Figure 2
Figure 2. A history of injury facilitates chronification of TMT-induced mechanical hypersensitivity. (A) Timeline for behavioral testing: Naive for CFA-primed mice were exposed to TMT for five-minutes, and von Frey (VF) paw withdrawal thresholds were repeatedly measured to investigate TMT-induced alterations in sensory thresholds. (B) VF raw withdrawal thresholds after TMT in the previously injured hind paw. (C) VF paw withdrawal thresholds after TMT in the right (not injured) hindpaw. (D) VF paw withdrawal thresholds 60 days after the single exposure to TMT. (E) VF paw withdrawal thresholds 180 days after the single TMT session. (F) To further characterize the nature of the persistent hypersensitive phenotype, we used a 45 second hotplate test 70 days after the TMT exposure. (G) Raster plots showing nociceptive responses (e.g., shaking, flinching) during the hot plate test. (H) Latency to exhibit the first nociceptive response on the hot plate. (I) Mice with a history of injury spent more time exhibiting nociceptive responses. (J) The frequency of nociceptive behaviors on the hotplate was also higher for CFA-primed mice. (K) Cumulative frequency of nociceptive responses across the 45 second hot plate test. Data displayed as mean +/- SEM. *** indicates p < 0.001.
Von Frey Paw Sensitivity After TMT
# Left
a <- Left_Data %>%
melt(id.vars = c("ID", "Condition")) %>%
na.omit()
anova_test(data = a, within = variable, dv = value, between = Condition, wid = ID)## ANOVA Table (type II tests)
##
## $ANOVA
## Effect DFn DFd F p p<.05 ges
## 1 Condition 1 14 46.740 8.10e-06 * 0.567
## 2 variable 9 126 21.226 1.93e-21 * 0.479
## 3 Condition:variable 9 126 30.857 7.38e-28 * 0.572
##
## $`Mauchly's Test for Sphericity`
## Effect W p p<.05
## 1 variable 0.017 0.548
## 2 Condition:variable 0.017 0.548
##
## $`Sphericity Corrections`
## Effect GGe DF[GG] p[GG] p[GG]<.05 HFe DF[HF]
## 1 variable 0.579 5.22, 73.01 2.56e-13 * 0.964 8.68, 121.48
## 2 Condition:variable 0.579 5.22, 73.01 4.62e-17 * 0.964 8.68, 121.48
## p[HF] p[HF]<.05
## 1 9.48e-21 *
## 2 6.13e-27 *# Right
a <- Right_Data %>%
melt(id.vars = c("ID", "Condition")) %>%
na.omit()
anova_test(data = a, within = variable, dv = value, between = Condition, wid = ID)## ANOVA Table (type II tests)
##
## $ANOVA
## Effect DFn DFd F p p<.05 ges
## 1 Condition 1 14 80.560 3.50e-07 * 0.621
## 2 variable 9 126 17.556 1.50e-18 * 0.473
## 3 Condition:variable 9 126 18.129 5.08e-19 * 0.481
##
## $`Mauchly's Test for Sphericity`
## Effect W p p<.05
## 1 variable 0.004 0.104
## 2 Condition:variable 0.004 0.104
##
## $`Sphericity Corrections`
## Effect GGe DF[GG] p[GG] p[GG]<.05 HFe DF[HF]
## 1 variable 0.486 4.37, 61.19 4.50e-10 * 0.733 6.6, 92.4
## 2 Condition:variable 0.486 4.37, 61.19 2.64e-10 * 0.733 6.6, 92.4
## p[HF] p[HF]<.05
## 1 3.62e-14 *
## 2 1.63e-14 *We measured von Frey (VF) paw withdrawal thresholds after TMT exposure to assess whether synthetic predator threat alters peripheral mechanical sensitivity (Figure 2A). A single five-minute exposure to TMT induced changes in paw withdrawal thresholds in the left (F9,126 = 21.23, p < 0.001, Figure 2B) and right (F9,126 = 17.56, p < 0.001, Figure 2C) hind paws that differed for pain-naive and CFA primed mice (Left hind paws: F9,126 = 30.86 p < 0.001; Right hind paws: F9,126 = 18.13, p < 0.001).
## Left Paws
a <- Left_Data %>%
melt(id.vars = c("ID","Condition","Baseline")) %>%
mutate(Perc_BL = value / Baseline * 100)
anova_test(data = a, dv = Perc_BL, between = Condition, within = variable, wid = ID)## ANOVA Table (type II tests)
##
## $ANOVA
## Effect DFn DFd F p p<.05 ges
## 1 Condition 1 14 87.365 2.14e-07 * 0.670
## 2 variable 8 112 6.793 3.03e-07 * 0.247
## 3 Condition:variable 8 112 26.260 1.90e-22 * 0.559
##
## $`Mauchly's Test for Sphericity`
## Effect W p p<.05
## 1 variable 0.015 0.114
## 2 Condition:variable 0.015 0.114
##
## $`Sphericity Corrections`
## Effect GGe DF[GG] p[GG] p[GG]<.05 HFe DF[HF]
## 1 variable 0.499 3.99, 55.91 1.59e-04 * 0.723 5.79, 81.02
## 2 Condition:variable 0.499 3.99, 55.91 2.86e-12 * 0.723 5.79, 81.02
## p[HF] p[HF]<.05
## 1 9.44e-06 *
## 2 7.74e-17 *a %>%
dplyr::group_by(Condition) %>%
pairwise_t_test(value ~ variable) %>%
filter(group1 == "Baseline")## # A tibble: 0 × 10
## # ℹ 10 variables: Condition <fct>, .y. <chr>, group1 <chr>, group2 <chr>,
## # n1 <int>, n2 <int>, p <dbl>, p.signif <chr>, p.adj <dbl>,
## # p.adj.signif <chr>## Right Paws
a <- Right_Data %>%
melt(id.vars = c("ID","Condition","Baseline")) %>%
mutate(Perc_BL = value / Baseline * 100)
anova_test(data = a, dv = Perc_BL, between = Condition, within = variable, wid = ID)## ANOVA Table (type II tests)
##
## $ANOVA
## Effect DFn DFd F p p<.05 ges
## 1 Condition 1 14 32.047 5.88e-05 * 0.553
## 2 variable 8 112 4.034 3.05e-04 * 0.117
## 3 Condition:variable 8 112 19.234 6.18e-18 * 0.388
##
## $`Mauchly's Test for Sphericity`
## Effect W p p<.05
## 1 variable 0.026 0.286
## 2 Condition:variable 0.026 0.286
##
## $`Sphericity Corrections`
## Effect GGe DF[GG] p[GG] p[GG]<.05 HFe DF[HF]
## 1 variable 0.596 4.76, 66.7 3.00e-03 * 0.94 7.52, 105.29
## 2 Condition:variable 0.596 4.76, 66.7 1.59e-11 * 0.94 7.52, 105.29
## p[HF] p[HF]<.05
## 1 4.34e-04 *
## 2 5.46e-17 *a %>%
dplyr::group_by(Condition) %>%
pairwise_t_test(value ~ variable) %>%
filter(group1 == "Baseline")## # A tibble: 0 × 10
## # ℹ 10 variables: Condition <fct>, .y. <chr>, group1 <chr>, group2 <chr>,
## # n1 <int>, n2 <int>, p <dbl>, p.signif <chr>, p.adj <dbl>,
## # p.adj.signif <chr>For pain-naive mice, TMT-induced mechanical hypersensitivity resolved three days after exposure (left hind paws: all p > 0.32; right hind paws: all p > 0.15, relative to baseline measurements). In contrast, CFA-primed mice exhibited a persistent state of mechanical hypersensitivity after TMT exposure that did not resolve. The ongoing mechanical hypersensitivity expressed by CFA-primed mice was present both in the previously injured (left) hind paw (all p < 0.012 relative to baseline measurements) and in the not-previously-injured right hind paw (all p < 0.001 relative to baseline measurements).
##
## Two Sample t-test
##
## data: L_60D by Condition
## t = 7.761, df = 12, p-value = 5.122e-06
## alternative hypothesis: true difference in means between group Naive and group CFA is not equal to 0
## 95 percent confidence interval:
## 0.4373241 0.7787102
## sample estimates:
## mean in group Naive mean in group CFA
## 1.0093144 0.4012973##
## Two Sample t-test
##
## data: R_60D by Condition
## t = 8.4717, df = 12, p-value = 2.082e-06
## alternative hypothesis: true difference in means between group Naive and group CFA is not equal to 0
## 95 percent confidence interval:
## 0.4831440 0.8177076
## sample estimates:
## mean in group Naive mean in group CFA
## 1.1013267 0.4509009Sixty days after the single TMT exposure, CFA-primed mice continued to exhibit lower paw withdrawal thresholds than pain-naive controls (F1,12 = 112.02, p < 0.001, Figure 2D), and the hypersensitivity did not differ between the right and left hind paws (p = 0.68).
##
## Two Sample t-test
##
## data: L_180D by Condition
## t = 9.2569, df = 9, p-value = 6.778e-06
## alternative hypothesis: true difference in means between group Naive and group CFA is not equal to 0
## 95 percent confidence interval:
## 0.4694201 0.7730487
## sample estimates:
## mean in group Naive mean in group CFA
## 0.9960839 0.3748495##
## Two Sample t-test
##
## data: R_180D by Condition
## t = 11.236, df = 9, p-value = 1.346e-06
## alternative hypothesis: true difference in means between group Naive and group CFA is not equal to 0
## 95 percent confidence interval:
## 0.5633834 0.8474259
## sample estimates:
## mean in group Naive mean in group CFA
## 1.0796716 0.3742669The hypersensitivity remained detectable among CFA-primed mice 180 days after the single exposure (F1,9 = 165.45, p < 0.001, Figure 2E) for both hind paws.
Hot Plate Test
# Latency to shake / flick
a <- data %>%
group_by(ID,CFA) %>%
filter(Behavior == "shake") %>%
filter(CFA != "CFA_1_day") %>%
summarise(
min=min(Start_clean)
)## `summarise()` has grouped output by 'ID'. You can override using the `.groups`
## argument.##
## Two Sample t-test
##
## data: min by CFA
## t = 1.2107, df = 12, p-value = 0.2493
## alternative hypothesis: true difference in means between group TMT_Only and group CFA_10_days is not equal to 0
## 95 percent confidence interval:
## -1.794543 6.282959
## sample estimates:
## mean in group TMT_Only mean in group CFA_10_days
## 12.094333 9.850125There was no group difference in the latency to first nociceptive response (p = 0.25 Figure 2G,H).
# Total time spent exhibiting nociceptive responses
a <- data %>%
group_by(ID,CFA) %>%
filter(Behavior == "shake") %>%
filter(CFA != "CFA_1_day") %>%
summarise(
sum=sum(Duration)
)## `summarise()` has grouped output by 'ID'. You can override using the `.groups`
## argument.##
## Two Sample t-test
##
## data: sum by CFA
## t = -3.1747, df = 12, p-value = 0.008
## alternative hypothesis: true difference in means between group TMT_Only and group CFA_10_days is not equal to 0
## 95 percent confidence interval:
## -1.7494097 -0.3254236
## sample estimates:
## mean in group TMT_Only mean in group CFA_10_days
## 2.753833 3.791250CFA-primed mice spent significantly more time displaying pain-related behaviors (t12 = 3.17, p = 0.008, Figure 2I)
# Frequency of nociceptive responses
a <- data %>%
group_by(ID,CFA) %>%
filter(Behavior == "shake") %>%
filter(CFA != "CFA_1_day") %>%
summarise(
n=n()
)## `summarise()` has grouped output by 'ID'. You can override using the `.groups`
## argument.##
## Two Sample t-test
##
## data: n by CFA
## t = -4.6446, df = 12, p-value = 0.0005657
## alternative hypothesis: true difference in means between group TMT_Only and group CFA_10_days is not equal to 0
## 95 percent confidence interval:
## -8.753410 -3.163256
## sample estimates:
## mean in group TMT_Only mean in group CFA_10_days
## 12.16667 18.12500and exhibited a higher frequency of pain responses (t12 = 4.64, p < 0.0001, Figure 2J,K) during the 45-second task.
In separate experiments, we replicated the same experimental setup, but exposed mice to the odor butyric acid (an odor that is repugnant but not fear-inducing) instead of TMT. We found that mice did not exhibit freezing behavior or mechanical hypersensitivity after butyric acid exposure (Figure S3), highlighting that the behavioral responses to TMT represent a specific fear response, and are not related to the other elements of the testing procedures.