Upregulation of TRPV1 immunohistochemical expression in L3-5 DRG neurons from FM model mice and reversal by EA.

<p>(A-C) Immunohistochemical staining showing TRPV1-positive cells (green) in control (A), FM (B), and EA groups (C). (D-F) Immunohistochemical staining showing TRPV4-positive neurons (green) in control (D), FM (E), and EA groups (F). (G, H) Proportions of immunopositive neurons. Con = Control; FM = acid induced fibromyalgia pain; EA = FM pain with electroacupuncture. DRG = dorsal root ganglion. Arrows identify immunopositive neurons. Scale bar = 50 μm.</p

Expression levels of pERK, pp38, and pJNK in L3-L5 DRG from TRPV1 null mice.

<p>(A) Western blots of DRG lysates probed for pERK, pp38, and pJNK. (B) Expression levels of pERK, pp38, and pJNK were unchanged at 15 min after injection. α-tubulin was the internal control. Con = Control; FM = acid induced FM pain.</p

Targeting TRPV1 and ERK signaling pathway attenuated mechanical hyperalgesia by von Frey filaments.

<p>(A) Mechanical responses of FM mice. (B) Mechanical responses of Trpv1^-/- mice. (C) Mechanical responses of FM mice pretreated with CZP. (D) Mechanical responses of FM mice pretreated with U0126. Mice were tested before injection (baseline, B), 4 hours after injection, day 1 (D1), day 5 (D5), day 6 (D6), and day 8 (D8). Red arrowheads indicate acid injection. **<i>p</i> < 0.01 compared to baseline. (n = 6 mice per group).</p

Protein expression of pERK in DRG neurons from Con, FM, and EA groups.

<p>(A-C) Immunohistochemical staining showing pERK-reactive cells in the L3-5 DRG of control (A), FM (B), and EA mice (C) 15 min after intramuscular injection of normal saline (control) or acid saline (FM and EA groups). (D-F) Immunohistochemical staining showing pERK-reactive neurons at 60 min after injection (red). (G, H) Proportions of immunopositive neurons. Con = Control; FM = acid induced fibromyalgia pain; EA = electroacupuncture. DRG = dorsal root ganglion. Arrows mean immuno-positive neurons. Scale bar = 50 μm.</p

The expression of pERK, pp38, and pJNK in lumbar SC.

<p>(A) Western blots of lumbar SC lysates. (B) pERK expression was increased at 15 min after acid injection and reversed by EA. This increase in pERK was not observed at 60 min after injection. (C) pp38 expression was not altered after acid injection and/or EA. (D) pJNK was not altered after acid injection and/or EA. α-tubulin was the internal control. Con = Control; 15 = 15 min after acid injection in FM group; 15EA = 15 min after acid injection in EA group. 60 = 60 min after acid injection in FM group; 60EA = 60 min after acid injection in EA group.</p

The expression of pERK, pp38, and pJNK proteins in L3-L5 DRG.

<p>(A) pERK, pp38, and pJNK kinases were measured by Western blot in lysates from DRG. (B) pERK expression was increased at 15 min after acid injection in FM mice and reversed by EA. No change in pERK expression was observed at 60 min after injection. (C) pp38 expression in DRG was not altered after acid injection and/or EA. (D) pJNK expression was not altered after acid injection and/or EA. α-tubulin expression was the internal control. Con = Control; 15 = 15 min after acid injection in FM group; 15EA = 15 min after acid injection in EA group EA. 60 = 60 min after acid injection in FM group; 60EA = 60 min after acid injection in EA group.</p

Upregulation of TRPV1 protein expression in DRG neurons from FM mice, upregulation of both TRPV1 and TRPV4 in lumbar spinal cord (SC) of FM mice, and reversal of overexpression by EA.

<p>(A) Western blots of DRG lysates showing TRPV1 upregulation in FM mice compared to control mice and reversal by EA. (B) Upregulation of TRPV1 protein in lysates from SC. (C) TRPV4 expression levels were unaltered in the DRG of Con, FM, and EA groups. (D) Upregulation of TRPV4 in the SC of FM mice and reversal by EA. β-actin was used as the internal control. (E, F) Proportions of immunopositive neurons. Con = Control; FM = acid induced fibromyalgia pain; EA = electroacupuncture. DRG = dorsal root ganglion. SC = spinal cord.</p

Electroacupuncture (EA) attenuated mechanical hyperalgesia induced by repeated intramuscular acid saline injection (fibromyalgia model, FM) as measured by von Frey filaments.

<p>(A) Mechanical responses of saline-injected control mice. (B) Mechanical responses of FM group mice. (C) Mechanical responses of FM mice pretreated with EA (FM + EA group). (D) Mechanical responses of FM mice pretreated by sham EA. Mice were tested before injection (baseline, B), 4 hours after injection, day 1 (D1), day 5 (D5), day 6 (D6), and day 8 (D8). Red arrowheads indicate acid injection; Blue dashes indicate EA. **<i>p</i> < 0.01 compared to baseline (n = 9 mice per group).</p

Electrophysiological properties of L3-5 dorsal root ganglion (DRG) neurons from Con, FM, and EA groups.

<p>(A) The action potential (AP) threshold was lower in the FM group than the control group. EA reduced neuronal excitation by increasing AP threshold, reversing the effect of acid injection. (B) FM induction decreased both AP rise and fall times compared to controls, effects also reversed by AE. (C) There were no significant group differences in AP amplitude and afterhyperpolarization (AHP) duration. (D) Data summary and statistical analyses.</p

Additional file 1 of Controlled release of Clenbuterol from a hydroxyapatite carrier for the treatment of Alzheimer’s Disease

Additional file 1: Pharmacological synergy and proposed mechanism of action of clenbuterol in Alzheimer’s diseas