Physiological and pathological impact of blood sampling by retro-bulbar sinus puncture and facial vein phlebotomy in laboratory mice

Abstract

Retro-bulbar sinus puncture and facial vein phlebotomy are two widely used methods for blood sampling in laboratory mice. However, the animal welfare implications associated with these techniques are currently debated, and the possible physiological and pathological implications of blood sampling using these methods have been sparsely investigated. Therefore, this study was conducted to assess and compare the impacts of blood sampling by retro-bulbar sinus puncture and facial vein phlebotomy. Blood was obtained from either the retro-bulbar sinus or the facial vein from male C57BL/6J mice at two time points, and the samples were analyzed for plasma corticosterone. Body weights were measured at the day of blood sampling and the day after blood sampling, and the food consumption was recorded automatically during the 24 hours post-procedure. At the end of study, cheeks and orbital regions were collected for histopathological analysis to assess the degree of tissue trauma. Mice subjected to facial vein phlebotomy had significantly elevated plasma corticosterone levels at both time points in contrast to mice subjected to retro-bulbar sinus puncture, which did not. Both groups of sampled mice lost weight following blood sampling, but the body weight loss was higher in mice subjected to facial vein phlebotomy. The food consumption was not significantly different between the two groups. At gross necropsy, subcutaneous hematomas were found in both groups and the histopathological analyses revealed extensive tissue trauma after both facial vein phlebotomy and retro-bulbar sinus puncture. This study demonstrates that both blood sampling methods have a considerable impact on the animals' physiological condition, which should be considered whenever blood samples are obtained.

Figure 1. Anatomical overview.

Blood samples were taken from either the facial vein (F. v.) or through the medial canthus of the eye to puncture the retro-bulbar venous sinus. Related anatomical structures in head are the extraorbital lacrimal gland (Ex), the parotid gland (Pa), the submandibular gland (Sm), the deep (Mas-d) and superficial masseter muscles (Mas-s), the superficial temporal vein (T.v.) and the external jugular vein (J. ex). The green dashed lines indicate the sections for histopathology. Nomenclature and anatomy are based on Popesko et al.

Figure 2. Body weights in mice subjected to facial vein phlebotomy (FVP, N = 12) and retro-bulbar sinus puncture (RSP, N = 12).

In 2a, pre-procedural (Day 1) and post-procedural (Day 2) measurements (mean ± SEM) are shown. Control mice (95% CI; 26.5 – 35.8 g) were also weighed at Day 1, but were euthanized in connection to blood sampling as trunk blood was obtained, why body weights were not available at Day 2 and therefore not shown. Mice sampled by FVP (repeated measures ANOVA, p < 0.001) and RSP (repeated measures ANOVA, p = 0.010) lost weight significantly after the blood sampling (2a). In 2b, the body weight loss (ΔBody weight, median with min. to max. values) is shown. The body weight loss was significantly greater in FVP mice than in RSP mice (Mann-Whitney U-test, p = 0.040). The lost blood volume in connection to blood sampling has not been included in the calculation of the body weight loss.

Figure 3. Food consumption during 24 hours after blood sampling in mice sampled by facial vein phlebotomy (FVP, N = 12) and retro-bulbar sinus puncture (RSP, N = 12).

In 3a, the total food consumption is shown. The lines indicate means. In 3b, the cumulative food consumption, recorded automatically in a feeding monitoring system during 24 hours, is shown; means (lines) ± SEM (light and dark grey areas). Blood samples were obtained at 4 and 6 pm. The final blood sample was obtained at the beginning of the dark period (grey box). No significant difference between groups was found (ANOVA, P = 0.101).

Figure 4. Plasma corticosterone levels in mice subjected to facial vein phlebotomy (FVP, N = 12), retro-bulbar sinus puncture (RSP, N = 12) and decapitation (Control, N = 4 at respective time points).

Shown are mean ± SEM. Plasma corticosterone levels of FVP mice were significantly increased at 4 pm compared to the control mice (ANOVA, p = 0.046) and at 6 pm compared to RSP mice (ANOVA, p = 0.001) and control mice (ANOVA, p < 0.001). Notice also the natural increase in circulating corticosterone concentrations as a result of the diurnal rhythm, as illustrated by the control mice.

Figure 5. Pathological changes in the cheeks and associated tissue.

Shown are examples of control sections (A-C) and sections from mice, which were subjected to facial vein phlebotomy (D-I). A) An overview is given, showing the temporo-mandibular joint, the mandible and masseter muscle. The area highlighted by the black box is shown in higher magnifications in B and C, respectively. D) Overview. E) The area highlighted by the dark blue box in D is shown in higher magnification, illustrating the puncture site after facial vein phlebotomy. Note the extensive hemorrhage with a central blood clot, deposited hair and diffuse polymorphonuclear cell infiltration. F) The area highlighted by the light blue box in D is shown in higher magnification. Note diffuse inflammation of the muscle, which involves the medial side of the mandible and the caudal end of the associated nerve (tentatively the lingual nerve). G) The puncture site after facial vein phlebotomy in another mouse, showing the puncture tract, created by the lancet, as evidenced by inflammatory cells and hair in the masseter muscle. H) A higher magnification of the area highlighted by the green box in G. Note the thrombus inside the vessel (possibly the facial vein), hair deposition and extensive cell infiltration. I) Muscle cell necrosis. All sections were stained with hematoxylin and eosin. Bars = 1000 µm in A and D. Bars = 500 µm in B and G. Bars = 100 µm in E, F and H. Bars = 50 µm in C and I.

Figure 6. Pathological changes in orbits and associated structures.

Shown are examples of control sections (A-C) and sections from mice, which were subjected to retro–bulbar sinus puncture (D-I). A) An overview is given, showing the palpebrae, eye and the Harderian gland as well as the ethmoid turbinates of the nasal passage. The cornea is artefactually folded. B) A higher magnification of the area in the black box in A. Shown is normal histology of the mouse Harderian gland. C) Higher magnification of the area lined by the green box in A, showing the caudal part of the retina photoreceptor segment and the pigmented epithelium, the nictitating membrane, Harderian gland and extraorbital muscle. D) Overview. Note the corneal thickening due to inflammation, retrobulbar inflammation and edema as well as Harderian gland necrosis. Note also the inflammatory foci, highlighted in the white box, which surround embedded hair. E) Higher magnification of the area in the white box in D, showing diffuse edema and inflammation in the retro-bulbar connective tissue as well as the inflammatory foci with embedded hair. F) The same section as in E with polarized light for identification of keratin from the embedded hair. G) Overview. H) The area marked by the blue box in G is shown in higher magnification. Note the partial necrosis of the Harderian gland, delineated by a clear demarcation line, which suggests ischemic necrosis. I) Higher magnification of the area marked by the light blue box in G, showing retro-bulbar inflammation of both the Harderian gland and extraorbital muscle and necrosis of the Harderian gland. All sections were stained with hematoxylin and eosin. Bars = 1000 µm in A and G. Bar = 500 µm in D. Bars = 200 µm in H and I. Bars = 100 µm in C, E and F. Bar = 50 µm in B.