Use of biophotonic imaging as a training aid for administration of substances in laboratory rodents

Dosing of experimental animals and the removal of blood are two of the most frequent procedures performed in biomedical research using live animals. Despite the apparently simple nature of these procedures, they can, if not correctly carried out, have significant effects on the welfare of the animals and the scientific value of the results. There are several methods by which research staff may obtain training in the administration of substances. These include practical demonstrations during teaching courses; observation of techniques; videos and educational computer programs and practising on recently killed animal cadavers or plastic animal models. Each method has its own advantages and disadvantages. A common factor encountered during training is the difficulty in assessing competency. This paper reports a pilot study on the use of bioluminescent imaging technology to assess competency in the administration of substances to rodents. Bioluminescence was rapidly detected after dosing of animals with a bioluminescent substance. However, living animals were required for a signal to be generated. The data presented suggest that this technology is ideal for use as a teaching aid and may also prove valuable in assessing the effectiveness of 'complex' and novel administration routes in 'realtime'.     

Efficient Cooperative Restraint Training With Rhesus Macaques

It is sometimes necessary for nonhuman primates to be restrained during biomedical and psychosocial research. Such restraint is often accomplished using a “primate chair.” This article details a method for training adult rhesus macaques to cooperate with a chair restraint procedure using positive and negative reinforcement. Successful training was accomplished rapidly in approximately 14 training days. The success of this training technique suggests that this method represents a refinement to traditional techniques. Further, this method worked effectively for animals previously deemed unfit for traditional pole-and-collar training.     

Principles of Rodent Surgery for the New Surgeon

For both scientific and animal welfare reasons, training in basic surgical concepts and techniques should be undertaken before ever seeking to perform surgery on a rodent. Students, post-doctoral scholars, and others interested in performing surgery on rodents as part of a research protocol may not have had formal surgical training as part of their required coursework. Surgery itself is a technical skill, and one that will improve with practice. The principles of aseptic technique, however, often remain unexplained or untaught. For most new surgeons, this vital information is presented in piecemeal fashion or learned on the job, neither of which is ideal. It may also make learning how to perform a particular surgery difficult, as the new surgeon is learning both a surgical technique and the principles of asepsis at the same time. This article summarizes and makes recommendations for basic surgical skills and techniques necessary for successful rodent surgery. This article is designed to supplement hands-on training by the user''s institution.     

Plasma corticosterone and restraint induced gastric pathology: age-related differences after administration of corticotropin releasing factor

Corticotropin releasing factor (CRF) or saline was administered i.p. to rats aged either 100 or 220 days, followed by either brief handling or water immersion restraint. Plasma corticosterone was measured 75 min. later. Age of the animals in itself was not a significant factor either for basal levels of plasma corticosterone or for extent of restraint induced gastric pathology. However after CRF administration, young but not older animals revealed a significant increase in plasma corticosterone levels, and post restraint gastric ulcerations were more severe in older than young animals. CRF significantly decreased the number of restraint induced ulcers in young rats, while the cumulative ulcer length was increased in older animals.     

Antagonism of specific corticotropin-releasing factor receptor subtypes selectively modifies weight loss in restrained rats

Rats exposed to 3 h of restraint stress on each of 3 days (RRS) lose weight on the days of RRS and gain weight at the same rate as controls after stress ends, but do not return to the weight of controls. RRS rats also show an exaggerated endocrine response to subsequent novel stressors. Studies described here tested the effects of corticotropin-releasing factor receptor (CRFR) antagonism on RRS-induced weight loss, hypophagia, and corticosterone release during mild stress in the postrestraint period. Weight loss was not prevented by either peripheral or third-ventricle administration of a CRFR1 antagonist, antalarmin, before each restraint. Antalarmin did, however, allow recovery of body weight in the poststress period. Third-ventricle administration of a CRFR2 antagonist, antisauvagine 30, had no effect in RRS rats but caused sustained weight loss in control animals. Surprisingly, third-ventricle administration of the nonselective CRFR antagonist, astressin, caused hypophagia and reversible weight loss in control rats. It had no effect in RRS rats. None of the antagonists modified the corticosterone response to RRS or to mild stress in the post-RRS period, but antalarmin suppressed corticosterone during the period of restraint in Control rats. These results suggest that CRFR1 activation is required for the initiation of events that lead to a prolonged down-regulation of body weight in RRS rats. The sustained reduction in body weight is independent of the severity of hypophagia on the days of restraint and of RRS-induced corticosterone release.     

Diagnosis of ecto- and endoparasites in laboratory rats and mice

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized animals. Before embarking on an examination of animals for parasites, two things should be considered. One: what use will be made of the information collected, and two: which test is the most appropriate. Knowing that animals are parasitized may be something that the facility accepts, but there is often a need to treat animals and then to determine the efficacy of treatment. Parasites may be detected in animals through various techniques, including samples taken from live or euthanized animals. Historically, the tests with the greatest diagnostic sensitivity required euthanasia of the animal, although PCR has allowed high-sensitivity testing for several types of parasite. This article demonstrates procedures for the detection of endo- and ectoparasites in mice and rats. The same procedures are applicable to other rodents, although the species of parasites found will differ.     

Ovariectomy and 17β-estradiol replacement in rats and mice: a visual demonstration

Estrogens are a family of female sexual hormones with an exceptionally wide spectrum of effects. When rats and mice are used in estrogen research they are commonly ovariectomized in order to ablate the rapidly cycling hormone production, replacing the 17β-estradiol exogenously. There is, however, lack of consensus regarding how the hormone should be administered to obtain physiological serum concentrations. This is crucial since the 17β-estradiol level/administration method profoundly influences the experimental results. We have in a series of studies characterized the different modes of 17β-estradiol administration, finding that subcutaneous silastic capsules and per-oral nut-cream Nutella are superior to commercially available slow-release pellets (produced by the company Innovative Research of America) and daily injections in terms of producing physiological serum concentrations of 17β-estradiol. Amongst the advantages of the nut-cream method, that previously has been used for buprenorphine administration, is that when used for estrogen administration it resembles peroral hormone replacement therapy and is non-invasive. The subcutaneous silastic capsules are convenient and produce the most stable serum concentrations. This video article contains step-by-step demonstrations of ovariectomy and 17β-estradiol hormone replacement by silastic capsules and peroral Nutella in rats and mice, followed by a discussion of important aspects of the administration procedures.     

Fundamental training for individuals involved in the care and use of laboratory animals: a review and update of the 1991 NRC Core Training Module

Public trust demands that individuals who do research, testing, or teaching with animals use humane, ethical, and scientifically sound methods. Furthermore, the Animal Welfare Act and the Public Health Service Policy require research institutions to provide basic training and to ensure that anyone who cares for and/or works with laboratory animals has the appropriate training or experience relevant to their job responsibilities. Institutions accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International must also provide training programs and ensure the qualifications of personnel. The primary goal of this training is to provide individuals with basic knowledge and to reinforce attitudes and behaviors that help to ensure humane animal care and use. This article provides an overview of the core training module outline and content from the 1991 report of the Institute for Laboratory Animal Research, Education and Training in the Care and Use of Laboratory Animals: A Guide for Developing Institutional Programs, as well as pertinent updates for introducing personnel to information regarding the care and use of laboratory animals. Both mandatory and suggested training topics are reviewed, including relevant regulations and standards, ethical considerations, humane methods of animal experimentation and maintenance, and other pertinent topics. Although the fundamental training course content and delivery will vary depending on the nature and complexity of an institution's animal care and use program, this basic training provides the foundation for more in-depth training programs and supports humane and ethical animal care and use.     

Institutional and IACUC responsibilities for animal care and use education and training programs

Training and instruction of personnel are important components of animal care and use programs because they help to ensure the health and welfare of the animals and the integrity of the research or testing results. Training also helps to promote the consideration of alternatives, recognition of animal pain and distress, appropriate use of pain-relieving agents, aseptic technique, pre- and post-procedural care, and personnel health and safety. While individuals who provide the care for or conduct research or testing in laboratory animals should take personal responsibility for ensuring that they have the skills to perform their duties, the institution is ultimately responsible for ensuring their competency. The institution is also responsible for providing the training or instruction that is required by federal legislation, regulations, and policies. The institutional animal care and use committee (IACUC) is responsible for ensuring, as part of their review of research activities, that the personnel are capable of performing the procedures described. The IACUC must also assess the institution's training program as part of their semiannual animal care and use program review and make recommendations regarding training to the institutional official. This article provides a comprehensive overview of the US regulatory mandates for training and personnel qualification.     

Corticosterone Assimilation by a Voluntary Oral Administration in Palatable Food to Rats

Drug delivery in research on nonhuman animals in the laboratory is still challenging because it is usually invasive and stressful. Stress-free voluntary oral drug administration in water lacks precise control of dose and timing of substance ingestion. Voluntary oral consumption of corticosterone has been previously successfully applied in mice using oat flakes, but protocols for oral corticosterone administration in rats remain unavailable. This study assessed the effectiveness of voluntary oral administration to rats of a palatable piece of bread soaked with corticosterone that can be rapidly prepared and is reliably dose- and timing-controllable. After three familiarization days, all rats ate the bread within 120 seconds of presentation, irrespective of the presence or absence of corticosterone or vehicle. Corticosterone plasma levels remained at basal levels with consumption of vehicle-containing bread, and they were significantly increased with corticosterone-containing bread. Hence, the method enabled corticosterone bodily assimilation while avoiding stress, making it a possible alternative for invasive and stressful procedures. This article includes a methodological refinement that lessens unnecessary discomfort to laboratory animals and is potentially suitable for acute and chronic protocol studies.     

运动员剧烈运动后血中应激免疫抑制蛋白的产生

我们曾经报道,大鼠或小鼠在束缚应激后血中产生了一种能抑制免疫功能的应激免疫抑制蛋白,（又称Ｎｅｕ－ｒｏｉｍｍｕｎｅｐｒｏｔｅｉｎ,ＮＩＰ,神经免疫蛋白）。本工作证明,运动员在大运动量的训练后血清中也产生一种能抑制淋巴细胞转化的物质,它的生化特性及分子量与前述大鼠和小鼠中的应激免疫抑制蛋白相同。在体外实验中,应激大鼠的血清培养人淋巴结细胞,获得了与大鼠实验相同的结果,即人淋巴结细胞也能产生应激免疫抑制蛋白。同时小鼠束缚应激的血清和大运动量的人类血清可以分别抑制人正常淋巴细胞和正常小鼠由ＣｏｎＡ诱导的淋巴细胞转化,以上结果表明,这种应激免疫抑制蛋白的种属特异性不强。     

Prior exposure to a single stress session facilitates subsequent contextual fear conditioning in rats. Evidence for a role of corticosterone

Previous studies showed that exposure of rats to chronic restraint stress for 21 days enhances subsequent contextual fear conditioning. Since recent evidence suggest that this effect is not dependent on stress-induced neurodegenerative processes, but to elevated training-elicited glucocorticoid release in chronically stressed animals, we aimed to explore here whether a single exposure to restraint stress, which is not expected to induce neuronal damage, would also affect contextual fear conditioning. We also questioned whether post-training corticosterone levels might be associated with any potential effect of stress on fear conditioning. Adult male Wistar rats were exposed to acute restraint stress for 2 h and, two days later, trained in the contextual fear conditioning task, under training conditions involving either moderate (0.4 mA shock) or high (1 mA shock) stress levels. The results showed that acute stress enhanced conditioned freezing at both training conditions, although data from the 1 mA shock intensity experiment only approached significance. Stressed animals were shown to display higher post-training corticosterone levels. Furthermore, the facilitating effect of prior stress was not evident when animals were trained in the hippocampal-independent auditory-cued conditioning task. Therefore, these findings support the idea that stress experiences preceding exposure to new types of stressors facilitate the development of contextual fear conditioning. They also indicate that not only repeated, but also a single exposure to aversive stimulation is sufficient to facilitate context-dependent fear conditioning, and suggest that increased glucocorticoid release at training might be implicated in the mechanisms mediating the memory facilitating effects induced by prior stress experiences.     

Susceptibility of laboratory and domestic animals to experimental infection with Potiskum virus

The biological characteristics of Potiskum virus, a hitherto undescribed virus isolated in Nigeria from the liver of a giant rat (Cricetomys gambianus), were studied by experimental infections of laboratory and domestic animals. The laboratory animal hosts used included mice, rats, rabbits and chicks. Suckling and weaning mice succumbed to fatal infection when infected with Potiskum virus by intracerebral or intraperitoneal routes. Infected mice had high titres of virus and mild histopathological lesions which were confined to the brain. Chicks also developed a fatal disease following subcutaneous or oral infections with Potiskum virus. In contrast, albino rats and rabbits failed to succumb to overt disease by subcutaneous and intraperitoneal routes of inoculation. Albino rats did not develop antibody but rabbits developed haemagglutination inhibiting, neutralising and complement fixing antibodies.     

Femoral Arterial and Venous Catheterization for Blood Sampling,Drug Administration and Conscious Blood Pressure and Heart Rate Measurements

In multiple fields of study, access to the circulatory system in laboratory studies is necessary. Pharmacological studies in rats using chronically implanted catheters permit a researcher to effectively and humanely administer substances, perform repeated blood sampling and assists in conscious direct measurements of blood pressure and heart rate. Once the catheter is implanted long-term sampling is possible. Patency and catheter life depends on multiple factors including the lock solution used, flushing regimen and catheter material. This video will demonstrate the methodology of femoral artery and venous catheterization of the rat. In addition the video will demonstrate the use of the femoral venous and arterial catheters for blood sampling, drug administration and use of the arterial catheter in taking measurements of blood pressure and heart rate in a conscious freely-moving rat. A tether and harness attached to a swivel system will allow the animal to be housed and have samples taken by the researcher with minimal disruption to the animal. To maintain patency of the catheter, careful daily maintenance of the catheter is required using lock solution (100 U/ml heparinized saline), machine-ground blunt tip syringe needles and the use of syringe filters to minimize potential contamination. With careful aseptic surgical techniques, proper catheter materials and careful catheter maintenance techniques, it is possible to sustain patent catheters and healthy animals for long periods of time (several weeks).     

Resistance training restores metabolic alterations induced by monosodium glutamate in a sex-dependent manner in male and female rats

Despite resistance exercises being associated with health outcomes, numerous issues are still unresolved and further research is required before the exercise can faithfully be prescribed as medicine. The goal of this study was to investigate whether there are sex differences in resistance training effects on metabolic alterations induced by monosodium glutamate (MSG), a model of obesity, in male and female rats. Male and female Wistar rats received MSG (4 g/kg body weight/day, s.c.) from postnatal day 1 to 10. After 10 days from MSG administration, the rats were separated into two groups: MSG-sedentary and MSG-exercised. At postnatal day 60, the animals started a resistance training protocol in an 80 degrees inclined vertical ladder apparatus and performed it for 7 weeks. Control rats received saline solution and were divided in saline-sedentary and saline-exercised. Resistance training restored all plasma biochemical parameters (glucose, cholesterol, triglycerides, aspartate aminotransferase, and alanine aminotransferase) increased in male and female rats treated with MSG. The MSG administration induced hyperglycemia associated with a decrease in the skeletal muscle glucose transporter 4 (GLUT4) levels and accompanied by deregulation in proteins, G-6Pase, and tyrosine aminotransferase, involved in hepatic glucose metabolism of male and female rats. MSG induced dyslipidemia and lipotoxicity in the liver and skeletal muscle of male rats. Regarding female rats, lipotoxicity was found only in the skeletal muscle. The resistance training had beneficial effects against metabolic alterations induced by MSG in male and female rats, through regulation of proteins (GLUT2, protein kinase B, and GLUT4) involved in glucose and lipid pathways in the liver and skeletal muscle.     

Can we optimise the exercise training prescription to maximise improvements in mitochondria function and content?

Background

While there is agreement that exercise is a powerful stimulus to increase both mitochondrial function and content, we do not know the optimal training stimulus to maximise improvements in mitochondrial biogenesis.

Scope of review

This review will focus predominantly on the effects of exercise on mitochondrial function and content, as there is a greater volume of published research on these adaptations and stronger conclusions can be made.

Major conclusions

The results of cross-sectional studies, as well as training studies involving rats and humans, suggest that training intensity may be an important determinant of improvements in mitochondrial function (as determined by mitochondrial respiration), but not mitochondrial content (as assessed by citrate synthase activity). In contrast, it appears that training volume, rather than training intensity, may be an important determinant of exercise-induced improvements in mitochondrial content. Exercise-induced mitochondrial adaptations are quickly reversed following a reduction or cessation of physical activity, highlighting that skeletal muscle is a remarkably plastic tissue. Due to the small number of studies, more research is required to verify the trends highlighted in this review, and further studies are required to investigate the effects of different types of training on the mitochondrial sub-populations and also mitochondrial adaptations in different fibre types. Further research is also required to better understand how genetic variants influence the large individual variability for exercise-induced changes in mitochondrial biogenesis.

General significance

The importance of mitochondria for both athletic performance and health underlines the importance of better understanding the factors that regulate exercise-induced changes in mitochondrial biogenesis. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research.     

Participation of the hypophyseal-adrenal cortex system in thrombin clearance during immobilization stress]

The examination carried out with thrombin marked by 131J resulted in a considerable increase of the thrombin clearance rate in healty male rats during the stress (caused by an immobilization lasting 30 minutes) and in an increase of thrombin deposits in the liver. A further increase of thrombin clearance occurred by the combination of immobilization and administration of ACTH. Contrary to ACTH the thrombin clearance is not stimulated in healthy animals by hydrocortisone. Thrombin clearance and thrombin deposits in the liver are lowered in adrenalectomized rats. In these animals the administration of ACTH does not result in an increase of thrombin clearance. The rate of thrombin clearance is normalized in adrenalectomized animals after administering hydrocortisone without as well as under conditions of stress. In adrenalectomized animals having received hydrocortisone as well as in healthy animals the administration of ACTH will results in an increase of thrombin clearance. From these experiments the conclusion can be drawn that ACTH will increase the intensity of thrombin clearance in stress and that hydrocortisone plays a transmitting part here.     

"Green odor" inhalation reduces the skin-barrier disruption induced by chronic restraint stress in rats: physiological and histological examinations

We investigated whether inhalation of green odor (a mixture of equal amounts of trans-2-hexenal and cis-3-hexenol) prevents the skin-barrier disruption induced by chronic restraint stress in rats. To this end, transepidermal water loss (TEWL) was measured as an index of the disruption of skin-barrier function, whereas light- and electron-microscope examinations were performed to observe histological changes in the skin of the stressed animals. In addition, the effects on TEWL induced by chronic administration of a glucocorticoid, dexamethasone (DEX), were examined. Chronic restraint stress (8 h per day for 14 days) increased TEWL (vehicle + stress group). This effect (and the chronic stress-induced increase in adrenal weight) was prevented in rats that inhaled green odor at the beginning of each day's restraint (2 h each day for 14 days; green odor + stress group). Electron-microscope studies revealed that rats in the green odor + stress group possessed sufficient intercorneocyte lipids to create an effective skin barrier, although these had apparently been decreased in the vehicle + stress group. Daily administration of DEX for 14 days increased TEWL. The present results suggest that chronic stress-induced disruption of the skin barrier in rats can be reduced or prevented by green odor (possibly at least in part through an inhibitory effect on the stress-induced activation of the hypothalamo-pituitary-adrenocortical axis).     

Evaluation of the micronucleus assay in bone marrow and peripheral blood of rats for the determination of cigarette mainstream-smoke activity

The mammalian in vivo micronucleus assay is widely used as part of the genotoxicity testing battery required during the development of new drugs. As such, the in vivo micronucleus assay has been used in a battery of assays for the assessment of cigarette ingredients or design modifications to help ensure that there is no increase in risk or any new risk introduced by these additions or modifications. The present series of studies was conducted to optimize and evaluate this assay for the assessment of the effects of mainstream smoke on the micronucleus frequency in the bone marrow and peripheral blood of rats. In a first experiment, the optimal conditions for performing the micronucleus assay in these tissues were determined. This was done by use of two compounds known for their micronucleus-inducing activity, i.e., the clastogen cyclophosphamide and the aneugen colchicine. In a second experiment, the effects of tube restraint on untreated control rats were investigated. In a third experiment, the optimal conditions were used to assess the clastogenic/aneugenic activity of cigarette smoke in Sprague-Dawley rats. The rat micronucleus assay in both bone marrow and peripheral blood is able to detect clastogenic and aneugenic activity. The flow cytometric determination of micronucleated cells in rat blood is at least as sensitive as determinations in bone marrow. No statistically significant differences were observed in micronucleus frequencies between rats with and without the additional stress of tube restraint; however, the cautious approach would be to use a fresh-air-exposed group (with tube restraint) as the negative control in inhalation experiments. Using the conditions identified as optimal in the above-mentioned experiments, the micronucleus assay was not able to detect effects induced by smoke from conventional cigarettes. Nevertheless, the micronucleus assay will remain a valuable tool as part of a testing battery used to investigate possible adverse effects related to product modifications.     

Reduction through education: the insight of a trainer

One of the articles contained within European Council Directive 86/609/EEC states that "Persons who carry out experiments or take part in them, and persons who take care of animals used for experiments, including duties of a supervisory nature, shall have appropriate training". In effect, this article stipulates that only competent individuals are allowed to work with laboratory animals. At least three groups of individuals can be identified with different responsibilities toward experimental animals: animal technicians, scientists, and veterinarians/animal welfare officers. The responsibilities and duties of the individuals within each of these categories differ. This paper focuses on the training of scientists. The scientist designs, and often also performs, animal experiments. Therefore, scientists must be educated to develop an attitude of respect toward laboratory animals, and must be trained so that, if an experiment must be performed with animals, it is designed according to the highest possible scientific and ethical standards. In The Netherlands, the law stipulates that scientists intending to work with animals must have completed a course in laboratory animal science. This compulsory course started in 1986. The Department of Laboratory Animal Science at Utrecht University is responsible for the national coordination of this course. Participants must have an academic degree (at the level of MSc) in one of the biomedical sciences, such as biology, medicine or veterinary medicine. Although the course is an intensive 3-week, 120-hour long course, which covers both technical and ethical aspects of laboratory animal experimentation, it cannot provide full competence. It is designed to provide sufficient basic training and knowledge to enable students to design animal experiments, and to develop an attitude that will be conducive to the implementation of the Three Rs. However, full competence will always require further training that can only be acquired as a result of practical experience gained while working in the field of laboratory animal research. Evaluations subsequent to the course have revealed that more than 98% of the students regard the course as indispensable for all scientists working in a research area where animal experiments are performed. They agree that the course not only contributes to the quality of experiments and to the welfare of animals, but also to a decrease in the number of animals used in experiments.