Journal: bioRxiv

Article Title: Microglia-derived IL-18 remodels hippocampal plasticity to constrain traumatic fear memory

doi: 10.64898/2026.05.04.721266

Figure Lengend Snippet: a , Schematic of PTSD model establishment. The SPS&S (Single Prolonged Stress & Shock) procedure, consisting of sequential restraint, forced swim, anesthesia, and foot shock. b–c , Contextual fear freezing in SPS&S-exposed and control mice at day 8 ( b ) and day 15 ( c ) post-stress. **P < 0.01, unpaired two-tailed t-test; n = 10 mice per group. d–e , Representative images showing IL-18-GFP fluorescence in sagittal brain sections from IL-18-GFP reporter mice at day 3 post-SPS&S. Images show whole-brain overview with magnified insets of the hippocampus and medial prefrontal cortex (mPFC) ( d ), and basolateral amygdala (BLA). IL-18-GFP specks are represented by green dots generated using IMARIS software. ( e ), scale bars,1mm (overview), 200 μm (inset, hippocampus), 100 μm (inset, mPFC and BLA). f , Quantification of mean IL-18-GFP fluorescent intensity across hippocampus, mPFC, and BLA in control and SPS&S groups. **P < 0.01, ns, not significant; two-way ANOVA with Bonferroni’s post hoc test; n = 3 mice per group. g , Schematic of the IL-18 luciferase reporter assay for quantifying IL-18 bioactivity in dissected brain regions. h , Luciferase activity in hippocampus, mPFC, and BLA from control and SPS&S mice. **P < 0.01, ns, not significant; two-way ANOVA with Bonferroni’s post hoc test; n = 3 per group. i , Schematic of hippocampal tissue collection and processing for IL-18 mRNA quantification by qPCR and protein quantification by ELISA at multiple post-stress time points. n = 3 mice per group. j , Temporal profile of hippocampal IL-18 mRNA levels from 0 to 15 days post-SPS&S. *P < 0.05, **P < 0.01 vs. 0 h; one-way ANOVA with Dunnett’s post hoc test; n = 7-11 per time point. k , Temporal profile of hippocampal mature IL-18 protein levels (normalized to total protein) from 0 to 15 days post-SPS&S. **P < 0.01 vs. 0 h; one-way ANOVA with Dunnett’s post hoc test; n = 7-11 per time point. l , Experimental timeline for intrahippocampal cannula implantation, SPS&S exposure, and daily PBS or IL-18 binding protein (IL-18BP) infusion, with fear memory assessed at days 8 and 15 post-stress. IL-18BP infusion began on day −3 to allow accumulation of antagonist before stress onset, ensuring complete neutralization of stress-induced IL-18. m–n , Contextual fear freezing at day 8 ( m ) and day 15 ( n ) in mice receiving intrahippocampal IL-18BP or PBS following SPS&S. *P < 0.05, **P < 0.01, ***P < 0.001; two-way ANOVA with Bonferroni’s post hoc test; n = 5-9 per group. o , Experimental timeline for intrahippocampal cannula implantation, SPS&S exposure, and daily IL18 infusion, with fear memory assessed at days 8 and 15 post-stress. p–q , Contextual fear freezing at day 8 ( p ) and day 15 (q ) in mice receiving intrahippocampal IL18 or PBS following SPS&S. *P < 0.05, **P < 0.01, ***P < 0.001; two-way ANOVA with Bonferroni’s post hoc test; n = 6–10 per group. Complete statistics are provided in Supplementary Table 1.

Article Snippet: IL-18 binding protein (IL-18BP; MCE) was dissolved in sterile PBS and stored at −80°C.

Techniques: Control, Two Tailed Test, Fluorescence, Generated, Software, Luciferase, Reporter Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Binding Assay, Neutralization

Journal: bioRxiv

Article Title: Microglia-derived IL-18 remodels hippocampal plasticity to constrain traumatic fear memory

doi: 10.64898/2026.05.04.721266

Figure Lengend Snippet: a, Representative confocal images of hippocampal sections from IL-18-GFP reporter mice at day 3 post-SPS&S. Scale bars, 10 μm. b, Experimental timeline for bilateral hippocampal AAV injection (day −21), SPS&S exposure (day 1), and contextual fear memory assessment (days 8 and 15). c, Contextual fear freezing at day 8 in mice receiving cell-type-specific AAV-shRNA targeting IL-18 in microglia (Iba1-shIL18), astrocytes (GFAP-shIL18), neurons (hSyn-shIL18 and CaMKII-shIL18), or scramble control (EF1-shCon), following SPS&S or control conditions. *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant Two-way ANOVA with post-hoc Bonferroni correction, n=7-11 mice per group. d, Same as c at day 15. e, Representative confocal images of hippocampal sections stained for IL-18R1. Scale bars, 10 μm. f, Experimental timeline for bilateral hippocampal AAV injection (day −21), SPS&S exposure (day 1), and contextual fear memory assessment (days 8 and 15) for IL-18R1 knockdown experiments. g, Contextual fear freezing at day 8 in mice receiving cell-type-specific AAV-shRNA targeting IL-18R1 in microglia (Iba1-shIL18R1), astrocytes (GFAP-shIL18R1), or neurons (hSyn-shIL18R1), or scramble control (EF1-shCon), following SPS&S or control conditions. *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant Two-way ANOVA with post-hoc Bonferroni correction, n=8-13 mice per group. h, Same as g at day 15. Complete statistics are provided in Supplementary Table 1.

Article Snippet: IL-18 binding protein (IL-18BP; MCE) was dissolved in sterile PBS and stored at −80°C.

Techniques: Injection, shRNA, Control, Staining, Knockdown

Journal: bioRxiv

Article Title: Microglia-derived IL-18 remodels hippocampal plasticity to constrain traumatic fear memory

doi: 10.64898/2026.05.04.721266

Figure Lengend Snippet: a, Experimental timeline for infusion tube installation (day −14), SPS&S exposure (day 1), daily intrahippocampal IL-18 or PBS infusion, and tissue collection for immunostaining at day 15. b, Representative confocal images of synaptophysin (yellow) immunostaining in hippocampal CA1 from PBS- and IL-18-treated mice under control and SPS&S conditions. Scale bars, 10 μm. c, Quantification of percent area synaptophysin staining across groups. ***P < 0.001, *P < 0.05, Two-way ANOVA with post-hoc Holm-Šídák, n=9-11 fields of view (FOVs) per group, from 3 mice per group. d, Quantification of normalized synaptophysin⁺ puncta density. Two-way ANOVA with post-hoc Holm-Šídák, n=9-11 fields of view (FOVs) per group, from 3 mice per group. e, Representative confocal images of VGLUT1 (green), Homer1 (red), and DAPI (blue) co-immunostaining in hippocampal CA1 from PBS- and IL-18-treated mice under control and SPS&S conditions. Scale bars, 10 μm. f, Quantification of normalized VGLUT1–Homer1 co-localization puncta. ***P < 0.001, Two-way ANOVA with post-hoc Bonferroni correction, n=18-21 fields of view (FOVs) per group, from 3 mice per group. g, Quantification of normalized VGLUT1⁺ puncta density. ***P < 0.001, Two-way ANOVA with post-hoc Bonferroni correction, n=18-21 fields of view (FOVs) per group, from 3 mice per group. h, Representative confocal images of WFA (yellow) and DAPI (blue) staining in hippocampal CA1 from PBS- and IL-18-treated mice under control and SPS&S conditions. Scale bars, 10 μm. i, Quantification of percent area WFA staining. **P < 0.01, *P < 0.05, Two-way ANOVA with post-hoc Bonferroni correction, n=6-13 fields of view (FOVs) per group, from 3 mice per group. Data are presented as mean ± SEM. Complete statistics are provided in Supplementary Table 1.

Article Snippet: IL-18 binding protein (IL-18BP; MCE) was dissolved in sterile PBS and stored at −80°C.

Techniques: Immunostaining, Control, Staining

Journal: bioRxiv

Article Title: Microglia-derived IL-18 remodels hippocampal plasticity to constrain traumatic fear memory

doi: 10.64898/2026.05.04.721266

Figure Lengend Snippet: a, Schematic of the TRAP2 engram-labeling system. AAV9-DIO-GFP was locally injected into the hippocampus of Fos-CreER T2 mice, enabling activity-dependent fluorescent labeling of SPS&S-activated neurons upon 4-OHT administration. b, Experimental timeline. DIO-GFP AAV was injected at day −14; mice received intrahippocampal IL-18 or PBS infusion 0.5h before SPS&S at day 1 and daily after, followed by intraperitoneal 4-OHT injection 1 hour later to complete engram labeling at day1. Brains were collected at day 15 for immunostaining. c, Representative confocal images of GFP (green, engram cells) and DAPI (blue) fluorescence in hippocampal sections from control and SPS&S-exposed mice under PBS or IL-18 treatment at day 15. Scale bars, 200 μm. d, Quantification of number of eGFP⁺ cells in hippocampus. ***P < 0.001, Two-way ANOVA with post-hoc Bonferroni correction, n=13 fields of view (FOVs) per group, from 3 mice per group. e, Schematic illustrating the synaptic markers (VGLUT1, Homer1, synaptophysin) analyzed within GFP⁺ engram cells. f, Representative confocal images of synaptophysin (yellow), DAPI (blue), and GFP (engram cell, white) immunostaining in hippocampal engram cells from PBS- and IL-18-treated mice under SPS&S conditions, scale bars, 10 μm. The magnified insets. Scale bars, 5 μm. g, Quantification of synaptophysin⁺ puncta contacts in engram cells per unit area (log scale). ns, not significant; unpaired two-tailed t-test; n=9 fields of view (FOVs) per group, from 3 mice per group. The extremely low baseline of engram cells in the control group precluded reliable quantification and thus precluded valid statistical comparisons. h, Representative confocal images of VGLUT1 (green), Homer1 (red), DAPI (blue), and GFP (engram cell, white) immunostaining within hippocampal engram cells from PBS- and IL-18-treated under control and SPS&S conditions, scale bars, 10 μm. The magnified insets. Scale bars, 5 μm. i, Quantification of VGLUT1⁺/Homer1⁺ co-localized puncta contacts in engram cells per unit area (log scale). ***P < 0.001, Unpaired Student’s t-test, n=14 fields of view (FOVs) per group, from 3 mice per group. j, Quantification of VGLUT1⁺ puncta contacts in engram cells per unit area (log scale). ***P < 0.001, Unpaired t-test with Welch’s correction, n=14 fields of view (FOVs) per group, from 3 mice per group. Extremely low baseline engram counts in the control group in panels i and j preclude reliable quantification (see also panel g ). Data are presented as mean ± SEM. Complete statistics are provided in Supplementary Table 1.

Article Snippet: IL-18 binding protein (IL-18BP; MCE) was dissolved in sterile PBS and stored at −80°C.

Techniques: Labeling, Injection, Activity Assay, Immunostaining, Fluorescence, Control, Two Tailed Test

Journal: Bioactive Materials

Article Title: 3D-MSCs apoptotic bodies-integrated conductive hydrogel mitigates spinal cord injury via immunoregulation and alleviating neuronal pyroptosis

doi: 10.1016/j.bioactmat.2026.01.043

Figure Lengend Snippet: Composite hydrogel promotes the polarization of BV2 cells to M2 types and alleviates PC12 cell pyroptosis in vitro inflammatory environment. (A) Representative western blots showing protein expression of iNOS and Arg-1 in each group, β-actin was utilized as a loading control. (B) Quantitative analysis of relative expression of iNOS and Arg-1. (C) Representative immunofluorescence images of CD68 positive and iNOS positive BV2 cells (scale bar: 20 μm). (D) Representative immunofluorescence images of CD68 positive and Arg-1 positive BV2 cells (scale bar: 20 μm). (E, F) Quantitative analysis of relative fluorescence intensity of iNOS and Arg-1. (G) Representative western blots showing the expression of NLRP3, Caspase-1, IL-1β, ASC, GSDMD-N, and IL-18 protein associated with pyroptosis, β-actin was utilized as a loading control. (H) PI staining of PC12 cells in each group (scale bar, 100 μm). (I) Quantitative analysis of PI staining of PC12 cells (n = 3). (J) Quantitative analysis of relative expression of NLRP3, Caspase-1, IL-1β, ASC, GSDMD-N and IL-18 (n = 3). The data are presented as the means ± SEMs (n = 3); ∗p < 0.05, indicates significant differences; ns, is not significant. Statistical analysis was performed using two-way ANOVA followed by Tukey's multiple comparison test.

Article Snippet: Enzyme-Linked Immunosorbent Assay (ELISA): The protein levels of IL-18 (Servicebio, Wuhan, China) and IL-1β (Servicebio, Wuhan, China) in the spinal cord tissue were detected using ELISA kits according to the manufacturer's protocol.

Techniques: In Vitro, Western Blot, Expressing, Control, Immunofluorescence, Fluorescence, Staining, Comparison

Journal: Bioactive Materials

Article Title: 3D-MSCs apoptotic bodies-integrated conductive hydrogel mitigates spinal cord injury via immunoregulation and alleviating neuronal pyroptosis

doi: 10.1016/j.bioactmat.2026.01.043

Figure Lengend Snippet: The composite hydrogel inhibits post-SCI pyroptosis in neurons. (A) Immunofluorescence images of residual neurons existing in the anterior horn of the spinal cord (scale bar, 500 μm and 200 μm). (B) Immunofluorescence image of Caspase-1 expression of neurons 3 days after SCI (scale bar, 20 μm). (C) Quantitative analysis of relative fluorescence intensity of Caspase-1 protein expression in neurons within the specified groups (n = 3). (D) Immunofluorescence image of GSDMD-N expression of neurons 3 days after SCI (scale bar, 20 μm). (E) Quantitative analysis of relative fluorescence intensity of GSDMD-N protein expression in neurons within the specified groups (n = 3). (F) Representative western blots showing the expression of NLRP3, Caspase-1, IL-1β, ASC, GSDMD-N, and IL-18 protein 3 days after SCI, GAPDH was utilized as a loading control. (G) Quantitative analysis of relative expression of NLRP3, Caspase-1, IL-1β, ASC, GSDMD-N and IL-18 (n = 3). The data are presented as the means ± SEMs (n = 3); ∗p < 0.05, indicates significant differences; ns, is not significant. Statistical analysis was performed using two-way ANOVA followed by Tukey's multiple comparison test.

Article Snippet: Enzyme-Linked Immunosorbent Assay (ELISA): The protein levels of IL-18 (Servicebio, Wuhan, China) and IL-1β (Servicebio, Wuhan, China) in the spinal cord tissue were detected using ELISA kits according to the manufacturer's protocol.

Techniques: Immunofluorescence, Expressing, Fluorescence, Western Blot, Control, Comparison

Journal: Research

Article Title: Neuromodulation and Copper Chelation Reverse Sleep Fragmentation-Aggravated Myocardial Ischemia–Reperfusion Injury by Targeting NET-Induced Endothelial Cuproptosis

doi: 10.34133/research.1266

Figure Lengend Snippet: Chronic SF exacerbated the inflammatory response and promoted the formation of NETs in MI/RI mice. (A to C) Plasma concentrations of IL-1β, IL-6, and IL-18 in mice were measured by ELISA ( n = 8 per group). (D to F) qRT-PCR assays were performed to determine mRNA levels of IL-1β, IL-6, and IL-18 in the hearts of MI/RI mice with SF ( n = 6 per group). (G) Representative images of immunofluorescence staining of NETs in myocardial tissues from Sham, SF + Sham, MI/RI, and SF + MI/RI groups (green: MPO, red: citH3, blue: DAPI) ( n = 6 per group). (H) Cell-free DNA (cfDNA) of each group ( n = 6 per group). (I) The MPO–DNA complex relative levels were assessed in mice ( n = 6 per group). Data are presented as mean ± SD. ### P < 0.001 versus Sham group; *** P < 0.001 versus SF + Sham group; $$ P < 0.01 versus MI/RI group; $$$ P < 0.001 versus MI/RI group. (J and K) Western blotting was used to analyze the expression of CXCR2 in NEs after treatment with EPI for 6 h ( n = 6 per group). Data are presented as mean ± SD. ### P < 0.001 versus Control group. C + EPI, Control + EPI. (L) Schematic diagram of coculture of EPI-induced NEs and CMECs exposed to H/R. CMECs were treated with hypoxia for 12 h and reoxygenation for 24 h and then cocultured with NEs for 6 h. (M) Statistical analysis of the number of migrated NEs after coculture with H/R-induced CMECs ( n = 6 independent experiments). (N and O) The cfDNA and MPO–DNA complex levels were assessed ( n = 5 independent experiments). (P) After coculture, the ultrastructure of NEs was observed via the electron microscope. Data are presented as mean ± SD. ### P < 0.001 versus Control + NEs group; *** P < 0.001 versus H/R + NEs group. Scale bars, 50 μm (G) and 2.5 μm (P). NEs, neutrophils; EPI, epinephrine.

Article Snippet: The plasma concentrations of IL-1β (EM0029, HUABIO), IL-18 (EM0034, HUABIO), and IL-6 (EM0004, HUABIO) were determined using ELISA kits per the supplier’s protocol.

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Immunofluorescence, Staining, Western Blot, Expressing, Control, Microscopy