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The effects of stress on the immune system and depression-like behaviors in mice Marcie Turner Introduction Advisor: Dr. Spilatro Effects of Stress on Leukocyte Counts Stress, a normal component of everyday life, has been shown to be associated with multiple physiological changes. The changes that occur in the body during times of acute stress enable the organism to cope immediately with a potential threat in the environment (Bartolomucci, 2007, 30). However, when a stressor is chronic, many damaging disruptions can take place in the body. For example, chronic stress has been associated with suppression of the immune system, particularly decreases in white blood cell counts and sensitivity to mitogens (Bartolomucci, 2007, 35). Increases in depression-like symptoms have also been associated with chronic stress (Bowers et al. , 2008, 106). In fact, decreased white blood cell concentrations have been linked to depression, indicating a relationship between the immune system, stress, and psychological states (Coe and Laudenslager, 2007, 1002). The purpose of this study was to observe and quantify the effects of stress on the immune system and psychological states by measuring physiological and behavioral changes in mice. Mice were subjected to chronic stress by restraint, and measurements were taken of their leukocyte populations, immune responsiveness, weight fluctuations, and depressionlike behaviors. It was hypothesized that chronic stress would have immunosuppressive effects and would be associated with increased depression-like behaviors in the mice. Methods It was predicted the experimental group would exhibit a significant decrease in their total leukocyte counts from pre-stress to post-stress. Mice were lightly anesthetized with ether, and blood was drawn from their submandibular veins using metal lancets. Blood was collected in heparin-coated vials. The blood was diluted and red blood cells were lysed using Unopette reservoirs. The white blood cells in the reservoir mixture were then counted using a hemacytometer to determine the total leukocyte count for each mouse. To determine the differential leukocyte counts, blood smears were created for each mouse. A drop of blood was smeared onto a cleaned glass microscope slide and allowed to dry. The slide was then stained with Wright-Giemsa stain and examined with a microscope. The percentages of lymphocytes and granulocytes counted in the first 100 observed leukocytes was applied to the total leukocyte count for each mouse to obtain the total number of lymphocytes and granulocytes. The average change in the total and differential leukocyte counts for the experimental group were then compared to the average change in the control Control Group Experimental Group p Value group. Eight C 57 BL 6 mice were divided into a control group and an experimental group. Prior to the experiment, the mice were allowed to habituate to their new environment for 7 days. Pre-measurements were then taken from each group. For the next 25 days, the experimental group was subjected to chronic stress by being placed in restraints for 4 hours a day. After the 25 -day stressing period, post-measurements were taken from each group. The average changes from the pre to post-stress measurements were then compared between the control and experimental groups. The pre and post-measurements taken from each group included weight measurements, total and differential leukocyte counts, and depression-like behavior measurements (the Porsolt Forced Swim Test). Measures of the mice’s delayed-type hypersensitivity (DTH) were taken as a post-stress only measurement after the 25 -day stressing period. DTH measurements are an indication of how well the mice’s immune systems are responding to a T-cell mitogen. Swelling at the site of the mitogen’s application indicates an immune response is occurring. The greater the swelling, the greater the immune response. Figure 1. Chronic stressing of mice. Mice were placed in plastic restraints for 4 hours/day for 25 days to induce stress. Effects of Stress on Weight Methods Materials and mice in the experimental group would not exhibit as It was predicted that Methods much growth as the control group by the end of the 25 -day stressing period. Results anesthetized with anhydrous ether and then weighed on a Mice were lightly mass scale. The average change in weight for the control group was then compared to the average change for the experimental group. Results Average weight change (grams) 0 ± 1654 -1500 ± 2596 0. 426 Average change in lymphocyte counts (cells/μl) -168 ± 981 -1709 ± 1243 0. 139 Average change in granulocyte counts (cells/μl) General Methodology Average change in total leukocyte counts Results (cells/μl) 168 ± 1436 209 ± 1501 0. 973 Table 2. Average change (post minus pre-stress) in total leukocyte counts, lymphocyte counts, and granulocyte counts of the control vs. the experimental group. For the total leukocyte counts, the control group did not see an average change, while the experimental group saw an average decrease of 1500 leukocytes/μl. For the differential leukocyte counts, the control group exhibited an average decrease of 168 lymphocytes/μl, while the experimental group had an average decrease 1709 lymphocytes/μl. The control group had an average increase of 168 granulocytes/μl, while the experimental group had an average increase of 209 granulocytes/μl. Using an independent-measures t-test, it was determined that there was no significant difference in the changes in total and differential leukocyte counts between the control and experimental groups. A B 2. 62 ± 1. 16 Experimental Group 2. 03 ± 0. 312 Figure 2. Images taken of the mice’s leukocytes. A: lymphocyte; B: neutrophil (a type of granulocyte); and C: granulocyte Effects of Stress on Depression-Like Behaviors Methods It was predicted the experimental group would exhibit a significant increase in depression-like behaviors from pre-stress to post-stress. Mice were subjected to the Porsolt Forced Swim Test to measure their depression-like behaviors (the amount of time they spend floating). Mice were individually dropped into a bucket filled with 8 inches of 24ºC water. The mice were allowed to swim for 6 minutes. During the first 2 minutes, no measurements were taken. However, during the last 4 minutes, the amount of time the mice spent floating was recorded. An increase in the amount of time spent floating indicates an increase in depression-like behaviors (Kurtuncu, et al. , 2005, 28). The changes in the amount of time the experimental group spent floating were then compared to the changes in the control group’s floating time. Results Control Group C Control Group Experimental Group p value -19. 7 ± 100. 1 22. 0 ± 47. 4 0. 489 p value 0. 364 Table 1. Average weight change (post minus pre-stress) of the control group vs. the experimental group. From pre-stress to post-stress the control group grew 2. 62 g on average while the experimental group grew an average of 2. 03 g. Using an independent-measures t-test, it was determined that there was no significant difference in the changes in weight between the control and experimental groups. Average change in time spent floating (seconds) Table 3. Average change (post minus pre-stress) in the amount of time spent floating in the control group and experimental group. On average, the control group saw a decrease of 19. 7 seconds in the amount of time they spent floating, while the experimental group saw an increase of 22. 0 seconds. Using an independentmeasures t-test, it was determined that there was no significant difference in the changes in frequency of depression-like behaviors (amount of time spent floating) between the experimental and control groups. Effects of Stress on Immune Functioning (Delayed-Type Hypersensitivity) Methods It was predicted the control group would exhibit greater immune functioning than the experimental group at the end of the 25 -day stressing period. Baseline thicknesses of the mice’s ears were measured with a pressure-gauge one day after the 25 -day stressing period. Mice’s right ears were then sensitized to 2, 4 dinitro-1 -fluorobenzene (DNFB), a T-cell mitogen, in an olive oil/acetone vehicle via a pipet. The left ears served as controls and were exposed to the vehicle only. Twenty-five days later, the mice’s right ears were again exposed to the DNFB and the left ears were exposed to the vehicle via pipet. For the next 7 days, the thicknesses of the ears were measured (Bowers, et al. , 2008, 107 -108). For the duration of the procedure, the experimental group continued to be placed in the restraints for 4 hours a day. The changes in ear thicknesses in the control group were then compared to the changes in the experimental group. Figure 3. Average ear Results thicknesses for the control group (teal) and experimental group (purple) for baseline, induction, and days 1 -7 post-induction. On average, the 1 2 3 4 5 6 7 li ctio thicknesses of the ears were greater se a ndu B I in the control group than the ne n experimental group. Using an independent-measures t-test for each day, it was determined that there was no significant difference in the increase in ear thickness between the control group and experimental group for any day (p=0. 451, 0. 159, 0. 396, 0. 224, 0. 499, 0. 280, 0. 393, 0. 435, respectively). Conclusions In conclusion, the obtained results do not support the hypothesis that chronic stress has immunosuppressive effects or is associated with increased depression-like behaviors in mice. Previous research that examined the effects of stress showed an association between stress and decreases in leukocyte counts (Bowers et al. , 2008, 109) and decreases in weight (Cyr et al. , 2007, 61). While the results of the present study indicate that there was not a significant effect of stress on the immune system or depression-like behaviors in mice, trends were observed: (1) The control group grew slightly more than the experimental group, (2) the experimental group exhibited a slight decrease in their total leukocyte counts, (3) the experimental group did not exhibit as much swelling in their ears as the control group during the DTH test (indicating a lower immune functioning in the experimental group), (4) and the experimental group showed a slight increase in the amount of time they spent floating during the Porsolt Forced Swim Test (indicating an increase in depression-like behaviors). It is important to reiterate, however, that none of these trends were statistically significant, and therefore the hypothesis is not supported. Future research building on the present experiment should aim to increase the statistical power of the study. The small sample size in this study and the large variation within this sample would make it difficult to detect a statistically significant difference between the control and experimental groups if one were to exist. In addition, future studies could attempt to increase the stress exposure of the mice and prevent habituation to the stressor, as these are potential reasons why to significant effect may not haveto those who assisted me in this I would like a express my utmost appreciation been observed. Acknowledgments project. I greatly appreciate all that Dr. Spilatro has done for me during my time at Marietta College, not only as my Capstone supervisor, but also as my academic advisor. I would also like to thank Dr. Doerflinger for assisting me with my statistical analyses. In addition, I greatly appreciate the support of the entire Biology Department and my fellow Capstone students. Literature Cited Bartolomucci, A. 2007. Social stress, immune functions and disease in rodents. Frontiers in Neuroendocrinology 28: 28 -49. Bowers SL, Bilbo SD, Dhabhar FS, Nelson RJ. 2008. Stressor-specific alterations in corticosterone and immune responses in mice. Brain, Behavior, and Immunity 22: 105 -113. Coe CL, Laudenslager ML. 2007. Psychosocial influences on immunity, including effects on immune maturation and senescence. Brain, Behavior, and Immunity 21: 1000 -1008. Cyr NE, Earle K, Tam C, Romero LM. 2007. The effect of chronic psychological stress on corticosterone, plasma metabolites, and immune responsiveness in European starlings. General and Comparative Endocrinology 154: 59 -66. Kurtuncu M, Luka LJ, Dimitrijevic N, Uz T, Manev H. 2005. Reliability assessment of an automated forced swim test device using two mouse strains. Journal of Neuroscience Methods 149: 26 -30.