Pain is appreciated as a multimodal experience in humans with both sensory-discriminative and affective-motivational components, and effective treatment of both acute and chronic pain appears to require consideration of both modalities.1,2 While the pharmacology and functional anatomy of these modalities display some overlap, they are nonetheless sufficiently distinct to merit separate consideration.3,4 The sensory-discriminative component of pain has proven to be relatively straightforward to model in rodents and serves as a valuable tool for validation of acute and chronic pain preclinical models. Sensory alterations produced by pain models in rodents are valuable end points for the investigation of potential therapies for sensory-discriminative abnormalities and for basic science investigations of the spinothalamic tract as well as descending pain inhibitory and facilitatory systems. Assessing the motivational-affective dimension of pain in preclinical rodent models has proven more difficult.5
Preclinical studies by using acute and chronic pain models overwhelmingly report the development of hypersensitivities to external stimuli. Frequently these studies report hindpaw hypersensitivity as the only behavioral end point, which is concerning because many compounds alleviate hypersensitivity in animals yet fail clinical trials.6 Since the primary complaint of neuropathic pain patients is ongoing spontaneous pain,7 preclinical studies should incorporate behavioral end points that assess nonevoked spontaneous pain in animals, the mechanism(s) of which may be unrelated to hypersensitivity.8
A number of behavioral end points have been described that reportedly measure affective dimensions of acute or chronic pain in rodents. Compared with assessing sensory abnormalities after acute and chronic pain, these measures are nonreflexive and nonevoked in nature, and include open field activity (OFA),9,10 food reinforcement (FR),11 conditioned place avoidance,12,13 conditioned place preference to analgesics,14 analgesic self-administration,15 intracranial electrical self-stimulation (ICSS),16,17 facial grimacing,18,19 hindpaw guarding,20,21 and weight-bearing bias.22,23 While a number of studies address the relationships between reflexive behaviors and some of these behavioral end points, the effect of pain induced by acute tissue damage and chronic nerve injury has generally not been evaluated within the same study. Furthermore, the influence of previous nerve injury on subsequent tissue damage on these measures has not been evaluated.
The goal of the current study was to assess the effect of acute pain after paw incision (INC) and the effect of neuropathic pain after L5/L6 spinal nerve ligation (SNL) on a variety of nonevoked behavioral end points hypothesized to decrease in the presence of ongoing spontaneous pain, and subsequently compare these effects to the traditional measure of mechanical hypersensitivity. To do this, rats were subjected to INC or SNL, and the effects on paw withdrawal threshold (PWT), ventral tegmental area (VTA) ICSS, FR, and OFA were assessed. Furthermore, it was hypothesized that chronic sensitization of nociception induced by SNL would exacerbate the effects of acute pain after INC to decrease the rate of these behavioral measures. To address this, the effects of INC in rats previously subjected to SNL were also assessed on these behavioral end points.
Subjects were 201 male, Fisher 344 rats weighing between 275 and 325 g at the beginning of the experiment (Harlan Laboratories, Raleigh, NC). Rats were group-housed unless otherwise indicated in a temperature and humidity-controlled room that was maintained on a reversed light–dark cycle (dark 05:00–17:00); this room was adjacent to the room in which behavioral experiments were performed. Food and water were continuously available except during behavioral experiments. All procedures were conducted according to guidelines of the Committee for Research and Ethical Issues of the International Association for the Study of Pain and were approved by the Animal Care and Use Committee of Wake Forest University School of Medicine, Winston-Salem, NC.
Spinal Nerve Ligation
SNL surgery was performed as previously described.24 Briefly, rats were anesthetized with inhaled isoflurane (2%) in oxygen and received penicillin G procaine (75,000 U, IM) to prevent infection. An incision was made through the skin and muscle of the lower back, and the left transverse process of the fifth lumbar vertebra was removed by using bone micro rongeurs. The fifth lumbar nerve was exteriorized and ligated with 4-0 silk suture. The sixth lumbar nerve was exteriorized from below the iliac bone at the sciatic notch and similarly ligated. Afterward, muscle layers were sutured with 4-0 chromic gut, the skin with 4-0 nylon suture, and exterior wounds dressed with antibiotic powder (Polysporin; Pfizer Healthcare, Morris Plains, NJ). This procedure lasted approximately 30 minutes. For sham-SNL surgery, rats were anesthetized but did not receive an incision.
Rats (N = 17, 8 for SNL, 9 for sham-SNL) were permanently implanted with electrodes into the VTA as previously described.16 Briefly, rats were anesthetized with pentobarbital (50 mg/kg, intraperitoneal) and received atropine methyl nitrate (10 mg/kg, intraperitoneal) to prevent bronchial secretions and penicillin G procaine (75,000 U, IM) to prevent infection. Rats were secured in a stereotaxic frame, and platinum bipolar stimulating electrodes (Plastics One, Roanoke, VA) were aimed at the left VTA (2.1 mm anterior to lambda, 0.6 mm lateral from the midline, and 8.5 mm), which were secured by 3 stainless steel screws embedded in dental acrylic on the skull surface. This procedure lasted approximately 30 minutes.
Rats were subjected to INC surgery as previously described.22 Briefly, rats were anesthetized with inhaled isoflurane (2%) in oxygen. A 1-cm incision was made in the left hindpaw near the heel, the plantaris muscle lifted and incised longitudinally with manipulation, and afterward, the skin sutured with 4-0 nylon suture. This procedure lasted approximately 10 minutes. For sham-INC surgery, rats were anesthetized but did not receive an incision.
Paw Withdrawal Threshold
Mechanical allodynia was assessed by using von Frey filaments (Touch Test Sensory Evaluators; Stoelting, Wood Dale, IL) as reported previously by using an up-down method.25 PWT was calculated for all animals by using Dixon nonparametric statistics as previously reported.26 PWTs of the midpaw were assessed 14 days after SNL to verify development of allodynia (PWT <4.0 g). PWTs of the heel region were assessed before and in the days after INC surgery to assess the effect of INC on PWT in the area surrounding the wound.
Ventral Tegmental Area Intracranial Self-Stimulation
Lever presses for electrical stimulation of the VTA were assessed as previously described.16 Rats were subjected to electrode implantation as well as SNL (N = 8) or sham-SNL (N = 9) surgery. After 14 days recovery from surgery, PWT was assessed, and rats were trained to lever press for VTA ICSS. Operant chambers were housed within sound- and light-attenuating enclosures equipped with a houselight, and ventilation fan were used (Med Associates Inc., St. Albans, VT). These chambers have a lever 5 cm above a grid bar floor, stimulus lamp 2 cm above the lever, and a tone generator. An ICSS stimulator controlled by computer software (Med Associates Inc.) that controlled all stimulation parameters, and data collection was located outside of the enclosure. A 2-channel swivel commutator (Model SLC2C, Plastics One) positioned above the operant chamber connected the electrodes to the ICSS stimulator via 25 cm cables (Plastics One). Illumination of the stimulus light indicated stimulus availability, and each lever press generated a 0.5-second train of rectangular alternating cathodal and anodal pulses (0.1-millisecond pulse durations). During stimulation, the stimulus light shut off, the houselight turned on, and a tone sounded. Responding during stimulation resulted in no further stimulation and was not recorded.
During training sessions, the frequency was held constant (150 Hz), and the intensity adjusted to maintain consistent responding. After this initial training (usually 1 or 2 sessions), frequency-response curves were generated. These 90-minute sessions consisted of six 10-minute components. Each component consisted of ten 60-second trials. Each trial began with a 5-second timeout followed by a 5-second priming period in which rats received 5 noncontingent stimulations and concluded with a 50-second response period. The current intensity remained constant (unique to each animal; based on the initial training sessions), and 10 frequencies (156–45 Hz, 0.06 log increments) corresponding to each trial were made available in descending order. A 30-minute timeout separated the third and fourth components. Initially, current intensity was adjusted during frequency-response sessions (when needed) to maintain high rates of responding for 4 to 5 of the frequencies presented. This was done so that any increases or decreases in responding after INC could be detected. For data analysis, components 2 and 3 were averaged and analyzed with Prism software (sigmoidal-dose response, variable slope; Graph Pad, La Jolla, CA). Component 1 was not included since it tended to be highly variable. Components 4 to 6 (after the 30-minute timeout) were also disregarded, because these components were only used to increase the speed of stable responding acquisition. Subsequently, these components4–6 remained in place after INC and sham-INC only to keep the experimental paradigm exactly the same during all experimental sessions. ICSS sessions were conducted daily, and once responding was stable (defined as when each EF50, the frequency maintaining 50% of maximal responding, of 4 consecutive sessions did not vary by >15% of the mean EF50 for those 4 sessions), rats were subjected to sham-INC surgery 4 hours before an ICSS session. The following day, rats were subjected to INC 4 hours before an ICSS session. Daily ICSS sessions continued in the days after INC, with PWT assessed 60 minutes before each session. Experimental sessions in which the baseline and postincision results were obtained were performed between 1 and 3 months after initial SNL or sham-SNL surgery, due to variability in the time needed for acquisition of the operant and time needed to perform stable session-to-session frequency-response curves. VTA ICSS sessions occurred during the dark phase of the light–dark cycle.
Food-Reinforced Operant Responding
Lever presses reinforced by delivery of standard rat chow pellets were assessed 24 hours/d, with no restriction on the maximum amount that could be earned, as described previously.11 Briefly, rats (N = 24) were housed in standard operant chambers within sound- and light-attenuating enclosures with a standard response lever, a stimulus light above the lever, and a magazine-type pellet dispenser (Med Associates Inc, St. Albans, VT). Initially, each lever press was reinforced by delivery of a 45-mg standard rat chow pellet (Bio-Serv Inc., Frenchtown, NJ), and the number of lever presses necessary for pellet delivery was increased to 10 across several sessions, where it then remained for the rest of the experiment. After 21 days, all rats showed stable responding (5 successive days where number of food pellets earned did not vary by >10% of the mean), and half of the animals were subjected to SNL and half sham-SNL surgery. After SNL or sham-SNL, all animals were housed in standard shoebox cages with ad lib access to food and water. Two weeks later, PWT was determined, and rats were returned to the operant chambers. After 10 days, all rats again showed stable responding (same criteria as previously described), and half of the SNL rats and half of the control rats were subjected to INC or sham-INC surgery and returned to the operant chamber after 2 hours. The number of food pellets delivered was determined hourly for 4 days after INC or sham-INC surgery alone. Responding during the 4-hour period was done during the dark phase of the light–dark cycle.
Open Field Activity
Open field exploratory activity was assessed as published previously.27 Rats (N = 160) were placed in commercially available chambers housed within sound- and light-attenuating enclosures equipped with a ventilator fan, and activity was measured by interruption of infrared beams 1 in apart in the X-Y direction as well as a second set placed 4 in above the floor surface in the X direction (Med Associates Inc). Sessions were 1 hour and occurred during the dark phase of the light–dark cycle. Rats were subjected to SNL (N = 80) or sham-SNL (N = 80) surgery as described above. PWT was assessed in all animals 2 weeks after SNL or sham-SNL surgery; half of the SNL and half of the sham-SNL animals were subjected to INC as described above, and the other half in each group had sham-INC surgery. Separate groups of animals (N = 8/group) were placed in open field chambers at 4, 24, 48, 72, or 96 hours after INC or sham-INC surgery, and the total distance traveled and number of rears were recorded. After the open field sessions, PWT was assessed for all animals as described above.
Rats were killed by carbon dioxide asphyxiation. Brains were rapidly removed, frozen in isopentane (−35°C), and stored at −80°C. Coronal sections (25 μm) around the electrode tract were obtained by using a cryostat to confirm electrode placement within the VTA (Fig. 1).
For all groups used for VTA ICSS, FR, and OFA, the effect of INC on PWT measured in either the heel region or midpaw was analyzed by using a 2-way repeated-measures analysis of variance (ANOVA) with surgical group (SNL or sham-SNL) and time after INC serving as the independent variables. For VTA ICSS, the EF50 (frequency at which rats emitted 50% of maximal responding for each session) and maximal response rate were calculated by using Prism 5.0 software (sigmoidal-dose response, variable slope; GraphPad Software, San Diego, CA) and served as the dependent measures. VTA ICSS baseline values for the intensity, EF50, and maximal response rates were compared between the 2 groups by using Student t test. To assess the effects of INC on VTA ICSS, reduction in EF50 was calculated by subtracting the EF50 for each session after sham-INC or INC from the average EF50 for the 2 baseline sessions immediately before sham-INC. The effect of INC on VTA ICSS was analyzed by using a 2-way repeated-measures ANOVA with surgical group (SNL or sham-SNL) and time after INC or sham-INC surgery serving as the independent variables. In a separate analysis, the SNL and sham-SNL groups were combined as a single group, and the effects of INC on VTA ICSS was analyzed by using a 1-way repeated-measures ANOVA with time after INC or sham-INC serving as the independent variables.
For FR, the effect of SNL and INC was analyzed by using a 2-way repeated-measures ANOVA with surgical group (sham-SNL + sham-INC, SNL + sham-INC, sham-SNL + INC, SNL + INC) and time after INC or sham-INC serving as the independent variables. For OFA, the effects of SNL and INC on distance traveled and number of rears were analyzed separately by using a 2-way ANOVA with surgical group (sham-SNL + sham-INC, SNL + sham-INC, sham-SNL + INC, SNL + INC) and time after INC or sham-INC surgery serving as the independent variables.
For all analyses, a 2-tailed P value of 0.05 or less was considered statistically significant, except for the analysis of the VTA ICSS data with the combined INC and SNL + INC groups in which a 2-tailed P value of 0.025 or less was considered statistically significant. Post hoc analyses were performed after 1-way ANOVA by using Dunnett t test and after 2-way ANOVA Tukey honest significant difference test. Data were analyzed for normality by using the D’Agostino and Pearson omnibus normality test and for equivalent residuals by using the Bartlett test for equal variances. All datasets passed both tests except for the PWT data in the heel region. PWT after INC in the heel region was subsequently analyzed by using the Kruskal-Wallis nonparametric test for the sham-SNL and SNL groups separately with respect to time after incision. Post hoc comparisons were made by using Dunn multiple comparison test for comparing differences in rank sum between time points. PWT was compared between sham-SNL and SNL groups at each time point by using the Kruskal-Wallis nonparametric test with corrected P value of 0.005 (to account for 10 individual comparisons at α level of 0.05). All statistical analyses were performed by using JMP software (version 5.0.1a, SAS Institute Inc., Cary, NC).
Effects of SNL and INC on PWT and VTA ICSS
In the animals used for VTA ICSS, PWT in the heel decreased significantly after INC in both sham-SNL (Kruskal-Wallis statistic 81.6, P < 0.0001) and SNL (Kruskal-Wallis statistic 74.1, P < 0.0001) groups (Fig. 2). In the sham-SNL group, PWT was significantly reduced from 4 to 96 hours after INC compared with baseline. In the SNL group, PWT was significantly reduced from 4 to 72 hours after INC compared with baseline. In both the sham-SNL and SNL groups, PWT was not significantly different from the 4-hour time point until 7 days after INC. Comparing PWT between sham-SNL and SNL groups and correcting for multiple comparisons, there were no significant differences between these groups at baseline or any time point after INC. In the midpaw region, PWT was lower in the SNL compared with sham-SNL group before INC (F(1,16) = 44.4, P < 0.0001) as expected; however, INC did not decrease PWT in the midpaw region compared with sham-INC in either the SNL or sham-SNL rats (F(5,47) = 0.7, P = 0.66).
For VTA ICSS, baselines values did not significantly differ between sham-SNL and SNL animals for either the intensity (sham-SNL = 177.8 ± 22.4, SNL = 217.5 ± 11.7 [mean ± SEM]; F(1,16) = 0.3, P = 0.06), the EF50 (F(1,16) = 0.2, P = 0.7) or maximal response rates (F(1,16) = 0.9, P = 0.3). Sham-INC had no effect on either the EF50 or maximal response rate for VTA ICSS in sham-SNL (F(1,17) = 0.03, P = 0.9; F(1,17) = 0.5, P = 0.5, respectively) or SNL (F(1,15) = 0.01, P = 0.9; F(1,15) = 0.01, P = 0.9, respectively) rats (Fig. 3A). In sham-SNL rats, INC significantly shifted the frequency-rate curve for VTA ICSS to the right, increasing the EF50 (F(4,44) = 3.4, P = 0.02) while having no effect on maximal response rates (F(4,44) = 1.0, P = 0.4) (Fig. 3B). The effect of INC lasted for only 4 hours, however, as the EF50 values were no different from baseline 24 hours after INC (P ≥ 0.05). The effect of INC in SNL rats on VTA ICSS was similar, with INC increasing the EF50 compared with baseline values (F(4,39) = 3.2, P = 0.03) while having no effect on maximal responding (F(4,39) = 1.3, P = 0.3) (Fig. 3C). As in the sham-SNL group, INC increased the EF50 for VTA ICSS 4 hours after surgery in SNL rats, with the EF50 returning to baseline values at 24 hours (P ≥ 0.05) (Fig. 3C). The effect of INC was not different between sham-SNL and SNL rats at the 4-hour time point, with surgery increasing the EF50 to a similar extent in each group (P > 0.05).
Since no differences were observed between sham-SNL and SNL rats for the effects of INC on EF50 or maximal responding at any time point, data from VTA ICSS sessions before and after INC across both groups (sham-SNL and SNL) were combined and analyzed with a corrected P value of 0.025. When analyzed as a single group, INC significantly increased EF50 for VTA ICSS for up to 2 days (F(4,84) = 6.5, P < 0.0001) while having no effect on maximal responding (F(4,84) = 2.0, P = 0.1) (Fig. 3B).
Effects of INC and SNL on FR
The effects of INC and SNL on PWT in the animals used for FR were similar to those found for the animals used for VTA ICSS. PWT was less in the SNL rats in the heel area compared with sham-SNL rats (F(1,80) = 6.9, P = 0.01) and was dependent on time after INC (F(3,80) = 9.6, P < 0.0001). Similarly to the groups used for VTA ICSS, PWT in the area of the incision was significantly less than baseline values at all time points (4, 24, 48, and 72 hours after surgery) up to the latest time tested (72 hours) after INC in both sham-SNL and SNL rats but was not affected by sham-INC (data not shown). PWT of the midpaw region was significantly reduced in SNL rats compared with sham-SNL rats but was not affected by INC compared with sham-INC, similar to the data obtained in the rats used for VTA ICSS (data not shown). INC significantly reduced PWT in the heel region compared with baseline during the time that FR was assessed (up to 3 days) (F(4,80) = 7.2, P < 0.0001).
SNL did not alter the number of food pellets earned daily compared with sham-SNL surgery when comparing the baseline values for each group before INC or sham-INC (F(1,23) = 0.0002, P = 0.99) (Fig. 4). INC significantly reduced the number of food pellets earned in both the SNL and sham-SNL groups (F(3,80) = 5.2, P = 0.008), and the effect was time-dependent (F(4,80) = 378, P < 0.0001). Compared with sham-INC, INC decreased the number of food pellets earned in the first 24 hours only. In contrast to VTA ICSS, combining data from SNL and Sham-SNL groups to assess the effects of INC did not change the statistical significance compared with assessing INC in either group alone. There were no significant differences between sham-SNL and SNL rats for the number of food pellets earned at any time after sham-INC (P = 0.96). Similarly, there were no significant differences between the sham-SNL + INC and SNL + INC rats for the number of food pellets earned at any time after INC (P = 0.76).
Effects of SNL and INC on OFA
The effects of INC and SNL on PWT in the animals used for OFA were similar to the animals used for VTA ICSS. PWT was less in the SNL rats in the heel area compared with sham-SNL rats (F(1159) = 6.9, P = 0.01) and was time-dependent after INC (F(4159)=23.2, P < 0.0001). Similarly to the groups used for VTA ICSS, PWT in the area of the incision was significantly less than baseline values at all time points up to the latest time tested (96 hours) after INC in sham-SNL and SNL rats but was not affected by sham-INC (data not shown). As was found with the other groups of rats, PWT of the midpaw region was significantly reduced in SNL rats compared with sham-SNL rats but was not affected by INC compared with sham-INC (data not shown).
Distance traveled was less in the SNL compared with the sham-SNL group (F(1,159) = 7.8, P = 0.006) and was dependent on time after INC or sham-INC (F(4,159) = 11.9, P < 0.0001). However, INC did not significantly reduce distance traveled relative to sham-INC in these animals (F(1,159) = 2.8, P = 0.1) (Fig. 5A). The only significant difference in total distance traveled was at the 4-hour time point in the SNL + INC rats (P ≤ 0.05). Rearing was also reduced in the SNL group compared with sham-SNL (F(1,159) = 13.1, P = 0.0004), as well as in the INC group compared with sham-INC (F(1,159) = 4.9, P = 0.03) (Fig. 5B). The effect of INC on rearing was also time-dependent (F(4,159) = 15.5, P < 0.0001). Compared with the sham-SNL + sham-INC group, rearing was reduced at all time points in the SNL + INC group and at all time points other than 4 hours after surgery in the SNL only group. As with the distance traveled, rearing was not reduced at any time after surgery in the INC only group, compared with sham-SNL + sham-INC animals (P > 0.05).
The current study evaluated the effects of INC, SNL, and their interaction on a number of nonevoked behaviors as well as the traditional PWT measure of mechanical hypersensitivity. Similar to literature reports, both INC and SNL produce robust mechanical hypersensitivity that lasts for days (INC) and weeks (SNL), but only INC decreases operant responding for VTA ICSS (up to 2 days) and FR (up to 1 day) whereas only SNL diminishes OFA (as much as 4 days). Surprisingly, the effects of INC in rats previously subjected to SNL were not exacerbated; in these decreases in PWT, VTA ICSS and FR were similar in magnitude and recovery time to INC alone. This indicates that INC and SNL differentially affect the nonevoked behaviors assessed in this study, which are affected to a much lesser extent in both magnitude and duration compared with PWT.
The present data agree with other studies comparing the effects of acute pain on nonevoked measures in rodents. We have previously reported that abdominal incision reduces FR and OFA with a time course similar that observed with INC in the present study.11,27 Similarly, acute abdominal pain induced by intraperitoneal injection of dilute acid decreases responding for VTA ICSS to that found with INC in the present study.17 The relatively short time course of effect of INC on VTA ICSS and FR is similar to hindpaw guarding behavior exhibited after INC,22 which corresponds to the time frame in which nociceptors exhibit increased spontaneous activity.28 Therefore, suppression of VTA ICSS and FR after INC are likely mediated by ongoing spontaneous pain acutely after INC that resolves after hours or days.
In the current study, the effects of INC on mechanical allodynia persisted longer than those of nonevoked spontaneous behaviors. The effect of INC alone or INC in SNL rats on decreasing VTA ICSS or FR was significant only at the 4-hour (VTA ICSS) or 24-hour (FR) time point after INC. Since SNL alone did not alter VTA ICSS, data from INC and SNL + INC rats were combined and analyzed, which revealed that INC significantly decreased lever pressing for VTA ICSS for up to 2 days. INC also produced mechanical hypersensitivity around the incision site in both groups to a similar degree, but the hypersensitivity did not begin to recover until day 7. The fact that INC inhibits VTA ICSS for a relatively short duration compared with mechanical allodynia suggests that mechanisms distinct from those that mediate hypersensitivity contribute to suppression of these nonevoked spontaneous behaviors.
Suppression of VTA ICSS and to a lesser extent FR by INC may result from increased spontaneous activity of nociceptive pathways that inhibit mesolimbic dopamine neurotransmission. One possible source for pain suppression of mesolimbic dopamine is the ascending spinoparabrachial pain pathway. The parabrachial nucleus (PBN) projects to the VTA and intra-PBN lidocaine attenuates noxious foot shock-induced inhibition of VTA dopamine neurons.29 Similarly, the amygdala receives direct ascending nociceptive input from the PBN30 and can modulate activity of VTA dopamine neurons through GABAergic projections to the VTA31 and glutamatergic projections to the nucleus accumbens.32,33 Future pharmacology studies by using direct injections into the PBN or amygdala after INC may be useful in elucidating this potential mechanism.
In contrast to INC, SNL did not disrupt VTA ICSS or FR. This is consistent with previous work showing that chronic neuropathic pain does not decrease feeding or locomotion measured in home cages in mice.10 The fact that SNL did not decrease responding for VTA ICSS is consistent with our previous work.16 Despite the inability to measure diminished VTA ICSS or FR after SNL, we and others have reported that SNL alters opioid reinforcement24 and renders intrathecal analgesics selectively reinforcing,14,34 suggesting that SNL induces an altered subjective state in rats. We and others have reported that only a select group of analgesics modify opioid reinforcement and produce conditioned place preference in SNL rats, further suggesting that the mechanisms by which nerve injury produces hypersensitivity and spontaneous pain differ.14,24
Though SNL did not decrease the operant, nonevoked measures assessed in the current study, the fact that rearing was selectively diminished by SNL and not INC was surprising. Previously, chronic constriction injury in mice did not affect locomotor activity, including rearing.35 Our previous studies found that abdominal incision decreases both distance traveled and rearing behavior; therefore, it was surprising that INC was without effect in the present study.27 In our previous work, however, exteriorizing and manipulating the intestines after laparotomy produced a greater effect on OFA than incision of the skin and abdominal wall.27 In the current study, however, the effect of SNL compared with INC on rearing behavior is clear; therefore, future studies evaluating whether other nerve injury models suppress rearing and determining whether the effect is sensitive to analgesics would be useful.
We expected that SNL would exacerbate the suppressive effects of INC on the nonevoked behavioral end points assessed. Instead, suppression of VTA ICSS or FR was similar in magnitude and duration after INC in SNL and sham-SNL rats. Similarly, PWT around the incision site (heel region rather than midpaw) did not differ between these groups. However, differences between these groups may be indistinguishable due to a floor effect. These findings are surprising since neuropathy produces significant peripheral and central sensitization36 that is expected to increase postoperative pain intensity and duration. Clinically, postoperative pain resolves at a slower rate in patients with chronic pain.37 For instance, in patients undergoing gynecological surgery, pain scores were higher in those with preexisting chronic pain in the perioperative period.38 Furthermore, the amount of pain present before surgery, regardless of its origin, directly correlated with the intensity of postoperative pain and patient-controlled analgesia morphine use.39
Though SNL did not exacerbate suppression of any of the behavioral end points after INC, it is possible that other types of chronic pain in rodents may display different characteristics. The clinical literature cited indicating that chronic pain exacerbates postoperative pain does not delineate between neuropathic and nonneuropathic chronic pain. Therefore, SNL, a type of deafferentation injury that nonetheless produces mechanical hypersensitivity, could influence postincisional pain in the hindpaw differently than chronic inflammatory pain. Furthermore, comorbid psychiatric conditions may underlie increased postoperative pain in chronic pain patients, since preexisting pain castrophizing and anxiety predict the intensity and duration of postoperative pain.40–42
It is also possible that any exacerbation of INC in SNL rats, or other types of future tissue injury, could depend on the time after nerve injury. In the current study, INC occurred at different times after SNL (approximately 1–3 months for VTA ICSS, approximately 3–4 weeks for FR, and approximately 2 weeks for OFA), yet at all these times, the effects of INC in SNL rats did not differ from INC in sham-SNL rats. It is possible that the effects of INC could be exacerbated in SNL rats at later (<3 months) time points after SNL, particularly if comorbid psychiatric conditions, such as stress or anxiety, contribute to increased postoperative pain.40–42 It is notable that in the current study, hypersensitivity was observed by 2 weeks after SNL and remained stable for months after nerve injury. This indicates that the presence of peripheral hypersensitivity in and of itself before incision is insufficient to prolong recovery from surgery.
A number of other nonevoked behavioral end points other than those assessed in the current study have been described, including conditioned place avoidance,12,13 conditioned place preference to analgesics,14 analgesic self-administration,15 facial grimacing,18,19 hindpaw guarding,20,21 and weight-bearing bias.22,23 For each of these nonevoked measures, and for traditional evoked measures such as PWT, pain is expected to increase and/or induce behavior. In contrast, the behavioral end points assessed in the current study (VTA ICSS, FR, and OFA) are predicted to decrease in the presence of ongoing pain. In fact, ICSS and feeding were previously shown to be acutely decreased in rats after intraperitoneal injection of lactic acid43; similarly, wheel running was shown to acutely decrease after intraplantar injection of complete Freund’s adjuvant (CFA).44 It is interesting to note that low doses of many anti-inflammatory and analgesic drugs reverse acute decreases in wheel running after intraplantar CFA, while having no effect on mechanical hypersensitivity,44 that suggest the 2 effects have different mechanisms and may be unrelated. To this end, assessment of pain-decreased behaviors may be advantageous since decreased behavior is frequently associated with pain in humans and animals.45
Collectively, the present study demonstrates that hypersensitivity to evoked noxious stimuli is more robust than other nonevoked spontaneous behaviors for both acute and chronic pain in rodents. Furthermore, the relatively short time course of effect of INC on nonevoked behaviors such as VTA ICSS and FR compared with the relatively long-lasting effect of hypersensitivity suggests different mechanisms may be responsible for evoked and nonevoked pain. One implication is that SNL, unlike INC, may not produce spontaneous pain of sufficient intensity to affect highly motivated behaviors such as VTA ICSS or FR. To this end, it is unclear whether the lack of reliable behavioral end points for spontaneous pain after neuropathy in rodents is due to the injury models not producing spontaneous pain, behavioral measures not being sensitive enough to detect spontaneous pain, or both. Therefore, continued development of novel chronic pain models and sensitive behavioral measures is needed.
Name: Eric E. Ewan, PhD.
Contribution: This author helped design the study, collected and analyzed data, and prepared the manuscript.
Attestation: Eric E. Ewan approved the final manuscript and attests to the integrity of the original data and the analysis reported in this manuscript.
Name: Thomas J. Martin, PhD.
Contribution: This author helped design the study, collected and analyzed data, and prepared the manuscript.
Attestation: Thomas J. Martin approved the final manuscript, is the archival author, and attests to the integrity of the original data and the analysis reported in this manuscript.
This manuscript was handled by: Jianren Mao, MD, PhD.
1. Katz J, Melzack R. Measurement of pain. Surg Clin North Am. 1999;79:231–52
2. Lumley MA, Cohen JL, Borszcz GS, Cano A, Radcliffe AM, Porter LS, Schubiner H, Keefe FJ. Pain and emotion: a biopsychosocial review of recent research. J Clin Psychol. 2011;67:942–68
3. Johansen JP, Fields HL, Manning BH. The affective component of pain in rodents: direct evidence for a contribution of the anterior cingulate cortex. Proc Natl Acad Sci U S A. 2001;98:8077–82
4. LaGraize SC, Labuda CJ, Rutledge MA, Jackson RL, Fuchs PN. Differential effect of anterior cingulate cortex lesion on mechanical hypersensitivity and escape/avoidance behavior in an animal model of neuropathic pain. Exp Neurol. 2004;188:139–48
5. Mao J. Translational pain research: achievements and challenges. J Pain. 2009;10:1001–11
6. Mogil JS, Crager SE. What should we be measuring in behavioral studies of chronic pain in animals? Pain. 2004;112:12–5
7. Backonja MM, Stacey B. Neuropathic pain symptoms relative to overall pain rating. J Pain. 2004;5:491–7
8. Gottrup H, Nielsen J, Arendt-Nielsen L, Jensen TS. The relationship between sensory thresholds and mechanical hyperalgesia in nerve injury. Pain. 1998;75:321–9
9. Martin TJ, Zhang Y, Buechler N, Conklin DR, Eisenach JC. Intrathecal morphine and ketorolac analgesia after surgery: comparison of spontaneous and elicited responses in rats. Pain. 2005;113:376–85
10. Urban R, Scherrer G, Goulding EH, Tecott LH, Basbaum AI. Behavioral indices of ongoing pain are largely unchanged in male mice with tissue or nerve injury-induced mechanical hypersensitivity. Pain. 2011;152:990–1000
11. Martin TJ, Kahn WR, Eisenach JC. Abdominal surgery decreases food-reinforced operant responding in rats: relevance of incisional pain. Anesthesiology. 2005;103:629–37
12. LaBuda CJ, Fuchs PN. Attenuation of negative pain affect produced by unilateral spinal nerve injury in the rat following anterior cingulate cortex activation. Neuroscience. 2005;136:311–22
13. Uhelski ML, Morris-Bobzean SA, Dennis TS, Perrotti LI, Fuchs PN. Evaluating underlying neuronal activity associated with escape/avoidance behavior in response to noxious stimulation in adult rats. Brain Res. 2012;1433:56–61
14. King T, Vera-Portocarrero L, Gutierrez T, Vanderah TW, Dussor G, Lai J, Fields HL, Porreca F. Unmasking the tonic-aversive state in neuropathic pain. Nat Neurosci. 2009;12:1364–6
15. Martin TJ, Ewan E. Chronic pain alters drug self-administration: implications for addiction and pain mechanisms. Exp Clin Psychopharmacol. 2008;16:357–66
16. Ewan EE, Martin TJ. Opioid facilitation of rewarding electrical brain stimulation is suppressed in rats with neuropathic pain. Anesthesiology. 2011;114:624–32
17. Pereira Do Carmo G, Stevenson GW, Carlezon WA, Negus SS. Effects of pain- and analgesia-related manipulations on intracranial self-stimulation in rats: further studies on pain-depressed behavior. Pain. 2009;144:170–7
18. Langford DJ, Bailey AL, Chanda ML, Clarke SE, Drummond TE, Echols S, Glick S, Ingrao J, Klassen-Ross T, Lacroix-Fralish ML, Matsumiya L, Sorge RE, Sotocinal SG, Tabaka JM, Wong D, van den Maagdenberg AM, Ferrari MD, Craig KD, Mogil JS. Coding of facial expressions of pain in the laboratory mouse. Nat Methods. 2010;7:447–9
19. Sotocinal SG, Sorge RE, Zaloum A, Tuttle AH, Martin LJ, Wieskopf JS, Mapplebeck JC, Wei P, Zhan S, Zhang S, McDougall JJ, King OD, Mogil JS. The Rat Grimace Scale: a partially automated method for quantifying pain in the laboratory rat via facial expressions. Mol Pain. 2011;7:55
20. Mao J, Hayes RL, Price DD, Coghill RC, Lu J, Mayer DJ. Post-injury treatment with GM1 ganglioside reduces nociceptive behaviors and spinal cord metabolic activity in rats with experimental peripheral mononeuropathy. Brain Res. 1992;584:18–27
21. Xu J, Brennan TJ. Guarding pain and spontaneous activity of nociceptors after skin versus skin plus deep tissue incision. Anesthesiology. 2010;112:153–64
22. Brennan TJ, Vandermeulen EP, Gebhart GF. Characterization of a rat model of incisional pain. Pain. 1996;64:493–501
23. King T, Rao S, Vanderah T, Chen Q, Vardanyan A, Porreca F. Differential blockade of nerve injury-induced shift in weight bearing and thermal and tactile hypersensitivity by milnacipran. J Pain. 2006;7:513–20
24. Martin TJ, Kim SA, Buechler NL, Porreca F, Eisenach JC. Opioid self-administration in the nerve-injured rat: relevance of antiallodynic effects to drug consumption and effects of intrathecal analgesics. Anesthesiology. 2007;106:312–22
25. Nichols ML, Bian D, Ossipov MH, Lai J, Porreca F. Regulation of morphine antiallodynic efficacy by cholecystokinin in a model of neuropathic pain in rats. J Pharmacol Exp Ther. 1995;275:1339–45
26. Chaplan SR, Bach FW, Pogrel JW, Chung JM, Yaksh TL. Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods. 1994;53:55–63
27. Martin TJ, Buechler NL, Kahn W, Crews JC, Eisenach JC. Effects of laparotomy on spontaneous exploratory activity and conditioned operant responding in the rat: a model for postoperative pain. Anesthesiology. 2004;101:191–203
28. Pogatzki EM, Gebhart GF, Brennan TJ. Characterization of Adelta- and C-fibers innervating the plantar rat hindpaw one day after an incision. J Neurophysiol. 2002;87:721–31
29. Coizet V, Dommett EJ, Klop EM, Redgrave P, Overton PG. The parabrachial nucleus is a critical link in the transmission of short latency nociceptive information to midbrain dopaminergic neurons. Neuroscience. 2010;168:263–72
30. Bernard JF, Besson JM. The spino(trigemino)pontoamygdaloid pathway: electrophysiological evidence for an involvement in pain processes. J Neurophysiol. 1990;63:473–90
31. Everitt BJ, Parkinson JA, Olmstead MC, Arroyo M, Robledo P, Robbins TW. Associative processes in addiction and reward. The role of amygdala-ventral striatal subsystems. Ann N Y Acad Sci. 1999;877:412–38
32. Brog JS, Salyapongse A, Deutch AY, Zahm DS. The patterns of afferent innervation of the core and shell in the “accumbens” part of the rat ventral striatum: immunohistochemical detection of retrogradely transported fluoro-gold. J Comp Neurol. 1993;338:255–78
33. Wright CI, Beijer AV, Groenewegen HJ. Basal amygdaloid complex afferents to the rat nucleus accumbens are compartmentally organized. J Neurosci. 1996;16:1877–93
34. Martin TJ, Kim SA, Eisenach JC. Clonidine maintains intrathecal self-administration in rats following spinal nerve ligation. Pain. 2006;125:257–63
35. Mogil JS, Graham AC, Ritchie J, Hughes SF, Austin JS, Schorscher-Petcu A, Langford DJ, Bennett GJ. Hypolocomotion, asymmetrically directed behaviors (licking, lifting, flinching, and shaking) and dynamic weight bearing (gait) changes are not measures of neuropathic pain in mice. Mol Pain. 2010;6:34
36. Suzuki R, Dickenson A. Spinal and supraspinal contributions to central sensitization in peripheral neuropathy. Neurosignals. 2005;14:175–81
37. Chapman CR, Davis J, Donaldson GW, Naylor J, Winchester D. Postoperative pain trajectories in chronic pain patients undergoing surgery: the effects of chronic opioid pharmacotherapy on acute pain. J Pain. 2011;12:1240–6
38. Magnani B, Johnson LR, Ferrante FM. Modifiers of patient-controlled analgesia efficacy. II. Chronic pain. Pain. 1989;39:23–9
39. Slappendel R, Weber EW, Bugter ML, Dirksen R. The intensity of preoperative pain is directly correlated with the amount of morphine needed for postoperative analgesia. Anesth Analg. 1999;88:146–8
40. Pinto PR, McIntyre T, Almeida A, Araújo-Soares V. The mediating role of pain catastrophizing in the relationship between presurgical anxiety and acute postsurgical pain after hysterectomy. Pain. 2012;153:218–26
41. Pinto PR, McIntyre T, Ferrero R, Almeida A, Araújo-Soares V. Predictors of acute postsurgical pain and anxiety following primary total hip and knee arthroplasty. J Pain. 2013;14:502–15
42. Pogatzki-Zahn EM, Englbrecht JS, Schug SA. Acute pain management in patients with fibromyalgia and other diffuse chronic pain syndromes. Curr Opin Anaesthesiol. 2009;22:627–33
43. Kwilasz AJ, Negus SS. Dissociable effects of the cannabinoid receptor agonists Δ9-tetrahydrocannabinol and CP55940 on pain-stimulated versus pain-depressed behavior in rats. J Pharmacol Exp Ther. 2012;343:389–400
44. Cobos EJ, Ghasemlou N, Araldi D, Segal D, Duong K, Woolf CJ. Inflammation-induced decrease in voluntary wheel running in mice: a nonreflexive test for evaluating inflammatory pain and analgesia. Pain. 2012;153:876–84
45. Negus SS, Bilsky EJ, Do Carmo GP, Stevenson GW. Rationale and methods for assessment of pain-depressed behavior in preclinical assays of pain and analgesia. Methods Mol Biol. 2010;617:79–91
46. Paxinos G WC, Paxinos G, Watson C. The Rat Brain in Stereotaxic Coordinates. 1998 New York, NY: Academic Press