Comparison of clinical pathology parameters with two different blood sampling techniques in rats:...

Comparison of clinical pathology parameterswith two different blood sampling techniques inrats: retrobulbar plexus versus sublingual vein

Andreas Mahl, Peter Heining, Peter Ulrich, Josef Jakubowski,Maria Bobadilla, Walter Zeller, Reinhard Bergmann, Thomas Singer &lothar MeisterNovartis Pharma AG, Preclinical Safety: Toxicology/Pathology, CH-4002 Basel. Switzerland

SummaryBlood samples were taken from the retrobulbar venous plexus or the sublingual vein of maleHanIbm:Wist rats to compare clinical pathology parameters between the two samplingtechniques. By analogy with a pharmacokinetic study, blood was sampled six times duringone day from unfasted animals. After 3 weeks of recovery, blood was taken from fastedanimals on a single occasion. In addition, prolactin and corticosterone levels were determinedto compare stress-related effects between the two sampling methods. Body weightdevelopment and food consumption were similar after single as well as after repeated bloodsampling for the two blood sampling techniques. Haemotological evaluation showed agradual decrease in erythrocyte count, haemoglobin concentration and haematocrit afterrepeated blood sampling. Repeated withdrawal of blood samples over 24 h corresponding toapproximately 22% of the total blood volume resulted in a decrease in red blood cellparameters by up to 30%. The withdrawal of approximately 10% of the total blood volumewas associated with a decrease in these parameters by up to 10% and should not be exceededfor animal welfare reasons and to allow a reliable evaluation of data in a study. Repeated bloodsampling was associated with an initial decrease in the number of white blood cells, mainlydue to a reduction in lymphocytes; white blood cell counts were slightly increased one dayafter. The decrease in lymphocytes and the increase in neutrophils after repeated samplingwere generally slightly more pronounced in the blood from the retrobulbar plexus than fromthe sublingual vein. Comparison of serum clinical chemistry data showed significantly higheractivities of creatine kinase and aspartate aminotransferase in samples from the retrobulbarplexus. These findings suggest a higher degree of tissue damage with blood sampling from theretrobulbar plexus than from the sublingual vein. Despite a large inter-individual variability,higher mean values of prolactin on each occasion and corticosterone after a single sample infasted animals indicate a higher stress associated with blood sampling from the retrobulbarplexus.

Keywords Single and multiple blood collections; rat; retrobulbar plexus versus sublingualvein; clinical pathology; distress

Blood samples from rats taken for determina-tion of clinical pathology parameters orexposure levels of the test article in toxicitystudies can be obtained by different methodsCorrespondence to: A. Mabl

such as bleeding from the retrobulbar venousplexus (also described as retroorbital plexusor periorbital sinusl, puncture of the sub-lingual, saphenous or tail vein and tail tipamputation. Blood sampling from the retro-

Accepted 31 May 2000 © Laboratory Animals Ltd. Laboratory Animals (2000) 34, 351-361

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352

bulbar venous plexus currently representsthe most widely used sampling method. TheJoint Working Group of Refinement(BVA/FRAME/RSPCA/UFAW 1993) does notrecommend this blood sampling techniquebecause there is a risk of inflicting both pri-mary and secondary tissue damage (Krinke etal. 1988, Van Herck et al. 1992, 1998, Le Netet al. 1994). In Switzerland, blood samplingfrom the retrobulbar venous plexus has beenrestricted by setting a minimal interval of 14days between two samplings from the samesite (Bundesamt fur Veterinarwesen 1995).In view of the need to investigate possibleadverse effects or monitor exposure levels inanimals by repeated blood sampling in toxi-city studies, bleeding from the sublingualvein was evaluated in a comparative studywith Wistar rats as an alternative to samplingfrom the retrobulbar venous plexus. In addi-tion, the effects of repeated blood samplingon clinical pathology parameters wereinvestigated in order to recommend themaximum blood amounts to be taken forpharmacokinetic evaluations. For conformitywith the Swiss animal welfare regulations,the study design was discussed with theanimal welfare officer of the company andapproved by the Cantonal Veterinary Office(Kantonales Veterinaramt).

Materials and methods

AnimalsA total of 30 male SPF HanIbm:WIST rats(breeder: Biological Research LaboratoriesLtd, Fullinsdorf, Switzerland) was used. Therats were 8 weeks old and their body weightsranged from 229-298 g at the start of thestudy (day 1l.

Animal husbandryThe animals were housed in pairs in type IIIMacrolon cages with metal grid flooringunder optimal hygienic conditions consistingof conventional husbandry with specifichygienic requirements in a controlled envir-onment. The room temperature was 22-23°Cand relative humidity 43-52 'Yo. Fluorescentlight provided a 12h light (06:30-18:30h) and12h dark cycle. The animals had free access

Laboratory Animals (2000) 34

Mahl et al.

to commercial rodent diet (KLIBA32-343-4rat/mouse maintenance diet from Klingen-talmuhle Ltd, Kaiseraugst, Switzerland;Batch No. 67/95), except for a fasting periodof approximately 24h before blood samplingon day 23. Tap water from the local watersupply was available from polyethylenewater bottles ad libitum. Before first bloodsampling, the animals were acclimatizedduring one week. Cleaning of the animalroom and cages was performed once weekly.Cage cleaning was done in the weeks of bloodcollection one day before sampling, roomcleaning after termination of blood samplingduring week 1 and one day before bloodsampling on day 23.

Clinical observations, body weights andfood consumptionThe animals were observed daily for abnor-mal behaviour. Special emphasis was placedon the observation during blood sampling forpossible injuries. Body weights of the ani-mals were determined before study start, ondays I, 2, 3, 5, 8, 23 and 24 (on sampling daysbefore blood collection), food consumption ofthe animal pairs for days -I, I, 2, 3-4, 5-7and 23 using a Mettler balance. All ratswere killed on day 24 with exposure tocarbon dioxide at an increasing concentra-tion.

Blood sampling method and samplehandlingSampling times were chosen by analogy witha pharmacokinetic study. Sampling startedon day 1 at 07:45 h. Blood samples were takenfrom the retrobulbar venous plexus or thesublingual vein after anaesthesia followinginhalation exposure with isofluraneIForene®, Abbot Laboratories S.A., Cham,Switzerland) in an inhalation chamberaccording to the schedule specified (Table 1).Puncture of the retrobulbar venous plexus ofthe right eye at the medial canthus was per-formed by skilled animal technicians usingglass micro-haematocrit tubes. Blood fromthe sublingual vein was obtained by puncturewith a sterile needle (Becton DickinsonMicrolance; 0.7 mm x 30 mm) according tothe method refined by Zeller et al. (19981.



Blood sampling: retrobulbar plexus vs sublingual vein

Table 1 Allocation of animals, blood sampling schedule and approximate sample volumes

353

Sampling from retrobulbar plexus Rats 01-10Sampling from sublingual vein Rats 16-25

Sample Day Time (vs TO) Haematology

1 0(07:45 h) 0.4ml2 1h 0.5ml3 2h 0.5ml4 4h 0.5ml5 7h 0.4ml6 2 24h O.4ml7 3 48h

Rats 1-30

8 23

•Evaluation of selected parameters

Endocrinology

O.4ml

O.4ml0.4ml0.4ml

0.4ml

Total volume

0.8ml0.5ml0.5mlO.5ml0.8ml0.8m'

Rats 11-15Rats 26-30

Clinical chemistry

0.8ml"O.5ml"O.5ml"O.5ml"0.8ml"0.8m'"

1.5ml

Where necessary, bleeding from the tonguewas stopped with a cotton tip, but withoutiron chloride impregnation as proposed byZeller et al. Blood sampling from the sub-lingual vein required slightly more time thanfrom the retrobulbar plexus by a factor of 1.2to 1.6 for most sampling time points with theexception of a few blood collections of thesame duration for both sampling techniques.Blood samples for haematology were takeninto 1.5ml Eppendorf tubes (Milian Instru-ments S.A., Plan-les-OutesjGeneva, Swit-zerland) impregnated with EDTA as ananticoagulant. The samples were stored atroom temperature and evaluated on the sameday the blood was sampled. For clinicalchemistry, 1.5ml Eppendorf tubes withoutan anticoagulant were used. The blood sam-ples were kept at room temperature (at least0.5 h) until they were centrifuged (1500 g for10min) for serum separation and then storedat approximately - 70°C until evaluated. Forendocrinology, blood samples were with-drawn into 1.5ml Eppendorf tubes with theaddition of sodium heparin (Liquemin®5000 i.u./ml; Roche pharma Ltd, Reinach,Switzerland) as an anticoagulant. Theblood samples were stored in ice water (atapproximately 4°C) during the samplingperiod and centrifuged (approximately 2000gfor 8min) at this temperature for separationof plasma which was then stored atapproximately -70°C until evaluated.

Haematology parameters: Erythrocyte count,haematocrit, haemoglobin, mean corpuscularhaemoglobin concentration, mean corpus-cular volume, mean corpuscular haemoglo-bin, percentage of reticulocytes, totalleucocyte count, differential leucocyte count,platelet count.

Red blood cell parameters and differentialleucocyte count were recorded using aTechnicon H*1analyser. Reticulocyte countswere determined with a FACScan. Slidepreparation was by the Uni-Smear SpinnerModel Coleman 90 (Perkin Elmer). In cases ofcoagula in blood samples, increased numbersof large unstained cells or of an abnormal celldistribution were detected by the TechniconH*1 analyser, the automated analysis wascomplemented by microscopic evaluation ofthe respective blood smear.

Clinical chemistry parameters: Glucose*,total protein*, albumin*, urea, cholesterol,triglycerides, total bilirubin, creatinine,creatine kinase*, aspartateaminotransferase *, alanine aminotransferase,alkaline phosphatase, a-amylase*, lipase*,sodium, potassium, calcium, magnesium,chloride, phosphorus, protein electro-phoresis*.

Parameters marked with * were deter-mined on days 1 and 2. All listed parameterswere determined on day 23. The data wererecorded using a Synchron CX5 analyser.




354 Mahl etal.

Endocrinology parameters: Corticosteroneand prolactin.

Prolactin levels were determined using arat-specific radioimmunoassay kit (Amer-sham). Corticosterone levels were deter-mined using a radioimmunoassay kit (IDS)intended for the measurement of humancorticosterone and validated in our labora-tory for the measurement of rat cortico-sterone in plasma samples.

Measurements were performed in agamma-counter COBRA 5005 (CanberraPackard). All samples were measured induplicate. Detection limits, intra-assay co-efficient of variation and inter-assay preci-sion profiles were determined in each assay.

Statistical analysisStatistical evaluation of the measured valueswas performed by repeated measures analysisto take into account the time course of theexperimental data. Univariate and multi-variate analyses were calculated.

retrobulbar plexus bleeding and -29.2%after sublingual vein bleeding). Bodyweightgain within the normal range was observedfrom days 3 to 8. The higher food intakeand body weight gain from days 23 to 24was a consequence of fasting before bloodsampling.

HaematologyThe rats were sampled for haematology fivetimes on day 1 and once on day 2. Conse-quently, as the time progressed, values for redblood cell parameters such as haematocrit,haemoglobin concentration and erythrocytecount (Figs 1-3) gradually decreased. Meanvalues of these parameters were lower on day1 by 4 or 5% for the 2nd sample, 7-9 or 9-10% for the 3rd sample, 10-11 or 14-15% forthe 4th sample, 4-12 or 12-19% for the 5thsample and on day 2 by 23 or 28-30% com-pared with the 1st sample from the retro-bulbar plexus and sublingual vein,

-.- Retrobulbar plexus-0- Sublingual vein

__ - Retrobulbar plexus

-0- Sublingual vein

16

ID

~ 12

::JEg 14

50

45

~I- 40U:c

35

3:T~~~~~~~-,-~~,~-~, -~,12 16 20 24

time (hours)

Fig 1 Haematocrit after single and repeated bloodsampling. Values are means ± SEM

1:r~__ ~~~,__ ~,__ ~,_~,__ ~,e 12 16 20 24

time (hours)

Fig 2 Haemoglobin concentration after single andrepeated blood sampling. Values are means±SEM

Results

Clinical observations, body weight andfood consumptionThere were no problems in taking therequired amount of blood, with the followingexceptions: coagulation prevented samplingof the full amount of blood from the retro-bulbar plexus in two animals on day 2. Fromthe sublingual vein, smaller volumes ofblood were obtained from two animals at the4th bleeding on day 1. Due to constrictedsublingual veins, no blood sampling wasperformed in two animals on day 3. Oneanimal died during the 4th anaesthesia onday 1. Small haematomas were evident onthe tongue at the site of venepuncture in 5animals on day 23.

Bodyweight development of the animalswas comparable following either samplingmethod and food intakes were almost iden-tical (data not presented). Following repeatedblood sampling, animals lost body weightwithin one day (-2.0% of initial body weightafter retrobulbar plexus bleeding and -2.4 %after sublingual vein bleeding) and consumedless food than on the day before (-26.4% after




Blood sampling: retrobulbar plexus vs sublingual vein 355

Fig 3 Erythrocyte count after single and repeatedblood sampling. Values are means± SEM

,24

,20

,16

,12

-..- Retrobulbar plexus

-0- Sublingual vein

time (hours)

Fig 5 White blood cell counts after single andrepeated blood sampling. Values are means ± SEM

,24

,20

,16

-.- Retrobulbar plexus

-0- Sublingualvein

,12

time (hours)


-0- Sublingual vain

::::;- 6000

~Qj 5000~'"'" 4000>.00

.s::;Cl. 3000E~

200:Ttime (hours)

Fig 6 Lymphocyte count after single and repeatedblood sampling. Values are means±SEM

-.- Retrobulbar plexus-0-- Sublingual vein

~ 7Q;u

~ 6o:c

"0

E'0~ 4~~g 3

~

~:~T---------,--,--,1'2 16 20 24

time (hours}

Fig 4 Reticulocytes (percentage of red blood cells)after single and repeated blood sampling. Values aremeans±SEM

-.- Relrobulbar plexus

-0- Sublingual vein

~ 85'"u

"0800

.2

.0

~ 75.s::;~~ 700

~'" 65

'">.0 6:T0

.s::;Cl.

E~+-~~~-~~,-~~,--~, --~, ~-~,

8 12 16 20 24

time (hours}

Fig 7 Lymphocytes (percentage of white blood cells)after single and repeated blood sampling. Values aremeans±SEM

increases in mean number and percentage ofneutrophils (Figs 8 and 9) on day 1 weregenerally slightly more pronounced in theblood from the retrobulbar plexus. This trendwas reversed from the 5th to the 6th sampleon day 2.

respectively. Increase in reticulocytes (Fig 4)became apparent by day 2 and was similarwith both sampling techniques. The uni-variate repeated measures analysis yielded nostatistically significant differences betweensamples from the sublingual vein and theretrobulbar plexus for the different haema-tological parameters. A multivariate analysisincluding all of the measured parametersat once would require a substantial largersample size.

White blood cell counts [Fig 5) showed aninitial decrease for the 2nd, 3rd and 5thsample from the retrobulbar plexus and forthe 2nd and 3rd sample from the sublingualvein. This was mainly due to a reductionof lymphocytes (Figs 6 and n For the 6thsample (day 2), mean white blood cell countswere slightly higher than those in the pre-vious samples. The decreases in mean num-ber and percentage of lymphocytes and the

Laboratory Animals (2000} 34



356 Mahl et al.

Fig 8 Neutrophil count after single and repeatedblood sampling. Values are means± SEM

~---~-a- Retrobulbarplexus-0- Sublingualvein

UlQ)

"&ocE 0

'£4i" 6

"0oo:0Q)

.'" 4.c3:'0~

~--~,--~,--~, -o 12 16 20 24

time (hours)

Fig 11 Monocytes (percentage of white blood cells)after single and repeated blood sampling. Values aremeans±SEM

24208 12 16

time (hour~)

2500

500

:J-2- 2000!!!.4i";;; 1500:ECoe 1000:;Q)c

.•....•- Retrobulbar plexus~ SubUngual vein

)~:

12 16 20 24 day 23fasted anif118/s

time (hours)

groups could be noticed for the total anddifferential white blood cell counts.

Clinical chemistrySignificantly higher mean values for creatinekinase activity (Fig 12)were evident in thesamples collected from the retrobulbarplexus compared with samples from thetongue vein (F = 0.0004). The univariaterepeated measure analysis yielded sig-nificantly higher mean values of aspartateaminotransferase activity (Fig 13) in samplesfrom the retrobulbar plexus (F = 0.032).Because of the significant interaction effectbetween the two blood sampling methodsand the time course, the two curves foraspartate aminotransferase activity could notbe considered to be parallel. Therefore, asingle t-test for each time point was appliedand corrected by Bonferronis adjustment:

Fig 12 Creatine kinase activity after single andrepeated blood sampling. Statistically significantlyhigher mean values with sampling from the retro-bulbar plexus (P= 0.0004). Values are means± SEM

3000

24

? 2000

'"01000

,24

20

,20

1612


-0- Sublingual vein

o

400

<=-.2- 300____ Retrobulbar plexus

!!!. -0- Sublingual vein4i~UlQ) 200>."0c0E 100

'£354i

""0 3000:0.~

25.c3: 20'0~ 15

~.c 10Coe:; 5Q)c

o· I I

8 12lime (hours)

Fig 9 Neutrophils (percentage of white blood cells)after single and repeated blood sampling. Values aremeans±SEM

The absolute number and percentage ofmonocytes (Figs 10 and 11)were marginallyhigher after sampling from the retrobulbarplexus than from the tongue vein for mosttime points on day 1. However, no statisti-cally significant differences between the two

time (hours)

Fig 10 Monocyte count after single and repeatedblood sampling. Values are means±SEM




Blood sampling: retrobulbar plexus vs sublingual vein 357

500

250_ Retrobulbar plexus

.-1200 -0- Sublingualvein

:v--150

~ -----1....

100III«

50

2500

i:~,J-0- Sublingual vein

12 16 20time (hours)

24 day 23fasted animals

12 16 20

time (hours)24 day 23

fasted animals

Fig 14 Glucose concentration after single andrepeated blood sampling. Statistically significantlyhigher mean values with sampling from the sublin-gual vein (P=O.002). Values are means±SEM

Fig 13 Aspartate aminotransferase activity aftersingle and repeated blood sampling. Statisticallysignificantly higher mean value with sampling fromthe retrobulbar plexus in 6th sample at 24 h(P=O.OOl). Values are means±SEM

day 23fasted animals

/4836

time (hours)

2412

125

J)____ Retrobulbar plexus

100 -o-Sublingual vein

75 V--I50

Fig 16 Prolactin concentration after single andrepeated blood sampling. Statistically significantlyhigher mean values with sampling from the retro-bulbar plexus (P=O.OOl). Values are means±SEM

EndocrinologyThe Bonferronis procedure was used to makean adjustment for multiple comparison ofcorticosterone and prolactin values. The twoP values for testing equality of the two bloodsampling methods had to be multiplied bytwo, resulting in P = 0.5 for corticosteroneand P = 0.001 for prolactin. Consequently,prolactin levels (Fig 16) were statisticallysignificantly higher for samples from theretrobulbar plexus than from the tongue vein.For corticosterone (Fig 17), there was a ten-

Fig 15 Amylase activity after single and repeatedblood sampling. Values are means±SEM

comparable to that of the 1st sample. Thiswas similar for both sampling techniques.The results of other clinical chemistry para-meters were similar with both samplingtechniques and did not show major variationsover the time course of the sampling, and aretherefore not presented in this paper.

day 23fasted animals

-11- Retrobulbar plexus

-0- Sublingual vein

12 16 20 24time (hours)

o

11

10

_ 8;::o 7E~ 6"'"oo.:!Ol

The 24h value was significantly higher in thesample from retrobulbar plexus (P=O.OOI),for all other time points no statistically sig-nificant differences existed. Slightly but sig-nificantly higher mean serum glucoseconcentrations (Fig 14) were evident in thesamples from the tongue vein compared withthe samples from the retrobulbar plexus(P =0.002). With both techniques, repeatedblood sampling on day 1 was associated withan increase in glucose concentrations. Themarkedly lower serum glucose values on day23 compared with days 1 and 2 with bothsampling techniques are considered to be aresult of fasting before blood sampling. Themean values of amylase activity (Fig 15)showed a decrease from the 1st to the 2ndsample and an increase with the 5th sample




358 Mahl et al.

Fig 17 Corticosterone concentration after single andrepeated blood sampling. Values are means±SEM

dency to higher values with sampling fromthe retrobulbar plexus on day 23 in fastedanimals, which affected approximately one-third of the animals. This difference was,however, not statistically significant due tothe high variation in the animals with theretrobulbar sampling.

Discussion

Repeated anaesthesia and blood sampling onthe same day resulted in body weight lossand decreased food consumption to a similarextent with both sampling methods. Thesefindings indicate a clear effect of the bloodsampling procedure on the general conditionof the animals, and one which might affectthe outcome of toxicity studies. There wasno relevant impairment of body weightdevelopment or food consumption after sin-gle blood sampling. Haematology data afterrepeated blood sampling showed a gradualdecrease in values for red cell parameterssuch as erythrocyte count, haemoglobinconcentration and haematocrit. Decreases inthese parameters by approximately 5% aftera 2nd sample or 7-10% after a 3rd samplewere considered unlikely to have an impacton toxicity studies. Assuming a total bloodvolume for the rat of between 6.5% of itsbody weight for a 300 g animal and 7.0% for a250g animal (Sato et al. 1985), the bloodvolume of approximately 1.8ml taken duringthe first three occasions corresponds toapproximately 10% of the total bloodvolume. Six blood samples from the sameanimal within 24 h, altogether nearly 4 ml,corresponding to approximately 22% of the

total blood volume, resulted in a decrease inred blood cell parameter values of up to 30%.This reduction may affect a study, especiallyif toxicity is related to red blood cells orthe test article binds to erythrocytes. Theslightly more pronounced decrease in redblood cell parameters in samples taken fromthe sublingual vein, especially after repeatedbleeding, might be related to some blood lossif compression of the sublingual vein aftersampling was not sufficient to stop thebleeding immediately. The similar increasein percentage of reticulocytes in the 6thsample (day 2) with both sampling methodsis considered to be an adaptive response torepeated blood sampling. White blood cellparameters revealed an initial decrease intota11eucocyte counts, mainly due to areduction in lymphocytes. This decrease inmean number and percentage of lymphocytesassociated with an increase in neutrophilswas observed with both sampling methods.Although without statistical significance,this was generally slightly more pronouncedin samples obtained from the retrobulbarplexus. Similar changes in white blood cellsafter repeated blood sampling from the sub-lingual vein have also been reported by Zelleret al. (1998). Dhabhar et al. (1995) showedthat acute stress induces rapid and reversiblechanges in the distribution of peripheralblood leucocyte subpopulations in the rat,consisting of significant decreases innumbers and percentages of lymphocytes andincreases in numbers and percentages ofneutrophils. Besides stress-related effects inthe present study, mechanical lesions due toblood sampling might also have contributedto these acute changes in white blood celldistribution. The markedly higher values ofcreatine kinase activity in samples from theretrobulbar plexus than in those from thesublingual vein suggest a more severe degreeof tissue damage during sampling from theretrobulbar plexus. Increased values of crea-tine kinase activity in samples from theretrobulbar plexus in comparison with othersampling sites were also reported by Hortonet al. (1986) and Matsuzawa et al. (1993,1994). Creatine kinase activity was markedlyhigher in samples collected after fasting onday 23 than without fasting on day I, with

/48 day 23

fasted animo/s12 24 36

time (hours)





both sampling methods. According to Mat-suzawa and Ishikawa (1993),creatine kinaseactivity may show great variations, caused byfactors that operate during animal handlingand feeding, sample collection and prepara-tion, and sample storage before assay.Increased activity of aspartate aminotransfer-ase was evident in samples from the retro-bulbar plexus on day 2 (6th samplej 24h) and,which was not statistically significant, on day23. The aetiology of this finding is unknownjhowever, a relationship with lesions causedby blood sampling cannot be ruled out.

Evaluation of serum glucose concentra-tions revealed a slight increase from the 1stto the 5th sample with both sampling tech-niques, which may be related to stress inconnection with repeated blood sampling.Armaria et al. (1986)showed that acutestress due to changes in the environment ofmale rats was associated with slight increa-ses in serum glucose levels, following asimilar trend as found with corticosterone. Inthe present study, mean serum glucose con-centrations were slightly, but statisticallysignificantly, higher in the samples from thesublingual vein than from the retrobulbarplexus. Comparison of individual glucose,corticosterone and prolactin concentrations,however, did not indicate a correlation.

Other clinical chemistry parametersshowed no significant differences betweenthe two sampling methods. Slight differencesin amylase activity between the two sam-pling techniques showed no consistent trend,and there was a high variation in individualvalues.

For comparison of stress-related effectswith the two sampling techniques, plasmaprolactin and corticosterone levels weredetermined in samples several times fromunfasted animals and on one occasion fromfasted animals. Krulich et al. (1974)reportedthat a variety of hormone levels are respon-sive to stress in the rat, with prolactinconcentrations being the most susceptible.Conybeare et ai. (1988)measured prolactinlevels which appeared to be lower in bloodsamples obtained by tail vein puncturewithout anaesthesia than in blood samplesfrom the retrobulbar plexus under bothhalothane and ether anaesthesia. In view of

359

the effect of additional stress due to anaes-thesia on prolactin levels, as demonstrated byWiersma and Kastelijn (1985),comparisonbetween these data is considered to bedubious. Corticosterone levels have beenfound to be increased in rats as a result ofstress after handling IDobrakovova et al.1993),restraint (Valleeet al. 1996)andchanges in the environment (Armario et al.1986).Haemisch et al. (1999)showed thatcorticosterone levels in blood samples col-lected from the tail vein rose in responseto the 1st sample and returned to baselinevalues in successive samples. In the presentstudy, there was a high inter-individual varia-tion in the endocrinology data. This is notsurprising, as the measured stress parametersprolactin and corticosterone are affected bymany factors such as handling of the animal,length and depth of anaesthesia and the bloodcollection procedure itself, in addition to theindividual condition of the animal,. There-fore, it is impossible to ensure comparableconditions for all animals.

Despite these insufficiencies, mean pro-lactin levels were significantly higher insamples obtained from the retrobulbar plexusthan in those from the sublingual vein andsuggest a higher acute stress associated withblood withdrawal from the retrobulbarplexus. There was no statistically significantdifference in mean corticosterone levels,although increased values were measured insamples from the retrobulbar plexus taken onday 23. In general, mean hormone levels insamples from the retrobulbar plexus of fastedanimals (day 23) were higher than those ofunfasted animals (days 1,2 and 3L whereasthe hormone levels in samples from thesublingual vein remained in the same rangefor fasted and unfasted animals. This findingsuggests that fasting may increase certainforms of stress. This is in accordance withthe observation of Kiss et al. (1994)that fooddeprivation potentiates the increase inplasma corticosterone levels in rats subjectedto immobilization stress.

ConclusionsBlood sampling from the retrobulbar plexusand from the sublingual vein resulted in




360

similar data, with the exception of a smallnumber of parameters. Differences betweenthe two sampling methods in haematologyand clinical chemistry suggest a higher stressassociated with the retrobulbar samplingprocedure, and this is further supported byendocrinology data. Based on these results,the decision was taken to replace bloodsampling from the retrobulbar plexus bybleeding from the sublingual vein in theToxicology Department of the former SandozPharma AG, Basel (now Novartis PharmaAG). Now}after 3 years of experience withblood sampling from the sublingual vein}it isapparent that this procedure is much easierto learn and to conduct. Furthermore, incontrast to blood sampling from the retro-bulbar vein plexus, it ensures a betterevaluation of toxicity with respect to eyeexaminations.

Acknowledgments The authors would like to thankPierre Wolber and Jean-Luis Hemmerlin for thetechnical support during the conduct of this study,and Brian Hunter for the review of this article.

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Comparison of clinical pathology parameters with two different blood sampling techniques in rats:...

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