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

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, fastresolving 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.

Ultrasonographic and histological evaluation of the effects of long-term carotid catheterization on cardiac function in NMRI mice.

Catheterization of laboratory mice is commonly performed in biomedical research to infuse substances and for blood sampling. One approach is to catheterize the right common carotid artery and advance the catheter until the tip is positioned in the aorta or the proximal brachiocephalic trunk. Owing to the small body size of the mouse, a catheter tends to occupy a great part of even the larger vessel lumens, and this may increase vascular resistance with potential pathophysiological impacts on the heart. The present study compared cardiac function of catheterized mice, with catheter tip placement in the brachiocephalic trunk, with sham-operated mice and non-operated control mice. During four weeks post-catheterization, M-mode echocardiography measurements of the thickness of the left ventricular anterior wall, left ventricular inner diameter and the thickness of the left ventricular posterior wall were performed. The left ventricular volume, ejection fraction and fractional shortening were calculated. Moreover, aortic recordings of the thickness of the medial and lateral walls as well as the inner diameter were measured. Terminally, histological analysis of the hearts was conducted, and body weights and heart weights were compared between groups. No effects on echocardiography parameters, histology, body weights or cardiac weights could be found between groups. In the present study, implantation of a carotid catheter with catheter tip placement in the proximal brachiocephalic trunk had minimal influence on cardiac and aortic physiology and did not induce significant cardiac changes.

Impact of heparin and short term anesthesia on the quantification of cytokines in laboratory mouse plasma.

BACKGROUND: Studies have reported that heparin may be unsuitable as an anticoagulant in human plasma samples when quantifying cytokines using multiplex bead array assays. For mouse samples, multiplex assays have been validated for serum and EDTA-plasma, but it remains to be elucidated whether heparin influences the quantification of cytokines, and if so - to what extent. Furthermore, laboratory mice are often anesthetized for blood sampling, which causes acute stress that may influence circulating cytokine concentrations and thus bias experimental results. The objectives of the present study were to identify whether specific cytokine concentrations varied between heparin-plasma, serum, and EDTA-plasma, and whether short isoflurane anesthesia would influence the concentrations of these cytokines in the circulation. Twenty-three acute phase and pro-inflammatory cytokines were quantified in matched serum, EDTA-plasma, and heparin-plasma samples from anesthetized and unanesthetized male NMRI mice using a multiplex assay. In addition, samples from unanesthetized mice were spiked with three levels of heparin. RESULTS: The concentrations of five out of 23 cytokines were significantly different between sample types, but only one cytokine (IL-17A) differed between heparin-plasma and serum. When further spiking the heparin-plasma with increasing concentrations of heparin, there was a significant effect on 11 cytokines, where the cytokine recovery could be correlated to the heparin concentration for ten of these cytokines. Anesthesia resulted in lower concentrations of G-CSF, but had no significant impact on the concentrations of the other 22 cytokines. CONCLUSION: In mice, heparin seems like a suitable anticoagulant for obtaining plasma for multiplex assays for the cytokines IL-1α, IL-1ß, IL-2, IL-6, IL-9, IL-12p40, IL-12p70, IL-13, G-CSF, GM-CSF, IFN-γ, KC, MCP-1, MIP-1α, MIP-1ß, RANTES and TNFα, but an effect of heparin in high concentrations should be considered for the cytokines IL-9, IL-12p40, IL-12p70, KC, MCP-1, MIP-1ß and RANTES. Short isoflurane anesthesia had significant impact on G-CSF, but none of the other cytokines.

Postsurgical food and water consumption, fecal corticosterone metabolites, and behavior assessment as noninvasive measures of pain in vasectomized BALB/c mice.

Recognition of pain and stress is a common challenge when working with laboratory mice. The aim of the current study was to identify noninvasive parameters to assess the severity and duration of possible pain and stress after vasectomy in BALB/c mice. Mice underwent isoflurane anesthesia with or without vasectomy. Body weight, food and water intake, and fecal corticosterone metabolites (FCM) were measured 3 d before and 3 d after the procedure. Behavior was recorded 1, 2, 4, and 8 h after the procedure. Food and water consumption and defecation were reduced postoperatively in the vasectomized group compared with mice given anesthesia only. FCM were elevated the first day after anesthesia in the control mice but not in the vasectomized group. Vasectomy resulted in behavioral changes that were not seen in the group that was anesthetized only. In conclusion, food and water consumption and pain-related behaviors, but not FCM, may be useful as noninvasive parameters to assess postoperative pain and stress in vasectomized mice.