Of further interest would be whether this response would subside upon a much longer period of common handling procedures. Pathway analysis demonstrated that handling procedures led to altered expression of immune function related genes with a general decrease in multiple Kaempferide complement system genes as well as a small number of additional immune function related genes. Collectively, these changes likely indicate a minor immune perturbation in the liver following handling procedures. The effects of handling laboratory animals and the resulting alterations in immune response has been documented previously. In general, mild and or acute stressors enhance humoral immune Echinacoside responses while severe or long-term stressors can be immunosuppressive. Stress-related changes in the functional capacity of the immune system have been discussed in a recent review article and include decreased immune cell numbers and functions, decreased response to vaccinations, and increased immunosuppressive pathways and proinflammatory cytokines. Effects of chronic stress on immune function have been well-documented in humans and include effects on both the innate and adaptive immune system. As summarized by Gouin, specific findings include attenuated responses to vaccination, poorer wound healing, exaggerated release of inflammatory mediators, and premature aging of the immune system. Endogenous glucocorticoids are essential for antibody responses and adrenalectomy or anti-glucocorticoid receptor treatment decreases IgM and IgG responses which can be restored by corticosterone replacement. Activation of the complement cascade occurs rapidly and is reported following acute psychological stress in humans with increased circulating levels of C3a, C5a, and Factor Bb. Thus, perturbation of immune system related genes in the liver and a post-handling increase in corticosterone levels at day 2 are consistent with previous reports of acute stress-related effects. Liver gene expression changes were measured in necropsied livers on the day following dose administration and tail vein blood collections. Therefore, the decreased expression of complement genes in light of an increase in other immune genes may reflect a transcriptional feedback loop. In conclusion, the results reported here highlight gene expression changes that occur in laboratory 
animals during routine handling procedures. These gene expression changes can be viewed as baseline changes upon which additional transcript level alterations resulting from vehicle and/or compound related effects are layered. One can envision the composition of these effects as depicted in Figure 1. This simple additive model illustrates how transcriptional effects at various levels on preclinical animals may add up. If control animals in a study are subjected to the same handling procedures these gene expression changes may be normalized as vehicle related changes are normalized. However, more complex models and interactions with vehicle and/or compound related gene changes cannot be excluded and may warrant further investigation. Synergistic or subtractive interactions with compound mediated effects may confound interpretation of gene changes in a study.