Y. H. Chun1, J. Alonso-Escalante3, B. P. Soares2, W. Fulton1, C. P. Sodhi11, D. J. Hackam1, I. W. Nasr1 1Johns Hopkins University School Of Medicine,Pediatric Surgery,Baltimore, MD, USA 2Johns Hopkins University School Of Medicine,Radiology,Baltimore, MD, USA 3Allegheny General Hospital,General Surgery,Pittsburgh, PA, USA
Introduction:
TBI induces a robust neuroinflammatory response that activates the innate immune system. We sought to investigate the effect of pharmacologically inhibiting the innate immune signaling pathways using a novel TLR4 inhibitor, C34 in a murine TBI model.
Methods:
A murine controlled cortical impact TBI model was used. Three experimental groups: (1)wild-type (WT) control, (2)WT+C34 (1-day treatment), (3)WT+C34 (7-day treatment). MRI measured lesion volume. Real-time PCR quantified cytokine gene expression. Flow Cytometry assessed microglial and monocyte activation. Behavioral testing was initiated on post-injury day (PID)7 and 28. Statistical analysis: Student’s T-test and One-way ANOVA.
Results:
Brain injury volumes in WT (2.1±1.687mm3, n=11) compared to WT with 7-day C34 treatment (3.2±2mm3, n=7) were not significantly different, However, the 1-day treatment with C34 (4.5±1.1mm3, n=7) had significantly larger lesion volumes on MRI on PID 7. On PID35, MRI showed no significant difference when C34 was given for 1 day; but lesion size is significantly increased when C34 was given over 7 days (p=0.01). The WT+C34 group had significantly decreased pro-inflammatory cytokines (TNFα, p=0.0010, IL-1, p=0.03) and anti-inflammatory cytokines (IL-10 p=0.0067, TGFβp=0.004) compared to WTs on PID 1. Apoptosis markers, Caspase (p=0.0001), Bad (p=0.0001) and Bax (p=0.02) were significantly downregulated in the treatment group on PID1; as were necroptosis markers MLKL (p=0.02), RIPK1 (p=0.0001), and RIPK3 (p=0.0001). There was no difference in gene expression on PID7 or PID35. Behavioral testing (water-maze) showed that the 1-day treatment group showed significant improvement in latency during week 1 testing and platform entries on week 4 testing (p=0.026). Flow Cytometry showed that infiltrating monocytes are significantly increased with C34 1-day treatment on PID 7 (p=0.0007). A difference in monocyte to microglial population ratio was detected only on PID 7 and not on PID 1 nor on 35.
Conclusion:
Our findings demonstrate that TLR4 plays a role in the early pathogenesis of the TBI-induced neuroinflammatory response. Early, short-term pharmacologic inhibition of the TLR4 pathway with C34 attenuates the inflammatory response and leads to improved neurocognitive outcomes compared to the prolonged 7-day treatment. Moreover, the increase in infiltrated monocyte population may play an important role in neurorecovery after TBI.
