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. 2022 Aug 22;15:992627. doi: 10.3389/fnmol.2022.992627

FIGURE 3.

FIGURE 3

Direct TLR4 activation with LPS and disulfide HMGB1 in the FSC model induces neuroimmune signaling leading to the loss of cholinergic neurons. (A) ELISA assessment of HMGB1 release in basal FSC media revealed that direct application of LPS (100 ng/mL; 24 h) caused an approximate 2.5-fold increase in media levels of HMGB1 relative to vehicle-treated CONs (n = 4 wells/group, two-tailed t-test). (B) Modified unbiased stereology revealed that direct application of the TLR4-specific disulfide redox form of HMGB1 (0.1 μg/mL; 24 h) to FSC media caused a 37% (±7%) reduction of ChAT + IR neurons relative to vehicle-treated CONs (n = 4 wells/group, two-tailed t-test). (C) Modified unbiased stereology revealed that direct application of LPS to FSC media caused an approximate 1.5-fold increase of TLR4 + IR cells in the basal forebrain relative to vehicle-treated CONs. Scale bar = 50 μm (n = 4 wells/group, two-tailed t-test). (D) Modified unbiased stereology revealed that direct application of LPS to FSC media caused an approximate 2.5-fold increase of pNFκB p65 + IR cells in the basal forebrain relative to vehicle-treated CONs (n = 4 wells/group, two-tailed t-test). (E) Immunofluorescent co-labeling analysis revealed an approximate 3.3-fold increase of ChAT + IR BFCNs that co-expressed activated pNFκB p65 + IR in the basal forebrain relative to vehicle-treated CONs (n = 4 wells/group, two-tailed t-test). Representative fluorescent photomicrographs of ChAT (red) and pNFκB p65 (green) colocalization in CON- and LPS-treated FSCs. White arrow heads = ChAT + IR neuron colocalization with pNFκB p65+ (yellow). Scale bar = 50 μm. (F) Schematic depicting experimental design for ex vivo LPS HMGB1 and TLR4 blockade experiments. FSCs were treated with NGF (100 μg/mL) until day ex vivo (DEV) 12. On DEV12, FSCs were treated with LPS (100 ng./mL) for 24 h in the absence of NGF in combination with either the HMGB1 inhibitor glycyrrhizin (Gly; 100 μM) or the TLR4 antagonist LPS-RS (100 ng/mL) until tissue collection on DEV13. (G) Reverse transcription PCR analysis revealed that direct application of LPS to FSC media caused a 54% (±10%) reduction of Chat gene expression relative to vehicle-treated CONs. While treatment with the HMGB1 antagonist glycyrrhizin (100 μM; 24 h) alone did not affect Chat gene expression in CON FSC, it blunted the LPS-induced loss of Chat mRNA gene expression relative to LPS-treated FSCs (n = 4 wells/group, 2 × 2 ANOVA with follow-up two-tailed t-tests). RTPCR analyses run in duplicate. (H) Modified unbiased stereological assessment revealed that direct application of LPS to FSC media caused a 43% 5%) reduction of ChAT + IR neurons relative to vehicle-treated CONs. Although treatment with the TLR4 antagonist LPS-RS (100 ng/mL; 24 h) did not affect ChAT expression in CON FSC, it blocked the LPS-induced loss of ChAT + IR neurons (n = 4 wells/group, 2 × 2 ANOVA with Tukey’s HSD). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01.