Figure 3 - Recovery from CFA Injury

Published Image

Figure 3. CFA injection produces mechanical hypersensitivity that resolves within 7 days in male and female mice. (A) Timeline of experimental testing. (B) Pain model to induce sensitization. (C) Representative images of Chaplan up-down von Frey measurements after CFA injection. CFA administration produces robust hypersensitivity at the site of injection that persists for at least 3 days and resolves within one week in both male (D, E) and female (H, I) mice. There were no changes in sensitivity of the contralateral (non-injected; right) hind paw during inflammatory pain and recovery from CFA injury in either males (F, G) or females (J, K). Data expressed as mean +/- SEM. \(***\) Indicates between-group difference where p < 0.001 and # indicates a within-subject difference from baseline where p < 0.05.

Statistical Analyses

# Select the left paws
left_paws <- rbind(female_left,male_left)

# Switch to long form
a <- left_paws %>% 
  melt(id.vars=c("ID","Sex","CFA"))

# Run RM anova on the 4 days of VF measuremenets
b <- anova_test(data=a, dv=value,wid=ID,between=c(CFA,Sex),within=variable,effect.size="pes")
knitr::kable(get_anova_table(b))

Effect	DFn	DFd	F	p	p<.05	pes
CFA	1	60	128.271	0.000	*	0.681
Sex	1	60	1.211	0.275		0.020
variable	3	180	99.726	0.000	*	0.624
CFA:Sex	1	60	1.314	0.256		0.021
CFA:variable	3	180	91.678	0.000	*	0.604
Sex:variable	3	180	2.651	0.050		0.042
CFA:Sex:variable	3	180	3.570	0.015	*	0.056

Significant main effects of CFA and timepoint.
Significant interaction between CFA and timepoint (F(3,180) = 91.67, p < 0.001)
Significant 3-way interaction between Sex, CFA and timepoint (F(3,180) = 3.57, p = 0.015)

# Run two way ANOVAs for males and females separately: 

## Males
res <- a %>%
  filter(Sex == "Male") %>%
  anova_test(dv=value,wid=ID,between=CFA,within=variable,effect.size = "pes")

There is a significant interaction between CFA treatment and time point (F(3,90) = 47.44, p < 0.001)

### Follow up for males: 
res <- a %>% 
  filter(Sex == "Male") %>%
  group_by(variable) %>%
  pairwise_t_test(value~CFA,p.adjust.method = "bonferroni")

tt(res)

variable	.y.	group1	group2	n1	n2	p	p.signif	p.adj	p.adj.signif
BL_L	value	Naive	CFA	16	16	0.5679999999999999	ns	0.5679999999999999	ns
hr_24	value	Naive	CFA	16	16	0.0000000000000122	****	0.0000000000000122	****
days_3	value	Naive	CFA	16	16	0.0000000146000000	****	0.0000000146000000	****
days_7	value	Naive	CFA	16	16	0.7670000000000000	ns	0.7670000000000000	ns

CFA-injected males have lower paw withdrawal thresholds than naive males 24 hours and 3 days post CFA administration (both p < 0.001).
There is no difference between the groups at baseline or 7 days post injection.

## Females 
a %>% 
  filter(Sex == "Female") %>%
  anova_test(dv=value,wid=ID,between=CFA,within=variable,effect.size="pes")

## ANOVA Table (type II tests)
## 
## $ANOVA
##         Effect DFn DFd      F                              p p<.05   pes
## 1          CFA   1  30 53.808 0.0000000359999999999999981061     * 0.642
## 2     variable   3  90 84.569 0.0000000000000000000000000427     * 0.738
## 3 CFA:variable   3  90 47.938 0.0000000000000000013200000000     * 0.615
## 
## $`Mauchly's Test for Sphericity`
##         Effect     W     p p<.05
## 1     variable 0.507 0.002     *
## 2 CFA:variable 0.507 0.002     *
## 
## $`Sphericity Corrections`
##         Effect   GGe      DF[GG]                    p[GG] p[GG]<.05   HFe
## 1     variable 0.754 2.26, 67.88 0.0000000000000000000289         * 0.819
## 2 CFA:variable 0.754 2.26, 67.88 0.0000000000000135000000         * 0.819
##       DF[HF]                      p[HF] p[HF]<.05
## 1 2.46, 73.7 0.000000000000000000000841         *
## 2 2.46, 73.7 0.000000000000001180000000         *

CFA-injected female mice also have lower paw withdrawal thresholds than naive males 24 hours and 3 days post CFA administration (both p < 0.001).

variable	.y.	group1	group2	n1	n2	p	p.signif	p.adj	p.adj.signif
BL_L	value	Naive	CFA	16	16	0.748999999999999999	ns	0.748999999999999999	ns
hr_24	value	Naive	CFA	16	16	0.000000000000000195	****	0.000000000000000195	****
days_3	value	Naive	CFA	16	16	0.000000594000000000	****	0.000000594000000000	****
days_7	value	Naive	CFA	16	16	0.446000000000000008	ns	0.446000000000000008	ns

CFA-injected males have lower paw withdrawal thresholds than naive males 24 hours and 3 days post CFA administration (both p < 0.001).
There is no difference between the groups at baseline or 7 days post injection.

# Follow up the significant 3-way interaction using piarwise comparisons by invstigating the effect of Sex on each day of testing split by CFA
res <- a %>%
  group_by(CFA,variable) %>%
  pairwise_t_test(value~Sex,p.adjust.method = "bonferroni")

tt(res)

CFA	variable	.y.	group1	group2	n1	n2	p	p.signif	p.adj	p.adj.signif
Naive	BL_L	value	Female	Male	16	16	0.4080	ns	0.4080	ns
Naive	hr_24	value	Female	Male	16	16	0.0254	*	0.0254	*
Naive	days_3	value	Female	Male	16	16	0.1390	ns	0.1390	ns
Naive	days_7	value	Female	Male	16	16	0.8710	ns	0.8710	ns
CFA	BL_L	value	Female	Male	16	16	0.5710	ns	0.5710	ns
CFA	hr_24	value	Female	Male	16	16	0.0170	*	0.0170	*
CFA	days_3	value	Female	Male	16	16	0.0288	*	0.0288	*
CFA	days_7	value	Female	Male	16	16	0.1940	ns	0.1940	ns

There was a sex difference in CFA-induced hypersensitivity both 24 hours (p = 0.017) and 3 days (p = 0.0288) post injection.
Female mice exhibited MORE sensitivity than males at the 24hour time point, and LESS sensitivity than males 3-days after CFA.

## Effect of day within each of the 4 groups (i.e., the same thing as the % BL stat..)
### Only read & interpret measurements relative to BASELINE

b <- a %>% group_by(CFA,Sex) %>% 
  pairwise_t_test(value~variable,p.adjust.method = "bonferroni")
tt(b)

Sex	CFA	.y.	group1	group2	n1	n2	p	p.signif	p.adj	p.adj.signif
Female	Naive	value	BL_L	hr_24	16	16	0.060299999999999999433786	ns	0.361999999999999988453681	ns
Female	Naive	value	BL_L	days_3	16	16	0.452000000000000012878587	ns	1.000000000000000000000000	ns
Female	Naive	value	hr_24	days_3	16	16	0.252000000000000001776357	ns	1.000000000000000000000000	ns
Female	Naive	value	BL_L	days_7	16	16	0.936000000000000054178884	ns	1.000000000000000000000000	ns
Female	Naive	value	hr_24	days_7	16	16	0.050599999999999999145128	ns	0.303999999999999992450483	ns
Female	Naive	value	days_3	days_7	16	16	0.405000000000000026645353	ns	1.000000000000000000000000	ns
Male	Naive	value	BL_L	hr_24	16	16	0.365999999999999992006394	ns	1.000000000000000000000000	ns
Male	Naive	value	BL_L	days_3	16	16	0.067000000000000003996803	ns	0.402000000000000023980817	ns
Male	Naive	value	hr_24	days_3	16	16	0.343000000000000027089442	ns	1.000000000000000000000000	ns
Male	Naive	value	BL_L	days_7	16	16	0.432999999999999996003197	ns	1.000000000000000000000000	ns
Male	Naive	value	hr_24	days_7	16	16	0.904000000000000025757174	ns	1.000000000000000000000000	ns
Male	Naive	value	days_3	days_7	16	16	0.285999999999999976463272	ns	1.000000000000000000000000	ns
Female	CFA	value	BL_L	hr_24	16	16	0.000000000000000000000110	****	0.000000000000000000000659	****
Female	CFA	value	BL_L	days_3	16	16	0.000000000000723000000000	****	0.000000000004340000000000	****
Female	CFA	value	hr_24	days_3	16	16	0.000000021800000000000000	****	0.000000130999999999999995	****
Female	CFA	value	BL_L	days_7	16	16	0.280000000000000026645353	ns	1.000000000000000000000000	ns
Female	CFA	value	hr_24	days_7	16	16	0.000000000000000000003480	****	0.000000000000000000020900	****
Female	CFA	value	days_3	days_7	16	16	0.000000000050700000000000	****	0.000000000304000000000000	****
Male	CFA	value	BL_L	hr_24	16	16	0.000000000000000000012500	****	0.000000000000000000075000	****
Male	CFA	value	BL_L	days_3	16	16	0.000000000000000101000000	****	0.000000000000000606000000	****
Male	CFA	value	hr_24	days_3	16	16	0.011700000000000000330291	*	0.069900000000000003796963	ns
Male	CFA	value	BL_L	days_7	16	16	0.338000000000000022648550	ns	1.000000000000000000000000	ns
Male	CFA	value	hr_24	days_7	16	16	0.000000000000000000000552	****	0.000000000000000000003310	****
Male	CFA	value	days_3	days_7	16	16	0.000000000000000003240000	****	0.000000000000000019400000	****

CFA administration produced a robust hypersensitivity in the injected paw.
There was no evidence of sensitivity in the contralateral (non-injured) paw.
CFA-induced sensitivity resolved within one week post injection.