxygenation, and emerging membrane technologies and applications.     

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.     

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.