Treatment with 750nmol C-176 post-injury rescues gait disturbances in mice 24h-post TBI
This study conducted a quantitative temporal gait analysis using to gauge alterations in neurobehavioral activity following TBI in mice. Changes in gait parameters were measured using DigiGaitâ„¢ (parameter definitions provided in Table 1). C-176 administration attenuated TBI-induced impairments in stride frequency (Fig 3E and Fig 4E) and forelimb stance width (Fig 3D), in addition to restoring time spent in forelimb and hindlimb stance phase and stride phase to values similar to sham and statistically longer than vehicle-treated TBI mice (Fig 3B-C and Fig 4B-C).
Furthermore, the vehicle treated TBI mice treated displayed an elevated gait symmetry ratio compared to sham (Fig 3F). A gait symmetry value of 1 indicates co-ordinated movement in both the left and right side of the body. The gait symmetry measured in mice treated with C-176 after TBI was closer to 1 compared to vehicle-treated mice and was not statistically different to sham mice (Fig 3F). The sham and vehicle treated TBI mice had similar hindlimb stance width and mice treated with C-176 after TBI exhibited a narrower hindlimb stance width (Fig 4E).

C-176 on STING expression after TBI

To evaluate the effect of single-dose C-176 administration on STING-mediated neuroinflammation, 2h and 24h post-TBI, protein and mRNA expression of STING (TMEM173) and downstream mediators TBK1 and IRF3 was measured using western blot and qPCR. Mice treated with C-176 did not display significant alterations to the expression of STING mRNA expression in the cortex and striatum when compared to vehicle-treated mice at both 2h and 24h post-TBI (Sup Fig 2F, Sup Fig 3F). IRF3 mRNA expression in the striatum of C-176 treated mice significantly increased 2h-post TBI when compared to vehicle-treated mice (Sup fig. 2G) and remained relatively unchanged 24h post-TBI (Sup Fig 3G). Western blot analysis revealed that there were no significant changes in cortical STING protein expression observed 2h-post TBI in C-176-treated mice compared to sham or vehicle-treated mice (Sup fig 2C). Interestingly, C-176 treated mice displayed significantly attenuated total TBK1 expression in the striatum 2h-post-TBI compared to sham (Sup Fig 2C). Both C-176 and vehicle treated mice had a reduction in the protein expression of STING and total and phosphorylated TBK1 in the cortex and striatum compared to sham 24h post-TBI (Sup Fig 3C-E).