Expression and distribution of aquaporin 8 in rat hepatocytes cold stored 72 hours in modified University of Wisconsin and bes-gluconate-sucrose solutions. Study of their correlation with water content

Since few data are availble on the genetic responses to low temperatures, we investigated if cold storage of hepatocytes (0 degree C, mUW or BGS solutions, 72 h) can affect gene expression and/or cellular localization of AQP8 and their correlation with water movements. Cold preserved hepatocytes showed a significant decrease in water content (P less than 0.05) but were able to regulate their volume when they returned to physiological conditions. These changes were not related to modulation in the expression and the pattern of distribution of AQP8 suggesting that other mechanisms are involved. The study of the quantitative changes in the expression of genes coding for liver specific proteins in cold preserved hepatic cells is of interest in order to develop new preservation methods or solutions that could contribute to maintain the utility of these cells when destined to be applied in clinical models.     

Cold preservation of endothelial cells in sucrose-based solution (SbS) and University of Wisconsin (UW) solutions: comparison of normoxic or hypoxic storage

Cold preservation of endothelial cells was studied, comparing primary endothelial cells (human umbilical vein endothelial cells - HUVECs) and a continuously growing cell line (ECV304 cells). Viability at the end of 24h cold preservation was measured by dye exclusion, whilst metabolism was assessed by Alamar blue conversion. Two preservation solutions were studied (UW solution) and sucrose-based (SbS) in both cell types. The response was similar in both cell types to preservation under normoxic conditions (with percentage dye exclusion maintained at about 80 percent in both preservation solutions) whereas under hypoxic conditions ECV304 were more sensitive to preservation in UW solution (dye exclusion reduced to 43.5+/-1.4 percent versus 73.6+/-14 percent (P<0.01). Metabolism assessed by Alamar blue conversion after cold preservation and rewarming was similar in both ECV304 and HUVECs after storage under normoxic conditions in UW solution, but in both cell types, metabolism was higher in SbS (P<0.05 and p<0.01) than in UW solution. Under hypoxic conditions, both cell types showed similar recovery of metabolism after storage in either UW or SbS. If the cells (in this case ECV304 under aerobic conditions) were stored for 24h and then allowed to rewarm in either of the respective preservation solutions (UW or SbS for 1h) before the Alamar blue test, metabolism was higher (p less than 0.01) in those exposed to SbS. UW solution and SbS provide similar protection for endothelial cells under hypoxic conditions, but SbS has some advantages under normoxic storage or if the cells experience variable temperatures in the presence of residual preservation solution at the end of cold preservation period.     

Ammonium detoxifying activity is maintained after 72 hours of cold preservation of rat hepatocytes in University of Wisconsin (UW) solution

The ammonium removal efficiency (ARE) and cell viability was investigated in freshly isolated rat hepatocytes exposed to increasing ammonium loads (0.1-2.0 mM). No difference was observed in both ARE and cell viability at the different ammonium concentrations tested. Storage of hepatocytes at 0 degrees C for 72 hours inhibited ammonium removal and urea synthesis. Rewarming of cells at 37 degrees C for 120 min was followed by an ARE fully comparable to freshly isolated hepatocytes. These data indicated that cold preservation of rat hepatocytes for 3 days in UW followed by a rewarming is associated with normal ammonium detoxification efficiency.     

Molecular imaging contrast media for visualization of liver function

Background and Aims

Diagnosis of liver disease has improved because of progress in imaging technology. Among the imaging methods, magnetic resonance imaging (MRI) has the advantage of a lack of radiation exposure, but the basis of the method (imaging of hydrogen atoms in water molecules) makes it hard to detect changes in tissue or the location of the diseased tissue in the liver. The aims of this study are to develop new contrast media for visualization of functional changes in the liver and to check the effectiveness of the media.

Methods

We developed a new molecular imaging contrast media that targets the asialoglycoprotein receptor (ASGP-R), a membrane protein that is specific to hepatocytes. We first checked the contrast media diameter and the cytotoxicity. Next, we examined the interaction of the media with ASGP-R through observation of fluorescein isothiocyanate (FITC)-labeled molecular imaging contrast media bound to normal hepatocellular ASGP-R using confocal laser scanning microscopy. Finally, we used MRI to observe hepatocyte interactions with the molecular imaging contrast media.

Results

The contrast media forms a nanoparticle of about 30 nm diameter in aqueous solution and the cytotoxicity is low. In vitro, the media has high specificity for ASGP-R in normal rat hepatocyte RLN-8 cells and this interaction was blocked by lactose (which has a similar molecular structure to that of galactose) and by an anti-ASGP-R antibody. The contrast media markedly enhanced T1-weighted images in MRI of normal rat hepatocytes compared to the signal strength for rat liver cancer cells.

Conclusions

We have shown that our new contrast media for molecular imaging of hepatocytes by MRI is effective in vitro.     

The assessment of viability in isolated rat hepatocytes subjected to cold or subzero non-freezing preservation protocols using a propidium iodide modified test

A rapid and simple assay (6 min, two steps) is described for determination of cell viability of hepatocytes subjected to cold preservation protocols. In this method, cells are incubated with the fluorescent marker propidium iodide (PI) and the fluorescence intensity is measured before (direct fluorescence--Fd) and after (total fluorescence--Ft) addition of digitonin, which allows the dye to enter the hepatocytes. The Fd originated from non-viable cells that have membrane damage and taken up PI. The Ft originated from all cells in the sample. The ratio between the two fluorescence values is used as an indicator of cell viability. The assay was challenged versus two classical viability tests: LDH retention and Trypan Blue exclusion. Our assay shows good correlation only with Trypan Blue test. In addition, a fluorescence confocal microscopy protocol was used to evaluate the possible toxicity of PI in hepatocyte suspensions.     

Reminiscence of 40-year research on nitrogen metabolism

This article summarizes my research over 40 years. The main theme of my work is nitrogen metabolism of amino acids, though later I focused on protein turnover in the cell. In the first years of my research work, I was busy dissecting the pathways involved in the metabolism of certain amino acids and their related enzymes. Then I became interested in the physiology and regulation of matabolism of these amino acids. For that, I used primary cultured hepatocytes, which contain many liver-specific enzymes. However, this play field was very rough around 1970 and hence I had to smooth them (differentiated) first. We discovered a specific growth factor (hepatocyte growth factor, HGF) in rat platelets. Exceptionally, I also worked on branched chain amino acids (valine, leucine and isoleucine). These amino acids are not efficiently metabolized in the liver, so I had to consider the physiology of extrahepatic tissues as well. Finally, I came across a huge protease complex, the proteasome. Whether these players, small amino acid metabolizing enzymes and the huge protease complex, danced well in harmony on my playground or not, I still do not know.     

Hypothermic renal preservation with a sucrose/ polyethylene glycol solution in a rabbit renal transplant model

Renal preservation at for 24 hours at hypothermia was studied in a rabbit model after flush cooling with sucrose-based solution (SBS), compared with a standard preservation solution (in this case, Marshall's Hypertonic Citrate solution - HCA). Polyethylene glycol supplementation to SBS (SBS-PEG) was also investigated. Renal function was measured by plasma creatinine assays during 1 months post transplantation, and pathology of the explanted kidneys was undertaken. Results showed that survival at 28 days was similar in all groups, (HCA - 3 out of 6; SBS - 2 out of 5; SBS-PEG - 3 out of 5), and there were no differences in recovery of plasma creatinine values. Histopathological evaluation of the grafts indicated that SBS preservation resulted in more severe damage after transplantation (P less than 0.05 in both corticomedullary region and medulla compared to HCA), whilst addition of PEG reduced the damage score to that seen with HCA. SBS can be used as a simple, inexpensive preservation solution for kidney cold storage provided that PEG is used as an additional colloid.     

A simple GC method for determination of cryoprotector diols 1,4-butanediol or 2,3-butanediol in isolated rat hepatocytes

We devised a simple method for determining the cryoprotectant agents 1,4-butanediol or 2,3-butanediol in isolated rat hepatocytes. After extraction of of hepatocytes with water (containing internal standard - ethylene glycol 1.25 mg/mL) the diol content was analyzed by gas chromatography. The method shows a linear response in the range 0.125 to 2.50 mg/mL for 1,4-butanediol and 0.25 to 3.75 mg/mL for 2,3-butanediol. The accuracy and precision of the method were evaluated and the coefficients of variation were found to be within = 6.0 %. The recoveries from hepatocyte samples containing 0.50, 1.00 and 2.00 mg/mL were 91.0 to 108 % for 1,4-butanediol and 80.6 to 100.3 % for 2,3-butanediol, respectively. This method allowed the determination of the intracellular concentration of diols in hepatocytes preserved for up to 120 hours at - 4 (C in UW solution + 8 % w/v 1,4-butanediol (or 2,3-butanediol).     

Tissue pH in human kidney transplants during hypothermic ischemia

Ex vivo NMR spectroscopy was used to investigate pH in 67 human kidney transplants. (1)H and (31)P spectra were recorded at 1.5 T during regular hypothermic storage in histidine-tryptophane-alpha-ketoglutarate (HTK) solution. Estimations of cytosolic pH from chemical shift differences between inorganic phosphate and phosphodiesters and of extracellular pH from the varepsilon1 and delta2 protons of histidine were based upon systematic titration studies. The possibility to predict acute tubular necrosis (ATN) by measuring pH was compared to results obtained with peak area ratios of phosphomonoesters (PME) and Pi and of the gamma-phosphorus of nucleoside 5'-triphosphate (gamma-NTP) and Pi. Cytosolic pH was 6.86+/-0.10 in kidneys showing immediate post-transplant function and 6.84+/-0.10 in those with ATN. Time-dependent studies demonstrated a monoexponential pH decay (velocity constant: 0.14+/-0.07 h(-1)). Extracellular pH varied between 7.40 and 7.15. Grafts with immediate function showed higher PME/Pi (2.24+/-0.57 vs. 1.77+/-0.50, p<0.05) and gamma-NTP/Pi (0.33+/-0.16 vs. 0.16+/-0.08, p<0.001). Intra- and extracellular pH can be monitored non-invasively during hypothermic transplant storage. The pH gradient between both compartments provides quantitative information about the buffer capacity of the preservation medium. Acidification is not a primary cause of ATN during regular HTK storage. The total nucleotide pool is a determinant of the reversibility of ischemic injury.     

四丁基溴化铵水合物浆在蓄冷空调中的应用前景

TBAB水合物浆作为适用于空调工况的新型两相潜热输送载冷剂,可以大幅度降低冷量输送的功耗。通过添加成核剂的方法来降低所需的过冷度,制备方法简单节能,而且蓄冷特性出色,相变蓄冷温度与溶液的浓度密切相关,可通过调节溶液的浓度,获得与空调冷冻水一致的相变温度。根据非牛顿流体的特点,综述了国内外关于TBAB水合物浆流变方程的选择,列出了表观粘度及传热系数的计算方法,并指出TBAB水合物浆应用于蓄冷空调中的优点。     

Mammalian hibernation: lessons for organ preparation?

The adaptations to low environmental temperatures exhibited in mammalian hibernation are many and varied, and involve molecular and cellular mechanisms as well as the systematic physiology of the whole organism. Natural torpidity is characterised by a profound reduction in body temperature and other functions lasting from a few hours to several weeks. Controlled reduction of heart rate, respiration and oxygen consumption is followed by the fall in body temperature. However, thermoregulation persists such that a decrease in ambient temperature below dangerous levels typically triggers arousal, and shivering and non-shivering thermogenesis from brown fat provide the heat to restore body temperature to normal levels. Many of the cellular mechanisms for survival are similar to those brought into play during medium-term storage of organs destined for transplantation. For example maintenance of ionic regulation and membrane fluxes is fundamental to cell survival and function at low body temperatures. Differences between hibernating and non-hibernating species are marked by differences in Na+/K+ transport and Ca++pumps. These in turn are probably associated with alterations in the lipoproteins of the plasma membrane and inner mitochondrial membrane. We have accordingly conducted a series of pilot studies in captured Richardson's ground squirrels kept in laboratory conditions as a model for hypothermic organ preservation. Tissue function was compared during the summer (non-hibernating season) with that in the winter when the animals could be: (i) in deep hibernation in a cold chamber at 4 degree C; (ii) maintained in an ambient temperature of 4 degree C but active and awake; or (iii) active at an ambient temperature of 22 degree C. The studies involved: whole animal monitoring of standard physiological parameters; whole organ (kidney) storage and transplantation for viability assessment; storage and functional assessment on an ex vivo test circuit with capacity for perfusion at normothermic and hypothermic temperatures; measurement of thyroid function; measurements of total nucleotides (ATP, ADP and AMP)and ratios by standard techniques after freeze-clamping of organs; similar nucleotide and pH measurements using31P-NMR as a non-invasive whole animal technique; and measurement of O2 uptake and gluconeogenesis using isolated renal tubules and isolated hepatocytes. Marked differences in cold tolerance were demonstrated between organs taken from hibernating versus non-hibernating individuals. In particular kidneys transplanted from animals in deep hibernation were capable of withstanding up to 72 hours of cold storage as compared with up to 24 hours in non-hibernating squirrels or in comparable sized rats. Adaptations which might provide valuable clues in our attempts to better preserve human organs for transplantation are explored in some depth in this report.     

Cold preservation of isolated hepatocytes in UW solution: experimental studies on the respiratory activity at 0 degrees C

To date, little attention has been paid to the role of the gas milieu in preservation solutions and its effect on cell viability. Dissolved O2 in the preservation media may be an important parameter to consider. In this study we polarographically measured the O2 concentration in air-equilibrated UW solution at 0 degrees C, as well as the respiratory activity of isolated hepatocytes cold-preserved in this solution up to 72 hours. To perform measurements at 0 degrees C, it was first necessary to characterize the sensor behavior at low temperatures. We verified that the sensor response is still linear at this temperature but the rate of response is significantly slower. The O2 solubility in UW-air solution at 0 degrees C was determined using a modified physical method and it was 410 microM O2, which, as expected, is lower than the solubility in water at the same temperature (453 microM O2). Isolated hepatocytes cold-stored in UW-air solution retained a measurable respiratory activity during a period of 72 hours. The O2 consumption rate was 0.48 +/- 0.13 nmol/O2/min/10(6) cells, which represents 1% of the control value at 36 degrees C (61.46 +/- 14.61 nmol/O2/min/10(6) cells). The respiratory activity and cell viability were well maintained during the preservation period. At present, preservation conditions need to be improved for cells to remain functionally active. Dissolved O2 may be required for energy re-synthesis but it also leads to an increment in reactive oxygen species. The O2 concentration in the preservation solution should be carefully controlled, reaching a compromise between cell requirement and toxicity.     

Study on the concentration difference heat pump using fusion and freezing processes of acetic acid-acetamide system

The concentration difference heat pump using fusion and freezing processes to generate a cold fluid has been investigated. This heat pump system utilizes the acetic acid-acetamide pair as working material, and consists of a cold fluid generating and a separation process. The operation at the cooling capacity of 3.52 kW (1 ton of refrigeration) has been investigated in this study. At the cold fluid generating process, solid acetic acid at 15°C is fused into an acetamide solution at 15°C, such that the temperature and the concentration of acetamide of the solution decreases. This dilute solution at lower temperature can be used to generate a cold fluid. The lowest attainable temperature of the solution has been investigated experimentally, and also calculated from the energy balance equation. The decreasing rates of the temperature have also been studied. At the separation process, continuous distillation is adopted to concentrate the dilute solution sent from the cold fluid generating process. The data which support the possibility of separation by continuous distillation are presented. The energy demand at the separation process is investigated theoretically.     

In vivo bone regeneration using a novel porous bioactive composite

Many commercial bone graft substitutes (BGS) and experimental bone tissue engineering scaffolds have been developed for bone repair and regeneration. This study reports the in vivo bone regeneration using a newly developed porous bioactive and resorbable composite that is composed of bioactive glass (BG), collagen (COL), hyaluronic acid (HYA) and phosphatidylserine (PS), BG-COL-HYA-PS. The composite was prepared by a combination of sol-gel and freeze-drying methods. A rabbit radius defect model was used to evaluate bone regeneration at time points of 2, 4 and 8 weeks. Techniques including radiography, histology, and micro-CT were applied to characterize the new bone formation. 8 weeks results showed that (1) nearly complete bone regeneration was achieved for the BG-COL-HYA-PS composite that was combined with a bovine bone morphogenetic protein (BMP); (2) partial bone regeneration was achieved for the BG-COL-HYA-PS composites alone; and (3) control remained empty. This study demonstrated that the novel BG-COL-HYA-PS, with or without the grafting of BMP incorporation, is a promising BGS or a tissue engineering scaffold for non-load bearing orthopaedic applications.     

大温差离心机组低负荷运行防喘振策略分析

稳态强磁场实验装置水蓄冷系统是国内外具有代表性的大流量大温差水蓄冷系统之一. 系统包含两个3000 m3 的蓄水罐, 为提升蓄冷量双罐均采用了自然分层技术. 由于该技术的特性, 在蓄冷后期, 离心式冷水机组的入口水温会偏低, 这导致系统新增的12 ℃ 大温差机组在试运行阶段多发低负荷喘振故障, 影响了制冷运行的稳定度. 本文首先对离心式冷水机组的喘振机理进行了阐述, 进而分析了在改造前针对该台冷水机组所做的防喘振优化措施; 通过热气旁通改造结合相关参数的合理设置, 避免机组喘振风险; 通过优化 DDC 控制, 优化机组减载能力, 避免了低负荷振动及噪声. 本次改造为系统的安全稳定运行提供了保障.     

肾脏冷灌注过程中的生物传热实验研究

在保护液亚临界相变温度范围进行肾脏器官的低温延时保存,是一个不同于传统低温保存的新方法。为保持细胞活性必须从器官摘取的第一步展开研究,用保护液对离体肾脏进行冷灌注是移植器官保存的重要环节,研究冷灌注过程中器官温度随时间变化的传热特征,探求提供与传统生物保存方法所不同的传热传质条件非常重要。用高倍率红外热象仪、热电偶探针、超声血流计等,对猪整体肾脏冷灌注过程进行了实时测试,得出动脉血管作为冷源时的肾脏温度动态变化曲线。实验结果表明,在1℃冷却液匀速灌注过程中肾脏温度的降低表现为非均匀变化,究其原因是由于肾脏内特定的组织结构引起。实验研究将对肾脏器官的三维温度场数值计算提供宝贵的实验依据。     

Ultrasonic propagation properties (@ 100 MHz) in excessively fatty rat liver

The effects on the ultrasonic propagation properties of livers of the addition of 1% orotic acid to rat diets were examined. In rats, dietary orotic acid exerts several effects on lipid metabolism; its overall consequence is that excessively high hepatic fat concentrations are built up over a short period of time, thus making this an ideal model to study the ultrasonic propagation properties as a function of sequential development of fatty liver. Over a 16-day period on the orotic acid diet, the supplemented rat liver lipid concentrations increased from a normal range of 2%-4% to the lower 20's%; hepatic water concentration decreased from a normal value of approximately 70% to approximately 50%; total protein concentration decreased slightly from a normal range of 17%-20% to 11%-16%; and rat liver weight increased from approximately 11 g to around 20 g. Ultrasonic attenuation coefficient and speed were assessed in liver tissue with the scanning laser acoustic microscope at 100 MHz. As hepatic lipid increased, ultrasonic attenuation at 100 MHz increased temporally from a normal range of 12-14 dB/mm to a maximum of 54 dB/mm and ultrasonic speed decreased from a normal range of 1553-1584 m/s to a minimum of 1507 m/s. Multivariant linear regression was used in the analysis of covariance to fit the least-squares estimates to the linear regression model. Strong correlates of ultrasonic speed with both water concentration and fat concentration in the liver were observed.     

Coolant choice for the central beryllium pipe of the BESⅢ beam pipe

In order to take away much more heat on the BESⅢ beam pipe to guarantee the normal particle detection,EDM-1(oil No.1 for electric discharge machining),with good thermal and flow properties was selected as the candidate coolant for the central beryllium pipe of the BESⅢ beam pipe.Its cooling character was studied and dynamic corrosion experiment was undertaken to examine its corrosion on beryllium.The experiment results show that EDM-1 would corrode the beryllium 19.9 μm in the depth in 10 years,which is weak and can be neglected.Finite element simulation and experiment research were taken to check the cooling capacity of EDM-1.The results show that EDM-1 can meet the cooling requirement of the central beryllium pipe.Now EDM-1 is being used to cool the central beryllium pipe of the BESⅢ beam pipe.