TLR4 signal ablation attenuated neurological deficits by regulating microglial M1/M2 phenotype after traumatic brain injury in mice  Xiaolong Yao, Shengwen Liu, Wei Ding, Pengjie Yue, Qian Jiang, Min Zhao, Feng Hu, Huaqiu Zhang  Journal of Neuroimmunology  Volume 310, Pages 38-45 (September 2017) DOI: 10.1016/j.jneuroim.2017.06.006 Copyright © 2017 Elsevier B.V. Terms and Conditions

Fig. 1 TLR4 deficit improves recovery of learning and memory after CCI. (A) On the first day of testing, animals in all groups could not find the platform. After initial trials, the TLR4 KO CCI mice showed consistently shorter latencies for locating the hidden platform from Day 3 to Day 5 compared with the WT CCI mice (comparison between sham and CCI groups on Day 3 to Day 5, p<0.05; comparison between WT CCI and TLR4 KO CCI groups on Day 3 to Day 5, ⁎p<0.05; n=12; t-test). (B) In the spatial probe test, TLR4-deficient animals crossed the location of the platform more frequently than the WT mice (⁎p<0.05; n=12; one-way ANOVA). (C) Representative views of the water maze for all three groups during the spatial probe test. Journal of Neuroimmunology 2017 310, 38-45DOI: (10.1016/j.jneuroim.2017.06.006) Copyright © 2017 Elsevier B.V. Terms and Conditions

Fig. 2 TLR4 deficient mice exhibited decreased edematous brain volume. (A) MRI of the brains 24h after CCI. (B) Quantitative analysis indicated that the TLR4 KO mice had smaller edematous volumes than the WT mice (⁎p<0.05; n=5, one-way ANOVA). Journal of Neuroimmunology 2017 310, 38-45DOI: (10.1016/j.jneuroim.2017.06.006) Copyright © 2017 Elsevier B.V. Terms and Conditions

Fig. 3 Absence of TLR4 affects microglial polarization after TBI on the protein level. (A-H) In TLR4 KO mice, the expression of iNOS and TNF-α decreased at 72h and 7d after CCI, whereas the expression of Arg-1 and VEGF at both time points (72h and 7d) increased (⁎p<0.05; n=5; one-way ANOVA). (I) ELISA was used to assess the amount of IL-4 around the CCI site, and the TLR4 KO group had higher IL-4 levels at 24h, 3d and 7d (⁎p<0.05; n=5; one-way ANOVA). Journal of Neuroimmunology 2017 310, 38-45DOI: (10.1016/j.jneuroim.2017.06.006) Copyright © 2017 Elsevier B.V. Terms and Conditions

Fig. 4 Absence of TLR4 affects microglial polarization after TBI on the mRNA level. (A-B) At the mRNA level, TLR4 KO mice not only had lower expression of M1 biomarkers such as iNOS at 72h and 7d compared to WT mice but also had lower expression of IL-1β at 24h (⁎p<0.05; n=5; one-way ANOVA). (C-D) Increased expression of Arg-1 and BDNF was detected in TLR4 KO mice (⁎p<0.05; n=5; one-way ANOVA). Journal of Neuroimmunology 2017 310, 38-45DOI: (10.1016/j.jneuroim.2017.06.006) Copyright © 2017 Elsevier B.V. Terms and Conditions

Fig. 5 TLR4 deficiency enhanced the migration of microglia after CCI. (A) Immunofluorescence of the brain after CCI showing DAPI (blue) and CD11b/c (red). (A-B) Quantitative analysis revealed that the number of activated microglia increased dramatically, peaking at day 3. Compared to WT mice, the TLR4 KO mice had more activated microglia around the TBI site at 3d and 7d post-TBI (⁎p<0.05; n=5; one-way ANOVA). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Journal of Neuroimmunology 2017 310, 38-45DOI: (10.1016/j.jneuroim.2017.06.006) Copyright © 2017 Elsevier B.V. Terms and Conditions

Fig. 6 TLR4 deficiency promotes microglial polarization toward the M2 phenotype in vitro. (A) TLR4 KO primary microglia polarize to a more obvious M2 phenotype in terms of morphology, exhibiting more process and a smaller ratio. (B) TLR4 KO primary microglia have lower expression of M1 biomarkers but greater expression of M2 biomarkers and BDNF than WT mice after stimulation by IL-4 (respectively, ⁎p<0.05; t-test). Journal of Neuroimmunology 2017 310, 38-45DOI: (10.1016/j.jneuroim.2017.06.006) Copyright © 2017 Elsevier B.V. Terms and Conditions

Fig. 7 TLR4 deficiency decreases Myd88/NF-κB but increases RAC-1 and AKT activity. (A–B, E–F) At 24h after CCI, Myd88 and NF-κB p65 were dramatically increased in WT mice compared to TLR4 KO mice (respectively, ⁎p<0.05; n=4; t-test). (C–D, G–H). Moreover, RAC-1 and AKT levels in brain tissue collected from post-CCI mice at 24h were also significantly increased compared to sham-operated WT and TLR KO mice. The expression of active RAC-1 and phospho-AKT in the brain was also investigated at 24h. Significant increases in RAC-1 and p-AKT levels were observed in the brains of TLR4 KO mice compared to WT mice subjected to TBI (respectively, ⁎p<0.05; n=4; t-test). Journal of Neuroimmunology 2017 310, 38-45DOI: (10.1016/j.jneuroim.2017.06.006) Copyright © 2017 Elsevier B.V. Terms and Conditions

Journal of Neuroimmunology 2017 310, 38-45DOI: (10. 1016/j. jneuroim Copyright © 2017 Elsevier B.V. Terms and Conditions