Increased susceptibility to lipid peroxidation in rabbit kidneys: a consequence of warm ischaemia and subsequent reperfusion

Rabbit kidneys were clamped and rendered warm ischaemic (WI) in situ for 60 and 120 min. They were then either removed immediately after the ischaemic insult or after reperfusion with blood for 60 min or 24 hr. Homogenates were assayed for phospholipid-Schiff base fluorescence (Ex. 360 nm, Em. 435 nm) and for diene conjugate formation by u.v. spectrophotometry (240 nm) as indices of lipid peroxidation. No alteration in tissue levels of Schiff base was evident immediately after WI but when the homogenates were incubated at 37 degrees C for 90 min, the rate of peroxidation was significantly elevated compared to controls (P less than 0.02 after WI of 60 min and P less than 0.001 after 120 min of WI). These values were still further elevated after reperfusion with blood for 60 min and 24 hr (P less than 0.001). Diene conjugates were raised after WI alone and further still after reperfusion. Thus an early index of lipid peroxidation (diene conjugation) suggested peroxidative damage during the warm ischaemic period itself, whilst detection of Schiff bases was only possible after in vitro incubation of the tissue. Both indices of oxygen-derived free radical damage were increased after reperfusion in vivo with blood and may relate to the degree of tissue damage sustained during ischaemia and reflow.     

Hypothermic reconditioning by gaseous oxygen persufflation after cold storage of porcine kidneys

BackgroundDelayed graft function still represents a major complication in clinical kidney transplantation. Here we tested the possibility to improve functional outcome of cold stored kidneys a posteriori by hypothermic reconditioning using retrograde oxygen persufflation (ROP) immediately prior to reperfusion.MethodsKidneys from female German Landrace pigs were flushed with Histidine–Tryptophan–Ketoglutarate (HTK) solution and cold-stored for 18 h (control).Some grafts were subsequently subjected to 90 min of retrograde oxygen persufflation (ROP) via the renal vein during cold preservation. Early graft function of all kidneys was assessed thereafter by warm reperfusion in vitro (n = 6, resp.).ResultsRenal function upon reperfusion was significantly enhanced by ROP with an approximately twofold increase in renal clearances of creatinine and urea. ROP also led to higher renal vascular flow rates, enhanced urine output and mitigated histological alterations.ConclusionIt is concluded that initial graft function can be improved by 90 min of hypothermic gaseous oxygenation after arrival of the preserved organ in the transplantation clinic.     

N-acetylaspartate: a literature review of animal research on brain ischaemia

The present review examines and discusses the changes in N-acetylaspartate (NAA) concentration in the ischaemic brain from the existing literature of animal research. By summarizing the current knowledge on NAA metabolic pathways and reviewing the data obtained in animal models of global and focal ischaemia, the following conclusions emerge from this compilation: (i) both magnetic resonance spectroscopy (MRS) and the absolute HPLC method of NAA quantification give converging information; (ii) decreases in brain NAA concentration in acute stroke can be considered as an index of neuronal loss or dysfunction, although NAA redistribution by glial cells and NAA trapping in cell debris restrict its use as a quantitative neuronal marker; (iii) further studies on NAA metabolism in pathologic brain are required before using NAA measurement in the chronic stage of ischaemia for evaluating neuroprotective strategies.     

Detection of ochratoxin A in porcine kidneys by a monoclonal antibody-based radioimmunoassay

By using an indirect enzyme-linked immunosorbent assay, eight monoclonal antibodies (MAbs) were selected. Mice were immunized with ochratoxin A that was conjugated to bovine serum albumin. The hybridoma cell line designated 10G2 was grown in tissue culture and as an ascites tumor. The MAb was characterized to be specific to ochratoxin A and of the immunoglobulin G (IgG) class. Subsequently, the ascites fluid of this hybridoma was used in a competitive solid-phase IgG radioimmunoassay on protein A-Sepharose CL-4B, with [14C]ochratoxin A as tracer. Porcine kidneys were extracted with 0.5% phosphoric acid in chloroform. A two-step cleanup was achieved on a Sep-Pak C18 cartridge and a Sep-Pak silica cartridge. Radioimmunoassay with MAbs coupled to protein A-Sepharose CL-4B allowed the detection of ochratoxin A in porcine kidneys at a concentration as low as 0.2 ng/g.     

The calm mouse: an animal model of stress reduction

Chronic stress is associated with negative health outcomes and is linked with neuroendocrine changes, deleterious effects on innate and adaptive immunity, and central nervous system neuropathology. Although stress management is commonly advocated clinically, there is insufficient mechanistic understanding of how decreasing stress affects disease pathogenesis. Therefore, we have developed a "calm mouse model" with caging enhancements designed to reduce murine stress. Male BALB/c mice were divided into four groups: control (Cntl), standard caging; calm (Calm), large caging to reduce animal density, a cardboard nest box for shelter, paper nesting material to promote innate nesting behavior, and a polycarbonate tube to mimic tunneling; control exercise (Cntl Ex), standard caging with a running wheel, known to reduce stress; and calm exercise (Calm Ex), calm caging with a running wheel. Calm, Cntl Ex and Calm Ex animals exhibited significantly less corticosterone production than Cntl animals. We also observed changes in spleen mass, and in vitro splenocyte studies demonstrated that Calm Ex animals had innate and adaptive immune responses that were more sensitive to acute handling stress than those in Cntl. Calm animals gained greater body mass than Cntl, although they had similar food intake, and we also observed changes in body composition, using magnetic resonance imaging. Together, our results suggest that the Calm mouse model represents a promising approach to studying the biological effects of stress reduction in the context of health and in conjunction with existing disease models.     

Variance reduction by simultaneous multi-exponential analysis of data sets from different experiments

The analysis of experimental data from the photocycle of bacteriorhodopsin (bR) as sums of exponentials has accumulated a large amount of information on its kinetics which is still controversial. One reason for ambiguous results can be found in the inherent instabilities connected with the fitting of noisy data by sums of exponentials. Nevertheless, there are strategies to optimize the experiments and the data analysis by a proper combination of well known techniques. This paper describes an applicable approach based on the correct weighting of the data, a separation of the linear and the non-linear parameters in the process of the least squares approximation, and a statistical analysis applying the correlation matrix, the determinant of Fisher's information matrix, and the variance of the parameters as a measure of the reliability of the results. In addition, the confidence regions for the linear approximation of the non-linear model are compared with confidence regions for the true non-linear model. Evaluation techniques and rules for an optimum experimental design are mainly exemplified by the analysis of numerically generated model data with increasing complexity. The estimation of the number of exponentials significant for the interpretation of a given set of data is demonstrated by using records from eight absorption and photocurrent experiments on the photocycle of bacteriorhodopsin. Offprint requests to: K.-H. Müller     

Sample Power and ExpDesign: tools for improving design of animal experiments

Proper experimental design, involving the correct number of animals, should be a basic skill for any scientist working with animals. The authors describe a university-developed and freely available tutorial program and an interactive computer-assisted learning program, both of which guide students through the steps necessary for designing animal experiments and estimating optimal sample sizes.     

Principles and practice in ethical review of animal experiments across Europe: summary of the report of a FELASA working group on ethical evaluation of animal experiments

This paper summarizes a more detailed report produced by the Federation of European Laboratory Animal Science Associations (FELASA 2005), which describes and explores a set of principles for the conduct of ethical review of laboratory animal use. It presents a synopsis of results from a questionnaire that elicited information on how each of 20 countries represented in FELASA currently approaches such ethical review. This information suggests that, although local practices differ, there is an emerging consensus on the key elements that any ethical review process should involve. Drawing on the questionnaire findings, this summary also includes a brief discussion to support and amplify a series of recommendations, covering the objectives of ethical review; legal requirements; the scope of work reviewed and the 'level' at which review is approached; general principles for the organization of ethical review processes; the factors considered in the review; needs for ongoing review after initial authorization; participants in the review process; wider impacts of the review process; and strategies that can help to ensure quality and consistency of review outcomes. For further information and examples of current practice, as well as more detailed discussion to support the recommendations, readers are urged to refer to the complete report, available at http://www.lal.org.uk/pdffiles/FELASA_ethics_FULL_Report. pdf or via: http://www.felasa.eu/recommendations.htm.     

Comparison of bovine udder skin with human and porcine skin in percutaneous permeation experiments

Rat and pig animal skin has been the most common replacement material for human skin for use in in vitro permeability experiments. Unfortunately, the permeability barrier of skin from laboratory animals is known to be relatively weak, due to significant follicular transport. Pig skin has been shown to be a suitable model for human skin. Unfortunately, it cannot be gathered from the regular slaughtering process, which makes it unsuitable for permeation experiments. We therefore studied the suitability of bovine udder skin, an untreated waste material of the butchering process, as a possible replacement material for use in in vitro permeability tests. We investigated the barrier strength of bovine udder skin against four different substances, and its histology and lipid profile, in comparison with pig skin and heat separated human epidermis. Pig and human skin were found to be equally permeable, whilst bovine udder skin seemed to exhibit a weaker, but less variable, barrier against caffeine, benzoic acid, testosterone, and flufenamic acid. The skin of all three species contained variable contents of the major lipid classes: cholesterol, ceramides, cholesterol ester, fatty acids and triglycerides. Morphological differences mainly comprised variations in the density of hair follicles. Based on these results, the amount of free fatty acids and triglycerides and the density of hair follicles seem to be important factors in the differences between the skin barriers in the three species.     

Use and misuse of an imprecise concept: alternative methods in animal experiments

The concept of inhumanity of Russell & Burch in 1959 with proposals for human procedures in experiments on animals by way of replacement, reduction and refinement (the 3 R's) and the revival of these by Smyth (1978) as alternatives to animal experiments are presented. Since then under the name 'alternative methods', replacement of in vivo by in vitro methods has found great public attention and promotion. It is argued that alternative methods are a fallacy, because in the progress of research a continual control and reinvestigation of findings on each system level is required until knowledge is complete, which makes in vivo experiments irreplaceable. True alternatives exist only for refinement within a system level. On the level of the organism, refinement would be in the care of animals and experimental procedure through alleviation of stress. The possibilities which refinement offers for animal welfare have been nearly forgotten and need promotion among researchers, animal technicians and attendants.     

Myocytes isolated from porcine coronary arteries: reduction of currents through L-type Ca-channels by verapamil-type Ca-antagonists.

Myocytes were enzymatically isolated from large epicardial arteries of the pig. In the cell attached configuration, we studied currents through L-type Ca-channels. At 22 degrees C, open channel conductance was 9 pS with 110 mM Ca2+ and 24 pS with 110 mM Ba2+ as charge carrier. According to the life time of the open state, 2 'modes' of gating are distinguished; mode 1 contributed time constants shorter than 1 ms, mode 2 those longer than 6 ms to the open time distribution. Mode 2 openings appeared spontaneously, more frequently with Ba2+ than with Ca2+ as charge carrier. The Ca-agonist Bay K 8644 (0.5 microM) facilitated the appearance of mode 2. Bath application of the phenylalkylamine D600 (1 microM) did not change the gating modes, but it reduced the channel openness by increasing the percentage of blank records. With whole cell recordings, we studied reduction of ICa by 1 microM D 600 at 3.6 mM [Ca2+] and 35 degrees C. At a holding potential of -45 mV, D 600 induced an 'initial block' of 35% (10% at -65 mV). Upon repetitive 1 Hz pulsing (170 ms to 0 mV) an additional, 'use-dependent' block developed with time. More negative holding potentials attenuated reduction of ICa by D 600, hyperpolarizations to -100 mV had an 'unblocking' effect. In regard to reduction of ICa, we compared the partially uncharged D 600 (membrane permeable) with the completely charged compound D 890 (membrane impermeable). When applied with the bath, 1 or 10 microM D 600 reduced ICa dose-dependently whereas D 890 was ineffective. When D 890 was applied via the patch electrode to the cytosol, it reduced ICa. We discuss that D 600 enters the cell in the uncharged lipid soluble form and reaches form the inside its receptor associated with the Ca-channel.     

Review. Establishing an experimental science of culture: animal social diffusion experiments

A growing set of observational studies documenting putative cultural variations in wild animal populations has been complemented by experimental studies that can more rigorously distinguish between social and individual learning. However, these experiments typically examine only what one animal learns from another. Since the spread of culture is inherently a group-level phenomenon, greater validity can be achieved through 'diffusion experiments', in which founder behaviours are experimentally manipulated and their spread across multiple individuals tested. Here we review the existing corpus of 33 such studies in fishes, birds, rodents and primates and offer the first systematic analysis of the diversity of experimental designs that have arisen. We distinguish three main transmission designs and seven different experimental/control approaches, generating an array with 21 possible cells, 15 of which are currently represented by published studies. Most but not all of the adequately controlled diffusion experiments have provided robust evidence for cultural transmission in at least some taxa, with transmission spreading across populations of up to 24 individuals and along chains of up to 14 transmission events. We survey the achievements of this work, its prospects for the future and its relationship to diffusion studies with humans discussed in this theme issue and elsewhere.