Damage to the isolated heart due to adrenaline perfused with a saline solution

Perfusion of the isolated heart with Krebs solution containing 5 and 20 microgram/ml of adrenaline induced cardiocyte micronecrosis. Perfusion with 0.5 microgram/ml of adrenaline induced no micronecrosis. Dispersion analysis showed a statistically significant effect of adrenaline concentrations on the degree of the cardionecrotic effect. The fact of micronecrosis appearance in the isolated heart during its perfusion with saline solution requires revision of the hypothesis on the leading role of blood factor in the realization of the cardionecrotic effect of adrenaline. The appearance of micronecroses with the action of adrenaline in concentrations which activate the mechanism of amines uptake by the heart myocytes speaks in favour of the casual relationship between the accumulation of the biogenic amines by myocytes and the development of their necrosis.     

Proteome analysis of isolated perfused organ effluent as a novel model for protein biomarker discovery

The discovery of novel serological biomarkers is critical for improving disease diagnosis and monitoring treatment response. Proteomic analysis of model systems, such as isolated cells in culture and patient plasma and serum, represents the current state-of-the-art. Here, we coupled proteomics with isolated organ perfusion, which allows a disease state to be studied in a physiologic, yet controlled, environment. Potential markers specific to the disease or to changes in the surrounding tissue may be discovered. The effectiveness of this model was evaluated using proteomic analysis of effluent fractions collected from isolated beating rat hearts during reperfusion after brief episodes of ischemia. The detection of clinical markers for myocardial ischemia in this effluent was robust and analytically straightforward, validating the potential of isolated organ perfusion in diagnostic protein discovery.     

Inotropic activity induced by carbamazepine-alkyne derivative in an isolated heart model and perfused to constant flow

Inotropic activity induced by carbamazepine-alkyne derivative in an isolated heart model and perfused to constant flow Introduction. Few data exist with respect to the effects of carbamazepine and its derivatives at cardiovascular level; furthermore, the molecular mechanisms and cellular site of action are still unclear. Objective. The effects induced by carbamazepine-alquine derivative on perfusion pressure, vascular resistance and left ventricular pressure were evaluated. Materials and methods. The effects of carbamazepine and carbamazepine-alquine on the perfusion pressure, vascular resistance and left ventricular pressure were examined in isolated rat hearts (Langendorff model). Results. Four results were obtained: (1) The carbamazepine-alquine derivative 10-9 mM increased the perfusion pressure and vascular resistance in comparison with the carbamazepine 10-9 mM; (2) the effect of carbamazepine-alquine derivative 10-9-10-4 mM on left ventricular pressure not was inhibited by metoprolol or prazosin at a dose of 10-6 mM; (3) nifedipine 10-6 mM blocked the effects exerted by the carbamazepine-alquine derivative 10-9-10--4 mM on left ventricular pressure, and (4) the carbamazepine-alquine derivative at dose of 10-9 mM increased the concentration of intracellular calcium over a time period of 3-18 min; nevertheless, in presence of nifedipine 10-6 mM this effect was inhibited significantly (p=0.005). Conclusions. The activity exerted by carbamazepine-alquine derivative on perfusion pressure, vascular resistance and left ventricular pressure involved activation of calcium channel type-L, brought indirectly changes in the intracellular calcium levels and subsequently induced a positive inotropic effect.     

Heat-inactivated coelomic fluid of the earthworm Perionyx excavatus is a possible alternative source for fetal bovine serum in animal cell culture

Fetal Bovine Serum (FBS) is used as a major supplement in culturing animal cells under in vitro conditions. Due to ethical concern, high cost, biosafety, and geographical as well as batchwise result variations, it is important to reduce or replace the use of FBS in animal cell culture. The major objective of this work is to evaluate the feasibility of heat-inactivated coelomic fluid (HI-CF) of the earthworm, Perionyx excavatus as a possible alternative for FBS in animal cell culture experiments. The coelomic fluid (CF) was extruded from the earthworm using electric shock method and used for the experiments. Electric shock method is a simple non-invasive technique, which has no harmful effect on earthworms. Mouse primary fibroblast and HeLa cell lines were used in this study. Among HI-CF, autoclaved CF and crude CF, the supplement of medium with HI-CF shows positive results. The processed HI-CF (90°C for 5 min) at 10% supplement in cell culture medium promote maximum cell growth but cells need the initial support of FBS for the attachment to the culture flask. Microscopic observation and immunofluorescence assay with actin and lamin A confirm that the cellular and molecular morphology of the cells is maintained intact. The HI-CF of earthworm, P. excavatus has shown better cellular viability when compared with FBS and making it possible as an alternative supplement to minimize the use of FBS.     

Porcine aortic organ culture: a model to study the cellular response to vascular injury

Organ cultures of porcine thoracic aorta were studied to define the characteristics of this system as a model to study the reaction of endothelial cells (ECs) and the underlying smooth muscle cells (SMCs) to injury. Both nonwounded and wounded cultures, the latter having had part of the endothelial surface gently denuded with a scalpel blade, were studied over a 7 d period by scanning and transmission electron microscopy. The results showed that the nonwounded ECs underwent a shape change from elongated to polygonal within 24 h in culture. In both nonwounded and wounded explants there was cell proliferation beneath the nondenuded endothelium so that by 7 d several layers of cells were present showing features of the secretory type of SMCs. This proliferation, however, did not occur if the endothelium was totally removed from the aorta. There was also evidence of gaps between the surface ECs, and by 7 d lamellipodia of cells beneath the surface were present in these gaps. Occasionally, elongated cells were seen to be present on the surface of the endothelium. In the wounded organ culture, cell migration and proliferation occurred extending from the wound edge and producing a covering of cells on the denuded area. There were also multilayered cells beneath the surface similar to the nonwounded area. Occasional foam cells were seen in the depth of the multilayered proliferating cells. The results indicate that organ culture of porcine thoracic aorta is a good model to study the reaction of ECs and underlying SMCs to injury.     

Inhibition of transducin by lithium: electrophysiological demonstration using the isolated retina

Recently Avissar et al. have established that Li+ can inhibit G proteins implicated in brain function. In order to investigate the effect of Li+ on transducin, the evolution of the electroretinogram (ERG) recorded on isolated rat retina has been studied in presence of lithium. Results indicate that 10(-5) M Li+ had no effect on ERG while 10(-3) M Li+, which corresponds to therapeutic blood levels, significantly decreases ERG amplitude. This effect being nearly totally inhibited by cholera toxin (75 micrograms/l), it is concluded that Li+ acts on transducin and so inhibits the visual transduction process.     

Biomarkers of oxidative damage to predict ischaemia-reperfusion injury in an isolated organ perfusion model of the transplanted kidney

Ischaemia-reperfusion (IR) injury is known to be a risk factor influencing both short and long-term graft function following transplantation. The pathophysiology of IR injury is suggested to involve elevated reactive oxygen species production resulting in oxidative damaged cellular macromolecules.

The objective of this study was to evaluate oxidative damage following IR using an isolated organ perfusion model of the transplanted kidney, in order to determine a simple, preferably non-invasive biomarker for IR injury.

Porcine kidneys were retrieved with 10 or 40 min warm ischaemic (WI) time and haemoperfused for 6 h on an isolated organ perfusion machine. ELISA was used to detect carbonyls, 8-isporostane and 8-hydroxy-2′-deoxyguanosine, representing protein, lipid and DNA damage respectively in pre and post reperfusion samples of plasma, urine and biopsy material.

Plasma carbonyl and 8-isporostane and were significantly increased in the 40 min group compared to pre-perfusion (0.96 ± 0.10 vs. 0.62 ± 0.06, P < 0.001 and 1.57(1.28–4.9) vs. 0.36(0.09–0.59), P < 0.05). The levels also correlated with creatinine clearance used to determine renal function (r = ? 0.6150, P < 0.01 and r = ? 0.7727, P < 0.01).

The results of this study suggest both plasma carbonyl and 8-isporostane to be reliable biomarkers to predict the level IR injury.     

Biomarkers of oxidative damage to predict ischaemia-reperfusion injury in an isolated organ perfusion model of the transplanted kidney

Ischaemia-reperfusion (IR) injury is known to be a risk factor influencing both short and long-term graft function following transplantation. The pathophysiology of IR injury is suggested to involve elevated reactive oxygen species production resulting in oxidative damaged cellular macromolecules. The objective of this study was to evaluate oxidative damage following IR using an isolated organ perfusion model of the transplanted kidney, in order to determine a simple, preferably non-invasive biomarker for IR injury. Porcine kidneys were retrieved with 10 or 40 min warm ischaemic (WI) time and haemoperfused for 6 h on an isolated organ perfusion machine. ELISA was used to detect carbonyls, 8-isporostane and 8-hydroxy-2'-deoxyguanosine, representing protein, lipid and DNA damage respectively in pre and post reperfusion samples of plasma, urine and biopsy material. Plasma carbonyl and 8-isporostane and were significantly increased in the 40 min group compared to pre-perfusion (0.96 +/- 0.10 vs. 0.62 +/- 0.06, P < 0.001 and 1.57(1.28-4.9) vs. 0.36(0.09-0.59), P < 0.05). The levels also correlated with creatinine clearance used to determine renal function (r = - 0.6150, P < 0.01 and r = - 0.7727, P < 0.01). The results of this study suggest both plasma carbonyl and 8-isporostane to be reliable biomarkers to predict the level IR injury.     

Interest of fed-batch culture for the production of a membrane-bound protein by an adherent animal cell

The production of a membrane-bound protein by culture and harvesting of r-CHO cells has been evaluated. While in batch culture, the maximal cell density is only maintained for a few hours, in a fed-batch one, the cell and enzyme concentrations, as well as the growth and production rates, can be kept constant during 100 hours. This stabilized phase facilitates the cell harvesting and provides a kinetic tool for testing chemical molecules as sodium butyrate.     

A mini organ culture as a model for studying the gallbladder epithelium of mouse

Summary— A mini organ culture of mouse gallbladder was developed as an alternative to primary cultures of epithelial cells of this organ. Small pieces of tissue were prepared and maintained in minimum essential Eagle medium with 10% foetal calf serum, for as long as 7 days. Qualitative and quantitative ultrastructural studies have been performed using electron microscopy. The viability of cells was evaluated by stereological quantification of endocytotic vesicles containing horseradish peroxidase and labelling of exocytotic glycoproteins with tannic acid. The morphology of tissue pieces during the 1st h of culturing and tissue isolated directly from animals exhibited no significant differences. However, after 4 h in culture degradative changes became evident in many cells. At that time, endo- and exocytosis were both dramatically reduced. After 24 h, the morphology, as well as endo- and exocytosis recovered and were comparable to the parameters of the tissue in vivo or after 1 h in culture. The endocytotic activity remained unchanged from day 1 to 7 of culturing, while the number of exocytotic vesicles gradually decreased after 2 days in culture. Our results prove that mini organ culture of gallbladder is morphologically and functionally comparable with the tissue in vivo and for studies of epithelium in culture it is more convenient than primary cultures.     

Development of a gut perfusion model as an alternative to the use of live fish

An alternative fish model with the principal aim of studying the interaction between fish pathogens and the intestinal tissue was developed. The preparation consisted of an excised gut tractus from rainbow trout (Oncorhynchus mykiss), perfused through cannulation of the aorta intestinalis ventralis with filtered and heparinized Cortland+dextran 1% as the perfusion fluid. This perfusion fluid was delivered by means of a drip. The perfused gut tractus was suspended in a circular bath filled with Ringer solution, which was aerated and kept at a constant temperature of 12 degrees C. Unperfused gut placed in Ringer solution at the same temperature served as the negative control. Perfusion was effective in maintaining the gut in a healthy condition for at least 60 min with only slight oedema and sloughing of the epithelium. Conversely, the unperfused gut revealed excessive tissue degeneration and severe necrosis.     

自记录动物挂杠持久度测试装置的研制

目的：研制一种检测动物挂杠持久度的实验装置,用于相关医药研究中的动物静态抗疲劳实验。方法采用51系列单片微机控制和记忆,光耦传感器、电磁水阀等组合控制电路和技术。结果成功研制出一种适用于医学及药学研究领域的带微机控制和自记忆功能的动物挂杠持久度实验装置,经28例小白鼠实验证明,效果良好。结论该实验装置为动物抗疲劳实验提供了一种全新的方法。