Sage Journals: Discover world-class research

Abstract

Summary

Rat serum or plasma creatine kinase (CK) activity is widely used to evaluate myopathic processes, to test the myotoxicity of different drugs, or to analyse the benefits of emerging gene therapies in some neuromuscular disorders. However, great variability is found in this determination. The aim of this study has been to control some factors of variation in order to reduce variability and increase the reproducibility of analytical data. 8–10-week-old Wistar-Han rats were used. The study consisted of four sequential phases. Phase I aimed to analyse the effect of ether and isoflurane as anaesthetic drugs. The objective of Phase II was to evaluate bleeding rats via retro-orbital sinus vs. tail vein. Phases III and IV were designed as two separate, repeated measure experiments on two factors: habituation to laboratory handling procedures in Phase III and gender in Phase IV. The repeated factor was the storage temperature of blood sample prior to centrifugation. Ether did not significantly increased the CK value. Using isoflurane, getting rats accustomed to laboratory handling procedures and whole blood refrigeration prior to centrifugation and serum separation resulted in statistically significant reduction in CK value and variability. Male rats showed significantly higher values than female rats. In the light of our findings, CK value and variability in rats may be minimized by choosing tail vein as site of bleeding, getting rats accustomed to laboratory handling procedures and maintaining whole blood refrigerated until centrifugation and serum separation.

Keywords

Creatine kinase blood sampling anaesthesia variability reduction refrigeration serum

Creatine kinase (CK; EC 2.7.3.2) is an intracellular enzyme present in a variety of striated and smooth muscles and in the brain; it is an important enzyme regulator of high-energy phosphate production and utilization in contractile tissues. CK is physiologically present in the plasma, indicating a permanent release from muscle cells, and it is one of the most widely used enzymes for the quantitative evaluation of myocardial infarction (Klein et al. 1973, Maroko & Vatner 1977, Visser et al. 1981), myopathic processes (Cardinet 1989), muscle damage following intramuscular injections (Lewis & Rhodes 1978) and in various other diseases. Although not all myopathies produce a rise in CK activity, it is increased as a result of muscle fibre destruction after mechanical trauma, toxic injury or alteration of enzymatic or structural proteins. CK elevation varies within disorders, with increases that may range from 2 to 100-fold the reference value.

Blood can be sampled from animals using different techniques with differing impacts on animal discomfort due to differences in handling, restraining, anaesthesia, invasiveness and the volume taken. The method of blood sampling can also affect the outcome of blood analysis. Orbital puncture is frequently used to obtain blood samples from rats. However, this technique is controversial because it possibly causes pain and distress to the animals (van Herck et al. 1992, 1999). The BVA/FRAME/RSPCA/UFAW joint working group has stated that orbital puncture is acceptable only as a terminal procedure while the animal is under anaesthesia (Morton et al. 1993). For non-terminal blood sampling in rats, the working group advises collecting a maximum of 0.5 mL of blood by puncturing the tail vein while applying a short-acting anaesthetic to ease the rats. In spite of this, retro-orbital puncture is still widely used. This may be due to the lack of either scientific proof of negative consequences when performed by trained staff or the lack of equally good alternatives.

CK activity has been reported to vary within animal species (Matsuzawa et al. 1993) and gender (Kaspar & Norris 1977, Loeb & Quimby 1999), and with age (Shibata & Kobayashi 1978, Cardinet et al. 1989, Matsuzawa et al. 1993), stress (Lefebvre et al. 1992), site of bleeding (Matsuzawa et al. 1993), puncture technique (Fayolle et al. 1992), handling procedures (Bacou & Bressot 1976, Sneddon et al. 1989, Lefebvre et al. 1992, Lefebvre et al. 1996, Yerroum et al. 1999), serum or plasma measurement (Shibata & Kobayashi 1978, Matsuzawa et al. 1993, Aktas et al. 1994), storage of the specimen and the analytical method used (Lev et al. 1994, Clark et al. 2003). Thus, for CK activity, values ranging from 100 U/L to 900 U/L have been observed in control rats (Hsu et al. 1995, Nosaka 1996, Reijneveld et al. 1996, Matsuda et al. 1998, Yerroum et al. 1999). However, in the majority of toxicological studies, preanalytical specifications and recommendations are described either poorly or not at all, making it difficult to standardize them on a repetitive basis. This matter may be of particular interest if it is recognized that the effect of mild myotoxins could possibly be masked as a result of omitting such factors of variation. Nowadays, with the performance of myotoxicity studies on new drugs and the animal testing of emerging cellular and gene therapies for the treatment of some muscular dystrophies, the analysis of CK variation could be one of the principal markers for evaluating potential new drug candidates or the course of a disease and its potential improvement with therapy.

The aim of this study has been to analyse a number of factors affecting CK activity in Wistar-Han rats, including anaesthesia, site of bleeding, handling, getting the rats accustomed to laboratory procedure techniques, whole blood sample storage prior to centrifugation and gender, in order to minimize variability and improve the reliability of the measurement of CK activity.

Materials and Methods

Design and phases of trial

The study consisted of four sequential phases in which the results of each stage conditioned the design of the following phase. Phase I aimed to analyse the effects on CK activity of the use of ether and isoflurane as anaesthetic drugs. The objective of Phase II was to compare two different blood sampling techniques: retrobulbar plexus vs. tail vein. Phases III and IV were designed as two separate, repeated measure experiments on two factors: habituation to laboratory handling procedures in Phase III and gender in Phase IV. The repeated factor was the storage temperature of blood sample prior to centrifugation. Phases I, II and IV were performed in one laboratory (Laboratory 1), whereas Phase III was carried out in another one (Laboratory 2). The study was undertaken in accordance with the ‘Real Decreto 1201/2005’ about animal welfare and its associated Codes of Practice.

The experimental design is summarized in Table 1.

Table 1

Phases of the study: factor studied at each phase and experimental conditions

Factor studied	N ^*	Sex	n ^†	Sampling procedure	Anaesthetic	Sampling day	Laboratory
Anaesthesia	10	♂	7	Retro-orbital	Ether	0,2,3,6,8,10,14	1
Ether vs. isoflurane				Retro-orbital	Isoflurane	0,2,3,6,8,10,14
Blood sampling technique	7	♂	7	Tail vein	Isoflurane	0,2,3,6,8,10,14	1
Tail vein vs. retro-orbital	5	♂	7	Retro-orbital	Isoflurane	0,2,3,6,8,10,14
Accustom and temperature of blood sample	5	♂	5	Tail vein	Isoflurane	0,2,4,7,9	2
	5	♂	6	Tail vein	Isoflurane	8,12,14,16,19,21
Sex and temperature of blood sample	5	♀	5	Tail vein	Isoflurane	0,3,5 17,18	1
	5	♂	5	Tail vein	Isoflurane	0,3,5 17,18

N = number of rats

†

n = number of samplings per rat

Animals

Eight-to-ten-week-old male and female HsdRccHan™:WIST rats (Harlan, Barcelona, Spain) were housed in individual Makrolon™ cages, allowing a one-week acclimatization period. All the animals were kept at a constant temperature (22°C) in a 12:12 h light–dark cycle, and were provided with food and water ad libitum. Carbon dioxide (CO₂) was used for euthanasia.

Handling and anaesthesia

To induce sedation with ether, this was first applied to an absorbent material and then placed in the anaesthetic chamber, being removed as soon as it lost consciousness. For the maintenance of sedation, the absorbent material soaked in ether was placed in an empty syringe cartridge and moved closer or further away from the animal's face to adjust the concentration.

When using isoflurane (Forene®, Deutsche Abbott, Delkenheim, Germany), rats were lifted by the base of the tail and rapidly introduced into the anaesthetic chamber and sedated with 5% isoflurane for 1 min. The anaesthesia was maintained using a polycarbonate mask with 2.6% isoflurane. As complementary material, a heated blanket was used to raise rat body temperature, lowered by the effect of the isoflurane. Blood sampling commenced approximately 5 min after the rat was placed on the electric blanket in order to permit vasodilatation of the tail veins. In Phases III and IV, rats were handled by the manipulator and placed in the anaesthetic chamber with oxygen for 1 min and then returned to the cage once a day for 1–2 weeks prior to samplings in order to get the rats accustomed to laboratory handling procedures.

Blood sampling procedure

Blood samples (0.5–1 mL) were taken from the retrobulbar venous plexus or the tail vein after anaesthesia on consecutive days (Table 1) using microhaematocrit tubes Na-hep BRAND® and 24G (0.7 × 19 mm) BD Insyte™ IV catheter respectively and placed in gel serum separator tubes (BD Microtainer™).

Processing of the blood samples

Blood samples from Phases I and II remained at room temperature (RT) for a minimum of 30 min until centrifugation. In Phases III and IV, 1 mL of whole blood was extracted from each rat and divided into two tubes to be stored at RT or refrigerated for a minimum of 30 min until centrifugation, after all samplings had been completed. In Laboratory 1, blood samples were centrifuged at 4°C for 15 min at 2778 g (4000 rpm) in a Heraeus Megafuge 1.0 R and serum was analysed in a Hitachi 911 spectrophotometer. In Laboratory 2, the serum was separated after centrifugation at 4°C for 15 min at 2799 g (3500 rpm) in a GS-6R Beckman centrifuge and then analysed at 37°C in an automatic Roche/Hitachi 917/MOD P spectrophotometer. CK measurement in both laboratories was performed according to IFCC recommendations for the measurement of CK (Horder et al. 1991) and the German Society of Clinical Chemistry (DGKC) using the reagent CK-Liquide, Roche.

Analysis of the results

In Phases I and II, the statistical analysis of results was based on the Student's t-test to assess the influence of anaesthesia and site of sampling on the CK level. In Phases III and IV, the results were obtained by repeated measure ANOVA. Statistical significance was established at α = 0.05. The analysis was performed using the SYSTAT version 9.0 statistical program.

Results

Results are summarized in Table 2.

Table 2

Creatine kinase (CK) activity results in rats in the several phases

Factor studied	N ^*	CK (U/L)^†
Anaesthesia	10	Ether		612 ± 208
Ether vs. isoflurane		Isoflurane		518 ± 248
Blood sampling technique	7	Tail vein		332 ± 132^a
Tail vein vs. retro-orbital	5	Retro-orbital		518 ± 248^a
Accustom and temperature of blood sample	10	Accustomed	140 ± 90^b	RT^‡>	129 ± 43^c
				4°C^§	97 ± 18^c
		Not accustomed	301 ± 276^b	RT	474 ± 304^d
				4°C	184 ± 111^d
Sex and temperature of blood sample	10	Male	144 ± 50^e	RT	189 ± 37^f
				4°C	107 ± 16^f
		Female	96 ± 26^e	RT	110 ± 28^g
				4°C	84 ± 17^g

Values with the same letter (denoted as a-g) are significantly different

N = number of rats

†

CK (U/L): Results are expressed as mean ± SD

‡

RT: Blood samples maintained at room temperature prior to centrifugation

4°C: Blood samples maintained refrigerated until centrifugation

Ether did not significantly increased CK values. A high variability in CK activity (coefficient of variation [CV] = 34% with ether and 48% when using isoflurane) was observed. For ethical and safety reasons isoflurane was used in the rest of the experiments.

Average CK activity was significantly higher when sampling from the retro-orbital sinus than when using the tail vein as the site of puncture, although high variability was still observed (CV = 48 and 40%, respectively).

We assessed the effect of refrigeration of the blood sample prior to centrifugation and the effect of habituating the rats to laboratory procedures over a period of one week. The mean CK value and variability were lower in rats accustomed to laboratory handing procedures. Moreover, refrigeration diminished CK mean value and variability in sera from both groups, although it was more pronounced in rats habituated to laboratory handling procedures than in those not habituated (CV = 19 and 33%, respectively).

Finally, the CK in samples from 10-week-old male and female Wistar-Han rats after being accustomed to handling procedures was determined. Mean CK activity was found to be lower in the male rats. There was also a significant difference between refrigerated and non-refrigerated blood samples for both sexes, with a greater reduction after refrigeration being observed in the male rats.

Thus, mean CK value of 10-week-old male and female Wistar-Han rats after being accustomed to handling procedures for two weeks, anaesthetized with isoflurane, sampled from tail vein and blood refrigerated prior to centrifugation and serum separation was shown to be 107 ± 16 and 84 ± 17 with a CV of 15 and 21%, respectively.

Discussion

The aim of this study has been to analyse a number of factors affecting serum CK activity in order to minimize variability and improve the reliability of the measurement of CK activity in neuromuscular research using rats. Some aspects affecting this determination might be:

Blood sampling site

Numerous blood collecting methods have been described for the rats that have been shown to have an impact on haematological and biochemical parameters (Dameron et al. 1992, Matsuzawa et al. 1994, Gummert et al. 1999, Mahl et al. 2000). Periorbital puncture may cause adverse effects including retro-orbital haemorrhage, corneal ulceration, damage to the optic nerve, fracture of the osseous orbit puncture of the vitreous body, necrotic dacryoadenitis of the Harderian gland and retinal degeneration (McGee & Maronpot 1979, Krinke et al. 1988, van Herk et al. 1992, Diehl et al. 2001). In spite of this, retro-orbital puncture is still widely used. This may be due to the lack of either scientific proof of negative consequences when performed by trained staff or the lack of equally good alternatives.

We found significantly higher values of CK activity in samples from the retro-orbital plexus than those from the tail vein that might suggest a more severe degree of tissue damage and a higher degree of haemolysis in such samples. Haemolysis in test specimens affects not only haematological parameters but also blood coagulation and various biochemical parameters such as CK (Hall 1991). Increased values and variability of CK in comparison with other sampling sites were also reported by other authors (Friedel et al. 1975, Horton et al. 1986, Matsuzawa et al. 1993). Moreover, according to endocrinology data higher stress is associated to the retrobulbar sampling procedure when it is compared with other sampling sites such as the sublingual vein (Mahl et al. 2000). Thus, puncture of the tail vein may represent a reasonable alternative to retro-orbital sampling for neuromuscular research purposes.

Habituation to laboratory handling procedures

Stress has been reported to be the factor that has the greatest effect on CK activity. Plasma CK activity in native rabbits increased by up to 10-fold if sampled every 2 h to 12 h but showed no alteration when the rabbits had been accustomed to laboratory handling procedures for two weeks (Lefebvre et al. 1992). The same phenomenon has been reported in pigs (Addis et al. 1974) but not in dogs or horses (Aktas et al. 1993, Volfinger et al. 1994). The physiological origin of this variation in CK activity appears to be a consequence of the emotional stress caused by handling, possibly associated with stress due to the vein punctures (Bacou & Bressot 1976). We have observed that there is a statistically significant reduction of CK activity in rats accustomed to handling procedures for one week, as previously reported in other animal species (Lefebvre et al. 1992).

Storage conditions

Blood sample storage conditions until centrifugation was another determining issue in CK activity observed in this experiment. We found significant increases in CK activity in non-refrigerated blood samples that might be attributed to contamination by CK released from platelets. In 1975, Shibata and Kobayashi demonstrated that substantial amounts of CK leaked out of rat platelets and suggested that CK might contribute to some reactions which consume ATP during aggregation, presumably donating the energy for shape change (Shibata & Kobayashi 1978, Shibata 1978). Importance of sample preparation after blood sampling in regards to platelets interference has also been reported by others (Matsuzawa et al. 1993) for lactate dehydrogenase (LDH), RBC, glutamate oxalate transaminase (GOT) and K measurement as well. The elevation of LDH and GOT and CK activities in whole blood was attributed to the liberation of these enzymes from platelets during the clotting process (Matsuzawa et al. 1993).

We suggest that CK might be liberated from rat blood platelets, and that this increases with the time allowed for clotting before centrifugation, as also occurs with LDH, malate dehydrogenase and GOT (Holmsen et al. 1969, Friedel & Mattenheimer 1970, Aktas et al. 1994). This increase might not be observed in other species such as dogs, as previously reported (Aktas et al. 1994). It could also be postulated that refrigeration may cause a reduction in rat platelet metabolism, possibly by reducing creatine phosphate (CP), as observed in 1975 by Shibata and Kobayashi when platelet rich plasma (PRP) was stored at 4°C for 45 min and the CP content in the rat platelets fell to 90% of the initial value (Shibata & Kobayashi 1978). Although it has not been confirmed, the elevated CK from platelets might be of the brain-type (CK-BB), as Meltzer and Guschwan (1972) only found this isoenzyme in rat platelets.

Some authors recommend the use of plasma in studies using laboratory animals, particularly rodents, in view of the marked release of intracellular substances from blood corpuscles, platelets in particular, during blood coagulation (Stevens & Gallo 1982, Matsuzawa et al. 1993).

Gender

Significantly lower CK activity was found in female than in male rats, as previously reported in Sprague-Dawley rats by some authors (Kaspar & Norris 1977, Loeb & Quimby 1999) and in contrast with published results of others (Matsuzawa et al. 1993). Sex differences in rats have been previously reported for other parameters such us haemoglobin concentration (Hb), haematocrit (Ht), white blood cell count (WBC), glucose, cholesterol, triglycerides and total protein (Matsuzawa T et al. 1993).

In the light of our findings, CK value and variability in rats may be minimized by choosing the tail vein as the site of bleeding, and getting rats accustomed to laboratory handling procedures and maintaining whole blood refrigerated until centrifugation and serum separation.

Footnotes

Acknowledgements

We would particularly like to thank Marian Ostáriz, from the Experimental Unit, for her continuous dedication to this research study and her technical assistance. We are also grateful to Arantxa Begiristain, from the Biochemistry Department, for the rapid analyses of the CK. We would also like to thank Dr Otaegui and Dr Ferre from the Center for Animal Biotechnology and Gene Therapy in Barcelona, and Dr Martín from the ‘Minimally Invasive Surgery Centre’ (CMI) in Cáceres, for their initial guidance. Finally, we are also grateful to all the members of the Experimental Unit for their constant support and collaboration.

References

Addis

, Nelson

, Ma

, Burroughs

(1974) Blood enzymes in relation to porcine muscle properties. Journal of Animal Sciences 38, 279–86.

Aktas

, Auguste

, Concordet

(1994) Creatine kinase in dog plasma: preanalytical factors of variation, reference values and diagnostic significance. Research in Veterinary Science 56, 30–6.

Aktas

, Auguste

, Lefebvre

, Toutain

, Braun

(1993) Creatine kinase in the dog: a review. Veterinary Research Communications 17, 353–69.

Bacou

, Bressot

(1976) Increased plasma creatine kinase activity in rabbits: effect of systematically repeated blood sampling. Experientia 32, 487–9.

Cardinet

(1989) Skeletal Muscle Function. Clinical Biochemistry of Domestic animals. 4th edn. New York: Academic Press, 462–95

Clark

, Youngman

, Palmer

, Parish

, Peto

, Collins

(2003) Stability of plasma analytes after delayed separation of whole blood: implications for epidemiological studies. International Journal of Epidemiology 32, 125–30.

Dameron

, Weingand

, Duderstadt

JM.

(1992) Effect of bleeding site on clinical laboratory testing of rats: orbital venous plexus versus posterior vena cava. Laboratory Animal Science 42, 299–301.

Diehl

, Hull

, Morton

(2001) A good practice guide to the administration of substances and removal of blood, including routes and volumes. Journal of Applied Toxicology 21, 15–23.

Fayolle

, Lefebvre

, Braun

(1992) Effects of incorrect venepuncture on plasma creatine-kinase activity in dog and horse. British Veterinary Journal 148, 161–2.

10.

Friedel

, Mattenheimer

(1970) Release of metabolic enzymes from platelets during blood clotting of man, dog, rabbit and rat. Clinica Chimica Acta 30, 37–46.

11.

Friedel

, Trautschold

, Gaertner

, Hellefeldmann

, Gaudssuhn

(1975) EinfluB verschiedener Methoden zur Blutgewinnung auf Enzym Akitivitaeten im Serum Kleiner Laboratoriumstiere. Zeitschrift für Klinische Chemie und Klinische Biochemie 13, 499–505.

12.

Gummert

, Otto

, Barten

, Morris

(1999) Effect of anaesthesia on whole blood lymphocyte proliferation assay in the rat. Immunopharmacology and Immunotoxicology 21, 267–76.

13.

Hall

(1991) Clinical pathology of laboratory animals. In: Animal Models in Toxicology ( Gad

, Chengelis

, eds). New York: Marcel Dekker, 765–811

14.

Holmsen

, Day

, Pimentel

(1969) Adenine nucleotide metabolism of blood platelets. V. Subcellular localization of some related enzymes. Biochimica et Biophysica Acta 186, 254–66.

15.

Horder

, Elser

, Gerhardt

(1991) Approved recommendation on IFCC methods for the measurement of catalytic concentration of enzymes. Part 7. IFCC method for creatine kinase. European Journal of Clinical Chemistry and Clinical Biochemistry: Journal of the Forum of European Clinical Chemistry Societies. 29, 435–56.

16.

Horton

, Olson

, Hobson

(1986) Femoral venipuncture for collection of multiple blood samples in the nonanesthetized rat. American Journal of Veterinary Research 47, 1781–2.

17.

Hsu

, Chen

, Lee

, Lin

, Kao

, Tsao

(1995) Mitochondrial alterations of skeletal muscle in a heat stress rat model. Proceedings of the National Science Council, Republic of China. Part B, Life Sciences 19, 233–9.

18.

Kaspar

, Norris

(1977) Serum chemistry values of normal dogs (beagles): associations with age, sex, and family line. Laboratory Animal Science 27, 980–5

19.

Klein

, Shell

, Sobel

(1973) Serum creatine phosphokinase (CPK) isoenzymes after intramuscular injections, surgery, and myocardial infarction. Experimental and clinical studies. Cardiovascular Research 7, 412–18.

20.

Krinke

, Kobel

, Krinke

(1998) Does the repeated orbital sinus puncture alter the occurrence of changes with age in the retina, the lens, or the Harderian gland of laboratory rats? Zeitschrift für Versuchstierkunde 31, 111–19.

21.

Lefebvre

, Jaeg

, Rico

, Toutain

, Braun

(1992) Variations of plasma creatine kinase in rabbits following repetitive blood sampling effects of pre-treatment with acepromazine, carazolol and dantrolene. European Journal of Clinical Chemistry and Clinical Biochemistry: Journal of the Forum of European Clinical Chemistry Societies 30, 425–8.

22.

Lefebvre

, Laroute

, Braun

, Lassourd

, Toutain

(1996) Non-invasive and quantitative evaluation of post-injection muscle damage by pharmacokinetic analysis of creatine kinase release (Review). Veterinary Research 27, 343–61.

23.

Lev

, Hendler

, Siebner

, Tashma

, Wiener

, Tur-Kaspa

(1994) Creatine kinase activity decrease with short-term freezing. Enzyme Protein 48, 238–42.

24.

Lewis

, Rhodes

(1978) Effects of IM injections on serum creatine phosphokinase (CPK) values in dogs. Veterinary Clinical Pathology 7, 11–13.

25.

Loeb

, Quimby

(1999) Clinical Chemistry of Laboratory Animals. 2nd edn. Philadelphia: Taylor & Francis

26.

Mahl

, Heining

, Ulrich

(2000) Comparison of clinical pathology parameters with two different blood sampling techniques in rats: retrobulbar plexus versus sublingual vein. Laboratory Animals 34, 351–61.

27.

Maroko

, Vatner

(1977) Altered relationship between phosphokinase and infarct size with reperfusion in conscious dogs. Journal of Molecular Medicine 2, 309–15.

28.

Matsuda

, Kimura

, Itokawa

(1998) Influence of selenium deficiency on vital functions in rats. Biological Trace Element Research 61, 287–301.

29.

Matsuzawa

, Nomura

, Unno

(1993) Clinical pathology reference ranges of laboratory animals. Working Group II, Nonclinical Safety Evaluation Subcommittee of the Japan Pharmaceutical Manufacturers Association. Journal of Veterinary Medical Science 55, 351–62.

30.

Matsuzawa

, Tabata

, Sakazume

, Yoshida

, Nakamura

(1994) A comparison of the effect of bleeding site on haematological and plasma chemistry values of F344 rats: the inferior vena cava, abdominal aorta and orbital venous plexus. Comparative Haematology International 4, 207–11.

31.

McGee

, Maronpot

(1979) Harderian gland dacryoadenitis in rats resulting from orbital bleeding. Laboratory Animal Science 29, 639–41.

32.

Meltzer

, Guschwan

(1972) Type I (brain type) creatine phosphokinase (CPK) activity in rat platelets. Life Sciences 11, 121

33.

Morton

, Abbot

, Barclay

(1993) Removal of blood from laboratory mammals and birds. Laboratory Animals 27, 1–22.

34.

Nosaka

(1996) Changes in serum enzyme activities after injection of bupivacaine into rat tibialis anterior. Journal of Applied Physiology 81, 876–84.

35.

Reijneveld

, Koot

, Bredman

, Joles

, Bär

(1996) Differential effects of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors on the development of myopathy in young rats. Pediatric Research 39, 1028–35.

36.

Shibata

, Kobayashi

(1978) Blood platelets as a possible source of creatine kinase in rat plasma and serum. Thrombosis and Haemostasis 39, 701–6.

37.

Shibata

(1978) Creatine phosphate in rat blood platelets. Thrombosis and Haemostasis, 39, 707–11

38.

Sneddon

, Minnaar

, Grosskopf

, Groeneveld

(1989) Physiological and blood biochemical responses to submaximal treadmill exercise in Canaan dogs before, during and after training. Journal of the South African Veterinary Association 60, 87–91.

39.

Stevens

, Gallo

(1982) Practical considerations in the conduct of chronic toxicity studies. In: Principles and Methods of Toxicology ( Hayes

, ed). New York: Raven Press, 53–77

40.

van Herck

, Baumans

, Stafleu

, Beynen

(1992) A questionnaire-based inventory of the orbital puncture method in The Netherlands. Scandinavian Journal of Laboratory Animal Science 19, 189–96.

41.

van Herck

, Baumans

, van Lith

, Beynen

(1999) Is orbital puncture justificable for the collection of non-terminal blood samples from rats? A literature review and making a stand. In: Orbital Puncture: A Non-Terminal Blood Sampling Technique in Rats ( van Herck

, ed). PhD Thesis. The Netherlands: Utrecht University, 117–52

42.

Visser

, Krill

, Muijtjens

, Willems

, Hermens

(1981) Distribution of enzymes in dog heart and liver; significance for assessment of tissue damage from data on plasma enzyme activities. Clinical Chemistry 27, 1845–50.

43.

Volfinger

, Lassourd

, Michaux

, Braun

, Toutain

(1994) Kinetic evaluation of muscle damage during exercise by calculation of amount of creatine kinase released. American Journal of Physiology 266, R434–41

44.

Yerroum

, Braconnier

, Chariot

(1999) Influence of handling procedures on rat plasma creatine kinase activity. Muscle and Nerve 22, 1119–21.

Minimizing creatine kinase variability in rats for neuromuscular research purposes

Abstract

Summary

Keywords

Materials and Methods

Design and phases of trial

Animals

Handling and anaesthesia

Blood sampling procedure

Processing of the blood samples

Analysis of the results

Results

Discussion

Blood sampling site

Habituation to laboratory handling procedures

Storage conditions

Gender

Footnotes

Acknowledgements

References