Fatty acid metabolism during hypothermic perfusion of the isolated dog kidney

Perfusion of isolated dog kidneys was performed at 8--12 degrees C using an albumin solution containing caprylic acid (about 6 mmol/l) and long-chain fatty acids (about 0.5 mmol/l). During 48 h the amounts of caprylic acid in the perfusate fell by about 3 mmol, whereas long-chain FFA increased by 0.2--0.3 mmol and small amounts of arachidonic acid appeared. [14C]palmitate or [14C]linoleate--when added--decreased by about 10% in the perfusate. The decrease was mainly due to exchange with kidney phospholipid fatty acids. Only about 0.4% was recovered as [14C]CO2. The amounts of total phospholipids in kidney tissue decreased by up to 10% during the perfusion (when 10% kidney weight gain was taken into account), whereas lysophospholipids, cholesterol, triglycerides and free fatty acids remained essentially unchanged. The distribution of fatty acids in the total phospholipid fraction was strikingly altered. The relative amounts of arachidonic acid increased, whereas all other major fatty acids decreased. Synthesis of arachidonic acid by chain elongation of perfusate [14C]linoleic acid could not be demonstrated, and no evidence was found for a significant increase of the total amounts of arachidonic acid. The possibility that arachidonic acid-containing phospholipids in the kidney are preferentially preserved during the hypothermic conditions is discussed.     

Release of platelet activating factor by the isolated kidney is not linked to the production of prostaglandins

In many isolated tissues, including glomerular mesangial cells and endothelial cells, the synthesis of platelet activating factor (PAF) occurs by remodeling the phospholipids so that the production of PAF results in the release of arachidonic acid with subsequent production of cyclooxygenase or lipoxygenase products. In some tissues, including the renal medulla, another pathway for PAF biosynthesis (the de novo pathway) has been found in which the production of PAF is not linked to the production of arachidonic acid products. We tested the hypothesis that the remodeling pathway was active in the release of PAF into renal venous effluent of the isolated kidney. Isolated rat kidneys perfused at constant flow with albumin-containing buffer were stimulated to produce prostaglandin by an infusion of angiotensin II or bradykinin. Some kidneys were also challenged with the calcium ionophore A23187. Perfusate was collected for bioassay of PAF and radioimmunoassay of prostaglandin (PG) E2; urine was collected for PAF bioassay. Angiotensin II (10(-9) to 10(-8) M) increased renal vascular resistance, and bradykinin (10(-8) to 10(-7) M) and A23187 (3 x 10(-6) M) reduced renal vascular resistance. PGE2 production was increased significantly by bradykinin and angiotensin II but not by A23187. Only A23187 increased the release of PAF into the perfusate. Urine PAF was not changed by any of the stimuli. These data indicate that the release of PGE2 by the isolated, perfused rat kidney can be dissociated from the release of PAF. The findings support the suggestion that PAF released by the kidney into the renal venous effluent is not produced by remodeling the lipids that are the source of renally released prostaglandins.     

超声造影分析正常移植肾皮质不同部位微循环灌注特点的研究

目的利用超声造影观测正常移植肾皮质不同部位的微循环灌注特征。方法入选病例为肾移植时间1年以上的20例患者,均知情同意,经随访证实移植肾功能均正常。常规超声检查结束后,采用声诺维团注法超声造影技术对移植肾实质微循环血流灌注进行实时监测,并利用Sono Liver定量分析软件进行分析。选择移植肾同一切面包膜下肾皮质(外层皮质,ROI1)、弓形动脉上方肾皮质(近髓皮质,ROI2)、中层皮质(ROI3)以及肾外皮下组织(参考区,ROIc)4个感兴趣区,获得相应的时间—强度曲线(TIC曲线)及定量参数:上升时间(RT)、达峰时间(TTP)和峰值强度(IMAX)。分析移植肾三个感兴趣区的微循环灌注特征。结果 TIC曲线显示:近髓皮质处最早开始显影,灌注强度迅速上升,迅速下降至一定灌注强度并缓慢消退;外层皮质最后显影,最早到达灌注峰值,并迅速下降至一定强度后缓慢消退;中层皮质介于两者之间。时间—强度定量参数:参考区、外层皮质、中层皮质和近髓皮质的RT分别为(25.29±12.75)s、(9.72±2.43)s、(10.45±2.55)s及(10.92±2.69)s;TTP分别为(35.88±19.07)s、(13.52±4.34)s、(13.73±4.47)s及(14.18±4.53)s;IMAX分别为(100.00±0)%、(1132.82±1170.93)%、(2032.93±2444.65)%及(2403.92±2981.03)%。参考区与皮质内3个感兴趣区的参数比较,差异有统计学意义(P0.05);外层皮质、中层皮质及近髓皮质的RT、TTP及IMAX呈逐渐增大之势,但差异无统计学意义(P﹥0.05)。结论超声造影能为临床监测移植肾的状况提供一种新的思路和方法。     

Evaluation of the transfusion effectiveness of various platelet concentrates by means of an in vitro perfusion technique

An in vitro perfusion technique was used for the assessment of the effectiveness of platelet concentrates (PCs) administered to patients with bone marrow failure. Denuded rabbit arterial segments mounted in annular chambers were exposed to nonanticoagulated blood drawn directly from the antecubital vein. This perfusion system allows the direct visualization of platelet-subendothelium interactions. The deposition of platelets on exposed subendothelium and the presence of fibrin were evaluated morphometrically. Perfusions were performed before and 10 and 120 minutes after the transfusion of PCs that had been obtained by apheresis and administered immediately after collection (A) or separated by centrifugation of blood units and transfused 24 hours after storage at 22 degrees C (B) or 4 degrees C (C). Bleeding times were also determined systematically. The deposition of platelets on the subendothelium increased significantly (p less than 0.01) 10 minutes after transfusions of A and B PCs, but the presence of fibrin did not differ from pretransfusion values. Two hours after the administration of A or B PCs, the improvement of platelet deposition persisted. By then, the presence of fibrin increased markedly (p less than 0.01). The deposition of platelets was not modified after the transfusion of C PCs, but the presence of fibrin was augmented significantly at both posttransfusion times. Comparisons between bleeding times and morphometric results pointed to a possible role of fibrin formation in contributing to primary hemostasis.     

预冻技术参数对血小板冻干保存的影响

本研究采用正交设计方案探讨冻结速率、退火速率、退火温度和退火持续时间4个预冻条件参数对血小板冻干保存的影响．冻干血小板再水化后的数值恢复率、细胞形态和结构、血小板活化和聚集力等指标分别用血细胞计数计、扫描电子显微镜、3色流式细胞仪及对凝血酶的聚集反应进行检测并综合评价．结果显示：在不同的预冻条件组合方式下，冻干血小板的数值恢复率在（91．3％-53．5）％范围内；实验各组血小板冻干制品的冰晶大小和形状有所不同；冻干再水化血小板的活化标记物PAC-1和CD62p的表达和分布与新鲜血小板较为接近，其中PAC-1表达率均维持在较低水平（0．03％-0．22％），而各组样本CD62p的表达水平存在差异．实验中根据血小板数值恢复率得到预冻条件的最佳理论组合是A2B1C1D3，即将血小板悬液先以20℃／min的速率冻结至-40℃维持2小时。再以1．5℃／min对搁板升温至-30℃后维持0．5小时，最后进入冻干程序直至结束。结论：冻结速率、退火速率、退火温度和退火持续时间对冻千血小板的数值恢复率均有作用，预冻条件参数的不同组合方式影响血小板冻干保存效果．     

Detection of HLA antibodies by platelet crossmatching techniques

Thirty-seven monospecific HLA antibodies directed against all common HLA-A and -B loci and reactive by the microlymphocytotoxicity assay (LCT) were tested against platelets carrying the corresponding antigen by three platelet crossmatch methods, the platelet immunofluorescence test (PIFT), platelet enzyme-linked immunosorbent assay (P-ELISA), and platelet radioimmunoassay (P-RIA). Positive reactions were obtained with the PIFT in 67 percent, the P-ELISA in 41 percent, and the P-RIA in 49 percent of 85 cell-serum pairs. The same cell-serum combinations gave 49 percent positive reactions in the lymphocyte immunofluorescence test. Three multispecific HLA antisera were positive in nine of nine cell-serum combinations by all four methods. Thirteen transfusions were given to eight patients with known HLA antibodies. All donor-recipient pairs were LCT positive, six were PIFT positive, and seven were PIFT negative. Three of seven PIFT-negative and none of six PIFT-positive transfusions were successful. Thus, platelet crossmatching is less sensitive than the LCT for the detection of complement-binding monospecific HLA antibodies. The platelet crossmatch, however, is able to identify some potentially successful HLA-incompatible donors for patients with multispecific HLA antibodies and limited access to HLA-identical donors.     

超声造影定量小鼠肾血流灌注的研究

目的评估超声造影定量小鼠肾脏血流灌注的可靠性。方法雌性小鼠7只,经颈静脉匀速泵入微泡造影剂,全程记录造影图像,脱机定量造影剂再充填曲线参数,软件自动计算声学造影平台强度、再充填曲线上升斜率以及两者乘积。比较小鼠两次造影结果的一致性,并评估造影强度对造影定量曲线拟合优度的影响。结果所有小鼠均取得良好的肾脏显影效果。全部造影定量分析曲线的拟合优度极佳,其决定系数高达0．945±0．050（P〈0．05）。在一定范围内,超声造影平台强度与超声造影定量曲线的拟合优度之间呈正相关（r=0．760,P=0．002）。同一只小鼠两次超声造影的肾脏血管密度、血流速度及肾血流量比较差异均无统计学意义（P〉0．05）。结论应用超声造影定量小鼠肾脏血流有良好的重复性,提高超声造影平台强度,对于改善定量分析结果有一定的帮助。     

肾脏CT灌注成像

CT灌注成像是在静脉团注对比剂后对选定层面进行同层动态扫描,以获得该层面内每一像素的时间-密度曲线,根据该曲线利用不同的数学模型计算出血流量、血容量、对比剂的平均通过时间、对比剂峰值时间、表面通透性PS图等参数,以此来评价组织器官的灌注状态.本文就肾脏CT灌注技术做一综述.     

超声造影评价移植肾皮质不同部位的微循环灌注

目的 探讨CEUS评价移植肾皮质不同部位的微循环灌注特点。方法 根据血清肌酐（SCr）值将40例移植肾患者分为SCr正常组和SCr异常组。经肘静脉团注SonoVue行移植肾灌注CEUS,采集动态图像。选择移植肾同一切面包膜下肾皮质（外层皮质,ROI₁）、弓形动脉上方肾皮质（近髓皮质,ROI₂）,获得相应时间-强度曲线及定量参数,包括到达时间（AT）、上升时间（RT）、达峰时间（TTP）、绝对达峰时间（ATTP）、平均渡越时间（MTT）。比较两组患者移植肾皮质不同ROI指标间差异。结果 正常移植肾的CEUS灌注期表现为近髓皮质首先开始显影,外层皮质随后显影;消退期表现为外层皮质首先开始消退,近髓皮质消退晚于外层皮质出现消退。除AT外,SCr异常组移植肾外层皮质的灌注指标较SCr正常组延迟（P<0.05）。SCr正常组与SCr异常组移植肾近髓皮质的灌注指标间差异无统计学意义。结论 移植肾外层皮质较近髓皮质能够更敏感地反映微循环灌注的改变。选择移植肾外层皮质作为ROI对于早期评价移植肾微循环改变具有重要意义。     

Comparison of cytologic and biochemical properties between liver cells isolated from adult rats by trypsin perfusion and those isolated by collagenase perfusion

Primary cultures of liver cells isolated from adult rats by trypsin and collagenase perfusion techniques were carried out to compare cytologic and biochemical properties between the differently prepared cells. Trypsin-dispersed cells consisted of comparatively smaller cells, whereas collagenase-dispersed cells consisted of larger cells. The cell attachment efficiency on culture day 1 was about twice as high in the liver cells prepared with collagenase than those prepared with trypsin. Mature hepatocytes isolated by collagenase perfusion could be maintained in the primary culture for a longer period than those isolated by trypsin perfusion. Epithelial-like clear cells started to grow much earlier in the primary culture of the trypsin-dispersed liver cells than in that of the collagenase-dispersed liver cells. Earlier proliferation of epithelial-like clear cells could not be induced by in vitro trypsinization of the collagenase-dispersed liver cells. Both kinds of enzymatically prepared liver cells showed albumin production and exhibited glucose 6-phosphatase (D-glucose-6-phosphate phosphohydrolase, EC 3.1.3.9, G6Pase) and tyrosine aminotransferase (L-tyrosine: 2-oxoglutarate amino-transferase, EC 2.6.1.5, TAT) activities for 1 week in the primary culture. Albumin production was higher in the liver cells prepared with collagenase than those prepared with trypsin, whereas G6Pase activity was almost the same between them. TAT activity up to culture day 2 was about 3-fold higher in the liver cells prepared with collagenase than in those prepared with trypsin. Combined supplementation of dexamethasone (1 X 10(-5)M) and insulin (10 micrograms/ml) consistently improved the cell attachment efficiency and was very effective in the maintenance of mature hepatocytes in both types. Furthermore, these hormones enhanced the albumin production and TAT activity in both types.     

Metabolism of acetaminophen by the isolated perfused kidney

Acetaminophen (APAP) produces proximal tubular necrosis in the Fisher 344 rat. This lesion may result from the covalent binding of reactive intermediates of APAP to cellular macromolecules when glutathione (GSH) is sufficiently depleted. Experiments were designed to evaluate the ability of the kidney to convert APAP to reactive electrophilic metabolites capable of depleting renal GSH by quantifying GSH concentrations in isolated perfused kidneys perfused with APAP. Perfusion without APAP reduced (3 X 10(-5) -3 X 10(-5) M) to the perfusion medium further reduced renal GSH content. Treatment of rats with polybrominated biphenyls enhanced the ability of 3 X 10(-8) M APAP to deplete GSH. In contrast, treatment with piperonyl butoxide reduced the depletion of GSH produced by 3 X 10(-5) M APAP. At 3 X 10(-5) M APAP, the glucuronic acid, sulfate and the mercapturic acid conjugates were excreted by the isolate perfused kidneys. After treatment with polybrominated biphenyls, mercapturic acid excretion increased 4-fold, whereas the glucuronic acid and sulfate conjugate excretions were unaffected. These data suggest that the kidney can produce an electrophilic metabolite of APAP which can combine with and deplete renal GSH. An electrophilic metabolite of APAP produced by the kidney may initiate APAP induced renal necrosis.     

超声造影评价横纹肌溶解致急性肾损伤肾的血流灌注实验

目的探讨超声造影定量参数评价横纹肌溶解致急性肾损伤大鼠肾血流灌注的变化。 方法将60只SD大鼠随机分为模型组和对照组,每组30只,模型组大鼠双后肢肌肉注射50%甘油生理盐水10 ml/kg,对照组大鼠双后肢肌肉注射0.9%生理盐水10 ml/kg。于注射后0.5 h、2 h、6 h、12 h和24 h五个时间点每组取6只大鼠行超声造影检查,获取肾皮质和髓质时间-强度曲线及相关定量参数［上升斜率（AS）、曲线下面积（AUC）、下降斜率（DS）、降半时间（DT/2）、峰值强度（PI）］,分析不同时间点肾超声造影特征。超声检查结束后下腔静脉取血检测血清尿素氮、肌酐、胱抑素C、肌酸激酶水平,肾组织固定后制备病理切片观察。采用t检验或Mann-Whitney U检验比较模型组和对照组间超声造影相关定量参数和血清生化指标的差异,采用单因素方差分析或Kruskal-Wallis H检验比较组内不同时间点各指标的差异。 结果与对照组相比,模型组0.5 h、2 h、6 h和12 h的血清肌酸激酶、尿素氮、肌酐及2 h、6 h血清胱抑素C显著升高,差异具有统计学意义（血清肌酸激酶：Z=-2.242,P=0.025;Z=-2.882,P=0.004;Z=-2.882,P=0.004;Z=-2.562,P=0.010;尿素氮：t=4.288,P=0.002;t=4.450,P=0.001;t=10.812,P＜0.001;t=5.260,P＜0.001;肌酐：t=6.327,P=0.001;t=10.577,P＜0.001;t=2.612,P=0.035;t=4.694,P=0.001;胱抑素C：t=3.460,P=0.009;t=2.391,P=0.038）;模型组0.5 h的皮质AUC明显升高［（2636.84±150.99）dB·s vs（2308.20±210.50）dB·s］,差异具有统计学意义（t=3.107,P=0.011）,2 h、6 h和12 h的皮质DS及6 h、12 h和24 h的皮质AS显著减小［DS：（0.16±0.05）dB/s vs（0.23±0.03）dB/s,（0.16±0.03）dB/s vs（0.23±0.04）dB/s,（0.16±0.05）dB/s vs（0.23±0.03）dB/s;AS：（0.54±0.22）dB/s vs（0.84±0.15）dB/s,（0.58±0.21）dB/s vs（0.86±0.20）dB/s,（0.67±0.20）dB/s vs（0.96±0.13）dB/s］,差异具有统计学意义（t=-3.342、-3.542、-3.226、-2.733、-2.318、-2.809,P=0.007、0.005、0.009、0.021、0.043、0.019）,12 h的皮质DT/2明显增加［（55.78±12.28）s vs（40.09±6.29）s］,差异具有统计学意义（t=2.789,P=0.019）;0.5 h、2 h、6 h和12 h的髓质AUC明显升高［（2382.40±189.84）dB·s vs（1910.82±140.13）dB·s,（2637.35±258.55）dB·s vs（1999.88±409.52）dB·s,（2424.63±409.39）dB·s vs（1910.47±263.59）dB·s,（2353.54±348.2）dB·s vs（1958.43±95.37）dB·s］,差异具有统计学意义（t=5.025、3.217、2.669、3.014,P=0.001、0.009、0.024、0.013）,2 h和6 h的髓质DS及AS显著减小［DS：0.13（0.07,0.15）dB/s vs 0.22（0.19,0.24）dB/s,0.13（0.11,0.16）dB/s vs 0.21（0.20,0.23）dB/s;AS：（0.44±0.22）dB/s vs（0.77±0.14）dB/s,（0.41±0.21）dB/s vs（0.80±0.10）dB/s］,差异具有统计学意义（Z=-2.892、-2.585,P=0.004、0.010;t=-3.063、-4.042,P=0.015、0.005）,2 h、6 h和12 h的髓质DT/2明显增加［（61.44±15.52）s vs（38.88±8.80）s,（54.60±12.47）s vs（37.59±8.71）s,（56.08±14.57）s vs（38.42±6.00）s］,差异具有统计学意义（t=3.105、2.724、3.003,P=0.011、0.021、0.013）。病理结果显示：对照组肾小球及肾小管结构均正常;模型组肾小球结构未见明显改变,部分肾小管刷状缘脱落,上皮细胞扁平化,0.5 h开始肾小管管腔扩张明显,以远曲小管为主,2 h开始出现肾小管管型,并且随着建模时间的增加,肾小管损伤逐渐加重,24 h最严重。 结论超声造影能够敏感地反映横纹肌溶解致急性肾损伤大鼠肾血流灌注的变化过程,具有一定的诊断价值。     

Controlled-rate versus uncontrolled-rate freezing as predictors for platelet cryopreservation efficacy

BACKGROUND: Cryobiologic variables responsible for cell injuries and freezing techniques applicable in medical cryopractice should be revised and/or reengineered for minimizing cryoinjuries and maximizing cell recovery. In this study, the efficacy of different cryopreservation protocols based on platelet (PLT) recovery was evaluated. STUDY DESIGN AND METHODS: PLTs (n = 33) were prepared from whole-blood units. Cell count and viability, PLT morphologic score (PMS), and hypotonic shock response were determined. PLT surface antigens were measured by flow cytometry. Controlled-rate (with compensated fusion heat) and uncontrolled-rate freezing methods combined with 6 percent dimethyl sulfoxide were used. RESULTS: PLT recovery was superior in the controlled-rate setting (91.0 +/- 5.5 vs. 86.0 +/- 6.5; p < 0.05). PMS was significantly better in controlled-rate freezing (p < 0.01). GPIb/CD42b expression was reduced in both freezing groups versus control. GP140/CD62p expression was significantly (p < 0.05) lower in the controlled-rate group and in both frozen groups was significantly higher than in the control groups. CONCLUSION: The use of strictly equalized (1 degrees C/min) controlled-rate freezing, combined with an intensified cooling rate (2 degrees C/min) during the liquid-to-solid-phase transition period, allows advanced quantitative and qualitative PLT recovery, even though the minor intergroup differences for some variables were observed.     

Evaluation of perfusion and viability in hypothermic non-beating isolated porcine hearts using cardiac MRI

The donor heart undergoes degradation during hypothermic storage. An assessment of donor heart preservation is typically done with histological or biochemical methods that are not feasible in the clinical setting. We describe a method to study the donor heart using cardiac perfusion MRI that is potentially feasible for clinical use. Standard cardiectomy was performed in the pig model and the hearts were stored in normal saline at 5 °C. Imaging was performed by using a rapid gradient-echo sequence (FLASH) with saturation-recovery preparation for T1-weighting in the short axis and horizontal long axis views. Approximately 80 serial images were acquired at a rate of 1/s during administration of 0.006 mmol/ml Gd–DTPA (500 ml, 1 l/min). Signal intensity vs. time curves were generated for each heart and slice imaged and compared to a 0.006 mmol/ml Gd–DTPA reference. H&E stained biopsies of the LV, RV, and septum were also obtained. The mean duration of heart storage (N=10) was 8.8 h (range 4.2–19.2 h). Histologically, no differences were seen in H&E stained biopsies among hearts at different storage times. However cardiac MRI revealed a decrease in perfusion units in each subsequent heart tested after 4.2 h. (R=0.49). Average peak up-slope was used as a surrogate measure for flow capacity through the microvasculature and peak contrast enhancement was used as a measurement of viable microvasculature. The 4 h heart had 83% peak contrast enhancement of the reference standard, as compared to 44% for the 19.2 h heart. The decrease in peak enhancement is directly related to the duration of storage time. No correlation of peak up-slope of the intensity curve to storage time was found. This new application of cardiac MRI in the donor heart is applicable to: (1) assessing marginal hearts, (2) evaluating donor heart preservation techniques, and (3) correlating pre- to post-transplant viability.