Sublingual and submandibular blood collection in mice: a comparison of effects on body weight, food consumption and tissue damage.

In a large number of animal experiments, blood collection is crucial for achieving the study aim. Requirements on sampling techniques used include their practicability, their effectiveness in terms of obtaining the desired blood volume, sample quality and low impact on animal's wellbeing. Numerous methods for blood collection from mice have been published. For large blood volumes, submandibular and sublingual bleeding was developed as alternatives to the retrobulbar bleeding method, which is considered controversial as it results in severe tissue damage. Only a few studies report the use of submandibular and sublingual techniques in mice. In particular, the degree of tissue damage or influence on animal's wellbeing due to submandibular bleeding in conscious mice has not yet been clearly assessed. This gap in the knowledge base has been filled by our exploratory study that revealed clear differences between both techniques. Defence movements during submandibular blood collection in conscious mice resulted in more complications and revealed insufficient blood sample quality due to the prolonged duration of blood collection. In addition, it is likely that these movements may have caused lesions to be more pronounced. Changes in red blood cell parameters (red blood cell count, haemoglobin and haematocrit), in glucose and in total protein concentrations observed in anaesthetized animals were most likely related to anaesthesia. Sublingually punctured mice gained significantly more body weight than submandibularly punctured mice, likely due to less severe tissue lesions and improved healing processes. Based on these results, we recommend the sublingual blood collection technique to be used in mice. However, if the submandibular bleeding technique is used, it should be performed in anaesthetized mice only.

Blood collection from the sublingual vein in mice and hamsters: a suitable alternative to retrobulbar technique that provides large volumes and minimizes tissue damage.

Blood examination is a key element in studies of laboratory animals. In rodents, retrobulbar venous plexus puncture is a commonly used method for obtaining a blood sample. Although this technique yields large volumes of blood, the disadvantage is that it can lead to severe tissue damage. The aim of the present study was to develop the puncture of V. sublingualis as a suitable alternative technique for drawing blood in mice and other rodents. In rats, this method has been established for collecting large blood volumes. During the first part of the study, the sublingual bleeding technique was developed for use in mice and hamsters. Guinea pigs, however, do not have a sublingual vein; therefore, in this species the method is not possible. In the second part of the study, retrobulbar and sublingual methods were compared using male CD-1 mice. When compared with the retrobulbar method, sublingual venepuncture showed less tissue destruction in mice, with a decreased mean severity in the histological examination. In conclusion, sublingual venepuncture can be recommended as a suitable, alternative blood collection technique, because of the reduced risk of tissue damage in mice and hamsters.

Effect of competitive marathon cycling on plasma N-terminal pro-brain natriuretic peptide and cardiac troponin T in healthy recreational cyclists.

For a further depiction of exercise-induced cardiac dysfunction, N-terminal pro-brain natriuretic peptide (NT-pro-BNP) and cardiac troponin T (cTnT) were measured in recreational cyclists (n = 29) during the Otztal Radmarathon 2004. In all subjects, NT-pro-BNP significantly increased from 28 +/- 21 to 278 +/- 152 ng/L immediately after the race (p <0.001), decreased again on the following day, and returned to baseline values 1 week later. The mean percentage increase in NT-pro-BNP was 1,128 +/- 803%. CTnT, negative in all subjects before the race, increased transiently in 13 athletes (45%), with levels ranging from 0.043 to 0.224 mug/L in 8 of them (28%). One day after competition, cTnT had normalized in all athletes. Because of the typical release of kinetics, the deflection of NT-pro-BNP is considered to be the adequate volume regulatory response of a hemodynamically stressed heart to prolonged strenuous exercise. The observed kinetics of cTnT substantiate a release from the free cytoplasmatic pool due to the half-life of cytosolic cTnT. In healthy cyclists, transient increases in NT-pro-BNP and cTnT are more likely to reflect cardiac fatigue than injury.

The smoker's paradox and the real risk of smoking.

No satisfactory explanations have been offered for the smoker's paradox, the greater short-term survival of smokers after a myocardial infarction nor for the large variations in the coronary risk rate for smoking ranging between 1 and 5.9. These discrepancies as well as the smoker's paradox may be caused by different baseline characteristics of smokers and nonsmokers, whereas the usually quoted coronary risk of 2 is derived from studies based on the assumption of equal baseline characteristics. As neither this assumption nor the possibility of unequal starting conditions have been tested, we examined the main cardiovascular risk factors in smoking and nonsmoking boys as near as possible to baseline, at the age of fourteen. This age appeared to be best suited, because boys starting to smoke early are most likely to become regular and heavy smokers. Of 336 boys, 39 had smoked 8.3+/-6.0 cigarettes/day for 15.5+/-11.2 months. Compared to nonsmokers, boys who started to smoke early had lower LDL cholesterol and alpha2-antiplasmin, greater handgrip strength, vital capacity and forced expiratory volume, better perfomance on bicycle ergometry and higher testosterone. The differences in total cholesterol, LDL cholesterol, vital capacity, handgrip strength, testosterone and alpha2-antiplasmin persisted after adjustment for age, body mass, and testosterone. In addition, the differences in perfomance on bicycle ergometry and forced expiratory volume persisted after adjustment for age. These favourable baseline characteristics of those starting to smoke early can explain the smoker's paradox. In addition, they suggest that the individual coronary risk in smokers is considerably higher than 2, because the assumption of equal baseline characteristics of smokers and nonsmokers cannot be upheld.

Optimising the design of preliminary toxicity studies for pharmaceutical safety testing in the dog.

A working party, comprising two animal welfare organisations and some 12 pharmaceutical companies in Europe, was established to minimise the use of the dog in safety testing. As first step, the participants defined the major objectives of preliminary dose-range finding/MTD toxicity studies in non-rodents, defined the principles and requirements for this study type and agreed on a proposal for an optimised study design, based on collective experience of conducting such studies in industry, involving an evaluation of 100 individual study data sets. The suggested study design is explained and described, and reflects current best practice in the pharmaceutical industry in Europe. The implementation of such an optimised design is believed to result in a reduction in the overall numbers of animals used for this purpose, without jeopardising the scientific rationale and usefulness of the studies for informing the conduct of later regulatory studies.

An approach to minimise dog use in regulatory toxicology: production of a best practice guide to study design.

The primary non-rodent species used in toxicology is the dog. It is generally agreed that, for ethical reasons, dog use should be reduced to the minimum consistent with maintaining the scientific quality of toxicology studies and ensuring human safety. Dog use in toxicology has been discussed widely, both from a scientific and ethical viewpoint, and there appears to be real potential for achieving significant reductions in the number of dogs used in pharmaceutical safety testing. An industry animal welfare initiative commenced in 2000, with the aim of evaluating and, where possible, putting into practice, scientifically valid approaches to minimise dog use in regulatory toxicology without increasing the use of other non-rodent species, such as non-human primates or minipigs. The study's Steering Group categorised potential reduction approaches into three distinct areas, one of which is the production of a best practice guide on aspects of study design, including: group sizes, use of control animals, single sex studies and design of maximum tolerated dose (MTD) studies. Information on current practice and experience within the pharmaceutical industry is now being analysed, and additional input is invited.

Preclinical safety evaluation using nonrodent species: an industry/welfare project to minimize dog use.

This review of the dog, the primary nonrodent species used in toxicology, and its use in the safety evaluation of pharmaceuticals, provides data on the number used in particular projects in an effort to establish a baseline from which some minimization can be measured. Opportunities for reduction and replacement, as identified by a European Industry/Welfare Steering Group, are discussed. The three distinct areas of potential approaches to minimize dog use are categorized as industrial cooperation/data sharing, achieving best practice in study design, and assessing the need for a particular study. The Steering Group prioritized the approaches based on the impact on the number of animals used, the impact on the welfare of the remaining animals, the potential for industry's acceptance of the scientific approach, the potential for regulators' acceptance of the validated approach, and the time/cost of evaluation or implementation. Examples of each category are presented, and the work needed to facilitate industry/regulatory change is discussed.

Physiological effects of an ultra-cycle ride in an amateur athlete - a case report.

The physiological effects of ultraendurance exercise are poorly investigated. The present case report describes the exercise intensity of ultraendurance cycling and its physiological impacts on various organ functions in an amateur cyclist performing the Ötztal Radmarathon twice en bloque in a circuit of 2 identical laps (distance 460 km; cumulative altitude difference 11,000 m). In a pre-race laboratory test the athlete's performance capacity was measured as the maximal aerobic power (VO2max= 70 ml.kg(-1).min(-1)), a maximal power output (5.7 W.kg(-1)) and lactate threshold of 89%. The overall intensity during the ride was moderate (HRmean = 131 b.min(-1); %HRmax = 0.71) and significantly declined during the course of the race. Extensive biochemical laboratory testing performed pre- and post-race excluded major exercise-induced organ disturbances. For further confirmation and better understanding of the physiological effects of ultra-cycle events future studies of larger athlete populations are required.