Discussion and Conclusions

Traumatic brain injury is often referred to as a ‘silent epidemic’, causing debilitating neurological deficits to millions of people globally each year (James et al., 2019). Treatments limiting the damage caused by secondary injury processes following TBI is an area of severe unmet medical need . Medical intervention is limited to stabilization of the patient via barbiturate coma and decompressive surgical intervention. The patient has little scope to improve neurological function and an extremely decreased quality of life . After injury the neural cells die from physical damage, eliciting a sequence of events that creates a neuroinflammatory microenvironment which prevents recovery. Neurodegeneration driven by complex, chronic and dysregulated neuroinflammation is particularly detrimental to the neurological recovery after TBI . cGAS-STING-IFN signalling is increasingly being appreciated as a driver of this neuroinflammation in rodent models of TBI . Here we examine if pharmacological inhibition of STING can attenuate neuroinflammation and neurodegeneration post-TBI and lead to neuroprotection and improved behavioural outcome.
We firstly identified that C-176 can successfully inhibit STING activation in glial cells with the use BV2 microglia-like cells (Fig 1). Importantly, this identified that C-176 was targeting STING activation. We then employed a single-dose administration of this STING inhibitor in our mouse model of mild-TBI. It was evident that single-dose administration of C-176 post-TBI in our mouse model was able to confer neuroprotection. The timepoint of 30 minutes was chosen in this study to represent the ‘golden hour’ in which intervention for TBI may have the most beneficial effects. It is important to note that in developed countries such as the England and Wales, the median time to intervention for patients post-severe and moderate TBI is 0.5-0.9 hours Mice treated with C-176 displayed significantly smaller lesion areas 24h-post TBI (Fig 2). These results are comparable with previous findings by our group using STING-/- mice, which highlighted that deletion of STING resulted in neuroprotection after TBI . These results are also consistent with the work conducted by Zhang et al (2022), where administration of C-176 in a rat model of severe TBI showed attenuated neuronal loss around the lesion site 24h-post TBI. Additionally, use of C-176 in a mouse model of stroke was also found to significantly reduce infarct size 24h after stroke modelling . Our neurobehavioral findings further confirm the neuroprotective capability of C-176 post-TBI. We found that TBI-induced gait deficits were reversed following C-176 administration (Fig 3 and 4). Studies using C-176 in rodent models of severe TBI and ischemic stroke have all found similar improvements in neurological and cognitive function suggesting that this compound could be beneficial in alleviating acute neurological symptoms .
Previous studies have identified microglia to key a key source of STING activity in the CNS . We were able to confirm that STING expression was predominately located in microglial cells post-TBI (Fig 5). We are the first to assess microglial morphology following administration of C-176 and found that administration of C-176 did not elicit any morphological changes in the perilesional microglia between C-176-treated mice and their vehicle counterparts (Fig 6.). Work conducted by Zhang et al (2022) found that administration of C-176 reduced the colocalization of STING in microglia and astrocytes 32 days post-severe TBI. This suggests that there may be more significant morphological and biochemical changes may be occurring in microglia at a timepoint later than the one used in the present study (24h-post TBI). This result also highlights that early administration of a STING inhibitor may be important in limiting the morphological and biochemical changes that occur in microglia after TBI.
Mild TBI and single-dose administration of C-176 were found to alter the neuroinflammatory profile in the cortex and striatum 2 and 24h-post TBI. The anti-inflammatory effects of the C-176 post-TBI were observed acutely post-injury and had most observable attenuation of expression of pro-inflammatory genes CXCL10 and TNF-α in the striatum 2h-post injury (Fig 7Aiii,v). Previous work by our group have demonstrated that hematopoietic cells play a key role in the neuroinflammatory response in this model. Neuroinflammation is a hallmark of various neurological disorders, including TBI . Activation of the STING pathway in microglia and astrocytes contributes significantly to the production of pro-inflammatory mediators, such as TNF-α, IL-1β, and IL-6, which exacerbate neuroinflammation . The STING pathway has been shown to amplify the inflammatory response by inducing the nuclear factor-kappa B (NF-κB) pathway, a master regulator of inflammation, and inflammasome activation . Future work would investigate if C-176 is acting through these peripheral cells to elicit the neuroprotective anti-inflammatory effect we have observed.
We did not observe changes to the total protein expression of STING 2h-following TBI or C-176 administration (Sup Fig 2). Interestingly we found significantly attenuated STING, TBK1 and p-TBK1 24h-post TBI. We did not however find any significant changes between the vehicle-treated TBI mice and the C-176 treated mice. Work conducted by Zhao et al (2022) using C-176 in a rat model of severe TBI found increased p-TBK1 and TBK1 expression in isolated hippocampus 24h-post TBI and C-176 was found to significantly decrease this p-TBK1 expression. We did observe an increase in the mRNA expression of STING (TMEM173) in the cortex and striatum of the vehicle-treated TBI mice 24h-post TBI and not at 2h-post TBI. Together this highlights the temporal sensitivity of STING activation in the CNS. Future studies should evaluate STING activation at later timepoints in addition to evaluating the expression in isolated hippocampus to further understand the effect of C-176 on STING activation in the CNS post-TBI.
Future studies will explore the pharmacokinetics of C-176 to address if its neuroprotective anti-inflammatory is occurring solely in the CNS or if it is acting on STING in peripheral immune cells. Furthermore, C-176 has been recorded to possess a short half-life when used in mice, thereby reinforcing the need to explore dosage frequency, concentration and timing to achieve optimal therapeutic effect (Haag et al., 2018). Future studies can provide more insight into the therapeutic window of using STING inhibitors by broadening the dosage concentration, frequency, timing, and route of administration tested in this mouse model.
This study has demonstrated a neuroprotective role of small-molecule inhibition of STING following mild TBI in mice, supporting additional studies investigating the therapeutic intervention of this pathway to address the severe unmet medical need of limiting the cell damage and functional deficits in TBI patients. The social and economic burden of brain injury is considerable to the community. New pharmacological tools are urgently needed to help delineate the neuroinflammatory pathways post TBI and determine the most advantageous therapeutic window for the acute treatment of TBI.