1. General Methodology Chronic Stress Restraint stress has been shown to be the most effective method to elicit a chronic stress response in mice 4. Sleep Deprivation The “Flower Pot Technique” was used to sleep deprive the mice. They were sleep deprived for 24 hours a day for 4 consecutive days. The platforms were not big enough for the mice to sleep on and the mice would not sleep in the water, thus a sleep deprivation state was achieved. Blood Collection In order to perform the total white blood cell count and the differential white blood cell count, it was necessary to collect blood from the mice. Figure 1. Mice were placed in plastic restraints for either 4 hours or 8 hours per day according to their assigned experimental group. The stressing occurred everyday for 25 days. Jennifer Valentic Advisor: Dr. Spilatro The effects of stress and sleep deprivation on the immune system of mice Literature Cited 1) Bartolomucci, A. 2007. Social stress, immune functions and disease in rodents. Frontiers in Neuroendocrinology 28: 28-49. 2) 2) Kemeny ME, Schedlowksi M. 2007. Understanding the interaction between psychological stress and immune-related diseases: A stepwise progression. Brain, Behavior, and Immunity 21: 1009-1018. 3) Benca R, Quintans J. 1997. Sleep and host defenses: a review. Sleep 20(11): 1027-1037. 4) 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. Conclusions Based on the previous research outlining the detrimental effects that stress and sleep deprivation can have on the immune system, it was predicted in this experiment that the order of most to least significant detrimental effects elicited by the experimental groups would be sleep deprivation, followed by 8 hour stress, then 4 hour stress. The results; however, did not show significant differences and therefore did not support the hypothesis. The failure to see significance might be explained by the small sample sizes, the large variation within each group, and the potential for an unknown environmental stimulus that was affecting the control mice in addition to the experimental mice, such as lighting or temperature of the living quarters. it is also possible that the mice, particularly in the stress groups, became habituated to the stressor and it no longer became a stressful event. Finally, there is a possibility that stress and sleep deprivation do not, in fact, have detrimental effects on the mouse immune system. A future experiments might include a better control group that would allow for more accurate comparisons to be made between the control and sleep deprivation groups. Control group data in this experiment was collected within the 25 day period, whereas the sleep deprivation data was collected within a 4 day period. Experimental Design Sixteen female C57BL6 mice were divided into four groups: In order to quantify the effect of each condition on the immune system, analytical tests were performed both before and after the stress or sleep deprivation conditions. Fluctuations in weight were examined and several white blood cell counts were performed, including a total white blood cell count as well as a differential white blood cell count that examined fluctuations in the populations of two categories of white blood cells: lymphocytes and granulocytes. In addition, immunocytology was performed which involved the labelling of B cells with fluorescently tagged antibodies. A delayed type hypersensitivity test was also attempted; however, despite several procedural modifications, the desired response was never achieved. Introduction In recent years, several fields of medicine, including psychology, neurology, neuroendocrinology, and immunology have been linked in a field called psychoneuroimmunology 1. This field was born out of research that suggests all of the aforementioned body systems are interconnected, specifically in regards to the body’s response to stress 1. Stress can come in many forms and can have a variety of effects on the body. If the stress only lasts several minutes to several hours, also known as acute stress, the body can experience immuno-activating effects 1. However, if the organism is continuously exposed to a stressor for an extended period of time, also known as chronic stress, the body can experience negative effects, such as immuno-suppression 1. In fact, there is a significant amount of literature that documents the negative effects that stress can have on the body’s innate and adaptive immune responses 2. For example, studies have shown that chronically stressed mice have a decreased leukocyte count and, as a result, a decreased immune response to pathogens 1. In addition to chronic stress, sleep deprivation has been shown to have similar detrimental effects on the immune system, leaving the organisms more susceptible to attack by invading pathogens 3. This experiment was a follow up of an experiment done by Marcie Turner (class of ‘09) who quantified the effects of 4 hour stress on immune system of mice. The purpose of this study was to determine the effects of chronic stress and sleep deprivation on the mouse immune system. It was predicted that the order of most to least significant detrimental effects on the immune system elicited by the experimental groups would be sleep deprivation, then 8 hour stress, then 4 hour stress. Effects of Stress and Sleep Deprivation on White Blood Cell Counts In order to perform the total white blood cell counts, blood was injected into Unopette reservoirs so that the red blood cells were lysed and the white blood cells could be easily counted using a hemacytometer. Table 2. The data was reported as the percent change in total white blood cells from before to after the stress or sleep deprivation. A one way ANOVA was performed and revealed that the p value was >0.05, thus there were no significant interactions between any of the groups. Blood smears were created to perform the differential white blood cell counts by smearing a small drop of blood across a microscope slide. The blood was allowed to dry and the slides were stained with Wright-Giemsa stain so that the nuclei of the white blood cells were distinguishable. The slides were viewed under a microscope and the first 100 white blood cells that were encountered were classified as a lymphocyte or a granulocyte.. Table 3. The data was reported as the average percent change in both lymphocytes and granulocytes from before to after following the stress or sleep deprivation. A one way ANOVA reported a p value of <0.05. Table 4. A Tukey Post-Hoc test was performed that compared the control group with each experimental group. This determined which interaction was responsible for the significant p value in Table 3. The test revealed that there was a significant change in lymphocytes and granulocytes in the sleep deprivation group. This method; however, did not allow conclusions to be drawn as to how sleep deprivation affected the total lymphocyte and granulocyte populations because a relative count was performed, as opposed to a total count of lymphocyte and granulocyte populations. Immunocytology Lysine charged slides were incubated with FITC conjugated CD19 antibodies in order to label B cell lymphocytes. An antifade solution, containing DNA staining DIAP, was applied post incubation. The slides were then observed using a dark field microscope. Definitive CD19 labelling of B cells was not observed; however DNA labelling revealed a variety of differently shaped nuclei. Acknowledgments I would like to express my utmost appreciation to Dr. Spilatro for his assistance as both my academic and capstone advisor and Dr. Brown for his assistance as our capstone class advisor. I would also like to thank Kate Knight for her assistance with my statistical analysis, the entire Biology Department, and my family, friends, and classmates for their continuous support. Control GroupStress Group 1 (4 hours) Stress Group 2 (8 hours) Sleep Deprivation Group 444 4 Figure 3. An animal lancet was used to puncture the facial vein in the mouse. Blood was collected in a heparin coated vial to prevent clotting. Figure 2. Platforms were built for the mice to sit on and 0.5 inches of water was placed in the bottom of the cage. Effects of Stress on Weight The mice were lightly anesthetized in ether and were weighed on an analytical scale. Table 1. The data was reported as a change in weight from before to after the stressing condition. A one way ANOVA was performed. The p value (0.530) is greater than the α value of 0.05, thus it can be determined that there were no significant fluctuations in the weight of the mice. Note: Sleep deprivation data was not included. The weight data for the control group was collected after the 25 day period, whereas the weight data for the sleep deprivation group was collected after the 4 day period, thus there was no true control data to which the sleep deprivation data could be compared. Control Group 4 Hour Stress 8 Hour Stress p value Average weight change (grams) 4.3653.4053.8520.530 AB 1 2 Figure 4. Images of leukocytes. A: Lymphocyte B. Types of granulocytes 1-Neutrophil. 2- Basophil C: Eosinophil Control Group 4 Hour Stress 8 Hour Stress Sleep Deprivation p value Average change in lymphocyte count (% change) 19.25% ±10.50% 27.25% ±11.09% 16.75% ±4.00% -16.25% ±12.71 <0.0005 Average change in granuolcyte count (% change) -19.25% ±10.50% -27.25% ±11.09% -16.75% ±4.00% 16.25% ±12.71 <0.0005 ControlLymphocytesControlGranulocytes 4 Hour Stress 0.686 4 Hour Stress 0.686 8 Hour Stress 0.985 8 Hour Stress 0.985 Sleep Deprivation 0.002 Sleep Deprivation 0.002 C Figure 4. Nuclei of 2 different white blood cells whose DNA was stained with DIAP. A. Solid, circular nucleus. B. Bilobed nucleus. A B One Way ANOVA of Weight Fluctuation Data One Way ANOVA of Total WBC Data Tukey Post- Hoc Test of Differential WBC Count Data One Way ANOVA of Differential WBC Data Control Group 4 Hour Stress 8 Hour Stress Sleep Deprivation p value Average change in total WBC (% change) -25.56% ± 27.00% -20.53% ± 30.70% -10.98% ± 58.48% -27.41% ±18.31 0.759 www.medipoint.com