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Pathological and immunological consequences of basic experimental animal

Dorte Bratbo Sørensen

Annually, millions of mice are used for experimental purposes and these experiments often include sampling of blood throughout the trial period. However, there is a lack of comparative studies investigating the impact of blood sampling on the mice with regards to both resulting tissue damages and immunological changes and stress. The overall aim of the project is thus to describe the local and systemic pathological and immunological changes and possible stress introduced in mice after blood sampling by six different basic procedures, namely retrobulbar venous plexus puncture (periorbital puncture), vena submandibularis puncture (cheek blood), vena sublingualis puncture (tongue blood), vena saphena puncture, lateral tail incision and amputation of tail tip.

All six blood sampling techniques are performed by skilled technicians and the mice are euthanasized after either 6, 10 or 24 hours or 2, 4, 6, 8, 10 and 12 days after the procedure. After euthanasia, a complete necropsy is performed and blood, selected internal organs and tissue samples from the local area of blood sampling are collected. The pathological damages induced as a consequence of the procedure and how fast these changes are normalized are described. The expression of major mediators of local acute inflammatory events and systemic inflammatory responses are analyzed using qPCR for gene expression profiling of 15 selected immune-related genes and ELISA (enzyme-linked immunosorbent assay) is used for quantifying relevant blood protein inflammation markers. Moreover, the Optical projection tomography (OPT) scanning technique is used to construct a complete three-dimensional image of the distribution of inflammatory cells surrounding the puncture site 6 and 24 hours and 10 days after sublingual vene puncture. Last, the level of stress hormone (corticosterone) is measured either 6, 12 or 24 hours after blood sampling.

This study will hence provide a comprehensive description of the effects of blood sampling in laboratory mice, forming the basis for a more detailed and useful guideline for choosing the optimal blood sampling technique for a specific study.

Millions of mice are used for testing on a global scale every year, and blood samples are taken from many of these mice. The choice of the best blood sampling method depends on the purpose of the test, as any impact on related parameters should be avoided, as well as on the degree of adverse effect on the mouse’s welfare. For this reason, it is important to know the impact of the various blood sampling methods on animal welfare and the immune system and the amount of tissue damage caused.

In this study, we tested six different frequently used methods for drawing blood from mice and observed the animals at various times for up to twelve days after blood sampling. At each time point, histopathological changes were assessed, both locally at the sampling site (including healing time) and systemically in selected organs (liver, kidneys, spleen and lungs). Systemic inflammation was assessed by measuring haptoglobin in the blood and locally by determining the gene expression of inflammatory markers such as S100A8, S100A9 and Cxcl2, IL-6, IL-1b, IL-33 and Nlrp3.

Animal welfare was assessed by measuring stress hormone levels, appetite and weight loss in the days following sampling. All blood sampling methods caused changes, but to a varying degree. Animal welfare generally appeared to be most affected by sampling in the head region whereas one of the methods for blood sampling from the tail generally resulted in the greatest impact on inflammatory parameters. This means that it is not possible to unambiguously identify “the best method,” but careful consideration should be given to which method is best suited for the test in question.


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