Abstract
When evaluating the effect of husbandry and biomethodologies on the well-being of laboratory mice, it is critical to utilize measurements that allow the distinguishing of acute stress from chronic stress. One of the most common measurements of stress in laboratory animals is the corticosterone assessment. However, while this measurement provides a highly accurate reflection of the animal's response to acute stressors, its interpretation is more prone to error when evaluating the effect of chronic stress. This study evaluated the use of the neutrophil:lymphocyte (NE:LY) ratio as an assessment of chronic stress in male and female C57Bl/6N mice as compared to serum corticosterone. One group of mice was exposed to mild daily stressors for 7 days, while the control group was handled with normal husbandry. The NE:LY ratio and serum corticosterone levels were significantly elevated in the chronically stressed mice, though a significant increase in corticosterone was only significant in males when compared by sex. The chronically stressed mice also demonstrated significantly fewer entries into the open arms and less time spent in the open arms of the elevated plus maze, suggesting that the mild daily stressors had induced a state of distress. The findings of this study confirm that the NE:LY ratio is a valid measurement for chronic stress in the laboratory mouse. However, these assays do not distinguish between distress or eustress, so behavioral and physiological assessments should always be included to determine a complete assessment of the well-being of the mouse.
This is a preview of subscription content, access via your institution
Access options



Similar content being viewed by others
References
Gouveia, K. & Hurst, J.L. Reducing mouse anxiety during handling: effect of experience with handling tunnels. PLoS One 8, e66401 (2013).
Jackson, E. et al. Aspen shaving versus chip bedding: effects on breeding and behavior. Lab. Anim. 49, 46–56 (2015).
Miller, A.L. & Leach, M.C. The effect of handling method on the mouse grimace scale in two strains of laboratory mice. Lab. Anim. 50, 305–307 (2016).
Pasalic, I. et al. Cage enrichment with paper tissue, but not plastic tunnels, increases variability in mouse model of asthma. Lab. Anim. 45, 121–123 (2011).
Poole, T. Happy animals make good science. Lab. Anim. 31, 116–124 (1997).
Rosenbaum, M.D., VandeWoude, S. & Johnson, T.E. Effects of cage-change frequency and bedding volume on mice and their microenvironment. J. Am. Assoc. Lab. Anim. Sci. 48, 763–773 (2009).
Selye, H. Stress and distress. Compr. Ther. 1, 9–13 (1975).
Stern, C.M. Corticotropin-releasing factor in the hippocampus: eustress or distress? J. Neurosci. 31, 1935–1936 (2011).
Pekow, C. Defining, measuring, and interpreting stress in laboratory animals. Contemp. Top. Lab. Anim. Sci. 44, 41–45 (2005).
Broom, D. & Fraser, A. Domestic Animal Behaviour and Welfare, 4th Ed. 58–69 (CAB International, 2010).
Zimprich, A. et al. A robust and reliable non-invasive test for stress responsivity in mice. Front. Behav. Neurosci. 8, 125 (2014).
Fitzpatrick, F. et al. Glucocorticoids in the nonobese diabetic (NOD) mouse: basal serum levels, effect of endocrine manipulation and immobilization stress. Life Sci. 50, 1063–1069 (1992).
Veissier, I. & Boissy, A. Stress and welfare: two complimentary concepts that are intrinsically related to the animal's point of view. Physiology & Behavior 92, 429–433 (2006).
Barden, N. et al. Antidepressant action of agomelatine (S 20098) in a transgenic mouse model. Prog. Neuropsychopharmacol. Biol. Psychiatry 29, 908–916 (2005).
Qin, M. & Smith, C.B. Unaltered hormonal response to stress in a mouse model of fragile X syndrome. Psychoneuroendocrinology 33, 883–889 (2008).
Kedia, S. & Chattarji, S. Marble burying as a test of the delayed anxiogenic effects of acute immobilisation stress in mice. J. Neurosci. Methods. 233, 150–154 (2014).
Swiergiel, A.H. & Dunn, A.J. Feeding, exploratory, anxiety- and depression-related behaviors are not altered in interleukin-6-deficient male mice. Behav. Brain. Res. 171, 94–108 (2006).
Lee, D.L. et al. Hypertensive response to acute stress is attenuated in interleukin-6 knockout mice. Hypertension 44, 259–263 (2004).
Lee, D.L., Webb, R.C. & Brands, M.W. Sympathetic and angiotensin-dependent hypertension during cage-switch stress in mice. Am. J. Physiol. Regul. Integr. Comp. Physiol. 287, R1394–1398 (2004).
van Bogaert, M.J. et al. Mouse strain differences in autonomic responses to stress. Genes Brain Behav. 5, 139–149 (2006).
Greenberg, N., Carr, J.A. & Summers, C.H. Causes and consequences of stress. Integr. Comp. Biol. 42, 508–516 (2002).
Ullman-Cullere, M.H. & Foltz, C.J. Body condition scoring: a rapid and accurate method for assessing health status in mice. Lab. Anim. Sci. 49, 319–323 (1999).
Pohorecky, L.A., Baumann, M.H. & Benjamin, D. Effects of chronic social stress on neuroendocrine responsiveness to challenge with ethanol, dexamethasone and corticotropin-releasing hormone. Neuroendocrinology 80, 332–342. (2004).
Barsy, B., Leveleki, C., Zelena, D. & Haller, J. The context specificity of anxiety responses induced by chronic psychosocial stress in rats: a shift from anxiety to social phobia? Stress 13, 230–237 (2010).
Davis, A., Maney, D. & Maerz, J. The use of leukocyte profiles to measure stress in vertebrates: a review for ecologists. Funct Ecol 22, 760–772 (2008).
Cupps, T.R., Edgar, L.C. & Fauci, A.S. Corticosteroid-induced modulation of immunoglobulin secretion by human B lymphocytes: potentiation of background mitogenic signals. J. Immunopharmacol. 4, 255–263 (1982).
Swan, M.P. & Hickman, D.L. Evaluation of the neutrophil-lymphocyte ratio as a measure of distress in rats. Lab Anim. (NY) 43, 276–282 (2014).
Harris, J.G., Flower, R.J. & Perretti, M. Endogenous corticosteroids mediate the neutrophilia caused by platelet-activating factor in the mouse. Eur. J. Pharmacol. 283, 9–18 (1995).
Kaufman, J. Diseases of the adrenal cortex of dogs and cats. Mod. Vet. Pract. 65, 429–434 (1984).
Munoz, M.C., Doreste, F., Ferrer, O., Gonzalez, J. & Montoya, J.A. Pergolide treatment for Cushing's syndrome in a horse. Vet. Rec. 139, 41–43 (1996).
Klein, B. et al. Activation of the mouse odorant receptor 37 subsystem coincides with a reduction of novel environment-induced activity within the paraventricular nucleus of the hypothalamus. Eur. J. Neurosci. 41, 793–801 (2015).
Guzman, Y.F. et al. Social modeling of conditioned fear in mice by non-fearful conspecifics. Behav. Brain. Res. 201, 173–178 (2009).
Gong, S. et al. Dynamics and correlation of serum cortisol and corticosterone under different physiological or stressful conditions in mice. PLoS One 10, e0117503 (2015).
HyLown Consulting, L. http://powerandsamplesize.com/Calculators/Compare-2-Means/2-Sample-Equality.
Hogg, S. A review of the validity and variability of the elevated plus-maze as an animal model of anxiety. Pharmacol. Biochem. Behav. 54, 21–30 (1996).
Charles Rivers Laboratories. C57BL/6 Mouse Clinical Pathology Data http://www.criver.com/files/pdfs/rms/c57bl6/rm_rm_r_c57bl6_mouse_clinical_pathology_data (2016).
Acknowledgements
This study was funded from internal sources. The author thanks Melissa Swan, Jessica Peveler, and Brittany Baker for their assistance with the data collection for this study.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
The author declares no competing financial interests.
Supplementary information
Supplementary information
Scatter plots of raw data used for summary statistics (PDF 206 kb)
Rights and permissions
About this article
Cite this article
Hickman, D. Evaluation of the neutrophil:lymphocyte ratio as an indicator of chronic distress in the laboratory mouse. Lab Anim 46, 303–307 (2017). https://doi.org/10.1038/laban.1298
Received:
Accepted:
Published:
Issue date:
DOI: https://doi.org/10.1038/laban.1298
This article is cited by
-
Effect of plasma-activated water on body weight, behavior, biochemical profile, intestinal microbiota, and liver and kidney histopathology in mice
Discover Applied Sciences (2025)
-
Effects of Protocatechuic Acid on Some Hematological Parameters, Brain-Derived Neurotrophic Factor and Brain Immunohistochemistry in Cisplatin-Administered Rats
Bratislava Medical Journal (2025)
-
Chronic Stress Disrupts Immune and Endocrine Axis, Inducing Persistent Behavioral Impairments in Male Rats: In Silico and In Vivo Insights
Neurochemical Research (2025)
-
Differential impact of pegfilgrastim, a recombinant human granulocyte colony stimulating factor, on the neutrophil count of male and female deer mice (Peromyscus maniculatus bairdii)
BMC Pharmacology and Toxicology (2024)
-
Characterization of white blood cell ratios in South American camelids presented at a veterinary teaching hospital
Scientific Reports (2024)


