Journal: Journal of Neuroinflammation

Article Title: α-Synuclein induces prodromal symptoms of Parkinson’s disease via activating TLR2/MyD88/NF-κB pathway in Schwann cells of vagus nerve in a rat model

doi: 10.1186/s12974-023-02720-1

Figure Lengend Snippet: Deposition of p-α-synuclein in SCs induces inflammatory responses via activating TLR2/MyD88/NF-κB signaling. A , B Representative confocal images and quantitative analysis of TLR2 in the rat left vagus nerve at 3 months post-injection. Scale bar = 10 μm. n = 6. C , D Representative confocal images and quantitative analysis of IL-1β in the rat left vagus nerve at 3 months post-injection. Scale bar = 10 μm. n = 6. Representative western blot bands ( E ) and the statistical graph ( F – J ) of total α-synuclein, p-α-synuclein, MyD88, p-NF-κB p65, and IL-1β in the rat left vagus nerve at 3 months post-injection. The protein levels were normalized to GAPDH and expressed as fold over AAV-EV. n = 4. K – M Representative immunohistochemical staining and quantitative analysis of TLR2 in sural nerves of controls and PD patients. Scale bar = 10 μm. n = 6. Data were presented as mean ± SEM. Statistical significance was analyzed using two-way ANOVA followed by Bonferroni’s multiple comparison test in B , D , and F – J , and Student’s t test in M . *** P < 0.001, **** P < 0.0001. ns, not significant

Article Snippet: In addition, AAVs carrying TLR2 sagRNA were employed to silence TLR2 gene expression (AAV2/6-CMV-NLS-SaCas9-NLS-3xHA-bGHpA-U6-sgRNA, 1.23 × 10 13 GC/mL) (Genechem Co., Shanghai, China).

Techniques: Injection, Western Blot, Immunohistochemical staining, Staining, Comparison

Journal: Journal of Neuroinflammation

Article Title: α-Synuclein induces prodromal symptoms of Parkinson’s disease via activating TLR2/MyD88/NF-κB pathway in Schwann cells of vagus nerve in a rat model

doi: 10.1186/s12974-023-02720-1

Figure Lengend Snippet: Silencing of TLR2 expression attenuates p-α-synuclein-induced AutD and vagus nerve lesions. SD rats were subjected to vagal injection with AAV-A53T or AAV-A53T as well as AAV-Cas9-TLR2 and then evaluated with gastrointestinal autonomic function 3 months later. A colonic transit time. B 12 h fecal pellet count and total weight ( C ). D 12 h fecal water content. E Representative images of intestinal blood flow detected by laser Doppler. F Average intestinal blood flow. G Representative images of the left vagus nerve conduction detected by the electromyography system. H Average vagus nerve conduction velocity. I Average amplitude of vagus nerve compound action potential. J Representative TEM images of the ultrastructure of the left vagus nerve in rats. Scale bar = 5 μm. Classification and counting numbers of non-myelinated axons ( K ) and myelinated axons ( L ) in left vagus nerve fibers. A – D : n = 12 per group; E – L : n = 6 per group. Data were presented as mean ± SEM. Statistical significance was analyzed using Student’s t test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ns, not significant; MA, myelinated axons; NMA, non-myelinated axons; MAA, Myelin anomalous alteration

Article Snippet: In addition, AAVs carrying TLR2 sagRNA were employed to silence TLR2 gene expression (AAV2/6-CMV-NLS-SaCas9-NLS-3xHA-bGHpA-U6-sgRNA, 1.23 × 10 13 GC/mL) (Genechem Co., Shanghai, China).

Techniques: Expressing, Injection

Journal: Communications Biology

Article Title: Age-dependent cerebral vasodilation induced by volatile anesthetics is mediated by NG2 + vascular mural cells

doi: 10.1038/s42003-024-07200-7

Figure Lengend Snippet: a Experimental design for spatially confined ablation of cortical NG2 + cells. NG2-Cre mice were injected with AAV2-FLEx-DTR-EGFP and AAV2-FLEx-EGFP (control) into the right and left cortex, respectively, on day −21. An i.p. injection of diphtheria toxin (DT) on day −7 ablated DTR-expressing NG2 + cells, and two-photon imaging on day 0 assessed changes in arteriole diameter upon SEVO 2.5%. b Images of the cortex stained for cleaved caspase-3. In the control cortex, EGFP + cells (green arrows) express low levels of cleaved caspase-3; in the ablation cortex, most DTR-EGFP + cells show high caspase-3 expression (white arrows), with a few exceptions (yellow star). c Viral transduction range measured by distribution of EGFP + fluorescence intensity (f.u., fluorescence unit) in the control (EGFP) and ablation (DTR-EGFP) cortex ( n = 3 mice per group; F (22, 88) = 1.278, P = 0.2096). ML, midline. d Expression of cleaved caspase-3 measured by fluorescent intensity in either side of the cortex (9 slices from three mice; from left to right, n = 107, 102, 123, and 118 cells; P = 0.0856, 0.2568, and < 0.0001 for others). e Immunofluorescence images of the cortex stained for virally transduced EGFP (green), antibody-labeled NG2 (red), and DAPI (blue) from the control and ablation cortex. f Density of NG2 + DAPI + cells in the control and ablation cortex ( n = 12 slices from three mice per group; P < 0.0001). g Two-photon images of cortical arterioles before and 15 min after SEVO exposure. Arrows indicate SEVO-induced dilation of arterioles in the control (EGFP) but not the ablation (DTR-EGFP) cortex. h Time course of SEVO-induced arteriole dilation in the control ( n = 64 segments from four mice) and ablation ( n = 58 segments from four mice) cortex of adult mice exposed to SEVO at time 0. BL, baseline under awake conditions. i Distribution of initial diameters of the sampled arterioles. There is no difference between the control and ablation cortex ( P = 0.9924). **** P < 0.0001; NS , not significant; by two-way ANOVA ( c ), two-tailed unpaired t- tests ( d ), two-tailed Mann-Whitney U test ( f ), two-tailed paired t -tests ( h ), or Kolmogorov-Smirnov test ( i ).

Article Snippet: To locally ablate NG2 + cells, adult NG2-Cre mice were injected with 0.2 μl of AAV encoding Cre-dependent diphtheria toxin receptor (DTR) (AAV2-FLEx-DTR-GFP; titer 1.3 × 10 13 gc ml −1 ; 124364; Addgene) into the right cortex, and a control reporter (AAV2-FLEx-GFP; titer 1.2 × 10 13 gc ml −1 ; 51502; Addgene) into the left cortex, at the coordinates described above.

Techniques: Injection, Control, Expressing, Imaging, Staining, Transduction, Fluorescence, Immunofluorescence, Labeling, Two Tailed Test, MANN-WHITNEY

Journal: Bone Research

Article Title: Endothelial PDGF-BB/PDGFR-β signaling promotes osteoarthritis by enhancing angiogenesis-dependent abnormal subchondral bone formation

doi: 10.1038/s41413-022-00229-6

Figure Lengend Snippet: The target sequences used for PDGFR-β knockdown lentivirus or siTLN1

Article Snippet: The pdgfrb shRNA virus (AAV2/9-TIE1p-EGFP-miR30shRNA(pdgfrb)) and the controls (AAV2/9-TIE1p-EGFP) were constructed by Obio Technology (China).

Techniques: Knockdown

Journal: Bone Research

Article Title: Endothelial PDGF-BB/PDGFR-β signaling promotes osteoarthritis by enhancing angiogenesis-dependent abnormal subchondral bone formation

doi: 10.1038/s41413-022-00229-6

Figure Lengend Snippet: PDGFR-β interacts with FAK and TLN1 in ECs. a Strategy for identifying PDGFR-β binding proteins using LC-MS/MS analysis. Bone morrow endothelial cells (BMECs) treated with PDGF-BB (300 ng·mL −1 ) for 24 h were subjected to immunoprecipitation (IP) with an antibody against PDGFR-β followed by LC-MS/MS analysis. b The PDGFR-β binding proteins FAK and TLN1 were identified with LC-MS/MS analysis. c PDGFR-β was immunoprecipitated from BMECs with an anti-PDGFR-β antibody. The presence of FAK, TLN1, and PDGFR-β in these immunoprecipitates was evaluated with immunoblotting. Western blot analysis of FAK, TLN1, and VEGF in the BMECs treated with different PDGF-BB doses (0-300 ng·mL −1 ) ( d ) or for different times at 0 h–36 h ( e ). Western blot analysis of FAK, TLN1, and VEGF in the BMECs treated with FAK inhibitors ( f ) or TLN1 siRNA ( g )

Article Snippet: The pdgfrb shRNA virus (AAV2/9-TIE1p-EGFP-miR30shRNA(pdgfrb)) and the controls (AAV2/9-TIE1p-EGFP) were constructed by Obio Technology (China).

Techniques: Binding Assay, Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation, Western Blot

Journal: Bone Research

Article Title: Endothelial PDGF-BB/PDGFR-β signaling promotes osteoarthritis by enhancing angiogenesis-dependent abnormal subchondral bone formation

doi: 10.1038/s41413-022-00229-6

Figure Lengend Snippet: Endothelial PDGFR-β promotes angiogenesis through the PDGFR-β/Talin1/FAK pathway. The efficiency of lentivirus transfection was verified using Western blot analysis ( a ) and qRT‒PCR ( b ). For pdgfrb silencing, 80958-1 exhibited optimum efficiency and was selected for the following experiments. c ELISA examination of VEGF expression in the CM of BMECs under different conditions. d – g Representative images and quantification analysis of tube formation in BMECs. Scale bar, 500 µm. h CCK-8 measurement of BMEC proliferation under different conditions. i , j Scratch wound assay showing BMEC motility under different conditions. Scale bar, 200 μm. Control = CM of BMECs from the normal control group; Over = CM of BMECs transfected with Lv-PDGFR-β; pdgfrb silencing = CM of BMECs transfected with Lv-si PDGFR-β; siTLN1 = CM of BMECs transfected with TLN1 siRNA; PF573228 = CM of BMECs treated with PF573228 (FAK inhibitor). * P < 0.05 and ** P < 0.01 compared to the over group. # P < .05 and ## P < .01 compared to the control group

Article Snippet: The pdgfrb shRNA virus (AAV2/9-TIE1p-EGFP-miR30shRNA(pdgfrb)) and the controls (AAV2/9-TIE1p-EGFP) were constructed by Obio Technology (China).

Techniques: Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing, CCK-8 Assay, Scratch Wound Assay Assay, Control

Journal: Bone Research

Article Title: Endothelial PDGF-BB/PDGFR-β signaling promotes osteoarthritis by enhancing angiogenesis-dependent abnormal subchondral bone formation

doi: 10.1038/s41413-022-00229-6

Figure Lengend Snippet: The primer sequences used to determine the genotypes of the mice

Article Snippet: The pdgfrb shRNA virus (AAV2/9-TIE1p-EGFP-miR30shRNA(pdgfrb)) and the controls (AAV2/9-TIE1p-EGFP) were constructed by Obio Technology (China).

Techniques: Sequencing