Abstracts

Rationale: Traumatic Brain Injury (TBI) is associated with significant physical, cognitive,
and psychosocial challenges. Our previous research has revealed heightened neural
stem cell (NSC) proliferation within the hippocampal dentate gyrus following
experimental concussive brain injury in rodent models. These newly generated neurons
exhibit aberrant migration, accelerated maturation, and premature circuit integration.
Suppression of post-TBI neurogenesis has shown promise in mitigating long-term
neurogenic loss and the onset of epilepsy. Toll-like receptor 4 (TLR4), an innate immune
receptor, is expressed in various central nervous system cell types, including neurons,
microglia, and astrocytes. TLR4 is also expressed in neural precursor cells in the adult
dentate gyrus. TLR4 activation contributes to neuroinflammation and
neurodegeneration, implicating its dysregulation in neurological disorders including TBI.
Our studies suggest that TLR4 inhibition can reduce memory deficits and risk for
epilepsy after TBI. Here we examined whether TLR4 signaling regulates neurogenesis
observed after brain injury.


Methods: Adult male and female C57BL6/J mice aged 8 to 10 weeks underwent
moderate lateral fluid percussion injury (FPI, at 1.8 atm) or sham surgery and were
examined 48 hours post-injury. EdU (5-ethynyl-2'-deoxyuridine), a thymidine analog,
was used to label proliferating cells. To assess the impact of FPI on neurogenesis, EdU
(50 mg/kg) was administered via three intraperitoneal injections after injury. The FDA-
approved TLR4 antagonist, CLI-095 (Tak 244, 0.5 mg/kg, i.p), or vehicle was injected 2
hours post-injury to assess the role of TLR4 signaling on neurogenesis. Animals were
sacrificed 48 hours post-injury. Brain slices from the vehicle and CLI-095 treated sham
and FPI mice were stained for EdU. Counts of Edu-positive cells were quantified using
Fiji software.


Results: EdU pulse labeling studies after FPI identified an increase in the number of
Edu labeled neurons in the dentate gyrus of mice 2 days after FPI, compared to sham
controls (numbers of Edu cells/section: sham: 6.034 ± 0.4107, FPI: 22.12 ± 0.9432,
p<0.05 by t-test, n=3 mice each). Treatment with CLI-095 reduced the post-injury
increase in Edu labeled cells to control levels.


Conclusions: Our findings demonstrate that treatment with the TLR4 antagonist CLI-
095 effectively reduces the abnormal increase in neurogenesis after TBI, restoring cell
proliferation levels to those observed in sham controls. These results suggest that
targeting TLR4 signaling may offer a promising therapeutic strategy to moderate TBI-
induced neurogenic abnormalities and reduce the risk of associated long-term
neurological deficits, such as epilepsy and cognitive impairments.

Funding: NIH R01NS097750 & R01NS069861 to V.S and CIRM TRANSCEND to R. J