Sustained pain-related depression of behavior: effects of intraplantar formalin and complete freund’s adjuvant on intracranial self-stimulation (ICSS) and endogenous kappa opioid biomarkers in rats

Preclinical procedures to evaluate pain and analgesia in laboratory animals play a key role in research on both neurobiology of pain and analgesic drug development [1‐3]. Two common chemical challenges for induction of sustained pain states in rodents are intraplantar administration of Complete Freund’s Adjuvant (CFA) and formalin. CFA is a heat-killed bacterial suspension that elicits an immune response at the site of its injection. For example, CFA administration in the hindpaw of rats or mice produces paw edema [4‐6] and increased local concentrations of inflammatory cytokines and trophic factors [7]. These and other inflammatory mediators contribute to peripheral and central sensitization of nociceptive neural pathways [8‐10], and this neural hypersensitivity correlates with a sustained behavioral hypersensitivity of withdrawal responses to mechanical or thermal stimuli [5, 11, 12]. These hypersensitive withdrawal responses often serve as a behavioral indicator of “pain”.

Formalin, in contrast, is an aqueous solution of formaldehyde, a cell toxin that cross links proteins to disrupt dynamic protein interactions critical to cell viability. Formalin functions as an acute irritant, and when administered into the hindpaw of rodents, it elicits transient behaviors such as flinching and paw-licking [13‐15, 12]. However, formalin also elicits a sustained inflammatory response characterized by edema and local release of inflammatory mediators [12]. Moreover, formalin also damages or kills cells, including primary nociceptors and other sensory neurons, and as a result, formalin-induced changes in behavior also include a neuropathic component. For example, formalin injection into the hindpaw of rodents has been shown to produce general necrosis at the site of injection, increased expression of the neuropathy-related transduction factor ATF-3 in dorsal root ganglion cell bodies, and increased spinal microglial activation to an extent similar to that produced by other neuropathy models [16, 12, 17]. Consistent with this evidence for long-lasting tissue injury, intraplantar formalin also produces hypersensitivity to mechanical and thermal stimuli, and as with CFA, this hypersensitivity is sustained for days or weeks and often serves as a behavioral indicator of pain [15, 18].

We have categorized behaviors such as hypersensitive paw withdrawal reflexes as “pain-stimulated behaviors,” which are defined as behaviors that increase in rate, frequency or intensity after delivery of a pain stimulus [1, 19]. However, pain states can also depress other behaviors, and treatment of pain-related behavioral depression is a common goal of human and veterinary medicine [20, 21]. Research on pain-related depression of behavior can be accomplished with procedures that measure “pain-depressed behaviors,” which can be defined as behaviors that decrease in rate, frequency or intensity after a pain stimulus [1, 19]. For example, intracranial self-stimulation is a procedure in which subjects perform an operant behavior (e.g. press a lever) to receive pulses of electrical stimulation that activate the mesolimbic dopamine system, and ICSS has been used to examine effects of various manipulations on brain reward function [22‐24]. Previous studies have reported relatively transient depression of ICSS by acute noxious stimuli including intraperitoneal (IP) injection of dilute acid or paw incision [25‐27]. The goal of the present study was to compare effects of intraplantar CFA and formalin as more sustained pain stimuli on ICSS in rats. We hypothesized that both stimuli would produce sustained depression of ICSS similar to their shared ability to produce sustained thermal and mechanical hypersensitivity. Pain-related depression of ICSS was evaluated for its sensitivity to reversal by the mu opioid analgesic morphine. In addition, pain-related depression of ICSS was evaluated for its relationship to central biomarkers of the endogenous kappa opioid system and its sensitivity to the kappa antagonist norbinaltorphimine (norBNI), because some other stressors depress behavior by activating central kappa systems [28, 29].

Figure 1 shows effects of intraplantar saline, CFA or formalin on paw thickness, body weight and mechanical sensitivity. All statistical results for this and other figures are presented in the figure legends and in Additional file 1. Intraplantar saline had no effect on paw thickness; however, both CFA and formalin produced significant and sustained increases in paw thickness (e.g. edema) within 24 hours, and these effects persisted for 7 days (Figure 1A,B). Body weight increased significantly in the saline-control group for the CFA rats (Figure 1C), but this increase was small, and weight gain was not significant in the control group for the formalin rats (Figure 1D). Administration of CFA, but not formalin, reduced body weight within 24 hours, and body weight in CFA rats remained below baseline for 7 days (Figure 1C,D). Saline had no effect on mechanical sensitivity; however, both CFA and formalin produced significant and sustained decreases in mechanical thresholds for eliciting paw withdrawal within 6 hours, and this hypersensitivity persisted for 7 days (Figure 1E,F).Figure 2 shows effects of intraplantar saline, CFA or formalin on intracranial self-stimulation (ICSS). Prior to intraplantar injections, the mean ± SEM baseline number of stimulation per component across all stimulation frequencies was 264 ± 13, and the mean ± SEM maximum control rate (MCR) at any one frequency trial was 58.9 ± 2 stimulations per trial. Intraplantar saline treatment did not alter ICSS at any time. CFA produced weak and transient depression of ICSS. Specifically, at 1 and 3 hr after injection, ICSS in the CFA-treated rats was significantly lower than ICSS in the saline-treated controls (Figure 2A); however, ICSS in CFA- and saline-treated rats did not differ after 3 hr, and within the CFA group, ICSS after CFA treatment never differed significantly from baseline ICSS before CFA. Figure 2C shows full frequency-rate ICSS curves at selected times in the CFA-treated group. At baseline before CFA administration, electrical brain stimulation maintained a frequency-dependent increase in ICSS. In this analysis, ICSS was significantly decreased at several brain-stimulation frequencies after 1 hr but not after 7 days. In contrast to CFA, formalin produced a more robust and sustained depression of ICSS (Figure 2B). Relative to the saline control group, formalin significantly reduced ICSS during the first 24 hr after treatment and again during Days 5-7 after treatment. Relative to the pre-formalin baseline within the formalin group, formalin significantly depressed ICSS during the first 10 hr and again on Day 7 after treatment. Figure 2D shows full frequency-rate curves at selected times in the formalin treated group. Formalin significantly depressed ICSS at multiple frequencies both 1 hr and 7 days after treatment.

Additional file 2: Figure S1 shows formalin effects on ICSS during the first 50 min after formalin administration. Formalin decreased ICSS throughout the first 50 min of testing, although the magnitude of this decrease was greatest from 20-50 min after treatment.

Additional file 3: Figure S2 shows CFA and formalin effects on ICSS at different lever heights in a subset of six rats from each group on Days 1, 3 and 7 after intraplantar treatment. In general, ICSS decreased as lever height increased, and this effect was largest on Day 1. CFA failed to significantly alter ICSS at any lever height on any day, whereas formalin depressed ICSS across all lever heights and days.Figure 3 shows effects of morphine on ICSS 8 days after treatment with intraplantar saline or formalin. In the saline-treated rats, cumulative dosing with 0.32-3.2 mg/kg morphine produced no significant effect on ICSS. Conversely, in the formalin-treated rats, baseline ICSS was depressed before morphine administration, and morphine produced a dose-dependent reversal of formalin-induced depression of ICSS. The lowest dose of 0.32 mg/kg morphine was sufficient to restore ICSS back to approximately baseline levels, and higher morphine doses produced a further facilitation of ICSS.Figure 4 compares ICSS performance over a 14-day period after treatment with (a) bilateral saline, (b) bilateral 0.5% formalin, (c) unilateral 5% formalin in one paw + unilateral saline in the other paw, and (d) bilateral 5% formalin. Prior to intraplantar injections, the mean ± SEM baseline number of stimulations per component across all stimulation frequencies was 274 ± 14, and the mean ± SEM MCR at any one frequency was 55.9 ± 3. As in the first experiment, bilateral 5% formalin depressed ICSS relative to bilateral saline treatment, and in this second experiment, the formalin effect persisted for 14 days. Unilateral 5% formalin decreased ICSS relative to saline controls only on Days 1 and 6, and bilateral 0.5% formalin did not alter ICSS relative to the saline controls.Figure 5 shows data to address the role of kappa opioid systems in mediating effects of CFA and formalin on ICSS. Neither CFA nor formalin significantly altered expression of PDYN or KOR mRNA in any brain area examined on Day 7 after intraplantar treatment (Figure 5A and B). Moreover, the kappa antagonist norBNI failed to alter ICSS in rats treated with intraplantar saline, and it also failed to block formalin-induced depression of ICSS in rats treated with intraplantar formalin (Figure 5C-E). Data in Figure 5C-E show results obtained on Day 8 after intraplantar injection and 22 hr after norBNI administration. Rats were also tested daily for another six days (Days 9-14 after intraplantar injection), and norBNI did not significantly alter either control ICSS or formalin-depressed ICSS on any day (data not shown).

This study compared effects of intraplantar CFA and formalin on a series of behavioral and physiological endpoints in rats. There were four main findings. First, in agreement with previous studies, both CFA and formalin produced similar degrees of paw swelling and mechanical hypersensitivity. Second, CFA and formalin produced different effects on ICSS. Specifically, CFA produced relatively weak and transient depression of ICSS, whereas formalin produced a more robust and sustained depression of ICSS that lasted as long as 14 days. Third, formalin-induced depression of ICSS could be reversed by morphine doses that had no significant effect on ICSS in saline-treated rats, suggesting that formalin effects on ICSS can be interpreted as an example of pain-related and analgesic-reversible depression of behavior. Finally, neither behavioral nor central biomarker data support a role for the endogenous kappa opioid system in mediation of formalin-induced depression of ICSS. These results suggest differential efficacy of sustained pain stimuli to depress brain reward function in rats as assessed with ICSS.