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

FIGURE 1.

FIGURE 1

In vivo and ex vivo reductions of ChAT + IR neurons and ChAT+somal shrinkage in the basal forebrain. (A) Schematic depicting experimental design for in vivo and ex vivo lipopolysaccharide (LPS) experiments. For the in vivo study, naïve Wistar rats received a single dose of LPS (1.0 mg/kg, i.p.) on postnatal day (P) 79 and tissue collected on P80. For the ex vivo studies, basal forebrain slice cultures (FSC) were treated with NGF (100 μg/mL) until day ex vivo (DEV) 12. On DEV12, FSCs were treated with LPS (100 ng/mL) or vehicle for 24 h in the absence of NGF until tissue collection on DEV13. (B) Modified unbiased stereology revealed that systemic administration of the proinflammatory TLR4 ligand LPS caused a 13% (±3%) reduction of ChAT + IR cholinergic neurons in the basal forebrain 24 h post-LPS treatment relative to vehicle-treated CONs (n = 8/group, two-tailed t-test). (C) Analysis of ChAT+ neuron somal size 24 h post-LPS treatment revealed a 24% (±2%) reduction in somal size of the remaining ChAT+ neurons relative to CONs (n = 8/group, two-tailed t-test). (D) Representative photomicrographs of ChAT + IR neurons in the basal forebrain of CON- and LPS-treated subjects. Note somal shrinkage of the remaining ChAT+ cholinergic neurons following LPS treatment relative to CONs. (E) Modified unbiased stereological assessment revealed that direct application of LPS to FSC media caused a 32% (±2%) reduction of ChAT + IR neurons relative to vehicle-treated CONs (n = 4 wells/group, two-tailed t-test). (F) Analysis of somal size revealed a 26% (±2%) reduction in somal size of the remaining ChAT+ neurons in LPS-treated FSC relative to vehicle-treated CONs (n = 4 wells/group, two-tailed t-test). (G) Representative photomicrographs of ChAT + IR neurons from CON- and LPS-treated FSCs. Note somal shrinkage of the remaining ChAT+ cholinergic neurons following LPS treatment relative to CONs. (H) Modified unbiased stereological assessment revealed that direct application of LPS to FSC media did not affect expression of NeuN + IR neurons. Time-course assessment (i.e., 6, 24, and 48 h) of NeuN + IR revealed that LPS treatment did not affect neuronal marker expression across time points, relative to CON at 24 h (one-way ANOVA with Tukey’s HSD). Representative photomicrographs of NeuN + IR neurons from CON- and LPS-treated FSCs at the 24 h time point. (I) Assessment of propidium iodide (PI) fluorescent immunoreactive cells revealed that direct application of LPS to FSC media did not increase PI + IR cells. Time-course assessment (i.e., 6, 24, and 48 h) of PI + IR revealed that LPS treatment did not increase cell death across time points, relative to CON at 24 h (one-way ANOVA with Tukey’s HSD). Scale bar = 50 μm. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01.