Comparison of Serial Blood Collection by Facial Vein and Retrobulbar Methods in C57BL/6 Mice

Abstract

Many biomedical research protocols for mouse models involve serial blood collection and analysis. Two common techniques for serial blood collection in this species are the retrobulbar (RB, also called retroorbital) and facial vein (FV) methods. However, previous studies comparing these methods typically evaluated collection at a maximum of 2 time points. Here we compared hematologic values, adverse clinical effects, and histopathologic lesions in mice bled either once or serially (6 times) by using the FV or RB method. Mice (n = 48) were divided into 4 groups: single FV, single RB, serial FV and serial RB. Mice in the single-collection groups underwent a single blood collection by the indicated method, whereas those in the serial-collection groups were sampled once weekly for 6 consecutive weeks. All animals were euthanized and necropsied 2 wk after their last blood collection. Compared with all other groups, the serial FV group experienced more serious clinical adverse events, including 33% mortality, convulsions, head tilt, and hemorrhage from the ear canal and nares. In addition, mice in the FV groups had a significantly greater acute body weight loss compared with mice in the RB groups. Histologically, mice in both serial-collection groups had an increased incidence of tissue lesions compared with their respective single-collection groups. Importantly, only mice in the serial FV group had life-threatening histopathologic lesions, including cerebral hemorrhage or ischemia. Given these data, we conclude that serial blood collection in mice causes increased incidence of tissue damage compared with single sampling, and serial blood collection by the FV method causes substantial morbidity and mortality compared with the RB method.

The Impact of Common Recovery Blood Sampling Methods, in Mice (Mus Musculus), on Well-Being and Sample Quality: A Systematic Review

“…In a retrospective study [26] with facial vein sampling performed serially, a mortality rate of 4/214 mice was observed (≈2%). Frolich et al 2018 [29] reported a substantially higher mortality rate of (4/12, 33%) when mice were serially sampled by the facial vein route. There was no associated mortality with the single-sample facial vein route or retrobulbar routes, or serial retrobulbar sampling [29].…”

“…The major physiological measures investigated were those reflecting fear or arousal, via the hypothalamic-pituitary-adrenal (HPA) axis response [2,[7][8][9][10][11]13,24,31,35,36,38] and the associated release of stored glucose [1,22,23,25,29,32,34,37]. Stress almost invariably activates the HPA axis, which, via a sequence of steps, leads to glucocorticoid production-the principle rodent glucocorticoid being corticosterone.…”

“…Other measures utilised are arguably less indicative of animal affective state, but may provide insight by inference-for example, the quantification of tissue damage through post-mortem or histology [1,4,8,24,[27][28][29]32,33,36,38] may imply pain or loss of function.…”

“…Only one study was at a low risk of selection bias for all three signalling questions used [27]. The generation of an adequate randomisation sequence was reported in only 8 studies [12,13,22,23,25,27,30,34], while 10 studies [1,2,4,8,11,28,29,32,36,38] stated that randomisation took place but failed to report the actual methods used, thus being assigned as "unclear". For the remaining studies, randomisation did not take place, or was not mentioned.…”

“…Mortality, expressed as a% of study sample based on conversion from the absolute figures, was presented in two studies [26,29]. Both of these studies examined the facial vein route, with [29] additionally comparing with the retrobulbar sample route. These studies evaluated serial sampling at intervals of up to one week.…”