Journal: bioRxiv

Article Title: Apolipoprotein E ε4 exacerbates microglia-mediated complement-dependent synapse loss caused by neuronal Tpk deficiency

doi: 10.1101/2025.01.16.633468

Figure Lengend Snippet: a, b. Representative images of Iba1 (green) and C1q (red) immunostaining ( a ) and quantification ( b ) in the cortices of mice. The results showed that the humanized APOE4 knock-in significantly aggravated the increase of c1q expression induced by neuronal Tpk deletion N = 5 mice per group. c, d. Representative images of Iba1 (green) and C3 (red) immunostaining ( c ) and quantification ( d ) in the cortices of mice. The results showed that the humanized APOE4 knock-in significantly worsened the increase of C3 expression induced by neuronal Tpk deletion. N = 5 mice per group. e, f. Representative images of Western blotting ( e ) and quantification ( f ) of C3aR protein in the cortices of mice. The results showed that the humanized ApoE4 knock-in significantly accelerated the increase of C3aR protein induced by neuronal Tpk deletion. N = 6 mice per group. The data represent mean ± SEM. * p <0.05, ** p <0.01, unpaired t -test. Scale bars, 10 μ

Article Snippet: The primary antibodies used were as follows: mouse anti-Synaptophysin (1:1000, MAB5258A4, Merck), mouse anti-PSD 95 (1:1000, MA1-046, Invitrogen), rabbit anti-C3aR (1:1000, sc-133172, Santa Cruz), mouse anti-Apolipoprotein E4 (1:1000, M067-3, MBL Life Science), rabbit anti-TPK1 (1:1000, ab230263, Abcam), rabbit anti-APP (1:1000, ab32136, Abcam), rabbit anti-BACE1 (1:1000, 5606, CST), mouse anti-Tau5 (1:1000, AHB0042, Invitrogen), rabbit anti-pTau (Thr181) (1:1000, 12885, CST), rabbit anti-pTau (Ser396) (1:1000, 9632, CST), mouse anti-pTau (Ser202, Thr205) (1:1000, MN1020, Invitrogen), rabbit anti-β-actin (1:2000, 4970, CST), rabbit anti-tubulin (1:2000, 2146, CST).

Techniques: Immunostaining, Knock-In, Expressing, Western Blot

Journal: CNS Neuroscience & Therapeutics

Article Title: C3 / C3aR Bridges Spinal Astrocyte‐Microglia Crosstalk and Accelerates Neuroinflammation in Morphine‐Tolerant Rats

doi: 10.1111/cns.70216

Figure Lengend Snippet: Experimental schematic diagram. (A) Experimental protocol and time course. (B) Animal experimental groups. The number of rats used for WB, qRT‐PCR, and IF was 5, 5, and 3, respectively. Compstain, a C3 inhibitor; i.t., intrathecal injection; IF, immunofluorescence; n , number of animals; PLX3397, a CSF1R inhibitor; qRT‐PCR, real‐time quantitative polymerase chain reaction; SB290157, a C3aR antagonist; Vehicle, 10% DMSO, 5% Tween 80 and 85% saline; WB, Western blot.

Article Snippet: The membranes were blocked with 5% non‐fat dry milk or 5% ( v / v ) bovine serum albumin dissolved in tris‐buffered saline and Tween‐20 buffer for 2 h at room temperature (22°C–24°C), and then incubated overnight at 4°C with following primary antibodies: mouse anti‐β‐actin antibody (1:10000; ABclonal, China), rabbit anti‐ionized calcium‐binding adapter molecule 1 (Iba1) antibody (1:500; ABclonal, China), rat anti‐C1qA antibody (1:500; Santa Cruz, USA), rabbit anti‐IL‐1α antibody (1:500; ABclonal, China), rabbit anti‐TNF‐α antibody (1:500 Elabscience, China), rabbit anti‐glial fibrillary acidic protein (GFAP) antibody (1:1000; ABclonal, China), rabbit anti‐C3 antibody (1:1000; ABcam, USA), rabbit anti‐serping1 antibody (1:1000; ABclonal, China), rabbit anti‐C3aR antibody (1:1000; ABclonal, China).

Techniques: Quantitative RT-PCR, Injection, Immunofluorescence, Real-time Polymerase Chain Reaction, Saline, Western Blot

Journal: CNS Neuroscience & Therapeutics

Article Title: C3 / C3aR Bridges Spinal Astrocyte‐Microglia Crosstalk and Accelerates Neuroinflammation in Morphine‐Tolerant Rats

doi: 10.1111/cns.70216

Figure Lengend Snippet: Chronic morphine treatment increased the expression of C3aR. (A, B) The results of qRT‐PCR and Western blot showed that chronic morphine treatment increased the expression of C3aR (** p < 0.01, **** p < 0.0001 vs. NS group, n = 5 per group). (C) The results of dual‐label immunofluorescence showed that C3aR was colocalized with NeuN, some with GFAP and Iba1 in the spinal dorsal horn of morphine‐tolerant rats (Scale bar: 100 μm, n = 3 per group, three sections per sample were measured). MT, morphine treatment; n = number of animals; NS, normal saline.

Article Snippet: The membranes were blocked with 5% non‐fat dry milk or 5% ( v / v ) bovine serum albumin dissolved in tris‐buffered saline and Tween‐20 buffer for 2 h at room temperature (22°C–24°C), and then incubated overnight at 4°C with following primary antibodies: mouse anti‐β‐actin antibody (1:10000; ABclonal, China), rabbit anti‐ionized calcium‐binding adapter molecule 1 (Iba1) antibody (1:500; ABclonal, China), rat anti‐C1qA antibody (1:500; Santa Cruz, USA), rabbit anti‐IL‐1α antibody (1:500; ABclonal, China), rabbit anti‐TNF‐α antibody (1:500 Elabscience, China), rabbit anti‐glial fibrillary acidic protein (GFAP) antibody (1:1000; ABclonal, China), rabbit anti‐C3 antibody (1:1000; ABcam, USA), rabbit anti‐serping1 antibody (1:1000; ABclonal, China), rabbit anti‐C3aR antibody (1:1000; ABclonal, China).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Saline

Journal: CNS Neuroscience & Therapeutics

Article Title: C3 / C3aR Bridges Spinal Astrocyte‐Microglia Crosstalk and Accelerates Neuroinflammation in Morphine‐Tolerant Rats

doi: 10.1111/cns.70216

Figure Lengend Snippet: SB290157 alleviated A1 astrocyte and microglia activation and attenuated morphine tolerance (MT). C3aR inhibitor SB290157 (10 μg/5 μL) was intrathecally injected 30 min before morphine administration twice daily for 9 days. (A) Pretreatment with SB290157 attenuated the development of MT (#### p < 0.0001 vs. Morphine + Vehicle group, *** p < 0.001, **** p < 0.0001 vs. NS + Vehicle group, n = 5 per group). (B, C) Pretreatment with SB290157 reduced the increased protein level of C3 and SERPING1 induced by chronic treatment of morphine (# p < 0.05, ## p < 0.01 vs. Morphine + Vehicle group, ** p < 0.01 vs. NS + Vehicle group, n = 5 per group). (D–G) Pretreatment with SB290157 reduced the increased protein level of Iba1, C1qA, TNF‐α and IL‐1α induced by chronic morphine treatment (# p < 0.05, ## p < 0.01, #### p < 0.0001 vs. Morphine + Vehicle group, * p < 0.05, ** p < 0.01, **** p < 0.0001 vs. NS + Vehicle group, n = 5 per group). n , number of animals; NS, normal saline; Vehicle, 10% DMSO, 5% Tween 80 and 85% saline. (H) The results of immunofluorescence staining showed that pretreatment with SB290157 reduced the expression of C3 and SERPING1 in the spinal cord of morphine‐tolerant rats (Scale bar: 100 μm, ## p < 0.01 vs. Morphine+ Vehicle group, ** p < 0.01, *** p < 0.001 vs. NS + Vehicle group, n = 3 per group, three sections per sample were measured).

Article Snippet: The membranes were blocked with 5% non‐fat dry milk or 5% ( v / v ) bovine serum albumin dissolved in tris‐buffered saline and Tween‐20 buffer for 2 h at room temperature (22°C–24°C), and then incubated overnight at 4°C with following primary antibodies: mouse anti‐β‐actin antibody (1:10000; ABclonal, China), rabbit anti‐ionized calcium‐binding adapter molecule 1 (Iba1) antibody (1:500; ABclonal, China), rat anti‐C1qA antibody (1:500; Santa Cruz, USA), rabbit anti‐IL‐1α antibody (1:500; ABclonal, China), rabbit anti‐TNF‐α antibody (1:500 Elabscience, China), rabbit anti‐glial fibrillary acidic protein (GFAP) antibody (1:1000; ABclonal, China), rabbit anti‐C3 antibody (1:1000; ABcam, USA), rabbit anti‐serping1 antibody (1:1000; ABclonal, China), rabbit anti‐C3aR antibody (1:1000; ABclonal, China).

Techniques: Activation Assay, Injection, Saline, Immunofluorescence, Staining, Expressing