Impact of vitamin E supplement in standard laboratory animal diet on microvascular manifestation of ischemia/reperfusion injury

Aimed at improving animal fertility and health, diets for farm and laboratory animals have over the last few years been supplemented with increasing amounts of the antioxidant vitamin E. We now demonstrate by intravital microscopy that feeding hamsters with a vitamin E-supplemented “standard” rodent diet (60 ppm vitamin E) significantly reduces the microvascular manifestations of ischemia/reperfusion injury when compared to animals fed a nonsupplemented diet. Postischemic leukocyte adhesion to venular endothelium was reduced from 770 ± 204 cells/mm² at 24 h after reperfusion in control animals on the nonsupplemented diet to 403 ± 105 cells/mm² in animals on the “standard” rodent diet (means ± SD, N = 7 animals per group, p < 0.01). Animals on the nonsupplemented diet showed a dramatic loss of capillary perfusion density until 7 days after reperfusion (to 21 ± 13% of preischemic baseline values), whereas this loss was significantly attenuated (to 71 ± 12% of preischemic values, p < 0.01) in animals on the “standard” rodent diet. No difference in the extent of reperfusion injury was seen between animals on the “standard” rodent diet and animals on diets with substantially higher vitamin E supplements (300 ppm–30.000 ppm). Besides underscoring the benefit of vitamin E in reducing the extent of ischemia/reperfusion injury, this study raises the concern that vitamin E supplements in “standard” laboratory animal diets may have a far-reaching impact on biomedical research by jeopardizing established animal models of disease.     

The development of a “microtitre” fluctuation test for the detection of indirect mutagens, and its use in the evaluation of mixed enzyme induction of the liver

The “Microtitre” Fluctuation test recently introduced for the detection of direct mutagens has been adapted for the detection of indirect mutagens through the incorporation of an “S9-mix” metabolic system. It compares favourably with Greens' original method for the detection of a range of chemical mutagens.

The technique has been employed in the evaluation of mixed enzyme induction using phenobarbitone and β-naphthoflavone (benzoflavone). as a safe substitute for Aroclor-1254. The post-mitochondrial preparations from rats induced with the combined inducers had a similar “metabolic competence” to those derived from Aroclor induced animals. Such a combination would therefore provide a useful alternative to Aroclor-1254 for routine screening.

It was found that the level of “S9” present in the metabolic system greatly affected the quantitative mutagenic response. This varied considerably from chemical to chemical and underlined the need for such preliminary investigations in routine screening.     

Period responses to Zeitgeber signals stabilize circadian clocks during entrainment

Circadian clocks with characteristic period (τ) can be entrained to light/dark (LD) cycles by means of (i) phase shifts which are due to D/L “dawn” and/or L/D “dusk” transitions, (ii) period changes associated with long-term light exposure, or (iii) by combinations of the above possibilities. Based on stability analysis of a model circadian clock it was predicted that nocturnal burrowing mammals would benefit less from period responses than their diurnal counterparts. The model further predicted that maximal stability of circadian clock is reached when the clock slightly changes both its phase and period in response to light stimuli. Analyses of empirical phase response curve (PRC) and period response curve (τRC) of some diurnal and nocturnal mammals revealed that PRCs of both diurnal and nocturnal mammals have similar waveform while τRCs of nocturnal mammals are of smaller amplitude than those of diurnal mammals. The shape of the τRC also changes with age and with increasing strength of light stimuli. During erratic fluctuations in light intensity under different weather conditions, the stability of phase of entrainment of circadian clocks appears to be achieved by an interplay between phase and period responses and the strength of light stimuli.     

Levels of citation of nonhuman animal studies conducted at a Canadian research hospital

The publication of scientific articles that receive few or no citations raises questions of the appropriate use of resources as well as ethics. In the case of animal research, the ethics issue extends beyond human patients to nonhuman animals, as the research subjects them to pain and, typically, to death. This study is a citation analysis of animal research conducted at Toronto's Hospital for Sick Children (HSC). Of the 594 publications (1990 to 1995) on animal research by affiliates of HSC, 29% received fewer than 10 citations in a 10-year period. We compare the research history of 13 “best ”and 13 “worst ”HSC scientists. Worst researchers continue to do infrequently cited research. Recommendations indicate how institutions and researchers can become more effective and accountable.     

Development of a new production system for weaned and growing pigs

The design of modem housing systems for farm animals calls for cooperation between scientists within different disciplines. This article describes the design of a new production system for weaned and growing pigs, as well as the analyses and working processes behind its development. The aim of the study was to develop an economically competitive system that also provided better welfare for the animals and a healthier environment for the workers. Analyses were carried out with emphasis on behavioral needs and building design and economy. The resulting system, the “safari system, ”consists of a number of straw flow pens of increasing size. Pigs are moved to larger and larger pens each week until slaughter. The main advantage is that pigs are not mixed during the growth period and that they are given more space, especially toward the end of the growth period when their space demand is high. Furthermore, the provision of straw for comfort and exploration and of wallow basins for cooling in the pens for the larger pigs were considered significant welfare improvements. An analysis revealed that the safari system would be economically competitive in comparison to traditional intensive systems.     

The Pragmatic Approach to Masking

This paper advocates the use of a pragmatic approach to the problem of masking in real-life situations involving an abrupt change in the timing of sleep, i.e. shiftwork and “jet-lag” situations. Although “pure” chronobiological research has pointed to the importance of taking masking effects into account, the techniques that it has provided for doing so are extremely difficult to apply in real-life situations. The approach advocated here is based on Wever's pioneering work, and involves estimating the normative endogenous and exogenous components of the circadian rhythm in body temperature. These estimates are then used to: (a) simulate the results of shiftwork studies; and (b) to “remove” the exogenous component in “jet-lag” studies to allow analysis of the estimated endogenous component. The simulated curves obtained cross-correlated extremely highly with published night-shift temperature curves, while the “removal” of the exogenous component resulted in very similar findings to those obtained in temporal isolation studies. It is concluded that this pragmatic approach to masking may prove extremely useful in interpreting the results of field studies of shiftwork and “jet-lag”.     

The Difference Between Activity When in Bed and Out of Bed. II. Subjects on 27-Hour “Days”

Nine healthy subjects have been studied while exposed to the normal alternation of light and dark, but with their sleep and activity pattern adjusted to a 27-h “day” for 17 imposed “days.” Rectal temperature showed clearly the competing influences of 27-h and 24-h components, and these were separated by the method of “purification.” The method indicated that the endogenous component had a constant amplitude throughout the experiment and remained entrained to solar (24-h) time; by contrast, the exogenous component followed the imposed 27-h “day” and increased rectal temperature in proportion to the amount of subjects' activity. Wrist movement was used to assess activity while in bed (attempting sleep) and out of bed (when naps were forbidden). While these results confirmed adherence of the subjects to the imposed 27-h “days,” they also showed that the dichotomy between “out of bed” activity and “in bed” inactivity depended on the phase relationship between endogenous (24h) and exogenous (27h) components. Thus, the dichotomy was highest and was equal to that during control days (with a conventional 24-h life-style) when the two components were in phase and lowest when the solar and imposed day were in antiphase. This was due to changes in activity, both during time spent in bed and out of bed.

We confirm that this protocol can produce valuable information about the properties of the circadian system in humans and the value of the process of purification of temperature data. We have established also that the very simple and noninvasive measurement of wrist movement, coupled with its use to calculate dichotomy indices, provides valuable information that both confirms and extends the results obtained from the more conventional (butalso more invasive) measurement of rectal temperature.     

The European Union ban on conventional cages for laying hens: history and prospects

Since the publication of Animal Machines (Harrison, 1964), there has been widespread public pressure in Europe-supported by European institutions-to “ban the battery cage. ”The European Union (EU) and national governments (particularly in Northern Europe) funded research on noncage systems for egg production and enriched cages. In 1986, the EU passed a Directive specifying a minimum size for cages, but public opinion-again particularly in the North-continued to require more. A market sector emerged that would pay more for noncage eggs. Denmark, Sweden, and Switzerland passed more stringent legislation than the rest of Europe. A 1999 Directive with details based on advice from the EU's Scientific Veterinary Committee will phase out conventional laying cages but allow enriched cages. Implementation depends on various factors, including negotiations in the World Trade Organization. In the next 10 years, however, major changes to the housing of most laying hens in Europe almost certainly will occur. Similar changes in other countries will follow. As in Europe, change probably will be piecemeal, affected both by public pressure and by all sectors of society: producers, retailers, consumers, legislators, and the media.     

Temperature profiles, and the effect of sleep on them, in relation to morningness-eveningness in healthy female subjects

There were 15 healthy female subjects, differing in their position on the “morningness-eveningness” scale, studied for 7 consecutive days, first while living a sedentary lifestyle and sleeping between midnight and 08:00 and then while undergoing a “constant routine.” Rectal temperature was measured at regular intervals throughout this time, and the results were subjected to cosinor analysis both before and after “purification” for the effects of physical activity. Results showed that there was a phase difference in the circadian rhythm of core temperature that was associated with the morningness score, with calculations that “morning types” would be phased earlier than “evening types” by up to about 3h. This difference in phase (which was also statistically significant when the group was divided by a median split into a “morning group” and an “evening group”) could not be attributed to effects of waking activity and existed in spite of the subjects keeping the same sleep-wake schedule. Moreover, it persisted when the subjects' data had been purified and when the data were obtained from the constant routine. That is, there was an endogenous component to this difference in phase of the core temperature. The morning group also showed a greater fall of core temperature during sleep; this was assessed in two ways, the main one being a comparison of constant routine and nychthemeral data sets after correction for any effects of activity. Even though the morning group was sleeping at a later phase of their circadian temperature rhythm than was the evening group, neither group showed a fall of temperature due to sleep that varied with time elapsed since the temperature acrophase. It is concluded that another factor that differs between morning and evening types is responsible for this difference. (Chronobiology International, 18(2), 227-247, 2001)     

Investigating fear in rainbow trout (Oncorhynchus mykiss) using the conditioned-suppression paradigm

Trout learned the operant task of pendulum-pressing for a food-reward in a mean of 4.3 sessions lasting 1 hr. In a separate phase, fish also learned—through classical conditioning—to associate a neutral light cue with an aversive stimulus. When again allowed to pendulum-press for food, after aversive classical conditioning, there was a drop in the rate of responding. The mean rate dropped from 3.6-2.9 responses per min. Most important, when the light-stimulus was superimposed on a steady bout of pendulum-pressing, trout ceased to press the pendulum and did not resume activity until termination of the light-stimulus (mean number of responses during a 3-min interval immediately prior to light-stimulus = 14.3 vs. during 3-min light-stimulus = 0.1). Psychologists have used this decrease in operant responding, or “conditioned emotional response,” as a tool to examine the psychological nature of this type of aversive conditioning. In this study, the fish demonstrated various results under this paradigm similar to those shown by “higher” nonhuman animals, therefore challenging the view of fish as unconscious, nonsentient animals.     

Opsin biosynthesis and trans-cis isomerization of aldehyde form chromophore in the blowfly Calliphora erythrocephala eye

The effect of eye carotenoid content, light conditions and retinoid supply on the biosynthesis of opsin, as well as the ability to isomerize of exogenous all-trans-retinal to 11-cis-retinal were investigated in the photoreceptors of blowfly Calliphora. SDS-PAGE of digitonin extracts from isolated rhabdom fractions of carotenoid-fortified and carotenoid-deficient animals revealed, on heavily loaded gels, that in both cases opsin forms faint minor bands similar to the patterns of “R⁻-flies” described as “vitamin A-deficient flies” (Paulsen and Schwemer, Biochim. biophys Acta 557, 358–390, 1979) or “carotenoid-deficient flies” (Paulsen and Schwemer, Eur. J. Biochem. 137, 609–614, 1983. Similar opsin patterns were obtained in flies subjected to continuous 72 h blue or yellow-green light or to a 12 h: 12 h white light:dark cycle, or total darkness, irrespective of the carotenoid content in their eyes. 11-cis-retinal and, to a lesser extent, all-trans-retinal, when included in the diet or painted on the cornea, were found to stimulate opsin biosynthesis in the dark. 11-cis-retinal in the dark or all-trans-retinal after illumination of the flies with blue light were the most effective, as compared with the effect of all-trans-retinal in the dark. Exogenous all-trans-retinal in the dark can be partially converted into a mixture of 11-cis-retinal (26%) and 13-cis-retinal (74%) by fly retina homogenate in vitro. It was concluded that Calliphora opsin biosynthesis is not strongly dependent on the carotenoid supply or on light: dark conditions and is triggered by the 11-cis aldehyde form of the chromophore, which can be produced in the fly retina either by an isomerase system in the dark or as a result of photoisomerization.     

The Relative Zeitgeber Strength of Lights-On and Lights-Off Is Changed in Old Mice

The daily activity pattern of old mice is characterized by a decreased amplitude, a phase advance, and less stable relationship between lights-off and the onset of the main activity maximum. When analyzing the possible causes of these changes, it must be remembered that the activity rhythm of laboratory mice is bimodal, with a main peak in the first half of the dark time and a secondary one shortly after lights-on. Thus it seems to be controlled by at least two circadian oscillators—an “evening oscillator” coupled more strongly to lights-off and a “morning oscillator” coupled to lights-on—though both oscillators are also coupled to each other. The objective of the present paper was to investigate the putative changes in the strength of these couplings in HaZ:ICR mice of different ages (adult animals of 20 weeks, n=12; old mice of 72 and 91 weeks of age, n=6 each) and kept in a 24h LD-cycle with a gradually reduced light:dark ratio.

In adult mice, lengthening the dark time caused the onset of the main maximum of activity to be delayed in relation to the time of lights-off, while the morning maximum of activity was advanced in relation to lights-on. On average, the sizes of the advance and the delay were equal. As a consequence, the activity pattern did not shift in relation to the middle of the dark time. Lengthening the dark time resulted in a bigger (on average, 1.5h) difference between the evening and morning activity onsets. Under short photoperiods (≤2h of light) the activity rhythm started to free run, and the difference between evening and morning activity onsets decreased again. The changes obtained in senile mice were similar. However, the limits of entrainment were reached with longer photoperiods compared to adult animals. Also, the phase delay of the activity onset in the evening was much less, nearly zero. As a consequence, the activity pattern as a whole phase-advanced in relation to the middle of the dark time.

A model was proposed in which lights-off triggers advances of the “evening oscillator,” lights-on delays the “morning oscillator,” and the two oscillators are coupled with each other. Though it was probably the case, decreased coupling strengths could not be shown with the present experimental approach. However, it was clearly evident that, with increasing age, the advancing effect of lights-off exceeded the delaying effect of lights-on.     

Time-Qualified Reference Limits (Chronodesms) for 24-Hour Blood Pressure Values: Classical and Bayesian

Background - This paper is a concrete example of the problems raised by the need of constructing the time-qualified reference limits (chronodesms) for blood pressure (BP), in order to clinically estimate the hemodynamic parameter in its intrinsic nychtohemeral variability. Methods - Assuming that the noninvasive ambulatory BP monitoring (ABPM) is the eligible technique for this need, it must be realized that the BP chronodems may be of two types, depending on the sample being used for their calculation. The first type may be regarded as “ a priori ” because of the fact that they are derived by a sample of normotensive subjects who are unavoidably recruited via “ causal ” sphygmomanometric measurements and reclassified as normotensive by comparing their ABPM to the fixed reference limits (monodesms) given by WHO (monodiagnosis). Therefore, the “ a priori ” BP chonodesms are by principle derived by subjects who could not be correctly classified as normotensive, their ABPM being not tested versus the time-varying physiological limits. The second type may regarded as “ a posteriori ” in virtue of the fact that they may be constructed on a sample which contemplates the previous subjects who result to be true normotensive via the reassessment of their ABPM versus the “ a priori ” BP chronodesms (chronodiagnosis). The “ a posteriori ” chronodesms may be regarded as biometrically reliable, whether the sample for their construction is additionally constituted by those subjects of the local population who have been erroneously monodiagnosed as hypertensive, while they result to be true normotensive via the chronodiagnostic comparison of their ABPM versus the “ a priori ” BP chronodesms. Results - The biometric reliability of the “ a posteriori ” BP chronodems is demonstrated by the fact that their upper limits are statistically significantly less pronounced due to the fact that they are provided by a sample which has been depured by the falsely monodiagnosed normotensives. Conclusions - The “ a posteriori ” BP upper chronodesms are the time-qualified reference limits which should be used in clinical practice for the chronodiagnosis of hypertension.     

Seeing the Light in Washington Chronobiology at the 3rd Annual Meeting of the Association of Professional Sleep Societies (Apss)

Chronobiology at the 3rd Annual Meeting of the APSS was, no doubt in the “Sign of Light”. Chronobiological research has certainly moved from DD to BL (bright light). Several factors contributed to the great interest and excitement regarding the effects of light on human beings. The Science publication of Czeisler et al., “Bright light induction of strong (type 0) resetting of the human circadian pacemaker”, which attracted the attention of the media, coincided with the scheduled APSS meeting. Also, a very well-attended one-day workshop in Bethesda, Maryland, on the biologic effects of light was organized by the Society for Light Therapy and Biological Rhythms the day before, and succeeded in raising interest and excitement over the subject. The presentations on the biologic effects of light on the circadian system focused on the phase resetting-response to light of the human circadian system, and on the practical implications of this resetting on seasonal affective disorders (SAD), in sleep-phase-delay and sleep-phase-advance syndromes as well as sleep disturbances associated with jet lag.     

Circadian control of migratory restlessness and the effects of exogenous melatonin in the brambling, Fringilla montifringilla

Circadian pacemakers control both “daytime” activity and nocturnal restlessness of migratory birds, and the daily rhythm of melatonin release from the pineal has been suggested to be involved in the control of migratory activity. To study the phase relations between the two activity components during entrainment and when free running, locomotor activity of bramblings (Fringilla montifringilla) was recorded continuously under a 12:12 “cool light” to “warm light” cycle (CL:WL, ca. 5000 K and ca. 2500 K, respectively) or blue light to red light cycle (BL:RL, maxima at 440 and 650 nm, respectively) at different irradiance ratios. Migratory activity was expressed primarily during the WL or RL phase of the light cycles. Under free-running conditions, the circadian periods τ correlated with the phase relations between day and night (migratory) activity components during preceding entrainment. Bramblings with migratory activity had significantly longer τ at constant light intensity than the same individuals without migratory activity. Birds with migratory activity reentrained faster after a 6h phase shift of the CL:WL cycle than birds without migratory activity. When exogenous melatonin was given in the drinking water (200 μg/mL 1% ethanol or 0.86 mM) to bramblings exposed to 12:12 CL:WL cycles with constant irradiance, the amounts of activity, which were initially higher during the WL phase of the light cycle, were suppressed to similar low levels during both light phases. The systematic changes in the amounts of activity during melatonin treatment were not correlated with consistent changes in entrainment status. The data support the hypothesis that changes in the amplitude and level of the daily melatonin cycle are involved in regulating migratory restlessness, by either allowing or inhibiting nocturnal activity. (Chronobiology International, 17(4), 471-488, 2000)     

Physiological changes in the Norway lobster Nephrops norvegicus (L.) escaping and discarded from commercial trawls on the West Coast of Scotland: 1. Body fluid volumes and haemolymph composition after capture and during recovery

The Norway Lobster Nephrops norvegicus (L.) was captured by trawling at depths of 120 to 150 m off the West Coast of Scotland using commercial fishing gears. Discarded animals (normally <25–35 mm carapace length) were sampled immediately after capture, and following emersion (exposure to air) on deck for 1 or 2 h. Their recovery from, and survival of, the stresses of being fished were examined in separate groups of animals transferred by SCUBA divers to underwater (u/w) cages on the seabed at a mean depth of 24 m. “Escaped” N. norvegicus were collected in a small-meshed (40 mm) “cover” around the cod-end and sampled without emersion. It was assumed that the physiological state of these animals would be similar to those passing through the cod-end meshes directly back into the surrounding sea during normal fishing operations. Groups of escaped animals were also transferred to u/w cages for recovery and survival studies. Control animals (creel-caught from the same fishing grounds) were sampled after recovery in u/w cages. Total body water content, haemolymph (blood) volume and composition were examined at various stages after capture and during recovery of control, discarded and escaped animals. Discarded animals showed significant reductions in total body water content compared to controls but recovered after 24 h. Controls that had been artificially damaged by puncturing also showed a reduction in water content. Escaped animals showed significantly higher mean water content compared to discards. Haemolymph volumes were also reduced in discarded animals suggesting that significant blood loss occurs during capture. There was also evidence of a shift of fluid from haemolymph to tissues in discarded animals. Pericardial (haemolymph) pressures were much reduced in discards compared to undisturbed control animals but showed significant recovery in surviving animals. Pressure reduction was less in escapes but was seen in control animals that had been exercised to exhaustion. Haemolymph osmotic concentrations, [Na⁺] and [K⁺] showed increases after discarding, notably after emersion, but haemocyanin [Hcy] and total haemolymph protein concentrations were less affected. The effect of these changes on longer term survival and recovery from the stress of capture are discussed.     

Some Introductory Comments on Silver Staining

Silver staining has become a versatile method for the visualization of specific cell structures and products. The similarity of the impregnation “nuclei” of reduced silver staining to the silver “specks” or “nuclei” of the latent image in photography is noted. “Physical” development (reduction of ionic silver in solution) in silver staining as compared to “chemical” development (reduction of ionic silver remaining in a silver halide crystal) in photographic procedures is briefly discussed.     

Detectability and criterion measures in temporal generalization

Computer simulation of performance on “normal” and “episodic” temporal generalization tasks was used to examine the relations between the theoretical parameters of models which fit temporal generalization data (“timing sensitivity” and “threshold”), and the d′ (detectability) and beta (decision criterion) measures of signal-detection theory. In general, changes in timing sensitivity altered d′, whereas threshold changes affected beta, supporting the assertion that the two sorts of variables (“sensitivity/detectability” and “threshold/criterion”) were psychologically equivalent. Cases where temporal generalization gradients were apparently contaminated by “random responding” could be treated by changes in beta, but cases in which temporal generalization gradients were not peaked at the standard posed severe problems for a simple signal-detection account, although existing models of temporal generalization performance could deal with them.     

Plasma Concentration of Nine Hormones and Neurotransmitters During Usual Activities or Constant Bed Rest for 34 H

Thyroid-stimulating hormone (TSH), Cortisol, melatonin, prolactin, luteinizing hormone (LH), delta-sleep-inducing peptide (DSIP), its phosphorylated form (P-DSIP), heart rate, and body temperature were measured every half hour during two 24-h periods in five normal men. τ-Amino-butyric acid (GABA) and 3-methoxy-4-hydroxyphenylglycol (MHPG) were measured less frequently. The first period, the “activity” condition, included usual daily activities. The second period, or “rest” condition, consisted of fasting, constant bed rest during 34 h, and partial light deprivation. Compared with the “rest” condition, the “activity” condition increased heart rate, temperature, LH, and TSH in most subjects, and Cortisol in two of five subjects. It retarded the onset of nocturnal Cortisol and melatonin secretion. The temporal pattern and the absolute values of the concentrations of DSIP, P-DSIP, MHPG, GABA, and prolactin showed no or minimal changes during the two conditions. In spite of the influence of the “activity” versus “rest” condition on several hormones, the mean concentrations as well as the temporal organization of their secretion into plasma were quite stable within each subject, whereas they varied much more between individuals. TSH, Cortisol, and melatonin values were also stable within an 8-month period in one subject who was studied on four occasions. The results illustrate that the patterns of hormones rhythms and their reactivity to changes in the environment are, to a large extent, specific to each subject.     

Visitors' effects on the welfare of animals in the zoo: a review

Since the 1970s, research about zoo visitors' effects on the welfare of nonhuman animals in captivity has intensified. Numerous studies have shown that characteristics such as visitor presence, density, activity, size, and position are associated with animal behavioral and—to alesser extent physiological—changes. Studies usually interpret these changes as negative (undesirable) or positive (enriching), but it remains unclear whether they significantly impinge on animal welfare. To make confident conclusions about visitors' effects necessitates more studies using (a) a wider range of animal groupings, (b) measures of stress, (c) visitor-animal variables, and (d) other methodological improvements In the meantime, in addition to further research, individual zoos need to emphasize (a) monitoring the stress indicators of their captive animals, (b) observing visitor behavior, and (c) ensuring that staffs are aware of the “visitor effect” concept.