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Hindlimb unloading (HU), widely used to simulate microgravity effects, is known to induce a stress response. However, as single-housed animals are usually used in such experiments, social isolation (SI) stress can affect experimental results. In the present study, we aimed to delineate stressful effects of 3-day HU and SI in mice. Three animal groups, HU, SI, and group-housed (GH) control mice, were recruited. A comprehensive analysis of stress-related markers was performed using ELISA, western blotting, and immunohistochemistry. Our results showed that blood corticosterone and activity of glucocorticoid receptors and cAMP response element-binding protein (CREB) in the hippocampus of SI and HU animals did not differ from GH control. However, SI mice demonstrated upregulation of the hippocampal corticotropin-releasing hormone (CRH), inducible NO synthase (iNOS), vesicular glutamate transporter 1 (VGLUT1), and glutamate decarboxylases 65/67 (GAD65/67) along with activation of Fos-related antigen 1 (Fra-1) in the amygdala confirming the expression of stress. In HU mice, the same increase in GAD65/67 and Fra-1 indicated the contribution of SI. The special HU effect was expressed only in neurogenesis attenuation. Thus, our data indicated that 3-day HU could not be characterized as physiological stress, but SI stress contributed to the negative effects of HU.

In search of stress: analysis of stress-related markers in mice after hindlimb unloading and social isolation