rhtimp 3  (R&D Systems)

Journal: Brain, behavior, and immunity

Article Title: Single-cell analysis of dorsal root ganglia reveals metalloproteinase signaling in satellite glial cells and pain.

doi: 10.1016/j.bbi.2023.08.005

Figure Lengend Snippet: Fig. 3. Transcriptional and protein expression of Timp3. (A) Schematic illustration of TIMP3′s inhibitory activity on matrix metalloproteinases (MMPs) and on disintegrin and metalloprotease 17 (ADAM17), also called TACE (tumor necrosis factor-α-converting enzyme). (B, C) RNAscope localization of Timp3, Mmp14 and Adam17 mRNA expression in DRG of Plp1-Cre/tdTomato mice, wherein Cre recombinase is expressed in satellite glial cells (SGCs) (B), and in naïve CD1 mice (C). Arrowheads indicate mRNA colocalization of Mmp14 and Adam17 with Timp3 in SGCs, * indicates neurons. Scale bars = 25 μm in (B) and 5 μm in (C). (D) PCR in mouse and human DRG tissues. Samples with omitted RT (reverse transcriptase) show no bands, confirming the specificity of the amplification. (E) Immunofluo rescence of TIMP3 in human DRG tissue. Scale bars = 50 μm. DAPI was used as counterstain. # indicates the fluorescent signal due to the presence of lipofuscins in human DRG neurons.

Article Snippet: We purchased recombinant human TIMP3 (Cat# 973-TM) and prosaptide Tx14 (Cat# 5151) from R&D Systems (Minneapolis, MN); recombinant mouse TIMP-1 (Cat# 593702) from BioLegend (San Diego, CA); an MMP14 inhibitor (NSC405020, Cat# 444295), MMP2 inhibitor (Cat# 444288), TACE/ADAM17 inhibitor (TAPI-2, Cat# 4444244), and R. Tonello et al.

Techniques: Expressing, Activity Assay, RNAscope, Reverse Transcription, Amplification

Journal: Brain, behavior, and immunity

Article Title: Single-cell analysis of dorsal root ganglia reveals metalloproteinase signaling in satellite glial cells and pain.

doi: 10.1016/j.bbi.2023.08.005

Figure Lengend Snippet: Fig. 4. Timp3 controls mechanical and thermal sensitivities in naïve mice. (A) Schematic illustration of the experiment showing the timeline of siRNA injections, pharmacological and biochemical studies. (B) Western blot representative image and quantification show that Timp3 siRNA significantly decreases Timp3 protein levels in DRGs tissues (n = 4). (C) Timp3 siRNA injections do not cause locomotor dysfunction in the Rota-rod test (n = 5). (D) Mechanical and thermal (von Frey, Hargreaves, and dry ice) allodynia induced by Timp3 siRNA compared to a control (Ctrl) non-targeting siRNA (2 µg of siRNA per delivery in the transfection agent PEI, n = 7). (E) Anti-allodynic effect of exogenous recombinant TIMP3 (rTIMP3, 100 ng/site, i.t.), general endogenous tissue inhibitor of MMPs (TIMP-1, 4 pmol/ site), MMP2 and MMP14 inhibitors (10 µg/site, i.t.), TACE/ADAM17 inhibitor (TAPI-2, 1 µg/site, i.t.), and a neutralizing antibody for TNF-α (5 µg/site, i.t.) on mechanical allodynia induced by Timp3 siRNA on day 2. (F) Anti-TIMP3 antibody (TIMP3 Ab, 10 µg/site, i.t.) induces mechanical allodynia compared to IgG control in male and female mice. BL = baseline. Data are expressed as mean ± SEM and statistically analyzed by two-tailed t-test (B, C, E) and Two-way ANOVA followed by Sidak’s post hoc test (D, F). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: We purchased recombinant human TIMP3 (Cat# 973-TM) and prosaptide Tx14 (Cat# 5151) from R&D Systems (Minneapolis, MN); recombinant mouse TIMP-1 (Cat# 593702) from BioLegend (San Diego, CA); an MMP14 inhibitor (NSC405020, Cat# 444295), MMP2 inhibitor (Cat# 444288), TACE/ADAM17 inhibitor (TAPI-2, Cat# 4444244), and R. Tonello et al.

Techniques: Western Blot, Control, Transfection, Recombinant, Two Tailed Test

Journal: Brain, behavior, and immunity

Article Title: Single-cell analysis of dorsal root ganglia reveals metalloproteinase signaling in satellite glial cells and pain.

doi: 10.1016/j.bbi.2023.08.005

Figure Lengend Snippet: Fig. 5. Recombinant TIMP3 protein reverses and prevents mechanical and cold allodynia in a mouse model of chemotherapy-induced neuropathic pain. (A) Schematic of the experiment showing the timeline of paclitaxel (PAX) or vehicle injections, pharmacological studies, and immunohistochemistry (IHC) analysis. (B) Representative image and (C) quantification of Timp3 protein in mouse DRG tissue 14 days after first injection of PAX or vehicle control (n = 5). (D, E) Paclitaxel- induced mechanical (von Frey) and (F) cold allodynia (dry ice) are significantly, dose-dependently reversed up to 6 h by single intrathecal administration of re combinant TIMP3 protein (rTIMP3, 3–100 ng/site) delivered at day 14 after chemotherapy injection (n = 4–5). (G) Schematic of experiment showing timeline of PAX injections concomitantly with rTIMP3 or PBS, behavioral tests, transcriptional, and histological analysis. (H, I) Repeated administrations of rTIMP3 (100 ng/site, i.t.) prevent paclitaxel-induced mechanical and cold allodynia (n = 5–6). BL = baseline. Data expressed as mean ± SEM, statistically analyzed by two-tailed t-test (C), two-way ANOVA followed by Sidak’s post hoc test (D, F, H, I), and one-way ANOVA followed by Tukey’s post hoc test (E). *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: We purchased recombinant human TIMP3 (Cat# 973-TM) and prosaptide Tx14 (Cat# 5151) from R&D Systems (Minneapolis, MN); recombinant mouse TIMP-1 (Cat# 593702) from BioLegend (San Diego, CA); an MMP14 inhibitor (NSC405020, Cat# 444295), MMP2 inhibitor (Cat# 444288), TACE/ADAM17 inhibitor (TAPI-2, Cat# 4444244), and R. Tonello et al.

Techniques: Recombinant, Immunohistochemistry, Injection, Control, Two Tailed Test

Journal: Brain, behavior, and immunity

Article Title: Single-cell analysis of dorsal root ganglia reveals metalloproteinase signaling in satellite glial cells and pain.

doi: 10.1016/j.bbi.2023.08.005

Figure Lengend Snippet: Fig. 6. Transcriptional analyses of TIMP3 signaling in cultured SGCs after paclitaxel treatment. (A) Schematic of the experi mental design used in cultured SGCs. (B) Representative image and quantification of immunofluorescence intensity of GFAP protein in SGC culture after 24 h of incu bation with paclitaxel (PAX, 300 nM) or vehicle control (Veh; n = 4). (C) Quantifi cation of SGC culture viability 24 h after PAX or Veh treatment (n = 6). (D) Quan tification of mRNA expression levels of Timp3, Mmp2, Mmp14 and Adam17 in SGC culture after PAX or Veh incubation (n = 6). (E) Heat map of mRNA expression of SGC and metalloprotease signaling markers in SGC culture after incubation with pro saptide Tx14 (1 µM) or pioglitazone (PGZ, 10 µM) and PAX compared to vehicle (n = 3). (F) Schematic illustrating the timeline of Tx14, PGZ, or PBS concomitantly treated with PAX, and the behavioral assay. Repeated injections of (G) Tx14 (10 µg/ site, i.t.) or (H) PGZ (100 µg/site, i.t.) prevent paclitaxel-induced mechanical allodynia (n = 6). BL = baseline. Data are expressed as mean ± SEM and statistically analyzed by two-tailed t-test (B, C, D), and Two-way ANOVA followed by Sidak’s post hoc test (G, H): *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: We purchased recombinant human TIMP3 (Cat# 973-TM) and prosaptide Tx14 (Cat# 5151) from R&D Systems (Minneapolis, MN); recombinant mouse TIMP-1 (Cat# 593702) from BioLegend (San Diego, CA); an MMP14 inhibitor (NSC405020, Cat# 444295), MMP2 inhibitor (Cat# 444288), TACE/ADAM17 inhibitor (TAPI-2, Cat# 4444244), and R. Tonello et al.

Techniques: Cell Culture, Immunofluorescence, Control, Expressing, Incubation, Behavioral Assay, Two Tailed Test