A simple and reliable refractometric method to determine the total solids concentration of the cervico-vaginal bovine mucus samples.

Cervico-vaginal mucus (CVM) is a viscoelastic substance continuously produced by secretory cells of the endocervix and the vagina of cows. Its physicochemical composition varies depending on the hormonal status of the estrous cycle. In veterinary medicine refractometry is a widely diffused technique to determine total solids (TS) content of biological samples, but there are not published data of CVM total solids from refractometric measures.

The Novel N,N-bis-2-Hydroxyethyl-2-Aminoethanesulfonic Acid-Gluconate-Polyethylene Glycol-Hypothermic Machine Perfusion Solution Improves Static Cold Storage and Reduces Ischemia/Reperfusion Injury in Rat Liver Transplant.

Organ transplantation is the treatment of choice against terminal and irreversible organ failure. Optimal preservation of the graft is crucial to counteract cold ischemia effects. As we developed an N,N-bis-2-hydroxyethyl-2-aminoethanesulfonic acid-gluconate-polyethylene glycol (BGP)-based solution (hypothermic machine perfusion [HMP]), we aimed to analyze the use of this solution on static cold storage (SCS) of rat livers for transplantation as compared with the histidine tryptophan ketoglutarate (HTK) preservation solution.

Proteome variation of the rat liver after static cold storage assayed in an ex vivo model.

Cold storage is a common procedure for liver preservation in a transplant setting. However, during cold ischemia, the liver suffers molecular alterations that can affect its performance. Also, deleterious mechanisms set forth in the storage phase are exacerbated during reperfusion.

DESIGN OF A SIMPLE SLOW COOLING DEVICE FOR CRYOPRESERVATION OF SMALL BIOLOGICAL SAMPLES.

Background: Slow cooling is a cryopreservation methodology where samples are cooled to its storage temperature at controlled cooling rates.

Objective: Design, construction and evaluation of a simple and low cost device for slow cooling of small biological samples.

Materials And Methods: The device was constructed based on Pye's freezer idea.

The effect of a hydrogen sulfide releasing molecule (Na2S) on the cold storage of livers from cardiac dead donor rats. A study in an ex vivo model.

Liver transplantation is currently the preferred treatment option for end-stage liver disease. Donation after cardiac death was a common practice in the early years of organ donation before brain death criteria were established. Those organs were subjected to variable periods of warm ischemia that might intensify cold ischemia/reperfusion injuries.

Hypothermic machine perfusion versus cold storage in the rescuing of livers from non-heart-beating donor rats.

The aim of this work was to compare the efficiency of cold storage (CS) and hypothermic machine perfusion (HMP) methods of preserving grafts excised from non-heart-beating donors that had suffered 45 minutes of warm ischemia. We developed a new solution for HMP to use in liver transplantation, based on BES, gluconate, and polyethylene glycol (BGP-HMP solution). After 24 h of HMP or CS, livers were reperfused at 37°C with Krebs-Henseleit solution with added dextran.

Delivery of the bioactive gas hydrogen sulfide during cold preservation of rat liver: effects on hepatic function in an ex vivo model.

The insults sustained by transplanted livers (hepatectomy, hypothermic preservation, and normothermic reperfusion) could compromise hepatic function. Hydrogen sulfide (H₂S) is a physiologic gaseous signaling molecule, like nitric oxide (NO) and carbon monoxide (CO). We examined the effect of diallyl disulfide as a H₂S donor during hypothermic preservation and reperfusion on intrahepatic resistance (IVR), lactate dehydrogenase (LDH) release, bile production, oxygen consumption, bromosulfophthalein (BSP) depuration and histology in an isolated perfused rat liver model (IPRL), after 48 h of hypothermic storage (4 °C) in University of Wisconsin solution (UW, Viaspan).

Organ Preservation: Current Concepts and New Strategies for the Next Decade.

SUMMARY: Organ transplantation has developed over the past 50 years to reach the sophisticated and integrated clinical service of today through several advances in science. One of the most important of these has been the ability to apply organ preservation protocols to deliver donor organs of high quality, via a network of organ exchange to match the most suitable recipient patient to the best available organ, capable of rapid resumption of life-sustaining function in the recipient patient. This has only been possible by amassing a good understanding of the potential effects of hypoxic injury on donated organs, and how to prevent these by applying organ preservation.

A modular perfused chamber for low- and normal-temperature microscopy of living cells.

An inexpensive modular perfused chamber (MPC) designed for low- and normal-temperature live-cell imaging is presented. The device consists of four lathed pieces of stainless steel assembled as a cylindrical open chamber that can hold either round or square glass coverslips. The chamber is connected to a thermal-bath operating with recirculation.

Heterologous ferredoxin reductase and flavodoxin protect Cos-7 cells from oxidative stress.

Background: Ferredoxin-NADP(H) reductase (FNR) from Pisum sativum and Flavodoxin (Fld) from Anabaena PCC 7119 have been reported to protect a variety of cells and organisms from oxidative insults. In this work, these two proteins were expressed in mitochondria of Cos-7 cells and tested for their efficacy to protect these cells from oxidative stress in vitro.

Principal Findings: Cos-7/pFNR and Cos-7/pFld cell lines expressing FNR and Fld, respectively, showed a significantly higher resistance to 24 h exposure to 300-600 µM hydrogen peroxide measured by LDH retention, MTT reduction, malondialdehyde (MDA) levels and lipid peroxide (LPO; FOX assay) levels.

A device to measure oxygen consumption during the hypothermic perfusion of the liver.

This work deals with the construction and performance of a device designed to measure the oxygen consumption by the liver during hypothermic perfusion in the rat model. Due to its simple design and the utilization of standard materials, it could serve to determine the role of oxygenation during hypothermic perfusion of the liver. The system consists of a reservoir containing the preservation solution, a peristaltic pump and an internal oxygenator made of silicone tube.

Subzero nonfreezing storage of rat hepatocytes using UW solution and 1,4-butanediol. II- functional testing on rewarming and gene expression of urea cycle enzymes.

Unlabelled: In the present study we have analyzed the viability and metabolic competence of isolated rat hepatocytes subjected first, to subzero nonfreezing storage (up to 120 h at -4 degrees C) in modified University of Wisconsin (UW) solution with 8% 1,4-butanediol, and then to a normothermic rewarming step (KHR media, 37 degrees C, up to 120 min, carbogen atmosphere). Results were compared with hepatocytes stored up to 120 h at 0 degrees C in modified UW solution and with freshly isolated hepatic cells. We have found that only cell suspensions stored in subzero nonfreezing conditions were able to finish the rewarming period with a viability comparable with the control group.

Morphological and biochemical analysis of human cardiac valve allografts after an increment of the cryostorage temperature.

Cryopreserved human cardiac valve allografts could suffer lethal damages if the temperature is elevated during cryostorage. This work describes the functional and morphological alterations suffered by human cardiac valve allografts after a gradual increment of the cryostorage temperature from -147 degrees C to -47 degrees C due to a technical failure. Three experimental groups of human pulmonary and aortic allografts were compared: fresh, cryopreserved (-147 degrees C) and cryopreserved with temperature changes from -147 degrees C up to -47 degrees C and back to -147 degrees C.

Subzero nonfreezing storage of rat hepatocytes using modified University of Wisconsin solution (mUW) and 1,4-butanediol. I- effects on cellular metabolites during cold storage.

Various cryopreservation techniques have been investigated to extend the storage of isolated hepatocytes; however, most have a reduced viability after rewarming due to ice crystal formation. Subzero nonfreezing conditions could theoretically reduce organ metabolism without damage due to ice crystal formation. In the present work we evaluated the viability and metabolic parameters of isolated rat hepatocytes preserved in subzero nonfreezing condition.

Construction and performance of a minibioreactor suitable as experimental bioartificial liver.

This work deals with the construction and performance of a hollow fiber-based minibioreactor (MBR). Due to its simple design and the utilization of standard materials, it could serve as a suitable tool to evaluate the behavior and performance of cold preserved or cultured hepatocytes in bioartificial liver devices. The system consists of 140 fiber capillaries through which goat blood is pumped at a flow of 9 mL/min.

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.

Efficient cold transfection of pea ferredoxin-NADP(H) oxidoreductase into rat hepatocytes.

We describe the use of a non-viral, polyethylenimine-based vector to transfect rat hepatocytes preserved under hypothermic storage. DNA sequences encoding Escherichia coli beta-galactosidase and pea ferredoxin-NADP(H) oxidoreductase (FNR), cloned into plasmids pCH110 and pKM4 respectively, were used. FNR was detected in the liver of animals transplanted with transfected cells; no reactivity was observed in endogenous parenchyma.

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.

Effect of cold preservation/reperfusion on glycogen content of liver. Concise review.

Livers cold preserved during variable periods of ischemia suffer functional, morphological and hemodynamic alteration, which are exacerbated when they are reperfused. One important injury is glycogen depletion during cold ischemia/ reperfusion. How liver can restore their energy during reperfusion is related with the preservation time, nutritional status of the donor, and the preservation solution used.

A simple and effective method to improve intrasplenic rat hepatocyte transplantation.

Transplanted hepatocytes integrate, survive, and express their specific functions in the liver parenchyma. The aim of this study was to determine whether a large number of hepatocytes could move from the spleen to the liver when the cells are injected together with sodium nitroprusside, and if the improved hepatocyte migration may be related with portal vein dilatation. Wistar rats were transplanted in the spleen with fluorescent-labeled hepatocytes alone or together with sodium nitroprusside.

The effect of rewarming media composition on the ammonia detoxification ability of cold preserved rat hepatocytes.

We examined how different media composition of rewarming solutions affected ammonium detoxification function, urea synthesis and the viability of hepatocytes after 72 hs of cold storage in UW solution. Freshly isolated rat hepatocytes were incubated at 37 C in a cell culture medium (MEM-E) with 3 mM glycine, 5 mM fructose and 2.5 mM adenosine (group 1) and in Krebs-Heinseleit buffer with 3 mM glycine, 5 mM fructose, 2 mM ornithine, 10 mM lactate and adenosine, that was used in two different concentrations: 2.

Effect of S-nitrosoglutathione (GSNO) added to the University of Wisconsin solution (UW): II) Functional response to cold preservation/reperfusion of rat liver.

Livers cold preserved in University of Wisconsin (UW) solution followed by reperfusion suffer ischemia/reperfusion injuries. Microcirculation is the primary target of damage, characterized by sinusoidal perfusion failure due, mainly, to morphological changes of sinusoidal endothelial cells. Here, we demonstrated that the addition of S-nitrosoglutathione (GSNO) to the UW solution before cold storage, as a nitric oxide (NO) donor, attenuated hepatic injuries.

The benefit of adding sodium nitroprusside (NPNa) or S-nitrosoglutathion (GSNO) to the University of Wisconsin solution (UW) to prevent morphological alterations during cold preservation/reperfusion of rat livers.

Cold liver preservation in the University of Wisconsin solution (UW) followed by reperfusion alters hepatic parenchyma and extra cellular matrix. In this study we analyzed the benefit of adding either 500 microM Sodium Nitroprusside (NPNa) or 100 microM S-nitrosoglutathione (GSNO) as Nitric Oxide (NO) donors to the UW solution to prevent hepatic injury. Wistar adult rat livers were stored in UW solution (0 degrees C) for 48Hs and reperfused (60 minutes) in the isolated perfused rat liver model (IPRL).