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PubMed1995

[The isolated perfused Bovine Udder Skin]

The isolated perfused Bovine Udder Skin (BUS) model from slaughter house material is an ex vivo skin model that can easily be maintained in culture at high vitality at least for 8 hours. The BUS model allows to study skin penetration and absorption as well as skin irritation almost under in vivo conditions. Moreover, the skin of the bovine udder is morphologically resembling human skin to a higher extent than the skin of rodents. Pharmacological and toxicological studies on raw materials and formulated products of interest to cosmetics and skin products industry reveal a high correlation to data obtained in humans. Therefore, the BUS model holds great promise since it allows to reduce the number of test animals in skin pharmacology and toxicology, since it provides information which cannot be obtained in other in vitro models and not even in human studies, e.g. pharmacokinetics and drug metabolism.

Journal Article↗
PubMed1992

The effect of cyanide on isolated perfused rat heart.

Using Langendorff isolated perfused rat hearts, we demonstrated direct effects of cyanide on the heart. A heart from a sacrificed male rat was placed on a Langendorff apparatus and perfused with Tyrode solution containing 2.0 mM NaCN (CN-TS), following with normal Tyrode solution (TS) at 37 degrees C. During perfusion we monitored the heart beat, and the state of inorganic phosphate (Pi), creatine phosphate (CrP) and adenosine triphosphate (ATP) by 31P-NMR spectrometer equipped with a surface coil probe. After the beginning of the CN-TS perfusion, the heart was initially excited, and ceased beating completely seventy seconds later. During CN-TS perfusion, the CrP immediately disappeared, but the ATP level was maintained considerably. The peak of Pi shifted to the right, indicating acidosis in the heart. These results suggest that the ATP level in a heart during exposure of cyanide is maintained by activation of anaerobic glycolysis in compensation for cellular oxygen utilization.

Adenosine Triphosphate↗
PubMed1978

The effects of phenformin in normal vs. diabetic isolated perfused rat liver.

In the isolated perfused liver system high concentrations of phenformin (0.93--1.24 mM) were required to reduce the greater than two-fold elevated rate of gluconeogenesis from L-[U-14C]lactate in acutely alloxan diabetic (48-hour) and chronically alloxan diabetic (7-day) rat livers to the slower rate of normal fed livers. At these phenformin concentrations, other hepatic functions such as substrate uptake and 14CO2 production were also inhibited. The livers were also in a very reduced state under these conditions as indicated by the elevated ratios of the redox couples lactate/pyruvate and 3-hydroxybutyrate/acetoacetic acid. The results are interpreted to indicate that if phenformin functions as an antidiabetic (hypoglycemic) agent by inhibiting hepatic gluconeogenesis to normal levels, it is also generally toxic to the liver under such conditions. The results are discussed in relation to current hypotheses of the mechanism of action of phenformin and to phenformin-associated lactic acidosis.

Animals↗
PubMed1995

Evaluation of a high sodium-low potassium cold-storage solution by the isolated perfused rat kidney technique.

The isolated perfused rat kidney (IPK) model was used to assess initial renal function after 24 h preservation in 3 different cold storage solutions: EuroCollins (EC), a solution prepared according to the formulation of Belzer's solution (High-K+ UW) and a high Na(+)-low K+ Belzer UW solution (High-Na+ UW). GFR and FRNa were measured after 24 h cold storage in each of the solutions during 60 min, and were compared to values obtained in a control group in which renal function was measured immediately after the kidneys had been harvested. ATP and CP were measured in fresh renal tissue, in kidneys preserved for 24 h in each solution, in control IPK, and in reperfused IPK after they had been preserved for 24 h. Main results showed that preservation in either solution caused a dramatic decrease in GFR and in FRNa within the first 60 min following reperfusion of cold-stored kidneys. However FRNa was significantly higher in the High-Na+ UW group. ATP and CP content were decreased to approximately 10% of basal values in all experimental groups after cold-storage. Normothermic reperfusion of IPK after cold-storage induced a restoration of ATP levels, but CP content decreased further. There was no significant difference in ATP and CP content between cold-storage solutions, nor any correlation between metabolic and functional parameters.

Adenosine↗
PubMed1995

[Pharmacologic action profile of crataegus extract in comparison to epinephrine, amirinone, milrinone and digoxin in the isolated perfused guinea pig heart].

Using isolated perfused guinea pig hearts experiments were performed to investigate the influence of crataegus extract LI 132 (Faros 300, CRA) in comparison to other inotropic drugs--epinephrine (adrenaline, ADR), amrinone (AM), milrinone (MIL) and digoxin (DIG)--on different functional parameters, with special emphasis on the effective refractory period of the myocardium. The simultaneous registration of appropriate parameters allowed to relate the effect on the refractory period to the inotropic, chronotropic, dromotropic and coronary actions of these compounds at each concentration level. All substances--with the exception of CRA--shortened the effective refractory period concentration-dependently besides their known other functional effects (max.: 1 x 10(-5) mol/l ADR by 38%, 7 x 10(-7) mol/l DIG by 26%, 1 x 10(-4) mol/l MIL by 13% and 5 x 10(-4) mol/l AM by 1.6%). Related to the positive inotropy the shortening was most effective under MIL (1.32 ms/mN), followed by AM (0.65 ms/mN), DIG (0.40 ms/mN) and ADR (0.28 ms/mN). On the contrary, CRA produced a prolongation of the effective refractory period by maximally 10% resp. by 2.54 ms/mN. Thus, the pharmacologic profile of CRA differs from that of other inotropic compounds mainly in this parameter (with potentially reduced arrhythmogenic risk).

Amrinone↗
PubMed1985

Reduction of the drug-induced nephrotoxicity by ATP-MgCl2. II. Effects on gentamicin-treated isolated perfused kidneys.

Drug-induced nephrotoxicity (NT) has become an increasingly significant clinical problem. An in vitro model of drug-induced NT was therefore developed using gentamicin and the effects of ATP-MgCl2 on reduction or prevention of NT were determined. To study this, non-pulsatile perfusion in isolated rat kidneys was maintained at 100 mm Hg during 2 hr of perfusion at 37 degrees C. The oxygenated Krebs-HCO3 perfusate contained 7.5 g/dl albumin as colloid, glucose, creatinine, amino acids, trace amounts of [3H]inulin and 125I-lysozyme, and either 0, 0.4, 0.8, or 1.2 mg/ml of gentamicin. In some studies, 2 mM ATP-MgCl2 was added with 0.8 mg/ml of gentamicin at 0 and 60 min of perfusion. During each 10-min clearance period, glomerular filtration rates, sodium absorption, water absorption, and fractional clearance of TCA-precipitable lysozyme were measured. The results indicate that renal perfusate flow, glomerular filtration rate, urinary flow and tubular absorption of protein (a sensitive indicator of tubular function), sodium, and water were affected by gentamicin in a dose-dependent manner. An isolated kidney preparation can therefore be used to study gentamicin-induced NT. Higher in vitro perfusate concentrations of the drug were needed, however, to acutely mimic the in vivo cumulative effects. Nonetheless, renal perfusate flow, glomerular filtration rate, and the depression in protein reabsorption which occurred with gentamicin treatment were markedly improved by simultaneous treatment with ATP-MgCl2. Thus, ATP-MgCl2 may be useful in reducing drug-induced nephrotoxicity.

Adenosine Triphosphate↗
PubMedDate not supplied

In transit metastases of malignant melanoma treated by high dose rTNF alpha in combination with interferon-gamma and melphalan in isolation perfusion.

To increase the therapeutic efficacy of recombinant tumor necrosis factor alpha (rTNF alpha) and reduce the systemic side effects, a protocol was designed using isolation perfusion of the limbs with hyperthermia for in transit metastases of melanoma. A triple combination of high dose rTNF alpha + recombinant interferon-gamma (rIFN-gamma) + melphalan was chosen because of a synergistic anti-tumor effect of rTNF alpha with rIFN-gamma and of rTNF alpha with alkylating agents reported in the literature. Twenty-nine patients of mean age 60 years (range 22-82 years) entered the study after informed consent and received a total of 31 isolation perfusions with the triple combination. There were 24 women and 5 men with multiple progressive in transit melanoma metastases of the lower limb (stage IIIa or IIIab). rTNF alpha at the unique dose of 4 mg was injected as a bolus in the arterial line, under mild hyperthermic conditions (40 to 40.5 degrees C) for 90 minutes. rIFN-gamma was given subcutaneously on days -2 and -1 and in the perfusate, with rTNF alpha, at the dose of 0.2 mg. Melphalan was administered in the perfusate at dose giving a concentration of 40 micrograms/ml. In all the 31 isolation perfusions performed in the triple combination protocol, in order to prevent a septic shock-like syndrome which had been encountered in 2 patients treated outside this protocol for sarcoma and carcinoma, the patients received dopamine continuous infusion at 3 micrograms/kg/min from the start of isolation perfusion and for 48 hours, and only showed mild hypotension and very transient chills and temperature.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed2001

Albumin-dependent digoxin transfer in isolated perfused human placenta.

OBJECTIVE: To determine the effects of albumin (BSA) concentration in perfusion medium on digoxin transfer in isolated perfused human placental cotyledon. STUDY DESIGN: Isolated placental cotyledons from 13 normal human placentas were dually perfused after cannulating artery and vein of the chorionic plate and piercing 4 catheters through the corresponding basal plate with M199 medium enriched with BSA and glucose. Flow rates were 12 and 6 ml/min in the maternal and fetal circuits, respectively. Digoxin was added to the maternal reservoir at a final concentration of 5.51 +/- 1.00 ng/ml. BSA in maternal and fetal perfusate was kept at 3 concentrations: 1, 3 and 5 mg/ml (Groups I, II, III). Transplacental passage of digoxin was calculated from repeated fetal and maternal perfusate samples collected over 3 hours in the 3 groups. Digoxin levels were measured by FPIA (TDx, Abbott). RESULTS: There was no transfer of digoxin from the maternal to fetal compartment when the concentration of BSA was 1 mg/ml. Increasing the concentration of BSA led to a substantial increase in the transfer of digoxin to the fetal compartment. Steady state levels of digoxin in the fetal compartment were 0.61 +/- 0.19 ng/ml at 3 mg/ml of BSA. CONCLUSION: Maternal and fetal serum concentration of BSA affect digoxin transfer in isolated perfused human placentas. Three mg/ml are considered to be the optimal albumin concentration.

Analysis of Variance↗
PubMed1980

Glucose stimulation of somatostatin and insulin release from the isolated, perfused rat pancreas.

Insulin and somatostatin release from the isolated perfused rat pancreas was studied under conditions of 50 and 300 mg/dl glucose as well as a linear 50-300 mg/dl glucose gradient. The glucose-stimulated response profile of somatostatin was nearly parallel to that of insulin in both the acute and gradient dose experiments. Antisomatostatin serum was without significant effect on glucose-stimulated insulin release. In spite of the marked, fifteenfold stimulation of somatostatin release (1.5 x 10-10 M in the perfusate effluent) by glucose, the concentration of somatostatin was insufficient to significantly alter glucose-stimulated insulin release in the isolated perfused rat pancreas.

Animals↗
PubMed1982

Oxygen-radical-mediated permeability edema and vasoconstriction in isolated perfused rabbit lungs.

Oxygen radicals have been implicated in the pathogenesis of permeability pulmonary edema. To determine directly if O2 radicals can cause increased alveolar-capillary membrane (ACM) permeability and low-pressure permeability edema, we chemically produced O2 radicals in the sale perfusates of isolated rabbit lungs. The O2 radicals generated by xanthine oxidase caused protein-rich edema and increases in lung perfusion pressures that were inhibitable by catalase (hydrogen peroxide scavenger) or dimethylthiourea (hydroxyl radical scavenger) but not by superoxide dismutase. To determine the effect of O2 radicals on ACM permeability without interference from increased perfusion pressures, we used papaverine to maintain baseline perfusion pressures during O2 radical exposure and then assessed ACM integrity by evaluating the response of isolated lungs to elevated outflow pressures (10 mmHg for 10 min). Under these conditions, increased ACM permeability manifested by weight gains and lavage albumin accumulations occurred in lungs treated with xanthine oxidase but not in control lungs. We conclude that O2 radicals can cause increased ACM permeability and vasoconstriction in isolated lungs.

Animals↗
PubMed2004

Isolated perfused liver model: the rat and guinea pig compared.

OBJECTIVE: Although the rat is the most commonly used species for the study of hepatic metabolism, the physiology of the guinea pig is closer to human physiology. We compared the model of isolated perfused guinea pig liver with the classic model of isolated perfused rat liver, especially with respect to amino acid metabolism. METHODS: After validation of an anesthetic mixture of ketamine, diazepam, and xylazine for the guinea pig, isolated perfused livers were harvested for both species. Three groups of animals were compared for the study of liver metabolic fluxes: 6-wk-old male Sprague-Dawley rats (R; 230 +/- 10 g, n = 5), young male Hartley guinea pigs (YG; 223 +/- 8 g, n = 6) matched to rats by liver weight, and adult male Hartley guinea pigs (AG; 389 +/- 5 g, n = 6) matched to rats by age. Results (mean +/- standard error of the mean) were compared by analysis of variance and Newman-Keuls tests. RESULTS: Both models displayed a satisfactory hepatic viability, but differences were noted, with higher portal flows (R: 3.1 +/- 0.3 versus YG: 4.5 +/- 0.3 and AG: 4.2 +/- 0.3 mL. min(-1). g(-1); P < 0.05, YG and AG versus R) and bile flows (R: 0.34 +/- 0.01 versus YG: 2.38 +/- 0.22 versus AG: 3.17 +/- 0.28 microL. min(-1). g(-1); P < 0.05, YG and AG versus R, and YG versus AG) and higher amino acid fluxes (P < 0.05) leading to greater nitrogen uptake (P < 0.05) in guinea pigs. We performed a second set of experiments to evaluate the influence of anesthesia and portal flow on this last parameter. In these experiments, rats were anesthetized with ketamine, diazepam, and xylazine and guinea pig livers were perfused at rat blood flow. Apart from a 50% anesthesia-related mortality for rats, bile flow and metabolic parameters were only slightly modified. However, some amino acid fluxes were statistically different (aspartate, serine, and histidine; P < 0.05), as confirmed by a higher transfer constant. CONCLUSION: Our results indicate that the isolated perfused guinea pig liver is a suitable model for the study of hepatic metabolism.

Amino Acids↗
PubMed1993

Gram-negative bacterial lipopolysaccharide impairs hyaluronan clearance in vivo and its uptake by the isolated, perfused rat liver.

The purpose of this investigation was to examine the effect of gram-negative bacterial lipopolysaccharide on hyaluronan concentration in blood plasma, hyaluronan removal from the blood and hyaluronan uptake by isolated, perfused rat liver. Intravenous administration of Escherichia coli lipopolysaccharide to rats markedly increased plasma hyaluronan concentration in a dose-dependent manner. One day after lipopolysaccharide challenge (0.1 or 1.0 mg per 100 gm body wt), plasma hyaluronan levels were 570.7 +/- 66.8 ng x ml-1 and 1,951.0 +/- 120.3 ng x ml-1, respectively, as compared with 94.2 +/- 12.2 ng x ml-1 in the time-matched control animals. Removal of intravenously injected hyaluronan (30 micrograms per 100 gm body wt) was suppressed 32% by lipopolysaccharide administration (100 micrograms per 100 gm body wt). At the same dose, lipopolysaccharide induced a severe inhibition (60% to 80%) of hyaluronan uptake by perfused livers isolated 3 or 24 hr after lipopolysaccharide administration. The inhibitory effect of lipopolysaccharide on hyaluronan uptake by the isolated, perfused liver was not abolished by pretreatment with either antibodies to tumor necrosis factor-alpha IgG or indomethacin, an inhibitor of the cyclooxygenase pathway. Continuous intravenous infusion of recombinant murine tumor necrosis factor-alpha for 18 to 20 hr did not affect plasma hyaluronan concentration. These data suggest that neither tumor necrosis factor-alpha, an early cytokine induced by lipopolysaccharide, nor prostaglandins are involved in the mechanism of lipopolysaccharide-induced inhibition of hyaluronan uptake by the perfused rat liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1997

Modulation of c-fos and egr-1 expression in the isolated perfused kidney by agents that alter tubular work.

The isolated perfused rat kidney provides a model of selective hypoxia to the medullary thick ascending limb. To investigate the relationship between immediate early gene expression and the extent of hypoxic damage, we determined expression of the immediate early genes (IEG) c-fos and egr-1 in isolated perfused kidneys during standard perfusion and after various measures shown previously to be protective. mRNA levels of c-fos and egr-1 were markedly increased in kidneys after 90 minutes of standard perfusion with Krebs-Henseleit buffer containing albumin. Gene expression was most prominent in the outer medulla followed by papilla and cortex, a pattern reflected by the immunohistochemical demonstration of a prominent accumulation of both egr-1 and c-fos polypetides mainly in the medullary thick ascending limb (mTAL). Protective measures known to minimize morphological damage to the mTAL, including hyperoncotic perfusion, perfusion with glycine, or perfusion with a mixture of amino acids, decreased mRNA levels of c-fos and egr-1 in the outer medulla (by 50% and 35%, respectively) and the papilla (by 60 and 30%, respectively). Renal cortex showed only minor changes. In contrast, prevention of tubular transport by perfusion with 1 mM ouabain increased mRNA levels of c-fos and egr-1 in the outer medulla by 100% and 60%, respectively. Ouabain also dramatically increased mRNA levels of both IEGs in two lines of cultured renal epithelial cells. Changes in the level and distribution of the protein products of these IEGs were not detectable in perfused kidneys by immunohistochemistry. Hypoxic injury of the kidney stimulates IEG expression even in the absence of reperfusion. Protection against hypoxic injury in the mTAL correlates with suppression of IEG mRNA levels when protection is provided by amino acids or hyperoncotic perfusion, but not when provided by inhibition of Na,K-ATPase, which stimulates IEG expression. We conclude that diminished IEG expression is not a necessary concomitant of protection against hypoxic injury.

Amino Acids↗
PubMed1984

Disposition of [4-14C]oestradiol-17 beta in the isolated perfused brain of the rat.

[4-14C] Oestradiol-17 beta was perfused through isolated brains of male and ovariectomized female rats. Two different perfusion media were used. The uptake of oestradiol-17 beta was higher in female brains, the highest concentrations being found in the hypophysis and hypothalamus. Oestradiol-17 beta was metabolized to a greater extent by female brains, the most important metabolite being oestrone. Additionally, 2-hydroxyoestradiol-17 beta, 6 zeta-hydroxyoestradiol-17 beta, and 7 alpha-hydroxyoestradiol-17 beta were found; 7 alpha-hydroxyoestrone and another polar metabolite could not be definitely identified. Quantitatively, 2-hydroxylation was no more important than hydroxylation at C atom 6 or 7.

Animals↗
PubMed1983

Haemodynamic effects of adrenaline on the isolated, perfused head of the dogfish 'pup' (Squalus acanthias).

The isolated, perfused head of the dogfish 'pup' (Squalus acanthias) maintained pressure:flow relationships near to those described for the in vivo adult for at least 3 h when perfused at a constant rate. The addition of 3% polyvinylpyrrolidone reversibly increased branchial resistance, and the postbranchial outflow (arterial + venous) equalled the inflow. 10(-5) M adrenaline reversibly reduced gill resistance (in some cases after a transient increase in resistance) and stimulated perfusate outflow from the dorsal aorta, at the expense of flow from the cephalic and branchial venous system. Phentolamine did not alter the effect of adrenaline on pressure and flow pattern; addition of propranolol inhibited both adrenaline effects and resulted in a slight increase in afferent pressure, indicating that alpha-adrenergic receptors are present, but that the dominant haemodynamic effects are mediated via beta-adrenergic receptors. The isolated, perfused 'pup' head may provide a vehicle for investigation of transport phenomena in the elasmobranch branchial epithelium.

Animals↗
PubMed1986

Uptake of lipophilic model compounds into the isolated perfused rat epididymal adipose tissue.

Uptake of xenobiotics into isolated perfused rat adipose tissue was studied. Aorta and vena cava were cannulated and ligations were placed so that only an epididymal fat pad was perfused. Perfusion experiments were performed in situ and nonrecirculating, for up to 350 min, with Krebs-Ringer bicarbonate buffer containing 4% serum albumin. The functionality of the preparation was tested by an after-perfusion with methylene blue as well as with the volume and mass balances. Formation of edema was not a problem under the experimental conditions used. The following model compounds were used at influx concentrations of 2 to 8 microM: thiopental, imipramine, chlorpromazine, 1,1-bis-(p-chlorophenyl)-2,2-dichloroethane (DDE) and 2,4,5,2',4',5'-hexachlorobiphenyl (6-CB). Uptake was determined during the experiments using the arteriovenous difference and after the experiments by direct determination in the perfused fat pad. All five model compounds were taken up readily. Rate of uptake tended to decrease initially and to reach a constant value. Only with 6-CB was the difference between initial and terminal rate considerable. Mean uptake fraction was: thiopental, 38 +/- 8%; imipramine, 69 +/- 4%; chlorpromazine, 85%; DDE, 56%; and 6-CB, 13 +/- 1%. Thus, imipramine and chlorpromazine, which do not accumulate in adipose tissue in vivo, are even taken up more rapidly into the isolated perfused adipose tissue than is thiopental. The difference between these two experimental situations is therefore not due to a permeability barrier, but rather to factors outside the adipose tissue, such as competing nonadipose tissues present in vivo only. For the neutral, insoluble and almost totally albumin-bound compounds, DDE and 6-CB, albumin may act as an additional binding competitor that inhibits adipose tissue uptake.

Adipose Tissue↗
PubMed1976

The uptake and subcellular distribution of radio-labeled metabolites of digoxin in the isolated perfused guinea-pig heart.

Cardiac glycosides like digitoxin and digoxin with three digitoxoside sugar residues have been reported to undergo step-wise degradation to yield the corresponding genins, and the importance of the digitoxoside side-chain for the pharmaco-dynamics property of cardiac glycosides has also been suggested. A sytematic study was therefore undertaken on the cleavage products of digoxin. Digoxigenin-bis-digiitoxoside (with two sugar residues) digoxigenin-monodigitoxoside (with one sugar residue) and digoxigenin (with no sugar) were compared with the parent compound, digoxin. The radio-labeled compounds were perfused through isolated guinea pig hearts using 10-7 M concentration in the perfusion medium for a fixed period of 64 min followed by an 8 min period of wash-out with normal medium. The uptake and sub-cellular distribution of the drugs were thereafter measured scintillation counting. All the compounds produced postive inotropic responses, the mondigitoxoside producing the greatest effect, digoxigenin next in order of inotropic response magnitude, the bis-digitoxoside produced the least effect, and digoxin was intermediate between the genin and bis-digitoxoside. The uptake of the monodigitoxoside was the highest, and in general, the quantitative uptake was related to the inotropic response. The greatest binding of each digitoxoside was found in the microsomal fraction. Both mechanical activity and uptake of all four drugs were uniformly reduced by an increase in potassium concentration in the perfusion medium.

Animals↗
PubMed1994

[Effects of E3123 on pancreatic injury in the isolated, perfused rat pancreas and in a pancreatic slice].

We studied the effect of E3123 on pancreatic injury induced in the isolated, perfused pancreas or in a pancreatic slice. Exposure of the isolated, perfused rat pancreas to trypsin-taurocholate or phospholipase A2 caused the leakage of pancreatic enzyme (lipase) into the perfusate. In trypsin-taurocholate-induced pancreatic injury, E3123 and nafamostat mesilate suppressed the leakage of lipase at concentrations of 0.1-1 microM and 1-10 microM, respectively. In phospholipase A2 induced pancreatic injury, E3123 at 10 microM significantly suppressed the enzyme leakage, and nafamostat mesilate had a weak suppressive effect. Exposure of a rat pancreatic slice to phospholipase A2 also caused the leakage of pancreatic enzyme, while the inhibition of enzyme leakage by E3123 was similar to that observed in the isolated, perfused pancreas; nafamostat mesilate was not effective. This finding suggests the possible involvement of a unique mechanism of action in the protection against pancreatic injury by E3123. Therefore, we studied the effect of E3123 on hemolysis by osmotic shock using rat red blood cells. E3123 demonstrated a potent protective effect against the hemolysis, suggesting that a membrane-stabilizing action may contribute to the protection E3123 affords against pancreatic injury.

Animals↗