Journal: Journal of Neuroinflammation

Article Title: The analgesic action of larixyl acetate, a potent TRPC6 inhibitor, in rat neuropathic pain model induced by spared nerve injury

doi: 10.1186/s12974-020-01767-8

Figure Lengend Snippet: Flow chart illustrating two experimental paradigms. a Single application. The catheterization was performed 4 days prior to SNI operation. LA at 3, 10, 30 μM was i.t. applied on day 5 post nerve injury. Mechanical and thermal tests were conducted 1 day prior to nerve injury and on POD 5 and 6. After drug application, mechanical tests were elaborately performed six times within 3 h, i.e., every 30 min a time. The cold allodynia was observed every 1 h, totally three times. b Multiple applications. The operation schedule for catheterization and SNI was the same as the single paradigm. LA at 30 μM, TRPC6 antisense or mismatch ODNs with or without supplementation of LA at 30 μM was i.t. applied daily from day 1 to 6 after SNI. Behavioral tests were performed pre- and post-drug application on day 5. On day 6, the drug was applied again and rats were sacrificed around 2.5 h post application after the final behavioral observation. Tissue collections for immunohistochemical (IHC) staining, Western blot (WB) and multiplex measurement (ELISA) were performed thereafter

Article Snippet: After being blocked in solution containing 5% nonfat milk and Tris-buffered saline with 0.02% Tween-20 (TBST) for 1 h at RT, the membranes were incubated overnight at 4 °C with following primary antibodies: goat anti-Iba-1 (1:200; Abcam, Cambridge, England), rabbit anti-GFAP (1:400; Abcam), rabbit anti-TRPC6 (1:200; Alomone, Jerusalem, Israel), rabbit anti-p38 (1:200; Cell Signaling, Danvers, MA, USA), rabbit anti-phosphorylated p38 (pp38, 1:200; Cell Signaling), and mouse anti-β-actin (1:5000; Sigma).

Techniques: Immunohistochemical staining, Immunohistochemistry, Western Blot, Multiplex Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Neuroinflammation

Article Title: The analgesic action of larixyl acetate, a potent TRPC6 inhibitor, in rat neuropathic pain model induced by spared nerve injury

doi: 10.1186/s12974-020-01767-8

Figure Lengend Snippet: Expression feature of TRPC6 and the influence of LA on the protein. a Western blot analysis showed that nerve injury increased the level of TRPC6 protein in the ipsilateral SDH, which was dose dependently downregulated by LA at 10 and 30 μM. Upper panel, typical blotting band. Lower panel, summarized analysis of TRPC6 in five groups. * p < 0.05 vs. sham + veh group; # p <0.05 vs. SNI + veh group; n = 5. b Typical picture of TRPC6 immunostaining in the SDH of vehicle treated SNI rat. Upper panel, TRPC6 IR exhibited cloudy like even distribution in the neuropil, with preference in laminae I and II. The intensity of TRPC6 in the superficial dorsal horn of the ipsilateral side appeared stronger than that in the contralateral side, mainly within the medial section (denoted by the white rectangle). Lower panel, control picture of TRPC6 staining after the antibody was pre-absorbed by the antigen. CC central canal, Ipsi ipsilateral side, Contra contralateral side, Ab antibody, Ag antigen. Scale bar, 200 μm. c Triple-labeled staining of TRPC6, Iba-1, and DAPI in cultured microglia. First panel, normal staining showing that TRPC6 was mainly distributed in the membrane, cytosol and the processes of Iba-1-labeled microglia. Second panel, normal cells with pre-absorbed TRPC6 antibody staining. Third panel, triple staining after the cells were challenged by LPS at 500 ng/mL for 24 h. Microglia showed the typical M1 phenotype with hypertrophied cell body and short but thick processes. TRPC6 and Iba-1 IR were greatly elevated in the cells. Last panel, co-incubation of the cell with 10 μM of LA and LPS markedly decreased TRPC6 and Iba-1 IR with the regression of the cell shape. Scale bar, 30 μm

Article Snippet: After being blocked in solution containing 5% nonfat milk and Tris-buffered saline with 0.02% Tween-20 (TBST) for 1 h at RT, the membranes were incubated overnight at 4 °C with following primary antibodies: goat anti-Iba-1 (1:200; Abcam, Cambridge, England), rabbit anti-GFAP (1:400; Abcam), rabbit anti-TRPC6 (1:200; Alomone, Jerusalem, Israel), rabbit anti-p38 (1:200; Cell Signaling, Danvers, MA, USA), rabbit anti-phosphorylated p38 (pp38, 1:200; Cell Signaling), and mouse anti-β-actin (1:5000; Sigma).

Techniques: Expressing, Western Blot, Immunostaining, Staining, Labeling, Cell Culture, Incubation

Journal: Journal of Neuroinflammation

Article Title: The analgesic action of larixyl acetate, a potent TRPC6 inhibitor, in rat neuropathic pain model induced by spared nerve injury

doi: 10.1186/s12974-020-01767-8

Figure Lengend Snippet: The changes of pain behaviors and p38 signaling after knockdown of TRPC6. a von Frey filament tests showing that i.t. application of TRPC6 antisense (AS) for 6 days significantly relieved mechanical hypersensitivity in SNI rats compared with mismatch (MM) treatment group. Supplementation with 30 μM of LA rescued the effect of sole mismatch while had no obvious influences on the effect of sole antisense. n = 6–8. b Acetone test showing that treatment with TRPC6 antisense also remarkably suppressed cold allodynia compared with mismatch group. This effect was almost equal to that of the co-administration groups. n = 6–9. c Western blot analysis demonstrated that application of antisense ODN caused significant decrease in the level of TRPC6, the efficacy of which was comparable to the other two groups. n = 5. d Typical picture of TRPC6 immunofluorescent staining illustrating the generally decreased intensity of TRPC6 IR after antisense treatment. CC central canal. Scale bar, 200 μm. e , f Western blot analysis demonstrated that phosphorylated p38 ( e ) other than p38 ( f ) was significantly suppressed in the antisense and the two co-administration groups. * p < 0.05, ** p <0.01, *** p < 0.001 vs. mismatch group, n = 5

Article Snippet: After being blocked in solution containing 5% nonfat milk and Tris-buffered saline with 0.02% Tween-20 (TBST) for 1 h at RT, the membranes were incubated overnight at 4 °C with following primary antibodies: goat anti-Iba-1 (1:200; Abcam, Cambridge, England), rabbit anti-GFAP (1:400; Abcam), rabbit anti-TRPC6 (1:200; Alomone, Jerusalem, Israel), rabbit anti-p38 (1:200; Cell Signaling, Danvers, MA, USA), rabbit anti-phosphorylated p38 (pp38, 1:200; Cell Signaling), and mouse anti-β-actin (1:5000; Sigma).

Techniques: Western Blot, Staining

Journal: Journal of Neuroinflammation

Article Title: The analgesic action of larixyl acetate, a potent TRPC6 inhibitor, in rat neuropathic pain model induced by spared nerve injury

doi: 10.1186/s12974-020-01767-8

Figure Lengend Snippet: The effects of TRPC6 knockdown and the co-administration with LA on pp38 immunofluorescent staining in SNI rats. a – d Mismatch group, e – h antisense group, i , j mismatch supplemented with LA at 30 μM, m – p antisense supplemented with LA at 30 μM. pp38 IR (in green) in the mismatch group were markedly increased in the medial two thirds part of the ipsilateral SDH, compared to the sporadic distribution in the contralateral side (data not shown). Most of pp38 IR were co-existed with Iba-1, suggesting the dominant localization in the microglia ( d ). Antisense or the two co-administrations caused prominent decrease of pp38 IR and Iba-1 IR (in red). d , h , l , p Illustrated the magnified images enclosed in c , g , k , o , respectively. Nerve injury caused the upregulation of pp38 IR in the soma and processes of microglia ( d ). But those in the processes were markedly reduced in the antisense and co-administration groups ( h , l , p ). Scale bar, 30 μm in d , h , l , p ; 100 μm in the remaining pictures

Article Snippet: After being blocked in solution containing 5% nonfat milk and Tris-buffered saline with 0.02% Tween-20 (TBST) for 1 h at RT, the membranes were incubated overnight at 4 °C with following primary antibodies: goat anti-Iba-1 (1:200; Abcam, Cambridge, England), rabbit anti-GFAP (1:400; Abcam), rabbit anti-TRPC6 (1:200; Alomone, Jerusalem, Israel), rabbit anti-p38 (1:200; Cell Signaling, Danvers, MA, USA), rabbit anti-phosphorylated p38 (pp38, 1:200; Cell Signaling), and mouse anti-β-actin (1:5000; Sigma).

Techniques: Staining