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.