Figure 4
Figure 4. Enhanced corticosterone production during TMT mediates chronification of mechanical hypersensitivity in CFA-primed mice. (A) Mice were injected with CFA and given 10 days to recover. 30 minutes before a single exposure to TMT, mice were injected with the corticosterone-synthesis inhibitor metyrapone (50 mg/kg). (B) Raster plots of freezing behavior during TMT exposure for metyrapone-treated mice. (C) Metrapone blocks freezing behavior during TMT and eliminates the group difference in freezing between CFA-primed and naive mice. (D) metyrapone blocks the increase in freezing across minutes with TMT. (E) There was no increase in the length of freezing episodes across the TMT session for metyrapone-treated mice. (F) Mechanical hypersensitivity after TMT in the left hindpaws. (G) Mechanical hypersensitivity after TMT in the right hidpaws.
Time Spent Freezing Behavior During TMT
Next, we administered the corticosterone synthesis inhibitor metyrapone to CFA-primed and pain-naive mice 30 minutes before a single five-minute TMT exposure to determine whether the hormonal stress response is necessary for TMT-induced freezing or mechanical hypersensitivity (Figure 4A).
b <- data %>%
filter(Behavior == "freeze") %>%
group_by(ID,Condition) %>%
summarise(
sum=sum(Duration),
Number=n(),
) %>%
mutate(Perc = (sum / 300)*100) %>%
mutate(Av_DUR = (sum / Number))## `summarise()` has grouped output by 'ID'. You can override using the `.groups`
## argument.##
## Two Sample t-test
##
## data: Perc by Condition
## t = 1.4134, df = 14, p-value = 0.1794
## alternative hypothesis: true difference in means between group Naive and group CFA is not equal to 0
## 95 percent confidence interval:
## -0.4533198 2.2054031
## sample estimates:
## mean in group Naive mean in group CFA
## 2.092833 1.216792Although metryapone does not alter general locomotor behavior in an open field (Figure S4), inhibition of corticosterone synthesis prevented TMT-induced freezing (Figure 4B) and eliminated the group differences in freezing between CFA-primed and pain-naive mice (p = 0.17, Figure 4C).
a <- data %>%
na.omit() %>%
mutate(Bins = cut(
Start_clean,
breaks = 5,
labels=c("1","2","3","4","5")
)) %>%
group_by(ID, Behavior, Condition, Bins) %>%
filter(Behavior == "freeze")
anova <- aov(Duration~Bins*Condition,data=a)
summary(anova)## Df Sum Sq Mean Sq F value Pr(>F)
## Bins 4 1.77 0.4415 1.234 0.300
## Condition 1 0.29 0.2934 0.820 0.367
## Bins:Condition 4 2.52 0.6291 1.758 0.142
## Residuals 120 42.93 0.3578Blocking the hormonal stress response also prevented the expected increase in freezing over the five-minute TMT exposure (p = 0.30, Figure 4D)
##
## Call:
## lm(formula = Duration ~ Start_clean * Condition, data = x)
##
## Residuals:
## Min 1Q Median 3Q Max
## -0.9888 -0.5259 -0.1586 0.3371 2.9247
##
## Coefficients:
## Estimate Std. Error t value Pr(>|t|)
## (Intercept) 1.0959277 0.0725218 15.112 <2e-16 ***
## Start_clean -0.0003900 0.0004045 -0.964 0.335
## ConditionCFA -0.0312178 0.1054500 -0.296 0.767
## Start_clean:ConditionCFA 0.0008662 0.0005866 1.477 0.140
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
##
## Residual standard error: 0.7025 on 756 degrees of freedom
## Multiple R-squared: 0.008431, Adjusted R-squared: 0.004496
## F-statistic: 2.143 on 3 and 756 DF, p-value: 0.09349and the extension of freezing episode duration across the session (p = 0.30, Figure 4E).
Raw VF scores
## Df Sum Sq Mean Sq F value Pr(>F)
## variable 4 4.772 1.1931 69.185 <2e-16 ***
## Condition 1 0.002 0.0025 0.145 0.705
## variable:Condition 4 0.055 0.0137 0.794 0.533
## Residuals 70 1.207 0.0172
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1## Df Sum Sq Mean Sq F value Pr(>F)
## variable 4 5.075 1.2686 74.424 <2e-16 ***
## Condition 1 0.043 0.0433 2.540 0.116
## variable:Condition 4 0.007 0.0019 0.109 0.979
## Residuals 70 1.193 0.0170
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1Despite the complete absence of TMT-induced freezing among metyrapone-treated mice, both CFA-primed and pain-naive mice exhibited significant bilateral mechanical hypersensitivity after TMT exposure (Left hindpaws: F4,70 = 69.18, p < 0.0001, Figure 4F; Right hindpaws: F4,70 = 74.42, p < 0.0001, Figure 4G).
VF Thresholds Relative to Baseline Measurements
# Left paws
a %>%
group_by(Condition) %>%
pairwise_t_test(value ~ variable, p.adjust.method = "fdr")## # A tibble: 20 × 10
## Condition .y. group1 group2 n1 n2 p p.signif p.adj
## * <fct> <chr> <chr> <chr> <int> <int> <dbl> <chr> <dbl>
## 1 Naive value BL_L L_3hr 8 8 9.95e-11 **** 9.95e-10
## 2 Naive value BL_L L_24hr 8 8 9.29e- 8 **** 2.32e- 7
## 3 Naive value L_3hr L_24hr 8 8 2.3 e- 2 * 3.29e- 2
## 4 Naive value BL_L L_3D 8 8 5.07e- 2 ns 6.34e- 2
## 5 Naive value L_3hr L_3D 8 8 3.23e- 8 **** 1.08e- 7
## 6 Naive value L_24hr L_3D 8 8 4.22e- 5 **** 7.03e- 5
## 7 Naive value BL_L L_7D 8 8 2.49e- 1 ns 2.77e- 1
## 8 Naive value L_3hr L_7D 8 8 2.68e- 9 **** 1.34e- 8
## 9 Naive value L_24hr L_7D 8 8 3.22e- 6 **** 6.44e- 6
## 10 Naive value L_3D L_7D 8 8 4.01e- 1 ns 4.01e- 1
## 11 CFA value BL_L L_3hr 8 8 2.08e-11 **** 1.04e-10
## 12 CFA value BL_L L_24hr 8 8 4.62e- 7 **** 9.24e- 7
## 13 CFA value L_3hr L_24hr 8 8 1.32e- 3 ** 1.89e- 3
## 14 CFA value BL_L L_3D 8 8 3.03e- 1 ns 3.37e- 1
## 15 CFA value L_3hr L_3D 8 8 3.61e-10 **** 1.20e- 9
## 16 CFA value L_24hr L_3D 8 8 1.10e- 5 **** 1.84e- 5
## 17 CFA value BL_L L_7D 8 8 6.32e- 1 ns 6.32e- 1
## 18 CFA value L_3hr L_7D 8 8 5.83e-12 **** 5.83e-11
## 19 CFA value L_24hr L_7D 8 8 1.08e- 7 **** 2.69e- 7
## 20 CFA value L_3D L_7D 8 8 1.35e- 1 ns 1.69e- 1
## # ℹ 1 more variable: p.adj.signif <chr># Right paws
b %>%
group_by(Condition) %>%
pairwise_t_test(value ~ variable, p.adjust.method = "fdr")## # A tibble: 20 × 10
## Condition .y. group1 group2 n1 n2 p p.signif p.adj
## * <fct> <chr> <chr> <chr> <int> <int> <dbl> <chr> <dbl>
## 1 Naive value BL_R R_3hr 8 8 2.06e-10 **** 1.03e- 9
## 2 Naive value BL_R R_24hr 8 8 3.28e- 7 **** 6.56e- 7
## 3 Naive value R_3hr R_24hr 8 8 1.59e- 2 * 2.28e- 2
## 4 Naive value BL_R R_3D 8 8 4.04e- 1 ns 4.48e- 1
## 5 Naive value R_3hr R_3D 8 8 2.24e- 9 **** 7.45e- 9
## 6 Naive value R_24hr R_3D 8 8 4.27e- 6 **** 7.11e- 6
## 7 Naive value BL_R R_7D 8 8 8.78e- 1 ns 8.78e- 1
## 8 Naive value R_3hr R_7D 8 8 1.34e-10 **** 1.03e- 9
## 9 Naive value R_24hr R_7D 8 8 2.06e- 7 **** 5.14e- 7
## 10 Naive value R_3D R_7D 8 8 3.24e- 1 ns 4.05e- 1
## 11 CFA value BL_R R_3hr 8 8 5.49e-12 **** 5.49e-11
## 12 CFA value BL_R R_24hr 8 8 6.60e- 8 **** 1.65e- 7
## 13 CFA value R_3hr R_24hr 8 8 1.94e- 3 ** 2.78e- 3
## 14 CFA value BL_R R_3D 8 8 2.22e- 1 ns 2.78e- 1
## 15 CFA value R_3hr R_3D 8 8 1.53e-10 **** 5.09e-10
## 16 CFA value R_24hr R_3D 8 8 2.82e- 6 **** 4.70e- 6
## 17 CFA value BL_R R_7D 8 8 6.49e- 1 ns 6.49e- 1
## 18 CFA value R_3hr R_7D 8 8 1.83e-11 **** 9.17e-11
## 19 CFA value R_24hr R_7D 8 8 2.62e- 7 **** 5.25e- 7
## 20 CFA value R_3D R_7D 8 8 4.39e- 1 ns 4.87e- 1
## # ℹ 1 more variable: p.adj.signif <chr>CFA-primed mice showed resolution of TMT-induced mechanical hypersensitivity within one week post-exposure (naive: both paws p > 0.27 relative to baseline measurements; CFA-primed: both paws p > 0.63), suggesting that the exaggerated hormonal stress response during TMT modulates the persistence of TMT-evoked mechanical hypersensitivity among mice with a history of CFA injury.