再次液氮冷冻保存对猪主动脉瓣膜细胞活性及组织结构的影响

目的了解再次冷冻保存对主动脉瓣膜细胞活性及组织结构的影响,探讨液氮冷冻保存的主动脉瓣解冻后再次冷冻保存使用的可行性.方法将猪主动脉瓣叶在抗菌处理后按随机数字表法分成三组,每组6个瓣叶,组Ⅰ作对照,组Ⅱ、组Ⅲ控制降温速率降至-80℃后在液氮中保存,1个月后融化解冻.组Ⅲ解冻并在室温下放置15分钟后更换保存液,再次降温至-80℃后放入液氮中保存,2个月后再融化解冻.采用XTT比色法测定各组瓣膜细胞活性,用免疫荧光组织化学染色、光学显微镜、透射电子显微镜行组织学检测.结果组Ⅱ冷冻保存后瓣膜细胞活性下降到组Ⅰ的63.97%,组织结构一定程度受损;组Ⅲ瓣膜细胞活性下降至组Ⅰ的38.60%,组织结构损害也进一步加重.结论液氮冷冻保存的猪主动脉瓣一经解冻融化,不宜再次冷冻保存使用.     

同种瓣的制作与临床应用

目的报告液氮深低温下保存同种带瓣血管的制作方法、组织活性及临床应用效果。方法制作同种瓣24个、抗生素灭菌、梯度降温后置于液氮中保存,并测定冷冻保存后同种瓣的组织活性。同种瓣临床应用5例,其中法洛四联症、肺动脉闭锁2例,先天性主动脉瓣狭窄1例,法洛四联症术后发生室间隔缺损残余漏伴肺动脉瓣重度关闭不全1例,Bentall术后发生感染性心内膜炎1例。结果抗生素灭菌、液氮深低温技术保存同种瓣具有良好的组织活性,糖代谢测定24h葡萄糖消耗大于16mg/dl,组织培养见成纤维细胞生长良好。临床移植5例均成功,术后随访3～8个月,同种瓣无狭窄或关闭不全。结论液氮深低温保存同种瓣安全可靠,临床应用早期效果良好。     

种植人体活性细胞的生物心脏瓣膜

目前,种植人体活性细胞的生物心脏瓣膜主要有组织工程心脏瓣膜和种植人体活性细胞的猪主动脉瓣两种。组织工程心脏瓣膜是在人体可吸收的聚二醇酸纤维支架上种植人体同种活性细胞,先种植成纤维细胞,再种植单层内皮细胞包裹瓣叶。种植人体活性细胞的猪主动脉瓣是在清除原有细胞的组织内重建人体同种活性细胞。清除新鲜猪主动脉瓣呐原有细胞的方法是将瓣膜先经高、低渗溶液处理,然后用酶溶液处理。细胞经培养分离后,将成纤维细胞植入经处理的瓣膜组织,再植入内皮细胞。种植人体活性细胞的生物心脏瓣膜不会促使受者产生有害的免疫反应,并具有再生能力。     

温缺血时间与同种心脏瓣膜细胞活性的关系

以１０具脑外伤死亡的健康男性的心脏的６０个同种主动脉、肺动脉瓣叶分为６组进行实验。经不同的温缺血时间灭菌并经过３个月的液氮深低温保存后，通过流式细胞计数测定细胞活性，同时行组织块贴壁培养及电镜观察。结果表明：温缺血时间在１２小时以内，细胞活性为９３．４９±２．３５％，较对照组无明显下降。灭菌２４小时，细胞活性为８９．８２±３．５２％，较对照组明显下降，Ｐ＜０．０１，并且可见形态上改变；冷冻后细胞活性进一步下降，但下降幅度减低，冻存３年后活细胞仍占８０％。结论为：温缺血时间对细胞活性有影响，时间延长与活性的下降成正相关。因此温缺血时间应控制在１２小时以内。     

经液氮保存有活性同种带支架瓣膜的实验研究

目的 检测经液氮保存的人同种带支架瓣膜的细胞活性和流体力学性能.方法 制作同种带支架瓣膜经液氮保存3个月后,利用流式细胞仪技术定量检测瓣叶细胞活性(实验组,n=6),采集新鲜同种瓣膜作为对照(对照组,n=6).利用国产脉动流实验装置,分别测试实验组21#、23#、25#同种带支架瓣膜的流体力学性能,测试各流量下的跨瓣压差、有效瓣口面积和回流百分比;并使用相应型号的国产Perfect牛心包生物瓣膜进行对比研究.结果 实验组内皮细胞死亡率与对照组比较差异无统计学意义(10.24%±1.71% vs.9.09%±2.72%,P=0.441);实验组平滑肌细胞死亡率与对照组比较差异无统计学意义(8.76%±1.82% vs.7.84%±0.59%,P=0.178);实验组总细胞死亡率与对照组比较差异无统计学意义(8.79%±1.44% vs.7.40%±0.49%,P=0.072).两种21#、23#、25#人工生物瓣膜的跨瓣压差对流量具有非常大的依赖性,随着流量的增大而增大.同种带支架瓣膜的跨瓣压差较Perfect牛心包生物瓣膜的大.两种各型号瓣膜的回流百分比均随测试流量的增大而略有减小,同样,同种带支架瓣膜的回流百分比较Perfect牛心包生物瓣膜的大.两种各型号瓣膜的有效瓣口面积均有随流量增加而增大的趋势,同种带支架瓣膜的有效瓣口面积较Perfect牛心包生物瓣膜的略小.结论 经液氮保存同种带支架瓣膜的细胞活性保存良好,流体力学性能较满意.     

两种方法冷冻保存异体髌腱重建膝关节交叉韧带比较

目的 观察程序冷冻液氮保存和-80℃深低温保存方法处理同种异体髌腱后重建膝关节交叉韧带的愈合过程并比较其差异.方法 将兔的1/2骨-髌腱-骨复合体经程序冷冻液氮保存和-80℃深低温保存2周后,观察冻存变化差异并行同种异体移植重建前交叉韧带,分别于术后3周和8周观察细胞毒反应、细胞活性、最大载荷和形态学变化等指标进行比较并与自体移植组对照. 结果 ①经程序冷冻液氮保存方法处理后,髌腱的最大载荷无明显下降,细胞活性得到了较好的保存,组织学观察冷冻损伤较-80℃深低温保存方法轻微.②程序冷冻液氮保存处理的移植物在术后未表现明显的排斥反应,且免疫反应随时间的推移而下降.③移植后3周,各组移植物的最大载荷差异无显著性,移植后8周,程序冷冻液氮保存组移植物的最大载荷优于-80℃深低温保存组,但和自体移植组相近.④和-80℃深低温保存组相比,程序冷冻液氮保存组的移植物在术后的愈合过程和组织学行为更接近于自体移植组.结论 ①经程序冷冻液氮保存和-80℃深低温保存方法冻存的兔异体髌腱重建膝关节交叉韧带后愈合过程和自体韧带移植重建过程相似.②程序冷冻液氮保存法优于传统的-80℃深低温保存法.     

不同复温方法对液氮保存人主动脉瓣活性影响的实验研究

寻找一种能量大程度保持液氮贮存人同种主动脉瓣活性的复温方法。方法同种主动脉瓣随机分为６组Ⅰ组为新鲜同种主动脉瓣,Ⅱ－Ⅳ组经液氮保存３月后,分别采用４２℃水浴复温,。（２）（１．０±０．２）℃／ｍｉｎ（Ⅲ组）,     

不同保存方式对嗅鞘细胞活性的影响

[目的]探索嗅鞘细胞最佳保存方式.[方法]20例对数生长期OECs,随机分为5组,分别加入10%DMSO、5%DMSOfi%HES、5%DMSO,冰箱降温或程控降温仪降温并液氮保存,定期复苏,通过观察细胞形态、MTT 比色法、台盼蓝染色法检测细胞活性.[结果]同种降温方式下,5%DMSO-6%HES组细胞活性优于另两组,差异显著;同种低温保护剂下,冰箱降温与程控降温仪降温并液氮保存相比,差异不显著;同种保存方式下低温保存半年,各组回收率差异不显著.复苏标本不洗涤,室温下放置不同时间,细胞活性均下降,其中10%DMSO组下降最大.[结论]推荐5%DMSO-6%HES作为OECs冻存低温保护剂;小样本保存应选用冰箱降温液氮保存方式,大样本保存可选用程控降温仪降温或冰箱降温,并液氮保存方式;OECs低温保存半年仍具有较好的细胞活性.     

冷冻保存同种带瓣管道的活性研究

目的　探讨液氮冷冻保存对带瓣管道的细胞活性的影响。方法　将 40份同种主、肺动脉管道 ,经修剪、抗生素液处理 ,液氮冷冻保存 4～ 12个月后取出 ,留取主、肺动脉壁片、二尖瓣作细胞培养、光镜和电镜检查 ,并取新鲜主、肺动脉壁片、二尖瓣 4份作对照。结果　冷冻保存 4～ 7个月的管道 ,于 4周后成纤维细胞长满视野 ,细胞收获数为 :二尖瓣 (67.64± 2 5 .60 )万～ (74.5 8±12 .3 4)万 ,主肺动脉壁片 (3 2 .45± 4.75 )万～ (3 8.67± 18.3 2 )万 ,冷冻 1年的管道极少量细胞生长 ,为 (3 0 .61± 17.3 3 )万和 (15 .64± 8.73 )万。保存 4～ 7个月组和新鲜组相比 ,差异无显著性 (P >0 .0 5 ) ,与保存 12组相比 ,差异有非常显著性 (P <0 .0 1)。病理改变见内皮细胞层消失 ,电镜下见成纤维细胞及平滑肌细胞形态正常 ,胶原纤维排列整齐。结论　液氮冷冻保存 7月内 ,带瓣管道仍具有较强的细胞活性。     

同种瓣内皮细胞再孵化的进展

自 195 6年首次使用新鲜的同种瓣治疗主动脉瓣关闭不全 ,至 1975年 ,因同种主动脉瓣置换的效果较差 ,瓣膜损坏率明显高于异种生物瓣 ,几近废弃。同年 ,Ｏ’Ｂｒｉｅｎ等创立了抗生素灭菌、控制降温速率、液氮深低温保存法 ,使瓣膜组织内成纤维细胞存活 ,瓣膜性能有较大的提高 ,同种瓣再次被广泛地应用于心脏外科。同种瓣的优点同种瓣有以下优点 :( 1)自然构形 ,最适宜的流体力学功能和中央无阻碍血流 ,故具有最好的血液动力学效应 ,跨瓣压差低 ,如同生理瓣膜。 ( 2 )无声响。 ( 3)退化慢 ,瓣叶较少发生钙化。 ( 4)血栓形成和栓塞发生率很低 …     

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.     

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.     

Immunomodulation in Transplantation with Photopheresis

Abstract: Photopheresis is a technique in which peripheral blood mononuclear cells, in the presence of a photoacti-vatable compound, are exposed extracorporeally to ultraviolet A light and reinfused, inducing a host autoregula-tory immune response. Experimental work and ongoing clinical studies are helping to define the role of this novel, safe, and non-toxic immunomodulating technology in the field of transplantation.