Effects of intraplantar administration of Complete Freund’s Adjuvant (CFA) on rotarod performance in mice

1 Introduction

Inflammatory pain is a common disease worldwide. Diseases such as rheumatoid arthritis and inflammatory bowel diseases can affect patient’s quality of life (such as limitation of movement and decrease in daily-life activities) with little improvement with current medications [1]. Thus, symptomatic treatment using analgesics, such as non-steroidal anti-inflammatory drugs (NSAIDS), are the most used strategy to relieve patients’ symptoms. This life-long strategy of treating inflammatory pain maybe associated with non-preferable side effects, and new novel drugs are needed to enhance clinical outcomes in those patients.

Preclinical studies using animal models are an integral step in the discovery of new analgesics. In these studies, administration of noxious stimuli produces behavioral changes in animals. These changes may be classified into pain-stimulated behaviors (i.e. behaviors that are increased in frequency or intensity after noxious stimulus, such as paw-withdrawal test [2]) and pain-depressed behaviors (i.e. behaviors that are decreased in frequency or intensity after noxious stimulus, such as locomotion) [3]. Combining these behavioral assays are helpful in screening the efficacy and potency of candidate analgesics in comparison to available analgesics. Animal behaviors that may be depressed after noxious stimuli include feeding [4], [5], locomotor activity [6], grip force [7], and others [8]. Also, it had been shown previously that chronic pain may be associated with general change in behavior [9]. Thus, decreases in locomotor activity are a major target to assess nociception and antinociception in animals [10].

Complete Freund’s Adjuvant (CFA) has been traditionally used to assess inflammatory pain in rodents. Subjects are usually injected with CFA (e.g. hindpaws) to induce behavioral changes that can be used to assess the degree of pain threshold. Several pain-stimulated behaviors were used previously to assess CFA-induced behavioral changes, such as paw withdrawal through von Frey filaments [11] and thermal hypersensitivity [12]. CFA injection can also produce pain-depressed behaviors, such as weight bearing [13], [14] and food consumption [2]. Nevertheless, CFA failed to produce reliable decreases in some behaviors, including pain-depressed electrical brain stimulation [2], and in an attention task [8].

In this study, we propose the use of rotarod as a new pain-depressed behavioral task to detect behavioral changes in mice after intraplantar injection of CFA in the hindpaw. In the past, rotarod was mainly used to assess motor coordination in rodents [15], [16]. Recently, it has also been used to assess behavioral outcomes after noxious manipulations in rodents, such as monosodium iodoacetate (MIA)-induced osteoarthritis [17], [18] and collagenase-induced central post-stroke pain [19]. In this study, we hypothesize that CFA will produce similar results, such that it will decrease rotarod performance in mice.

2 Materials and methods

3 Results

3.1 Rotarod experiments

Figure 1 shows the effect of unilateral intraplantar injection of vehicle or CFA on rotarod performance. In the vehicle group, the time spent on the wheel increased from trial to trial (within days) and from day to day (between days). The lowest score was during the first trial on day −1 (mean±SEM was 24.17 s±10.8 s), and the maximum score was during the third trial on day 1 (mean±SEM was 190 s±43.5 s). In the CFA group, similar results to vehicle group were obtained during day −1 and day 0 (before and after CFA injection). However, CFA produced a decrease in rotarod performance on day 1, that was significant on trials 1 and 3 when compared to vehicle group. Finally, injection of haloperidol during day 1 produced a robust and significant decrease in rotarod performance, which returned back to normal on day 2. Two-way ANOVA results were as follows: There was a significant main effect of time [F(11.165)=20.36; p<0.0001], non significant effect of treatment [F(2.15)=0.7505; p=0.4891], and significant time by treatment interaction [F(22.265)=5.523; p<0.0001]

Fig. 1:

Rotarod performance before and after saline, CFA, or haloperidol administration. Separate groups of mice were injected with intraplantar saline (saline group), intraplantar CFA (CFA group), or intraperitoneal haloperidol (Haloperidol group). CFA or saline were administered (black arrow) directly after the third trial on day 0. Rotarod speed started at four RPM and accelerated until it reached 40 RPM within 5 min (cut-off). Abscissa: number of trial on the indicated day. A trial means a separate attempt of subject to stay balanced over the moving rotarod before falling. Ordinate: time (in seconds) until falling. Asterisks (*) indicate significance vs. vehicle (p<0.05) within that trial. Each value represents mean±SEM of six mice.

Figure 2 shows a summary data with all trials on the same day combined together. Vehicle group demonstrated significant improvement on rotarod each day. CFA and haloperidol group did not produce any significant changes in rotarod performance compared to vehicle during days −1, 0 before injection, and 0 after injection. However, on day 1 (24 h after injection), both CFA and haloperidol groups produced a significant decrease in rotarod performance compared to the vehicle group. Two-way ANOVA results were as follows: There was a significant main effect of time [F(3.45)=30.94; p<0.0001], non significant effect of treatment [F(2.15)=0.7505; p=0.4891], and significant time by treatment interaction [F(6.45)=10.11; p<0.0001]. Ibuprofen (32 mg/kg, i.p.) was tested for its antinociceptive property, and it enhanced rotarod performance compared to vehicle-treated group as shown in Fig. 3. Two-way ANOVA results were as follows: There was no significant main effect of time [F(1.12)=3.334; p=0.0928], no significant effect of treatment [F(1.12)=2.223; p=0.1618], and significant time by treatment interaction [F(1.12)=9.296; p=0.0101].

Fig. 2:

Average rotarod performance across all trials within the same day. Data shown in Fig. 1 were averaged across all mice on the same day. Data on day 0 was separated into two data points to represent before and after saline or CFA injections (black arrow). Pound keys (#) indicate significant difference between days within the same treatment (p<0.05). Asterisks (*) indicate significance vs. vehicle within the same day (p<0.05).

Fig. 3:

Rotarod performance after 32 mg/kg ibuprofen CFA-treated mice on day 1. Subjects were injected with ibuprofen or vehicle subsequently at the end of the third trial, and 60 min before the beginning of the 4th trial on days 1. Abscise and ordinates are similar to Figs. 1 and 2. Pound key (#) indicates significant difference between treatment groups (p<0.05).

Figure 4 shows the long term effect of CFA in the sub-chronic groups. CFA administration did not produce any significant change in rotarod performance after 7 and 14 days when compared to the control group. Two-way ANOVA results showed no difference between groups (p=0.4785).

Fig. 4:

Average rotarod performance across all trials in sub-chronic experiment. Rotarod performance was evaluated after 7 and 14 days from CFA administration. Abscissa: Day after intraplantar CFA injection of trial on the indicated day. Ordinate: time (in seconds) until falling. Each value represents mean±SEM of five mice.

3.2 Mechanical allodynia

Figure 5 shows the effect of intraplantar injection of vehicle or CFA on mechanical allodynia at times points that are similar to the rotarod experiment. The mean±SEM of threshold at day −1 was 2.17±1.5 and 1.73±1.2 for vehicle and CFA, respectively. Vehicle injection did not significantly change thresholds compared to preinjection results. On the other hand, CFA produced significant decreases in threshold on day 0 and day 1 compared to preinjection. Also, thresholds after CFA administration were significantly different from vehicle on day 1. Two-way ANOVA results were as follows: significant main effect of time [F(3.30)=7.18; p=0.0009], non significant effect of treatment [F(1.10)=4.849; p=0.0523], and non significant time X treatment interaction [F(3.30)=0.8413; p=0.4820].

Fig. 5:

Mechanical allodynia measured post intraplantar CFA or saline administration. Von Frey filaments were used to assess nociceptive threshold before and after saline or CFA injections. Abscissa: time from saline or CFA injection in hours. Ordinate: paw withdrawal threshold from von Frey filaments in grams. Asterisks (*) indicate significance vs. −24 h within group (p<0.05). Pound key (#) indicates significance between groups at 24 h time point after multiple t-tests. Each value represents mean±SEM of six mice.

4 Discussion

The current study tested the effect of intraplantar CFA injection on mice in two different behavioral tests, including rotarod and mechanical allodynia. There were three main findings in this study. First, CFA decreased pain threshold and increased sensitivity to von Frey filaments in mice. Second, locomotor function can be improved by daily exposure to rotarod, and this improvement can be attenuated by acute injection of CFA. Finally, CFA did not produce significant change in rotarod after 2 weeks from injection. Collectively, our findings could encourage the use of rotarod assay to measure acute inflammatory nociception in rodents as a useful tool in preclinical studies.

The sensitivity to von Frey filaments after CFA injection is consistent with previous findings, and our data replicate these findings in rats [6] and mice [11], [22]. Similarly, CFA produced similar effects in other pain-stimulated behavioral tests such as hypersensitivity to thermal stimuli [12]. However, as indicated previously, traditional assays of pain-stimulated behaviors are vulnerable of producing false-positive results, such that drugs that suppress locomotion may produce similar results. Thus, we suggest that adding rotarod experiment may help eliminate such false-positive results. One limitation of this study is the absence of mechanical allodynia results for sub-chronic group. Nevertheless, previous studies showed that the CFA decreases von Frey threshold for long periods that may last up to 35 days post CFA administration in both rats and mice [23], [24].

CFA produced attenuation of rotarod performance in our study. This was consistent with other assays of locomotion. For example, Kandasamy and others reported recently that CFA decreased voluntary wheel running in male and female rats [6], [25]. Also, our data is consistent with previous findings using different noxious stimuli. For example, one study showed that rotarod performance was decreased compared to the sham group after intra-articular injection of MIA [17], although a similar study using MIA did not show the same effect [18].

The data collected in this study will provide an objective assay to detect analgesic properties of new candidate medications that are void of any motor impairment to the subjects. Many drugs showed promising analgesic effects in preclinical studies, but failed as analgesics in clinical studies. A comparison between new medications and currently available analgesics (opioid or non-steroidal anti-inflammatory drugs) is necessary to confirm antinociceptive properties in preclinical studies. Previous studies showed that morphine, but not gabapentin, can reverse Mono-iodoacetate (MIA)-induced motor dysfunction, without production of motor impairment [7], [17]. This suggests that morphine can be more tolerable than gabapentin by osteoarthritic patients. Similarly, this study showed that ibuprofen attenuated the CFA-induced impairment of rotarod performance. Demonstration that ibuprofen fails to block haloperidol-induced depression of rotarod performance would confirm antinociceptive effects.

One advantage of using rotarod to assess nociception in rodents is the ability to dissociate between non-specific effects of drugs vs. its specific analgesic effect. For example, pregabalin and other drugs produced antinociceptive effect in neuropathic pain model in rats had also an effect on rotarod performance in naïve rats [26]. Thus, rotarod can be helpful to eliminate false positive results from other pain-induced behaviors in rodents. The rotarod protocol used in this study was chosen based on previous publications using similar parameters (i.e. 4–40 RMP; 300 s duration) [15], [27]. These studies showed that rotarod performance (or latency to fall) is increased with each trial, suggesting a strong learning component of this task. This is also obvious in this study in both acute (Vehicle group only) and sub-chronic studies (both vehicle and CFA groups). Thus, the use of restricted training history on rotarod would dissociate CFA effect in comparison to extensive history of rotarod training.

Our study tested the acute effect of CFA on rotarod, and our data is limited to 24 h post injection. CFA did not affect the rotarod performance 30 min after CFA administration, and that can be related to two factors. First, the inflammatory process after such short time is not enough to produce measurable outcomes to the pain threshold, and this is consistent with previous findings [6]. Second, subjects may be still under the influence of the general anesthetic isoflurane that was used to enhance CFA injections, which may interfere with subjects’ behavior. Consequently, we investigated a longer time point duration of CFA to determine its long-term effect. Our results in the sub-chronic experiment showed no long-term effect of CFA on rotarod performance. This effect does not encourage the use of rotarod to evaluate long term effect of inflammatory nociception. Similar results were reported in other preclinical assays that produced transient depression in animal behavior after inflammatory pain in rodents, such as intraplantar formalin injections [2].

The main effect of CFA on rotarod performance was maximized after 24 h (or day 1) post-CFA injection. Specifically, CFA effects primarily manifest as a “failure to improve” subjects’ performance more than a “depression” of behavior from the pre-CFA baseline. For example, there was no statistical significance in the rotarod performance in all trials on day 1 in CFA group when compared to trial 1 on day 0 within group. On the other hand, the same analysis revealed a significant increase in rotarod performance in vehicle-treated group, indicating an “improvement” in the subjects’ locomotor activity in the vehicle group. These findings are different than previous reports of the effect of a noxious stimulus to depress established behaviors, such as wheel running and electrical brain stimulation [2], [6]. Taken together, we presume that learning new tasks or activities during painful conditions may be delayed in comparison of pain-free states. Finally, in addition to a modest effect size, CFA seems to have a relatively short time course compared to other CFA-induced depression, such as voluntary wheel running [6] and CFA-induced mechanical allodynia (this study). This difference in duration could be interpreted to the learning component in rotarod task versus unconditioned behaviors, such as wheel running and paw withdrawal.