ELEVATED MIDBRAIN INTERLEUKIN-6 AND IMPAIRED GOAL-DIRECTED BEHAVIOR IN OLD C57BL/6J MICE AFTER HINDLIMB UNLOADING
Authors: Matsubara *Yosuke , Mizuno Keita , Ikeda Yoshiki , Yakabe Mitsutaka , Ogawa Sumito 
Published: February 2025

Abstract

Background
Disuse syndrome can cause not only a decline in physical function in the older people but also psychiatric symptoms such as depressive state. Although the effects of hindlimb unloading, an in vivo model of disuse syndrome, on physical functions have been widely studied, there is insufficient evidence regarding emotional state in old animals.

Aim
This study focuses on the effects of disuse due to hindlimb unloading on emotional state in old C57BL/6J mice.

Method
Old Male C57BL/6J mice (20-21 month old) were subjected to 2-weeks of hindlimb unloading and the following behavioral tests; open field, elevated plus maze, sucrose splash and nest building. Age- matched male C57BL/6J mice without hindlimb unloading were used as sham mice. Interleukin-6 (IL-6) expression in midbrain was measured by ELISA and immunohistochemistry.

Results
Old mice 7-days after hindlimb unloading displayed the decrease of nestlet score compared to sham mice in the nest building test. In this study, we also observed the reduction of locomotor activity in the open field test and the elevated plus maze test; however, no significant increase in anxiety-like behavior was observed. In the sucrose splash test, the duration and the number of grooming were not altered by hindlimb unloading. IL-6 expression in the midbrain was elevated at 7-days after hindlimb unloading and was co-expressed in microglia in the ventral tegmental area (VTA).

Discussion
Several disuse models have been developed and used to evaluate physical function, but emotional state has not been fully examined. Interestingly, we found that hindlimb not only impaired physical function, but also impaired even goal-directed behavior (nest building) after 7-days of reloading in old mice. To elucidate the mechanisms underlying the impairment of goal-directed behavior, we examined changes in the mid brain from old mice 7-days after hindlimb unloading. As a result, we detected elevated expression of IL-6 in midbrain by ELISA. In addition, we observed the co-expression IL- 6 and Iba-1 in the VTA that constitutes the brain reward circuit by immunohistochemistry. It is possible that the elevated IL-6 after hindlimb unloading is derived from microglia. On the other hand, previous study has reported increased systemic IL-6 expression by hindlimb unloading (Yakabe et al., 2018). We are currently elucidating the relationship between increased IL-6 in the VTA and decreased goal-directed behavior, and the cause of increased IL-6 expression. We expect that in vivo model of hindlimb unloading using old mice is useful for evaluating the drug efficacy against the psychological symptoms that occur in disuse syndrome in frail older people. Recently, several studies have reported that a traditional Japanese herbal medicine known as Kampo medicine attenuated physical and psychological symptoms associated with aging (Matsubara et al., 2022, Otsuka et al., 2023). We will also evaluate the efficacy of Kampo medicine in this model.

References
1.Yakabe, M. et al. (2018) "Inhibition of interleukin-6 decreases atrogene expression and ameliorates tail suspension-induced skeletal muscle atrophy." PLoS One 13(1), e0191318. doi: https://doi.org/10.1371/journal.pone.0191318
2.Matsubara, Y. et al. (2022) "Ninjin'yoeito, a traditional Japanese medicine, attenuates age-related deficits of muscle performance, self-care motivation, and body temperature in C57BL/6 mice." Bioscience, Biotechnology, and Biochemistry 86(7): 895-901. doi: https://doi.org/10.1093/bbb/zbac052
3.Otsuka, S. et al. (2023) "Analysis of the Effects of Ninjin’ yoeito on Physical Frailty in Mice." International Journal of Molecular Sciences 23(19), 11183. doi: https://doi.org/10.3390/ijms231911183