Time-dependent Pathologic and Inflammatory Consequences of Various Blood Sampling Techniques in Mice

Abstract

We compared 6 frequently used mouse blood-sampling methods (lateral tail incision; tail-tip amputation; sublingual, submandibular, and saphenous vein puncture; and retrobulbar sinus puncture during isoflurane anesthesia) with regard to induction of local and systemic inflammation, stomach contents, weight changes, and corticosterone levels at 6 h to 12 d after sampling. Local inflammation was assessed through histopathology and assessment of the expression of inflammation and tissue damage–related genes (S1008/9A, Cxcl2, Il1b, Nlrp3, Il6, and Il33) in sampled tissue. Systemic inflammation was assessed through quantification of plasma haptoglobin levels, measurement of blood Il1b expression, and evaluation of histopathologic changes in lung, kidney, liver, and spleen. Apart from slight, transient increases in plasma haptoglobin levels after lateral tail incision, retrobulbar sinus puncture, and saphenous vein puncture, no other signs of systemic inflammation were found. Mice subjected to retrobulbar sinus puncture, sublingual puncture, or isoflurane anesthesia only showed the highest plasma corticosterone concentrations. Retrobulbar sinus puncture had the largest effect on body weight loss. Retrobulbar sinus puncture, sublingual puncture, and submandibular puncture only showed minor and in, most cases, fast-resolving inflammation. By contrast, blood sampling by lateral tail incision, tail-tip amputation, or saphenous vein puncture caused tissue damage and inflammation locally at the sampling site, which resolved more slowly compared with head-region sampling techniques, according to results from pathologic and gene expression assessments. Expression of S1008/9A, Cxcl2, Il1b, and Nlrp3 increased 10- to 1000-fold and did not return to baseline until day 6 after sampling or later and did not resolve after tail-tip amputation within the 12-d observation period. Increased expression of genes involved in inflammation and tissue repair correlated with histopathologic changes and may thus represent a quantitative supplement to histopathology. In conclusion, none of the tested methods for obtaining blood samples from mice is superior, according to simultaneous immunologic, histopathologic, and animal welfare–related parameters.

In animal studies, an important part of the study design is to optimize the implementation of 3R principles, which include the replacement of animals with in vitro methods, when possible, reduction in the number of animals within the framework of the study, and refinement of the study so that the animals involved experience the best welfare possible.²⁰ Mice are used worldwide for scientific purposes and are often exposed to basic procedures, including blood sampling. To comply with 3Rs principles, it is vital to use the method having the least negative effect on animal welfare (that is, refinement). To reduce the number of animals needed for a study (that is, reduction), the optimal blood sampling method in relation to for example impact on inflammatory parameters and histopathologic changes must be used. The optimal method for blood sampling in a given study may depend on the purpose of the study. In some studies, an inflammatory response to blood sampling is highly undesirable, whereas in other studies, the extent of tissue damage or rate of recovery may be pivotal. Therefore, the effects of the blood sampling on the pathomorphology and induced local and systemic inflammatory changes—both acute and long-term—may be of importance.

Several studies assessing the influence of blood sampling on the physiology and welfare of the animals have been performed to identify the optimal method, but often such studies only compare 2 methods evaluating only a few parameters assessed shortly after sampling.^{1,3,6,11,12,21,22}

The present study is quite comprehensive in terms of the number of methods included, duration of the observation period, and number of variables measured. We aimed at comparing 6 blood-sampling methods used for mice, namely 1) lateral tail incision, 2) amputation of the tail tip, 3) retrobulbar vein puncture (also often referred to as ‘periorbital vein puncture’), 4) sublingual puncture, 5) submandibular puncture, and 6) saphenous vein puncture. As an earlier study demonstrated that mice healed within 10 d after blood sampling, the present project had a duration of 12 d.⁷ We chose C57BL/6NTac mice for the study as this strain is widely used in research applications such as immunology and metabolic disease including diet-induced obesity as well as for generation of genetically engineered models.

We monitored the development of histologic changes at the local sampling site and in selected internal organs (liver, kidneys, spleen, and lungs) and assessed systemic inflammation by measuring the level of acute-phase protein haptoglobin,^5,18 IL6, and IL1β in plasma. In addition, gene expression of major mediators of locally acute inflammatory events, including neutrophil-attracting chemokines (S100A8, S100A9, and Cxl2), proinflammatory cytokines (Il6, Il1b), and endogenous danger-associated molecular pattern molecules (Gp96, Il1a, Il33, and Hmgb1) were assessed. Animal welfare was evaluated by measuring changes in body weight, stomach contents, and plasma corticosterone levels after blood sampling.

Materials and Methods

Ethical statement.

All procedures were performed in accordance with EU Directive 2010/63/EU and Danish legislation. The study was approved by the Danish national authority, the Animal Experimentation Inspectorate, under the Ministry of Environment and Food of Denmark, and performed under license number 2012-15-2934-00256. All mice were anesthetized with a mixture of fentanyl, fluanisone, and midazolam prior to euthanasia.

Animals.

A total of 228 barrier-bred C57BL/6NTac female mice were procured from Taconic (Lille Skensved, Denmark), which provides health monitoring according to FELASA health monitoring guidelines. The mice were 7 wk old on arrival, were randomly allocated into 6 treatment groups and 3 control groups, and acclimated for 1 wk prior to test start. The mice were housed in groups of 4 in transparent type III cages (425 × 266 × 185 mm, Tecniplast, Buguggiate, Italy) with aspen bedding (Tapvei, Peakna, Estonia). Moreover, the cages were supplied with Enviro-dri nesting material (Shepherd Specialty Papers, Watertown, TN), cotton squares, cardboard tunnels, and DesRes mouse house (LBS Biotechnology, Surrey, UK); cages were cleaned twice each week. The mice were fed a commercial diet (no. 1324, Altromin, Germany) without restriction and provided with tap water. The mice were housed at 21 ± 1 °C with a 12:12-h light:dark cycle (lights on, 0600).

Study design.

Six blood-sampling methods (treatments) were included in the study: lateral tail vein incision, amputation of the tail tip, retrobulbar vein puncture (under isoflurane anesthesia), sublingual vein puncture, submandibular vein puncture, and saphenous vein puncture. Each group (n = 4) underwent blood sampling by using 1 of the 6 test methods and terminal blood sampling just prior to euthanasia. Mice in treatment groups were euthanized at 1 of 9 time points: 6 or 10 h or 1, 2, 4, 6, 8, 10, or 12 d after the initial blood sampling. Prior to the terminal blood sampling and euthanasia, all animals were anesthetized by using a mixture of fentanyl and fluanisone (0.315 and 10 mg/mL, respectively; (Hypnorm, VetaPharma, Leeds, United Kingdom) and midazolam (5 mg/mL; Dormicum, Roche, Basel, Switzerland) diluted in sterile water. The terminal blood sample was taken from the retrobulbar sinus in all mice (contralateral to the eye used for sampling in mice exposed to retrobulbar puncture at test sampling). The anesthetized mice were euthanized by using 20% pentobarbital intraperitoneally. Half of the animals in each group (n = 2) were used for histopathologic evaluation, and the second half (n = 2) were used for real-time quantitative PCR analysis. The mice were weighed prior to test sampling and at euthanasia. All blood collection was done between 0800 and 1000. In addition, 8 mice were anesthetized with isoflurane and euthanized after either 6 or 24 h (n = 4 in each group) and included as control animals to assess the possible effects of isoflurane anesthesia itself. Another 4 mice were euthanized without prior bleeding and acted as overall control animals.

After euthanasia, tissue from the blood sampling site, liver, kidney, spleen, and lungs was collected for histopathology and gene expression analysis.

Blood sampling treatment

Except for the retrobulbar sinus puncture, each of the test blood-sampling methods was performed on unanesthetized mice; mice exposed to retrobulbar sinus puncture at treatment were anesthetized by using 4% isoflurane delivered by mask from a precision vaporizer (Anesthesia Work Station for Rodents, Hallowell EMC, Pittsfield, MA) in accordance with guidelines from the Danish National Authority. All procedures were performed by experienced animal technicians, with each blood-sampling method allocated to a single trained technician. When test sampling was performed, access was confirmed by collecting 50 to 100 μL of blood, but no further analysis of these samples was done. At euthanasia, all animals were terminally bled. All terminal blood samples were collected in heparin-coated 1.5-mL microfuge tubes and placed on ice; 50 µL was immediately transferred to a 1.7-mL cryotube containing 200 µL lysis–binding solution (catalog no. AM 1830, MagnaMAX 96 Total RNA Isolation Kit, ThermoFisher Scientific, Waltham, MA) and stored at –20 °C until RNA extraction. The remaining blood was centrifuged for 10 min at 4000 × g, and plasma was transferred to 1.5-mL microfuge tubes and stored at –80 °C until further analysis.

Lateral tail incision.

The mouse was restrained in a V-trap, and lateral tail incision was performed on one side of the tail, approximately 2 cm from the tail base, by using a scalpel.

Amputation of the tail tip.

The mouse was restrained in a V-trap, and 1 mm of the tail tip was amputated by using a scalpel. The blood sample was collected in a microhematocrit tube.

Retrobulbar sinus puncture.

The mice were anesthetized with 2% to 4% isoflurane delivered in 100% oxygen by using a face mask and then were placed on one side on a table. A microhematocrit tube was inserted in the medial canthus of the orbit and gently pressed and rotated until blood appeared in the tube.

Sublingual puncture.

The mouse was restrained by the scruff of the neck, and one of the sublingual veins was punctured by using a 25-gauge cannula. Exposure of the vein was not attempted.

Submandibular puncture.

The mouse was restrained by the scruff, and the submandibular vein was punctured approximately 3 mm caudal and 1 mm dorsal to the lateral tactile hair of the right cheek by using a 23-gauge cannula.

Saphenous vein puncture.

The mouse was restrained in a modified 50-mL syringe barrel and shaved distal to the knee. As the veins were compressed at the thigh to provide stasis, the lateral saphenous vein was punctured by using a 25-gauge needle.

Assessment of animal welfare related parameters.

All mice were weighed twice: just prior to test sampling and at euthanasia. The change in body weight was calculated for each mouse. At euthanasia at 6, 10, and 24 h after test sampling, the weight of the stomach contents was calculated for all treatment groups by weighing the dissected stomach before and after the stomach contents were flushed out. Plasma corticosterone concentrations were used as a parameter for acute stress and were quantified at 6, 10, 24, and 48 h after bleeding by using a competitive ELISA (catalog no. EIA-4164, DRG Diagnostics, Marburg, Germany) according to the manufacturer's instructions.

Expression of inflammatory genes.

The site of blood sampling was dissected and placed in RNAlater (ThermoFisher Scientific) for RNA stabilization and stored at –20 °C until RNA extraction. The following samples were dissected: lateral tail incision, 8 to 10 mm of the whole tail; amputation of the tail tip, 3 to 4 mm of the tail tip; retrobulbar puncture, the Harderian gland; sublingual puncture, the whole tongue, cut at the throat; submandibular puncture, muscle tissue of the cheek (approximately 0.5 cm²) after removal of the skin; and saphenous vein puncture, muscle tissue distal to the knee (approximately 0.7 cm²) after removal of the skin.

RNA isolation and cDNA synthesis.

Tissues were homogenized in lysis buffer (MagMAX-96 RNA Isolation Kit, ThermoFisher Scientific) by using glass beads and the FastPrep-24 instrument (MP Biomedicals, Solon, OH). Total RNA from homogenized tissues was extracted by using MagMAX Express (ThermoFisher Scientific) and the MagMAX-96 RNA Isolation Kit (ThermoFisher Scientific). Total RNA from homogenized tissues was extracted by using the MagMAX-96 Blood RNA Isolation Kit (ThermoFisher Scientific). cDNA from approximately 500 ng total RNA was produced by using High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific) according to the manufacturer's instructions.⁸

Gene expression analyses by qPCR.

Gene expression of the genes listed in Figure 1 (assays purchased from ThermoFisher Scientific) was analyzed (StepOnePlus, ThermoFisher Scientific) by using Universal Fast Thermal Cycling settings (ThermoFisher Scientific) and TaqMan Fast Universal PCR Mastermix (ThermoFisher Scientific). Relative fold change in gene expression compared with control was calculated according to the cycle threshold (C_T) method. The expression of target genes was normalized to Actb as the reference gene:

Figure 1.

Genes for which expression was tested.

$$\Delta C_T=C_{T\left(target\right)}=C_{T\left(reference\right)}$$

Fold change in gene expression was calculated as 2^-∆∆CT, where

$$\Delta \Delta C_T=\Delta C_{T(sample)}-\Delta C_{T\left(calibrator\right)}$$

For tissues, the mean ∆C_T of samples from control mice was used as the calibrator. For blood samples, ∆C_T for each mouse at test sampling was used as the calibrator.¹⁹

Quantification of plasma proteins related to inflammation.

Plasma concentrations of haptoglobin, IL6, and IL1β were quantified by ELISA (DuoSet ELISA kit, R and D Systems, Minneapolis, MN) according to the manufacturer's instructions. Detection limits were 140, 40, and 40 pg/mL plasma for haptoglobin, IL6, and IL1β, respectively.

Assessment of histopathologic changes.

The abdomen of the euthanized mouse was opened and the entire mouse immersion-fixed in 10% neutral buffered formalin. After fixation, the head, tail, and hindlegs were decalcified for 1 wk in a solution containing 3.3% formaldehyde and 17% formic acid. The site of blood sampling (including the entire globe in cases of retrobulbar blood sampling) and the lungs, liver, spleen and kidneys were collected for histopathology. Tissues were processed through graded concentrations of alcohols and xylene, embedded in paraffin wax, sectioned, and stained with hematoxylin and eosin. For each mouse, 2 stained sections (thickness, 4 to 5 µm) of internal organs (lung, spleen, kidney, liver) and 2 from the local sampling site were evaluated. In selected cases of acute inflammation, additional sections of the local sampling area were stained with phosphotungstic acid hematoxylin for demonstration of fibrin. In selected cases of chronic inflammation, additional sections of the local sampling area were stained with Masson trichrome for visualization of collagen. In cases in which it was difficult to visualize suspected fibrin or collagen from slides stained with hematoxylin and eosin, additional sections were selected for staining with phosphotungstic acid hematoxylin and Masson trichrome. All tissue samples from the local areas (sites of blood sampling) were evaluated histopathologically, and the internal organs were examined for any lesions related to local changes (for example, embolic material, infarcts). All sections were evaluated by the same pathologist and confirmed by a second pathologist.

Statistics.

Statistical analysis was performed by Prism version 5.03 (GraphPad Software, San Diego, CA) and Minitab version 17.3.1.0 (Minitab, State College, PA). Plasma protein concentrations, body weight, and corticosterone were analyzed by using 2-way ANOVA and Bonferroni post tests. The Dunnett post hoc test was used to compare gene expression with controls. The Tukey post hoc test was used for pairwise comparisons of body weight. Data for stomach contents were not normally distributed and were analyzed by using the Kruskal–Wallis test. Correlations were analyzed according to Spearman nonparametric 2-tailed analysis. P values lower than 0.05 were considered statistically significant.

Results

Body weight and stomach contents.

Overall, significant differences in body weights between time points (P < 0.001) and between treatment groups (P = 0.004) were found. No interaction between treatment group and time was found. All puncture methods resulted in significant weight loss at 6 and 10 h after puncture compared with other times points (P < 0.001 for all comparisons); weight did not differ between the 6- and 10-h time points (Figure 2 A). Significant differences in body weight between puncture methods were found at 6 h (P = 0.030), day 2 (P = 0.012), and day 10 (P = 0.004) after puncture. Tukey pairwise comparison indicated that at 6 h, mice that had undergone retrobulbar sinus puncture had lost significantly (P = 0.015) more weight than those sampled through saphenous vein puncture. On day 2, the tail-tip amputation group had lost significantly (P = 0.010) more weight than those sampled through saphenous vein puncture, which had not lost weight. On day 10, mice in the retrobulbar sinus group had lost significantly more body weight compared with saphenous vein group (P = 0.008) and submandibular group (P = 0.024), which had not lost weight. Sublingually sampled mice lost significantly (P = 0.020) more body weight than the saphenous vein group.

Figure 2.

Changes in body weight and weight of stomach contents after blood sampling. The mice were weighed prior to blood sampling and at the indicated time points afterward. (A) Average deviation from the initial weight for each of the 6 different methods (n = 4 at each time point). (B) The weight of stomach contents at 6, 10, and 24 h after blood sampling (n = 4 at each time point. (C) Correlation between weight of stomach contents and changes in body weight at 6, 10, and 24 h (n = 72). Data are given as mean ± 1 SD (bars); *, P < 0.05; †, P < 0.01; ‡, P < 0.001.

No significant differences in the weight of stomach contents were found among the 6 treatment groups. Overall significant differences in stomach content were found among all groups at 6 h compared with 10 h (P = 0.004), 10 h compared with 24 h (P = 0.001), and at 6 h compared with 24 h (P = 0.001), confirming that the weight of stomach contents was lowest at 10 h after blood sampling (Figure 2 B). Weight gain and the weight of stomach contents were positively correlated (Figure 2 C).

Plasma corticosterone levels.

Overall, significant differences in data between time points (P = 0.017) and between treatment groups (P < 0.001) were found. No interaction between treatment group and time was found. In general, mice that underwent retrobulbar sinus puncture or sublingual puncture had significantly higher plasma corticosterone concentrations than tail tip–amputated mice (P = 0.017 and P = 0.033, respectively) and saphenous vein–punctured mice (P = 0.003 and P = 0.005, respectively). Mice that underwent isoflurane anesthesia only had significantly higher plasma corticosterone concentrations than those in the lateral tail (P = 0.012), tail tip amputation (P = 0.003), and saphenous vein (P = 0.001) groups. Of note, isoflurane anesthesia alone resulted in corticosterone levels comparable to those in retrobulbar sinus–punctured mice (Figure 3 A).

Figure 3.

Plasma corticosterone levels after blood sampling. (A) Corticosterone level (ng/mL; mean ± 1 SD; n = 4 per group) in plasma from mice euthanized at 6 h to 2 d after blood sampling. The horizontal lines indicate the mean ± 2 SD of control mice. Corticosterone levels in mice that were isofluorane treated but not punctured are indicated at 6 h and 24 h (isoflurane). (B). Correlation of weight gain and corticosterone plasma level in mice after 6 h to Day 2 after blood sampling (n = 96). Data are given as mean ± 1 SD (bars); *, P < 0.05; †, P < 0.01; ‡, P < 0.001.

The expected negative correlation between corticosterone concentration and weight gain was confirmed (P = 0.015, Figure 3 B), indicating that the level of acute stress (as assessed through plasma corticosterone) and eating behavior were associated.

Systemic effects.

No histomorphologic changes were found in the lungs, kidneys, liver, or spleen in any of the groups. As a measure of systemic inflammation, the plasma concentration of the acute-phase protein haptoglobin was determined (Figure 4). Compared with levels in control animals, an increase was seen at 24 h after blood sampling by lateral tail incision (P < 0.001), retrobulbar sinus puncture (P < 0.01), and saphenous vein puncture (P < 0.001). Plasma IL1β and IL6 levels were below the detection limit in all samples (data not shown). Regarding other time points and bleeding methods, only a few, individual samples showed nonsignificantly increased levels of haptoglobin (Figure 4). For all sampling methods except submandibular puncture, the expression of Il1b in blood showed a decreasing trend at 10 h (statistically significant [P < 0.05] for lateral tail incision only), indicative of a transiently reduced blood neutrophil concentration (Figure 4).

Figure 4.

Systemic inflammation after venepuncture. (A) Plasma levels of haptoglobin (mean ± 1 SD, n = 4 per group) at 6 h to 12 d as determined by ELISA. (B) Expression of Il1b in blood at 6 h to 12 d after venipuncture as measured by qualitative PCR analysis. Relative quantification (RQ) is the ratio between the expression in blood from mice euthanized at the indicated time point to the average expression among nonbled animals. *, P < 0.05; †, P < 0.01; ‡, P < 0.001.

Effects at the sampling site.

For most sample methods (except submandibular and saphenous vein puncture), histopathologic effects were still present at the end of the experiment (Table 1). However, there were substantial differences in the course of inflammation and tissue healing between the various sampling methods.

Table 1.

Summary of histopathologic findings.

	Blood sampling method
Histopathologic finding	Lateral tail incision	Tail tip amputation	Sublingual puncture	Submandibular puncture	Retrobulbar sinus puncture
Completion of migration of epithelial skin cells	Day 4	No complete healing	Not applicablea	Day 4	Not applicablea
Acute inflammation	Until day 4	Until day 6	Until day 1	Until day 2	Until day 1
Micro abscesses	No	No	Yes (Day 2 +8)	No	No
Trichogranuloma	No	No	Yes	Yes	Yes
Muscular necrosis and regeneration	Yes	No	Yes	Yes	Yes
Evidence of tissue trauma on day 12	Yes	Yes	Yes	None identifiedb	Yes

Data for saphenous vein puncture could not be obtained due to technical problems.

^aBecause no epithelial skin cells are found in the oral cavity or tissues surrounding the globe, this is a nonapplicable parameter for sublingual vein and retrobulbar sinus puncture.

^bFor mice that underwent submandibular puncture, no lesions were identified on day 12. The tissues appeared normal and without signs of pathologic processes.

Lateral tail incision.

Epidermal cell migration was visible at the wound surface at 10 h, and the wound was completely closed on day 4 (Figure 5 A and C). Hypertrophic epithelial cells were found throughout the study period (6 h through day 12). Epidermal edema was present from 6 h until day 8. In dermis and subcutis, extravascular erythrocytes were visible until day 6, and edema and lymphangiectasia were present throughout the experiment. Neutrophil infiltration was noted at the beginning of the wound-healing process, but on day 4, mononuclear cells dominated. On day 2, fibroblasts started to proliferate, and on day 4, newly formed connective tissue was present, whereas granulation tissue first appeared on day 8 (Figure 5 D). Necrosis and inflammatory cellular infiltration of muscle fibers showed that the tail muscles were affected (Figure 5 B); some cases showed muscular regeneration.

Figure 5.

Histopathologic lesions at the site of blood sampling. (A–D) Lateral tail incision. (E and F) Tail tip amputation. (A) Wound involving epidermis, dermis, and subcutis at 10 h after sampling (bar, 150 μm). (B) Muscular necrosis (arrow) at 10 h after sampling (bar, 75 μm). (C) Epidermis reestablished by a narrow layer of epidermal cells (arrow) with inflammation underneath at day 4 after sampling (bar, 150 μm). (D) Closed epidermal lesion with loose granulation tissue (gt) still present at day 12 after sampling (bar, 75 μm). (E) Epidermal proliferation and necrosis and subcutaneous inflammation with bleeding at the tail tip at 2 d after sampling (bar, 150 μm). (F) Epidermis still not closed (arrow) at day 12 after sampling (bar, 150 μm). Hematoxylin and eosin stain. Magnification: 100× (A, C, E, and F); 200× (B and D).

Expression of the genes for neutrophil-attracting chemokines S100A8/A9 (results shown for S100A8) and Cxcl was strongly increased in affected tissues, peaked at day 4 to 6 with a greater than 1000fold increase for Cxcl2 (P < 0.001), and returned to control levels at day 8 (Figure 6). Similar expression profiles were seen for the 2 cytokine genes Il6 and Il1b and for Nlrp3, which encodes a key inflammasome component in myeloid cells and is strongly induced on NFκB activation (P < 0.01; Figure 8).¹⁰ Expression of Il33, which is especially prominent in endothelial and epithelial tissue, increased after 10 h, peaked at day 4, and returned to background levels at day 8 (P < 0.001; Figure 6).^14,15 Together, the expression profiles reflected well the course indicated by microscopic data. Il1a exhibited the same profile as Il33 (not shown). The other genes listed in Figure 1 did not change in expression (data not shown).

Figure 6.

Local expression of genes encoding the cytokines IL1b and IL6 and the inflammasome subunit NLRP3 in tissue from the site of sampling at 6 h to 12 d after blood sampling (n = 2 per time point). Gene expression was measured through qualitative PCR analysis and shown as the change relative to the expression in samples from nonbled animals (relative quantity, RQ). (A) Relative expression of Il1b. (B) Relative expression of Il6. (C) Relative expression of Nlrp3. *, P < 0.05; †, P < 0.01; ‡, P < 0.001.

Figure 8.

Local expression of genes encoding the chemokine Cxcl2 and the alarmins A1008S and IL33 in tissue from the site of sampling at 6 h to 12 d after blood sampling (n = 2 per time point). Gene expression was measured through qualitative PCR analysis and shown as the change relative to the expression in samples from nonbled animals (relative quantity, RQ). (A) Relative expression of Cxcl2. (B) Relative expression of A1008S. (C) Relative expression of Il33. *, P < 0.05; †, P < 0.01; ‡, P < 0.001.

Tail-tip amputation.

Epidermis was ruptured at all time points, although migration of epidermal cells was first visible on day 2 (Figure 5 E and F). Hypertrophy of migrating epithelial cells was seen throughout the wound-healing process (6 h through day 12). Epidermal edema was present on days 2, 4, 6, 10, and 12. Dermal and subcutaneous bleeding was apparent from 6 h to day 2. Neutrophils were seen in samples from 6 h through day 12, whereas mononuclear cells were predominated on day 8 and thereafter. Fibroblasts started to proliferate on day 4, newly formed connective tissue emerged on day 6, and granulation tissue was visible on day 8. The musculature of the tail was not involved.

In mice that experienced tail-tip amputation, Cxcl2 expression was induced at 6 h, peaked (greater than 1000-fold increase; P < 0.01) on day 2, dropped rapidly to approximately 10-fold above background, and remained increased at this level for the remainder of experiment (Figure 6); this progression was quite comparable to that after lateral tail incision. Expression of S100A8/A9 was increased (albeit nonsignificantly) after 10 h and, like Cxcl2 expression, remained increased during the entire experiment. At 6 h after blood sampling, Il1b expression was induced, peaked at day 2 (greater than 3000fold higher than control), and remained increased throughout the experiment (significant [P < 0.05] only at day 2; Figure 8). Similar to Il1b, Nlrp3 expression increased during the first 2 d (P < 0.01) but then dropped to slightly over the background level from day 6 onward, reflecting the generally low numbers of infiltrating myeloid cells. As for lateral tail incision, Il33 expression after tail-tip amputation was increased from 24 h until day 8 (Figure 6), reflecting the prolonged wound-healing process. The other genes listed in Figure 1 did not change in expression (data not shown).

Retrobulbar sinus puncture.

Lesions were observed in the tissue surrounding and supporting the eye, including the Harderian gland, but not within the structures of the eye (Figure 7 A). Bleeding was present in the cavity around the eye, in the Harderian gland, in the connective tissue behind and around the eye, and in the musculature at 6 and 10 h and, sporadically, at 24 h (Figure 7 A). In addition, necrosis of the Harderian gland and the muscles behind the eye was visible at these time points. Diffuse cellular infiltration was seen primarily in the connective tissue behind the globe and occasionally in the musculature and gland. At 24 h, cellular infiltration was dominated by neutrophils and, from day 2 onward, by macrophages. Fibroplasia was seen behind the eye on day 4 and thereafter. In 2 cases (1 case each on days 6 and 10), massive necrosis, mononuclear cell infiltration, and fibroplasia were evident in the Harderian gland. In 6 cases, throughout the experiment, trichogranulomas (that is, contamination with hair) were seen in the connective tissue behind the eye adjacent to the Harderian gland.

Figure 7.

Histopathologic lesions at the site of the blood sampling. (A) Retrobulbar puncture. (B–D) Sublingual puncture. (A) Inflammatory cells and bleeding (b) were seen in the Hardarian gland (h) at 10 h after sampling (bar, 75 μm). (B) A microabcess (m) in the tongue on day 2 after sampling (bar, 75 μm). (C) Trichogranuloma formed by a hair (arrow) and surrounded by inflammatory cells at 24 h after sampling (bar, 75 μm). (D) Myxomatous tissue (arrow) deep in the tongue at day 6 after sampling (bar, 150 μm). Hematoxylin and eosin stain; magnification: 100× (D); 200× (A–C).

Expression of Cxcl2, Il6, Il1b (approximately 100fold for each gene), and Nlrp3 (approximately 10fold) was induced in the Harderian gland at 6 h and later (significantly only for Il6 at 6 h) and normalized on day 6 (Figures 6 and 8). By contrast, mRNA levels of S100A8/A9 were similar to controls throughout the study (Figure 6), indicating the distinctive role of the 2 chemokines Cxcl2 and S100A8/A9 in the Harderian gland. Il33 was not significantly increased from background level at any time point nor were any of the other genes listed in Figure 1 (data not shown).

Sublingual puncture.

The musculature of the tongue was affected in all samples, and necrosis of muscle fibers became visible during the first 2 d (6 h through day 2). As soon as 10 h after blood sampling, regeneration of muscle fibers was initiated, and regeneration could be seen throughout the experiment. Leukocyte infiltration was present throughout the study period, first dominated by neutrophils and then by mononuclear cells at 24 h and later. In both mice euthanized on day 2 and in 1 euthanized on day 8, microabscesses were present (Figure 7 B). In addition, one of the day 2 mice had a trichogranuloma (Figure 7 C). Edema was seen throughout the experiment, whereas bleeding was present from the beginning and disappeared at day 6. Edematous newly formed connective tissue was present in 1 mouse each on days 6, 8, and 12 (Figure 7 D).

Expression of most of the tested genes (Figure 1) in the tongue after sublingual puncture was unaffected at all time points. Interestingly, the 2 potential ‘danger molecules,’ encoded by Il1a (data not shown) and Il33, were expressed at low levels in 2 waves in all mice that underwent sublingual puncture, reaching nadir (P < 0.001 for both genes) at days 2 and 10, respectively, compared with controls (Figure 6).

Submandibular puncture.

By day 4, the epidermis was closed. After 6, 10, and 24 h and on day 2, bleeding were observed in the subcutis and dermis and occasionally in the musculature. Neutrophil infiltration was seen at 6 and 10 h. At 24 h and on day 2, mononuclear cells predominated. One mouse euthanized on day 4 had a trichogranuloma, and on day 8, muscular regeneration at the sampling site was evident.

Mild, nonsignificant induction of Cxcl2, S100A8/A9, Il6, Il1b, and Nlrp3 was observed in the cheek muscle within the first 24 h after submandibular puncture (Figures 6 and 8), thus reflecting the early and relatively mild changes seen on histology. S100A8/A9 yielded the greatest fold-change (approximately 100fold) in expression, whereas the remaining genes only increased by 8-to 30-fold relative to controls.

Saphenous vein puncture.

Muscular bleeding and minor inflammatory infiltrates were the only abnormalities found after saphenous vein puncture. This minimal morphologic change may be due to imperfect sampling of tissue, given that fixation of the mouse with open abdomen made the site of puncture difficult to recognize macroscopically during sampling for histology.

Compared with the 2 tail-puncture methods, the expression of all tested genes were rapidly increased after saphenous vein puncture, but dropped already after 4 to 6 d to background level (Figures 6 and 8). Cxcl2 and Il1b showed the highest fold-induction (approximately 1000fold; P < 0.05 on day 2 for Cxcl2), whereas S100A8/A9 (P < 0.001 at 10 h), Nlrp3, and Il6 peaked at approximately 100-fold higher mRNA levels compared with controls. IlL33 expression did not show any alteration during the 3 d evaluated.

Discussion

In general, the findings indicated that venipuncture methods involving the head region affect body weight and corticosterone levels to a greater extent than methods at the tail and leg regions. In contrast, methods involving the tail or hindleg caused greater and prolonged inflammation and tissue damage.

In general, our observations were largely in accordance with earlier studies, but this study is the first to compare 6 sampling methods. Furthermore, our current study is the first to assess tissue effects at sampling sites by studying alterations in the expression of inflammatory genes.

Mice are nocturnal animals and, under normal circumstances, eating will be reduced during daytime, consequently explaining much of the decrease in weight after sampling that we observed. Still, we found a slight method-dependent difference in body weight. Increased weight loss and higher corticosterone levels were most prominent hours after sampling in mice that underwent retrobulbar or sublingual puncture compared with those sampled from the tail or saphenous vein. Reduced eating behavior due to pain or discomfort relating to the head region of the animal could be a possible explanation for the loss in body weight, even though no significant difference in stomach content was found between the treatment groups. In contrast, the sampling methods involving the tail and saphenous vein caused marked local inflammation, as seen by histologic inspection as well by increased expression of various genes related to inflammation and tissue damage (Table 1). Regarding systemic effects, only modest signs of systemic inflammation and no distant tissue damage were found.

Regardless of the blood-sampling method, all mice showed high levels of corticosterone. Because all animals were anesthetized with a mixture of Hypnorm and Dormicum prior to the second blood sample, it could be suggested that this anesthesia caused the general increase in corticosterone in our study and, in retrospect, perhaps another anesthetic should have been used.¹⁷ Nevertheless, the corticosterone levels in mice sampled by retrobulbar puncture (under isoflurane anesthesia) or sublingual puncture and those exposed to isoflurane anesthesia only were significantly higher after 6 h than those of the tail amputation and saphenous vein groups. No significant difference was found between mice test sampled by retrobulbar puncture (under isoflurane) and those given isoflurane anesthesia without blood sampling. These findings indicate that the retrobulbar puncture and isoflurane anesthesia alone are particularly and equally stressful. Whether the stress resulting from accessing the retrobulbar sinus is mainly due to the effect of the anesthesia or the puncture as such is unknown. However, isoflurane anesthesia reduces nest building behavior in mice, and they find reexposure to isoflurane to be aversive.^9,16

The findings regarding the retrobulbar and sublingual puncture methods are in accordance with the data for weight gain, which indicate that the decrease in body weight is greatest in mice test sampled by using these methods.

Acute-phase proteins represent systemic markers of infection, inflammation, and stress,⁴ and serum levels of the acute-phase protein haptoglobin have previously been shown to be a useful systemic marker for infection in mice¹⁹ and of tail shock in rats.⁵ However, only lateral tail incision, retrobulbar sinus puncture, and saphenous vein puncture showed any effects on haptoglobin levels, which were transiently and slightly increased at approximately 24 h.

Il1b is expressed by myeloid cells, of which a high number also serves as a systemic sign of a local infection.¹³ Expression of Il1b in whole blood is an accurate proxy for the whole-blood neutrophil level. Il1b expression levels were not significantly increased in mice after any of the puncture methods. By contrast, at 10 h after lateral tail incision, Il1b expression dropped below baseline, indicative of neutrophil recruitment from blood to tissues. Given that the IL6 and IL1β plasma levels were below the detection limit at all time points and that none of the mice showed pathomorphologic organ change, these subtle effects on Il1b expression demonstrate that none of the blood-sampling methods induced marked systemic effects. Histopathologic changes at the local site could still be found at the end of the experiment for most sampling methods (except submandibular and saphenous vein methods). Comparing the effects on muscle tissues between the 2 tail-sampling methods revealed that the muscles of the tail were affected only by lateral tail incision (seen as regeneration). In addition, muscular regeneration and necrosis were evident in animals that received submandibular and retrobulbar punctures. Furthermore, muscular regeneration was very fast in the tongue, as expected.¹²

Acute inflammation (defined as the presence of neutrophils with lack of regeneration) lasted for 2 to 4 d after lateral tail incision and 4 to 6 d for tail-tip amputation. This pattern was in excellent accordance with the results obtained by measuring the expression of genes involved in the acute-phase response, specifically Cxcl2, which encodes a chemokine for neutrophil recruitment expressed in injured or infected tissue, and A1008/9S, which encode alarmines and likewise are expressed in injured and infected tissue, thus substantiating the inflammatory process.² These 2 genes were increasingly upregulated until day 2 to 4 after lateral tail blood sampling and then dropped rapidly thereafter; the expression pattern of the 2 genes after puncture of the saphenous vein was similar to that after the lateral tail method. In addition, Il1b and Nlrp3, reflecting the abundance of myeloid cells in the affected tissue, showed an expression pattern similar to the other genes, thus confirming the presence of numerous neutrophils, other granulocytes, and macrophages. Expression of Il33 showed a clear increase in samples from the lateral tail incision group that peaked at day 4 to 6, corresponding to the pathomorphologic changes indicative of growth and migration of epidermal cells at 24 h and wound closure on day 4.

All of the pathomorphologic changes observed at the local blood-sampling site represented a normal healing process (from acute inflammation to healing), ultimately restoring the normal tissue architecture at the sampled site. However, normal healing can be considered a pathologic process, involving an inflammatory reaction with the infiltration of leukocytes and the expression of specific molecules such as chemokines and cytokines. Therefore, our current findings show that different blood-sampling techniques have different healing processes, at both the pathomorphologic and cellular levels. For the submandibular, sublingual, and retrobulbar methods, acute inflammation was present only until day 2. This finding was reflected by the expression patterns of inflammatory genes. In particular, submandibular puncture was associated with clear but decreasing expression of inflammatory genes during the first 24 h after blood sampling. The sublingual puncture method resulted in the development of microabscesses composed of neutrophils that accumulated in small, well-demarcated foci in some of the mice.

Contamination with hairs introduced into the tissues by the puncture may result in a granulomatous inflammatory reaction (trichogranuloma). In the current study, such granulomas were found in tongue, cheek, and eye tissues (Table 1). This contamination may have induced the increased expression of Il1b seen in the blood but was not reflected in the local expression of any genes measured. The presence of a piece of hair deep within the injection site may negatively influence healing.¹² Because tissue for gene expression analysis was sampled from different animals than those sampled for pathologic inspection, we cannot exclude that none of these mice had hair contamination. Overall, the inflammation evoked by blood sampling in the head region was lower and resolved faster than that induced by methods involving the tail or hindleg.

To our knowledge, real-time quantitative PCR analysis to assess local inflammation has not been used in previous studies comparing the consequences of various blood-sampling techniques. The effects on the expression of genes related to inflammation and tissue regeneration are highly relevant, because this method may offer an alternative, sensitive, and highly quantitative way to address tissue changes. Moreover, the long-term effect of various blood sampling techniques on, for example, pathologic processes at the injection site may differ in regard to wound repair and resolution of inflammation. Our results demonstrated that expression levels of Cxcl2, Il1b, A1008/9S, Nlrp3, and Il33 were in accordance with the damage and healing revealed through pathologic inspection. The RT qPCR thus constitutes a quantitative supplement to traditional pathologic evaluation. Even though we saw significant increases in the expression of most genes only after lateral tail incision, the gene-expression profiles in many other tissues showed a pattern of increased expression. However, because we assessed only 2 mice for each time point, large differences in expression between methods and low variation between animals from the same group are required to obtain significant differences.

In the present study, we aimed at comparing the inflammation induced after a single bleeding event by using 1 of 6 bleeding techniques. Whether multiple, serial bleeds would have resulted in the same differences, we cannot deduce from the current data. However, because the bleeding methods involving tail or hindleg produced the greatest inflammation, these procedures are likely to induce equivalent or even increased or prolonged inflammation after repeated bleedings.

In summary, of the 6 investigated blood-sampling methods, retrobulbar sinus puncture and sublingual puncture methods, which both involve the head region of mice, seemed to affect the welfare of the animals the most. The difficulty in visualizing the sublingual vein in unanesthetized mice likely added to the negative effects; if this method is to be used in mice, anesthesia should be considered for animal welfare reasons. However, using anesthesia may affect the animal's immunologic response. In contrast to venous access sites in the head region of the mice, methods involving the tail or saphenous vein resulted in more prolonged local inflammation and tissue damage but only minor or no signs of systemic inflammation. Blood sampling procedures clearly can lead to pathologic and immunologic changes and thereby possibly to uncontrolled variation and alteration of standard parameters. Therefore, to optimize studies to reflect the principle of reduction, an awareness of the pathologic and immunologic changes introduced by a basic experimental procedures is important when designing experiments and when evaluating the results. In addition, it is of importance to consider the influence of the various methods on animal welfare to advance the principle of refinement. This study emphasizes that the knowledge of the effects of the procedures on the animals is highly important, because seemingly simple standard procedures that are apparently similar in type may have large and dissimilar effects on various parameters. Furthermore, researchers need to well consider the choice of blood sampling method, because all techniques may influence the data obtained. Identification of the optimal method for blood sampling in mice is indeed a very difficult task that demands further comparative studies, including studies on the effects of multiple sampling, which is often used in pharmacokinetic–pharmacodynamic studies.