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. Author manuscript; available in PMC: 2021 Jul 15.
Published in final edited form as: Behav Brain Res. 2020 May 16;390:112698. doi: 10.1016/j.bbr.2020.112698

C57BL/6 Substrain Differences in Formalin-Induced Pain-Like Behavioral Responses

Esad Ulker a, Martial Caillaud a, Trusha Patel a, Alyssa White a, Danyal Rashid a, Mashael Alqasem a, Aron H Lichtman a, Camron D Bryant b, M Imad Damaj a
PMCID: PMC7375808  NIHMSID: NIHMS1595169  PMID: 32428630

Abstract

Substantial evidence from preclinical models of pain suggests that basal and noxious nociceptive sensitivity, as well as antinociceptive responses to drugs, show significant heritability. Individual differences to these responses have been observed across species from rodents to humans. The use of closely related C57BL/6 inbred mouse substrains can facilitate gene mapping of acute nociceptive behaviors in preclinical pain models. In this study, we investigated behavioral differences between C57BL/6J (B6J) and C57BL/6N (B6N) substrains in the formalin test, a widely used tonic inflammatory pain model, using a battery of pain-related phenotypes, including reflexive tests, nesting, voluntary wheel running, sucrose preference and anxiety-like behavior in the light/dark test at two different time points (1-h and 24-h). Our results show that these substrains did not differ in reflexive thermal and mechanical responses at the 1-h time point. However, B6N substrain mice showed increased sensitivity to spontaneous pain-like behaviors. In addition, B6N substrain continued to show higher levels of mechanical hypersensitivity compared to controls at 24-h. indicating that mechanical hypersensitivity is a more persistent pain-related phenotype induced by formalin. Finally, no sex differences were observed in our outcome measures. Our results provide a comprehensive behavioral testing paradigm in response to an inflammatory agent for future mouse genetic studies in pain.

Keywords: Genetic variability, Rodents, C57BL/6 substrains, Formalin model, Inflammatory pain, Reflexive tests

1. INTRODUCTION

Pain is defined by the International Association for the Study of Pain as an unpleasant sensory and or emotional experience associated with actual or potential tissue damage [1] and is one of the most common reasons patients seek medical attention. There is a high degree of individual variation in pain, likely due to multiple complex environmental and genetic factors [2]. There is growing evidence that several genes play a critical role in determining pain sensitivity, pain reporting and susceptibility to developing chronic pain and their response to surgical pain [3]. However, the study of pain in humans is challenging, since pain is a subjective complex sensory, motivational, and behavioral trait influenced by many factors such as gender, genetics, race and outcome measures of the pain experience [4]. Over the last two decades, rodent models have proven useful for identifying genetic factors that influence pain. Mouse models provide greater control over genetic background, environmental influences, previous history, and noxious stimulus parameters, compared to human studies. Findings from mouse genetic studies will help in the development of safer and more individualized analgesics [5].

Recently, Bryant et al. reported enhanced sensitivity to formalin-induced inflammatory nociceptive behaviors (paw licking in late phase or phase II) and paw edema in C57BL/6J (B6J) versus C57BL/6NCrl (B6N) substrains[6]. The formalin test involves an injection of formalin into the plantar surface of an animal paw and produces specific behaviors like paw lifting, flinching, licking, and vocalization in a biphasic manner. Most studies report only pain like-behaviors in phase I (early phase) (0–5 min) which results from the direct activation of primary nociceptive afferents and phase II (10–40 min) which involves, at least in part, inflammation-induced central sensitization in the dorsal horn of the spinal cord [7]. However, paw behaviors such as lifting and licking may not always indicate pain because neuronal hyperactivity can persist long after formalin injection with no paw phenotypic responses [8,9].

In the present study, we examined formalin-induced differences in pain-associated behaviors in female and male B6J and B6N mice at 1-h and 24-h post-formalin injection. Furthermore, we included several behavioral outcome measures in addition to the traditional paw diameter and paw licking that encompass not only reflexive responses such as mechanical and thermal sensitivity but various spontaneous and affective-like responses. These additional measures were chosen based on items from the Brief Pain Inventory (BPI)(short form) [10] which is the most widely used scale in the clinical setting such as general and daily life activities, walking ability, and enjoyment of life [11]. We also included two innate behaviors to assess the general well-being of rodents as surrogate markers of pain-like behaviors, namely, the reduction in nesting behavior and the reduction in motor performance/motivation using the voluntary wheel running test. These tests exploit “evolutionary-conserved rodent behaviors that are not essential for survival in a laboratory setting”. It has thus been suggested that these behaviors could model aspects of the “activities of daily living” in humans[12]. We also measured the decrease in sucrose preference as a measure of anhedonia-like behaviors as well as anxiety-like behavior in the light-dark box test.

2. MATERIALS AND METHODS

2.1. Animals

Adult (8–10 weeks of age at the start of the experiments) male and female C57BL/6J (B6J) (The Jackson Laboratory, Bar Harbor, ME) and C57BL/6NCrl mice (B6N) (Charles River Laboratories, Wilmington, MA). Animals’ body weights were B6J/1-h/Male 26.1±0.4 gr, B6J/1-h/Female 20.4±0.4 gr, B6J/24-h/Male 26.0±0.4 gr, B6J/24-h/Female 20.4±0.4 gr, B6N/1-h/Male 26.1±0.4gr, B6N/1-h/Female 20.2±0.3 gr, B6N/24-h/Male 26.2±0.4 gr, B6N/24-h/Female 20.5±0.6 gr. Mice were housed in a 21°C humidity-controlled Association for Assessment and Accreditation of Laboratory Animal Care–approved animal care facility at Virginia Commonwealth University. The rooms were on a 12-h light/dark cycle (lights on at 7:00 AM, off at 7:00 PM). Mice were acclimated for two weeks after their arrival into the facilities. Animals received food (Teclad, Envigo) ad libitum before and during the experiment. For testing, they were placed in individual cages with two 15 ml sippers with tap water. All mice were acclimated to their new cages 2 days before the experiments. All experiments were performed during the light cycle and were approved by the Institutional Animal Care and Use Committee of Virginia Commonwealth University. All studies were conducted by the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals.

2.2. Formalin injection

Each mouse received an intraplantar injection of 20 μL of vehicle (physiologic saline) or formalin (2.5%) to the right hindpaw. Mice were tested in a series of behavioral assays at 1-h and 24-h post-formalin injection.

2.2.1. 1-hour post-formalin injection

Paw diameter was measured before the formalin injection. After 1 hr post-injection, mice were transported into the light-dark behavior (LDB) room and acclimatized for 30 min. After the LDB test, paw diameters were measured again. Mice were subjected to mechanical sensitivity test, thermal sensitivity test, wheel running test, nesting test, and sucrose preference test in this order.

2.2.2. 24-hour post-formalin injection

A new cohort of experimentally naïve mice was used for the 24-h experiments. Paw diameter was measured before the injections. After 24-h mice were transported to the LDB room and acclimatized for 30 minutes. After the LDB test, paw diameters were once again measured. Mice were subjected to mechanical sensitivity test, thermal sensitivity test, wheel running test, nesting test, and sucrose preference test in this order.

2.3. Paw edema

The thickness of the right paw was measured both before and after formalin injection following the LDB assay using a digital caliper (Traceable Calipers, Friendswood, TX, USA). Data were recorded to the nearest ±0.01 mm and expressed as a change in paw thickness.

2.4. Light Dark Boxes

The light-dark box is made of a small, enclosed dark chamber (36cm length × 10cm width × 34cm height) with an opening (6 × 6 cm) extending to a larger, light compartment (36cm length × 21cm width × 34cm height). Illumination levels of light and dark side were 850 lumens and 5 lumens, respectively. The light-dark box was also inside an enclosed sound-attenuating box. The mice were acclimated to the test room for 30 minutes before testing. Mice were placed in the corner of the light chamber; their head orientation was towards the opening and allowed to freely explore the box for 5 min. The total time spent in the light chamber, latency to first enter the dark side, and total number of transitions was recorded for 5 min by video monitoring and results reported by ANY-MAZE software (Stoelting Co., Wood Dale, IL).

2.5. Mechanical Sensitivity

Mechanical withdrawal thresholds were determined with the up-and-down method [13] with slight modifications. Mice were placed in clear plastic chambers (9 × 10 cm) with mesh metal flooring and allowed to acclimate for 15 min before testing. A series of calibrated von Frey filaments (Stoelting, Inc., Wood Dale, IL, USA) with logarithmically increasing stiffness ranging were applied to the injected paw. Paw withdrawal, licking, and shaking was considered as positive responses. The mechanical sensitivity was expressed in grams.

2.6. Thermal Sensitivity

Thermal sensitivity was measured via the Hargreaves test. Mice were placed in the same clear plastic chambers (9 × 10 cm) on a glass surface and allowed to acclimate before testing. The radiant heat source was directed to the plantar surface of the paw which received the injection. The same responses (paw withdrawal, licking, and shaking) were chosen to determine withdrawal latency. The cut-off for this test was 20 seconds. Three measurements were taken from the paw which received the injection and average latency was calculated

2.7. Wheel Running

Wheel running was assessed in polycarbonate activity wheels (diameter 21.6 cm; width 6 cm) with a steel rod axle. The wheel could only be turned in a single direction. Multiple activity cages were contained within a testing room. The number of rotations completed was measured over 1 hand turned into a distance traveled value (meters) by multiplying rotations completed by ‘0.68’.

2.8. Nesting Test

The nesting procedure was adapted as previously described by Oliver et al. [14]. All previous nesting material was removed from the home cage before conducting the nesting assay. For each cage, two compressed cotton nestlets were divided into half, yielding four rectangular pieces of equal size. The nestlet pieces were then placed in each of the four corners of the cage. The mice were allowed 120 min to nest, after which a nesting consolidation score was recorded (1: no nestlet piece grouped, 2: nestlet pieces grouped in one or two pair, 3: 3 nestlet pieces grouped, 4: all nestlet pieces grouped, 5: all nestlet pieces grouped and completely shredded).

2.9. Sucrose Preference

Mice had free access to two 15 ml sipper tubes containing tap water for 2 days as a baseline. Mice were then exposed to two 15 ml sipper tubes, one with tap water and the other with 2% sucrose solution for two hours after the nesting test was done. At the end of two hours, sucrose preference was calculated by (sucrose consumption/total fluid consumption) × 100 and sucrose bottle was removed. We conducted the sucrose preference test at the very end as our preliminary studies showed that it interfered with the nesting test.

2.10. Cumulative Behavioral Changes

Cumulative behavioral changes were calculated from reflexive and non-reflexive responses. Reflexive responses included mechanical and thermal sensitivity assays; non-reflexive responses were composed of LDB, wheel running, nesting and sucrose preference assays. The changes were reported as percentile changes from their corresponding control groups.

2.11. Statistical Analysis

Data were analyzed using the GraphPad software, version 8.3.0 (GraphPad Software, Inc., La Jolla, CA) and are expressed as the mean ± S.E.M. Normality and homoscedasticity of all data sets except nesting and number of transitions were confirmed by using the Shapiro–Wilk and Levene tests, respectively. Preliminary results did not show any sex differences and thus males and females were pooled together (Supplementary Tables 1 and 2). Nesting and number of transitions were analyzed with Kruskal Wallis [planned comparison (Dunn’s test)]. All other tests were analyzed with 2-way ANOVA [post hoc analysis (Tukey’s test)]. Data are expressed as the mean ± S.E.M. of 10 mice/per sex/per group for all tests. The p values less than 0.05 were considered significant.

3. RESULTS

3.1. Paw Diameter

Paw diameter reflects formalin-induced edema. There was a significant effect of Substrain and Treatment on paw diameter in both time points (Fsubstrain(1-h)(1,76)=12.81, p<0.001; Ftreatment(1-h)(1,76)=1243, p<0.0001; Fsubstrain(24-h)(1,76)=8.198, p<0.01; Ftreatment(24-h)(1,76)=942.6, p<0.0001) An interaction of these two factors was only observed in 1-h timepoint (Finteraction(1-h)(1,76)=11.22, p<0.01; Finteraction(24-h)(1,76)=2.703, p=0.1043) (Figures 1a and 2a). Vehicle treated groups did not differ from each other at any time point (Figure 1a; B6J-VEH vs B6N-VEH, p=0.9984 and Figure 2a; B6J-VEH vs B6N-VEH, p=0.9492). The B6J substrain showed significantly more edema than the B6N substrain at both time points (Figure 1a; B6JFOR>B6N-FOR, p<0.0001, and Figure 2a; B6J-FOR>B6N-FOR, p<0.05). At the time of the injection, there was no difference in body weights between substrains (Supp. Figure 1: BW)

Figure 1:

Figure 1:

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Formalin’s effect on the 1-h groups.

The difference in paw diameters (a), light-dark boxes (b), mechanical sensitivity (c), thermal sensitivity (d), wheel running (e), nesting scoring (f), and sucrose preference (g) in vehicle and formalin treated B6J mice. Data expressed as the mean±S.E.M of 10 mice/per sex/per group. *, p<0.05; ****, p<0.0001; #, p<0.05; ####, p<0.0001

Figure 2:

Figure 2:

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Formalin’s effect on the 24 h groups.

The difference in paw diameters (a), light-dark boxes (b), mechanical sensitivity (c), thermal sensitivity (d), wheel running (e), nesting scoring (f), and sucrose preference (g) in vehicle and formalin treated B6N mice. Data expressed as the mean S.E.M of 10 mice/ per sex/per group. ****, p<0.0001; #, p<0.05; ##, p<0.01

3.2. LDB

The LDB assay serves as a model for anxiety-like behaviors. Two-way ANOVA revealed main effects of Treatment (Ftreatment(1-h)(1,76)=4.821, p<0.05 and Ftreatment(24-h)(1,76)=4.035, p<0.05) and Substrain (Fsubstrain(1-h)(1,76)=24.25, p<0.0001 and Fsubstrain(24-h)(1,76)=21.19, p<0.0001) but not for interaction (Finteraction(1-h)(1,76)=2.777, p=0.0998 and Finteraction(24-h)(1,76)=2.777, p=0.0998)(Figure 1b and 2b).

Formalin treatment did not alter B6J/1-h group’s time spent in light compared to its vehicle group (Figure 1b; B6J-VEH vs B6J-FOR, p=0.9820) but the B6N/1-h formalin group significantly spent less time in light compared to its vehicle group (Figure 1b; B6N-FOR>B6N-VEH, p<0.05) and to the B6J/1-h formalin group (Figure 1b; B6J-FOR>B6N-FOR, p<0.0001) also.

The B6N/24-h vehicle group spent less time on the light side compared to the B6J/24-h vehicle group (Figure 2b; B6J>B6N, p<0.05).

The B6J/24-h group was also not affected by formalin injection compared to its vehicle group (Figure 2b; B6J-VEH vs B6J-FOR, p=0.7979). The B6N/24-h group was no longer significant at this time point (Figure 2b; B6N-VEH vs B6N-FOR, p=0.2252). The B6N/24-h formalin group also spent less time on the light side (Figure 2b; B6J-FOR>B6N-FOR, p<0.01).

Latency to first enter the dark side did not differ between substrains and formalin did not affect it (Ftreatment(1-h)(1,76)=0.7016, P=0.4049 and Ftreatment(24-h)(1,76)=0.9244, p=0.3394; Fsubstrain(1-h)(1,76)=0.06693, p=0.7966 and Fsubstrain(24-h)(1,76)=0.9303, p=0.3378; Finteraction(1-h)(1,76)=0.06447, p=0.8002 and Finteraction(24-h)(1,76)=0.6730, p=0.4146).

Number of transitions between light and dark side was not affected after formalin injection but B6J substrain showed increased number of transitions compared to B6N substrain (Supp. Table 3).

3.3. Mechanical Sensitivity

Mechanical sensitivity was evaluated via von Frey after 2.5% formalin injection. Two-way ANOVA showed significant effects of Treatment for both time points (Ftreatment(1-h)(1,76)=280.9, p<0.0001 and Ftreatment(24-h)(1,76)=131.1, p<0.0001). Effect of Substrain was significant only at the 24-h timepoint (Fsubstrain(1-h)(1,76)=0.9165, p=0.3414 and Fsubstrain(24-h)(1,76)=9.459, p<0.01). There was no interaction between Substrain and Treatment for neither timepoint (Finteraction(1-h)(1,76)=0.1488, p=0.7007, and Finteraction(24-h)(1,76)=3.122,p=0.0813)(Figure 1c and 2c).

VEH groups did not differ significantly. (Figure 1c; B6J-VEH vs B6N-VEH, p=0.9775, and Figure 2c; B6J-VEH vs B6N-VEH, p=0.7913) at neither timepoint.

Formalin treatment caused mechanical sensitivity in both substrains (Figure 1c; B6JFOR>B6J-VEH, p<0.0001, and Figure 2c; B6N-FOR>B6N-VEH, p<0.0001). Formalin treated groups showed different trends at different time points. B6J/1-h and B6N/1-h groups did not differ from each other (Figure 1c; B6J-FOR vs B6N-FOR, p=0.7781) but B6J/24-h had slightly higher mechanical threshold than B6N/24-h group (Figure 2c; B6J-FOR vs B6N-FOR, p<0.01).

3.4. Thermal Sensitivity

Thermal sensitivity was evaluated on the Hargreaves test after 2.5% formalin injection. 2-way ANOVA indicated significant effects of Treatment, subtrain and their interaction at the 1-h time point (Ftreatment(1,76)=80.29, p<0.0001; Fsubstrain(1,76)=5.12, p=0.0265 and Finteraction(1,76)=4.3, p=0.0415)(Figure 1d). At 24-h timepoint, only effect of substrain remained (Fsubstrain(1,76)=1.989, p<0.0178; Ftreatment(1,76)=1.989, p=0.1626 and Finteraction(1,76)=0.4379, p=0.6082)(Figure 2d).

VEH groups were different from each other at 1-h timepoint but not at 24-h (Figure 1d; B6J-VEH<B6N-VEH, p=0.0156, and Figure 2d; B6J-VEH vs B6N-VEH, p=0.6524).

Formalin treatment caused thermal sensitivity in the 1-h groups (Figure 1d; B6JFOR>B6J-VEH, p<0.0001, and B6N-FOR>B6N-VEH, p<0.0001). B6J/1-h and B6N/1-h FOR groups did not differ from each other (Figure 1d; B6J-FOR vs B6N-FOR, p=0.9993). In the 24-h time point, all the groups treated with FOR showed a total recovery relative to VEH (Figure 2d; B6J-VEH vs B6J-FOR, p=0.8903, and B6N-VEH vs B6N-FOR, p=0.9703). Again, there was no substrain difference in formalin treated groups at this time point (Figure 2d; B6J-FOR vs B6N-FOR, p=0.1155)

3.5. Wheel Running

Wheel running test was chosen as a pain-associated phenotype as it reflects motor capabilities and the motivation to run. Treatment nor substrain did not affect wheel running at either timepoint (Ftreatment(1-h)(1,76)=1.337, p=0.2511 and Fsubstrain(1-h)(1,76)=0.01156, p=0.9147; Ftreatment(24-h)(1,76)=0.8262, p=0.3663 and Fsubstrain(24-h)(1,76)=0.3104, p=0.5790)(Figure 1e and 2e).

3.6. Nesting

The nesting test was performed to assess daily activity. Data was analyzed with planned comparison. Vehicle treated groups showed no substrain differences at any timepoints (Figure 1f; B6J-VEH vs B6N-VEH, p=0.2739, and Figure 2f; B6J-VEH vs B6N-VEH, p>0.99).

Formalin injection did not affect their nesting performance in any of the groups treated with formalin in any timepoints (Figure 1f; B6J-VEH vs B6J-FOR, p>0.99, and B6N-VEH vs B6N-FOR, p>0.99)(Figure 2f; B6J-VEH vs B6J-FOR, p>0.99, and B6N-VEH vs B6N-FOR, p>0.99). The B6N/1-h formalin treated group showed less nesting activity than the B6J/1-h formalin group Figure 1f; B6J-FOR>B6N-FOR, p=0.0005). This effect was not seen in the 24-h timepoint (Figure 2f; B6J-FOR vs B6N-FOR, p=0.2860).

3.7. Sucrose Preference

2-way ANOVA indicated no effect of Treatment or Substrain on sucrose preference at either timepoint (Ftreatment(1-h)(1,76)=0.8668, p=0.3548 and Fsubstrain(1-h)(1,76)=3.476, p=0.0598; Ftreatment(24-h)(1,76)=0.06719, p=0.7962 and Fsubstrain(24-h)(1,76)=0.8100, p=0.3710)(Figure 1g and 2g). There were no statistical differences in the total fluid intake between the different groups (Supplementary Table 1: FC)

3.8. Behavioral Cumulative Changes

Formalin’s effects on the reflexive and non-reflexive test as behavioral cumulative changes were reported as percentile change from their corresponding control groups. Paw diameter measurement was not included because it is not a behavioral response.

Reflexive Responses

Reflexive responses included mechanical and thermal sensitivity assays. Mixed effects analysis revealed significant effects of Substrain, Timepoint and their interaction in reflexive responses (Fsubstrain(1,39)=21.30, p<0.0001; Ftimepoint(1,39)=42.89, p,0.0001; Finteraction(1,39)=5.142, p=0.0291)(Figure 3a).

Figure 3:

Figure 3:

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Cumulative behavioral changes after formalin injection.

The percent change in reflexive responses (a) and non-reflexive responses (b). Data expressed as the mean S.E.M of 40 and 80 results/ per sex/per group in figures (a) and (b). **, p<0.01; ****, p<0.0001; ##, p<0.01; ####, p<0.0001

A posthoc test using Tukey’s test showed B6J and B6N substrains had significantly higher changes at 1-h and showed partial recovery at 24-h (Figure 3a; B6J/1-h>B6J/24-h, p<0.001, and B6N/1-h>B6N/24-h, p=0.0054).

Substrain differences were not observed at 1-h (Figure 3a; B6J/1-h vs B6N/1-h, p=0.6740) but at 24-h timepoint, the B6N substrain had fewer changes compared to the B6J (Figure 3a; B6J/24-h>B6N/24-h, p<0.0001) time point, indicating less recovery at 24-h in B6N.

Non-reflexive Responses

Non-reflexive responses included LDB, wheel running, nesting and sucrose preference. Analysis of non-reflexive responses revealed significant interaction of substrain and timepoints (Fsubstrain(1,79)=10.16, p=0.021) but not timepoint (Ftimepoint(1,79)=0.2858, p=0.5944) and there was no interaction (Finteraction(1,79)=3.925, p=0.0510)(Figure 3b).

The B6J substrain did not show any difference in different timepoints (Figure 3b; B6J/1-h vs B6J/24-h, p=0.3253). The B6N/1-h group showed higher changes but compared to B6N/24-h it was not significantly different (Figure 3b; B6N/1-h vs B6N/24-h, p=0.8065).

The B6N/1-h group showed higher changes at 1-h timepoint compared to the B6J/1-h group (Figure 3b; B6N/1-h>B6J/1-h, p=0.017) but this difference was not observed for the 24-h groups (Figure 3b; B6N/24-h vs B6J/24-h, p=0.7174).

4. DISCUSSION

Recently, several studies have suggested genetic variability plays an important role in pain perception in mice and humans [15] [16]. Indeed, a large heterogeneity of response to reflexive or non-reflexive tests has been reported in different mouse strains across various models of chronic pain [15][17]. These differences have been reported in substrains of C57BL/6 with various pain models [6,18]. Thus, in this study, we studied the effect of intraplantar formalin injection in these two C57BL/6 substrains and assessed pain-like behavior measurements at 1-h and 24-h after injection. Reflexive measurements (von Frey and Hargreaves tests) were supplemented by a battery of non-reflexive tests, i.e. nesting, wheel running, sucrose preference and Light Dark Boxes. Also, we used a “cumulative behavioral % change” based on reflexive and non-reflexive responses to compare responses between the two substrains.

We recently reported that C57BL/6 substrains differ in their paw licking behavior and edema after intraplantar injection of formalin (2.5%) [6]. Similarly, our results confirmed that the B6J substrain showed more edema than the B6N substrain at both timepoints post-injection. After the initial phases of formalin responses subsided, we supplemented our behavioral outcomes with a battery of tests at 1-h and 24-h time points. Our data showed a similar increase in mechanical and thermal hypersensitivity of the injected paw in both substrains at 1-h post-formalin injection. The B6N substrain showed greater anxiety-like behaviors in the LDB test compared to the B6J substrain at 1-h that dissipated by 24-h. The higher degree of baseline anxiety-like behaviors reported with B6N substrains [18] (also observed in our results - Figure 1b) in the LDB test may have contributed to the effects seen at 1-h post-formalin injection. Interestingly, Pitzer et al found that the two substrains did not show changes in anxiety-like behaviors on day 1 in the elevated plus-maze and day 3 in the LDB in the CFA chronic inflammatory pain model [19].

Interestingly, a dissociation was observed between mechanical and thermal responses at 24-h post-formalin injection. While paw edema and mechanical responses persisted, there was no thermal hypersensitivity in neither substrain after 24-h post-formalin. Thus, formalin-induced mechanical hypersensitivity persists longer than other behaviors such as thermal hypersensitivity and paw licking. Indeed, a study in rats reported similar results as mechanical hypersensitivity persisted for a long term after the injection of formalin [8]. Similar results were also observed in models of chronic inflammatory pain [complete Freund’s adjuvant (CFA) model] [6] and in the spared nerve injury (SNI) chronic neuropathic pain model in the B6J substrain. Interestingly, Cobos et al. reported a difference in the onset of mechanical and cold hypersensitivity and that this temporal divergence was associated with major differences in global gene expression in dorsal root ganglia [20]. The mechanisms mediating the difference in the time course of mechanical versus thermal pain sensitivity between the B6 substrains in the formalin pain model are unknown but could involve differences in behavioral and molecular adaptations between the two substrains after formalin injection. For example, when Li et al. [19] and Fan et al. [20] investigated the effects of pain history on subsequent formalin-evoked pain behaviors in rats, they found that animals with a history of chronic inflammatory pain (CFA injection) developed persistent pain-like behaviors. Additionally, Fan et al. showed that this enhanced responses to formalin injection involve p38 MAP-Kinase pathway activation in the prelimbic cortex [20].

There were no significant substrain differences in the remaining non-reflexive behaviors that we measured post-formalin (nesting, wheel running, and sucrose preference). Thus, the impact of pain on mobility, anxiety, physical, and social activities observed in humans seems to be more limited in C57BL/6 mice [19,20,21]. Also, the hypersensitivity observed with reflexive tests is much more prolonged than the behaviors observed with non-reflexive tests[24]. This is explained in particular by different neuronal circuits using the spinothalamic tract and locally by the inflammatory “soup” and the release of neurotrophins [24,25].

Lastly, data from reflexive and non-reflexive test results were each pooled together, and we calculated the”cumulative behavioral % changes” which allowed us to compare the global pain-like responses between the two substrains. This percentile change showed there were no differences in reflexive responses at 1-h and both substrains showed partial recovery over time. However, after 24 hours, the B6N substrain showed more pain-associated behaviors than the B6J substrain. In the non-reflexive responses, the B6N substrain showed more percentile changes, especially at the 1-h timepoint. Overall, these results show a phenotypic difference between B6J and B6N strains in the manifestation of pain-like behaviors in the formalin model.

Using a B6J × B6N-F2 cross and quantitative trait locus (QTL) mapping, we recently identified polymorphic regions of the genome containing genetic variants underlying B6 substrain differences in sensitivity to acute thermal nociception as measured in the hot plate test [6]. This study provides direct genetic evidence that the genetic background of the different substrain and transgenic mice must be carefully chosen and taken into account in the interpretation of phenotypic responses and indicates that a similar approach could be employed to identify causal genetic factors underlying the B6 substrain differences in formalin-induced pain-associated behaviors in the present study.

Studies with the formalin test, widely used as an indication of so-called tonic pain, commonly use observations on Phase I and II based on paw licking for experimental drug testing [26] and gene mapping [27]. In fact, reflexive and affective measures are not widely used in the formalin test studies. However, our results suggest that reliance on paw edema and paw licking behavior alone is not sufficient to represent the range of formalin-induced pain-like behaviors. In support, the degree of paw licking and edema do not correlate with non-reflexive and spontaneous measures. Furthermore, our results highlight the importance of studying additional time points beyond the typical 1-hour post-formalin injection. Thus, formalin’s effects on separate tests may be minimal or discordant but the consolidation of multiple tests into cumulative behavioral changes might better reflect formalin’s effects on pain behaviors. Other parameters must also be taken into account such as the age of the mice, the time of testing (light-dark cycle), and also the dose of formalin used with the implementation of a dose-response. Vendor differences should be seen as another source of variability [28,29]. This variable can cause a difference in the behavioral traits measured in mice [30] caused by possible genetic drift and/or environmental factors such as alterations in the gut microbiome of the animals [31].

Supplementary Material

MMC 1

Supplementary Table 1: 3-way ANOVA results of parametric results.

BW: Body weight, PD: Paw Diameter change, LT: Time spend in light zone, VF: Mechanical sensitivity, HG: Thermal sensitivity, WR: Wheel Running, SP: Sucrose Preference, FC: Fluid consumption

MMC 2

Supplementary Table 2: Planned comparison of sex differences in nesting and number of transitions.

MMC 3

Supplementary Table 3: Planned comparison of number of transitions.

Funding:

This work was supported by the National Institutes of Health [grant R01CA221260 (MID and CDB)] and National Institute on Drug Abuse (grant T32DA007027).

Footnotes

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

MMC 1

Supplementary Table 1: 3-way ANOVA results of parametric results.

BW: Body weight, PD: Paw Diameter change, LT: Time spend in light zone, VF: Mechanical sensitivity, HG: Thermal sensitivity, WR: Wheel Running, SP: Sucrose Preference, FC: Fluid consumption

NIHMS1595169-supplement-MMC_1.docx (24.1KB, docx)
MMC 2

Supplementary Table 2: Planned comparison of sex differences in nesting and number of transitions.

NIHMS1595169-supplement-MMC_2.docx (18.3KB, docx)
MMC 3

Supplementary Table 3: Planned comparison of number of transitions.

NIHMS1595169-supplement-MMC_3.docx (17.7KB, docx)

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