Journal: The Journal of Neuroscience

Article Title: Citric Acid in Drug Formulations Causes Pain by Potentiating Acid-Sensing Ion Channel 1

doi: 10.1523/JNEUROSCI.2087-20.2021

Figure Lengend Snippet: Citrate potentiates ASIC-dependent pain behavior. A–C , Each mouse was injected with either PBS, pH 7.4 ( A, C ), or citrate, pH 7.4 ( B ) in one hindpaw and then PBS, pH 5.5 ( A ); or citrate, pH 5.5 ( B ); or citrate, pH 7.4 ( C ), in the other hindpaw, or vice versa. The presence (+) or absence (o) of a withdrawal reflex was recorded; n = 4–6 per group, n.s. p > 0.05, * p < 0.05, two-tailed Mann–Whitney U test, in which (+) was ranked as 1 and (o) was ranked as 0. D–F , Each mouse was injected in either hindpaw with PBS, pH 7.4 or 5.5, as described for A–C , with or without amiloride (100 μ m ). The presence (+) or absence (o) of a withdrawal reflex was recorded; n = 4–14 per group, n.s. p > 0.05, *** p < 0.001, two-tailed Mann–Whitney U test, in which (+) was ranked as 1 and (o) was ranked as 0. G , Each mouse was injected with PBS, pH 5.5, in one hindpaw and then citrate, pH 5.5 (25 m m ), in the other hindpaw, or vice versa. The relative withdrawal intensity between the two hindpaws was compared and recorded: (+) for greater, (=) for equivalent, and (−) for lesser withdrawal response; n = 7 per group, * p < 0.05, two-tailed Mann–Whitney U test, in which (+) was ranked as +1, (=) was ranked as 0, and (−) was ranked as −1. H , Summary of the results from . n.s., Not significant.

Article Snippet: Sodium citrate tribasic dihydrate and capsaicin were purchased from Sigma-Aldrich (catalog #S4641 and #M2028, respectively), amiloride was purchased from Alomone Labs (catalog #A-140), 10% neutral buffered formalin was purchased from TONYAR BIOTECH.

Techniques: Injection, Two Tailed Test, MANN-WHITNEY

Journal: The Journal of Neuroscience

Article Title: Citric Acid in Drug Formulations Causes Pain by Potentiating Acid-Sensing Ion Channel 1

doi: 10.1523/JNEUROSCI.2087-20.2021

Figure Lengend Snippet: Citrate also potentiates ASIC lacking ASIC1 subunit in mouse primary sensory neurons. A , Primary sensory neurons isolated from the mouse dorsal root ganglia were perfused in a bath solution, pH 7.4, containing no citrate, and each stimulus consisted of a 3 s exposure (at 30 s intervals) to a test solution, pH 5.5, with or without citrate (25 m m ) or amiloride (100 μ m ). B , Summarized result from A . C , D , Similar to B , except the primary sensory neurons were from mice lacking ASIC1 ( ASIC1 −/− ; C ) and ASIC2 ( ASIC2 −/− ; D ), respectively. In B–D , error bars indicate the mean ± SEM; n = 5 per group, ** p < 0.01, *** p < 0.001, one-way ANOVA with a post hoc Tukey's test.

Article Snippet: Sodium citrate tribasic dihydrate and capsaicin were purchased from Sigma-Aldrich (catalog #S4641 and #M2028, respectively), amiloride was purchased from Alomone Labs (catalog #A-140), 10% neutral buffered formalin was purchased from TONYAR BIOTECH.

Techniques: Isolation

Journal: The Journal of Neuroscience

Article Title: Citric Acid in Drug Formulations Causes Pain by Potentiating Acid-Sensing Ion Channel 1

doi: 10.1523/JNEUROSCI.2087-20.2021

Figure Lengend Snippet: Concentrated citric acid at pH 3.5 but not at pH 5.5 induces paw-licking behavior that is ASIC1 independent. A , Each mouse was injected with either 5% formalin; 30 m m citrate, pH 5.5; or 100 m m citrate, pH 5.5 or 3.5, into one hindpaw, and the duration of paw licking over a period of 30 min was recorded; n = 6 per group, n.s. p > 0.05, *** p < 0.001, one-way ANOVA with a post hoc Tukey's test. B , C , Each mouse was injected with either 5% formalin, with or without amiloride (100 μ m ; B ) or 100 m m citrate, pH 3.5, with or without amiloride (100 μ m ; C ) into one hindpaw, and the duration of paw licking over a period of 30 min was recorded; n = 6 per group, n.s. p > 0.05, two-tailed Student's t test. D , wild-type mice (WT) or mice with homozygous ASIC1 deletion ( ASIC1 −/− ) were injected with 100 m m citrate, pH 3.5, into one hindpaw, and the duration of paw licking over a period of 30 min was recorded; n = 6 per group, n.s. p > 0.05, two-tailed Student's t test. n.s., Not significant.

Article Snippet: Sodium citrate tribasic dihydrate and capsaicin were purchased from Sigma-Aldrich (catalog #S4641 and #M2028, respectively), amiloride was purchased from Alomone Labs (catalog #A-140), 10% neutral buffered formalin was purchased from TONYAR BIOTECH.

Techniques: Injection, Two Tailed Test

Journal: The Journal of Neuroscience

Article Title: Citric Acid in Drug Formulations Causes Pain by Potentiating Acid-Sensing Ion Channel 1

doi: 10.1523/JNEUROSCI.2087-20.2021

Figure Lengend Snippet: Acetic acid but not citric acid induces writhing behavior that is ASIC1 independent. A , B , Each mouse received intraperitoneal injection of either 1% acetic acid, pH 3.5; 30 m m citric acid, pH 5.5; or 100 m m citric acid, pH 3.5; and the numbers of abdominal contractions ( A ) and body contortions ( B ) over a period of 10 min were recorded; n = 5 per group. n.s. p > 0.05, *** p < 0.001, one-way ANOVA with a post hoc Tukey's test. C , D , Each mouse received intraperitoneal injection of 1% acetic acid, pH 3.5, with or without amiloride (100 μ m ), and the numbers of abdominal contractions ( C ) and body contortions ( D ) over a period of 10 min were recorded; n = 5 per group, n.s. p > 0.05, two-tailed Student's t test. E , F , wild-type mice (WT) or mice with homozygous ASIC1 deletion ( ASIC1 −/− ) received intraperitoneal injection of 1% acetic acid, pH 3.5, and the numbers of abdominal contractions ( E ) and body contortions ( F ) over a period of 10 min were recorded; n = 6 per group, n.s. p > 0.05, two-tailed Student's t test. n.s., Not significant.

Article Snippet: Sodium citrate tribasic dihydrate and capsaicin were purchased from Sigma-Aldrich (catalog #S4641 and #M2028, respectively), amiloride was purchased from Alomone Labs (catalog #A-140), 10% neutral buffered formalin was purchased from TONYAR BIOTECH.

Techniques: Injection, Two Tailed Test