2.3.2. Spared nerve injury surgery
Spared nerve injury surgery was performed as described by Decosterd and Woolf.18 Briefly, mice were anesthetized and prepared as described above for CCI surgery. After exposure of the sciatic nerve, the tibial and common peroneal nerve branches were ligated using silk sutures and transected while leaving the sural nerve intact. As with CCI surgery, the overlying musculature was closed using (1) 4-0 sterile silk suture and the animals recovered from anesthesia within 5 minutes.
2.4. Marble-burying assay
Mice were placed in Plexiglas cages (internal dimensions: 33 cm long × 21 cm wide × 19 cm high) with ∼3 cm of wood chips (7090 Teklad Sani-Chips; Envigo, Somerset, NJ) on bottom and 20 marbles were spaced in grid-like manner (4 × 5). Marbles used in these experiments were clear, with exception to Digital Supplement Figure 2B (available online at http://links.lww.com/PAIN/A549) where black marbles were used, but this did not noticeably influence digging behavior, or the data obtained. After the 20-minute test period, subjects were carefully removed to minimize disturbance to the bedding. Marbles at least 75% covered with bedding were considered buried. In the model characterization studies, mice were recorded using Anymaze software (Stoelting Co, Wood Dale, IL) to determine time spent digging. Digging time was scored by an experimenter blinded with respect to group. Digging was operationally defined as use of the hind paws to kick, or forcefully move, the woodchip bedding. This scoring was highly reliable, as a correlation of r = 0.98 was observed between 2 independent scorers. The experimenters were blinded with respect to surgical and treatment condition throughout all experiments. For the pharmacological assessment studies that examined both pain-evoked behaviors and marble burying, the order of testing was von Frey, hot plate, and then marble burying was analyzed last. For these studies, time spent digging was not captured. To minimize the number of mice needed to complete these studies, mice were tested with drug or vehicle on both day 3 and day 6 after CCI, in a counterbalanced fashion.
2.5. Assessment of pain-stimulated behavior
Mechanical allodynia and thermal hyperalgesia were used to assess nociceptive behavior after sham or CCI surgery (see above). Before surgery, mice were habituated to the testing environment. Next, von Frey monofilaments (North Coast Medical, Morgan Hills, CA) were used to establish BL responses to light mechanical touch and to assess the development and presence of allodynia after surgery.29 Specifically, the mice were placed on top of a wire mesh screen, with spaces 0.5 mm apart and habituated for approximately 30 minutes on 4 consecutive days before surgical procedures commenced. Mice were unrestrained, and singly placed under an inverted Plexiglas basket (8 cm diameter and 15 cm height), with a wire mesh top to allow for unrestricted air flow. The von Frey test uses a series of calibrated monofilaments, (2.83-4.31 log stimulus intensity) applied randomly to the left and right plantar surface of the hind paw for 3 seconds, using a modified “up-down” method.10 Each paw was stimulated 5 times with each filament, starting with the 3.61 log stimulus intensity filament and increasing until the mouse responded 5 out of 5 times.29 Lifting, licking, or shaking the paw was considered a response. Three or more responses out of 5 stimulations at the 3.61 log stimulus intensity filament were coded as a positive response. If a positive response was detected, at the 3.61 log stimulus intensity filament, lower weight filaments, starting at 2.84 and then sequentially increasing were used to assess the sensory threshold for each paw until the paw responded 5 out of 5 times. After completion of allodynia testing, the mice were placed on a heated (52°C) enclosed Hot Plate Analgesia Meter (Columbus Instruments, Columbus, OH). The latency to jump or lick/shake a hind paw was assessed. A 30-second cutoff time was used to avoid potential tissue damage.22 For each assay, testing was performed in a blinded fashion. For dose–response analysis of each drug, data from the same sham-vehicle and CCI-vehicle groups are included in each appropriate graph depicting marble burying, mechanical allodynia, and thermal hyperalgesia.
2.6. Data analysis
All data are presented as mean ± standard error (SEM). For allodynia testing, psychometric behavioral analysis was performed to compute the log stiffness that would have resulted in the 50% paw withdrawal rate, as previously described.50, 67 Briefly, thresholds were estimated by fitting a Gaussian integral psychometric function to the observed withdrawal rates for each of the tested von Frey hairs, using a maximum-likelihood fitting method.49,71 Pearson correlations were conducted to examine the relationship between marble burying and digging time, and these data were transformed in a best-fitting curve analysis. Data were analyzed using appropriate inferential statistical analysis of t tests, 1-way analysis of variance (ANOVA), or 2-way ANOVA.13 Except where indicated, the Tukey test was used for post hoc analyses of significant 1-way ANOVAs. Multiple comparisons after 2-way ANOVA were conducted with Bonferroni post hoc comparison.
3.1. Chronic constriction injury surgery reduces marble-burying and digging behaviors
The first experiment tested whether CCI surgery would alter marble burying and time spent digging compared with mice that had undergone sham surgery. As shown in Figure 1, mice in the CCI group buried fewer marbles than mice in the sham control (P < 0.01, Fig. 1A), and displayed a reduction in time spent digging (main effect of surgery, P < 0.01, main effect of time, P < 0.05, Fig. 1B) compared with controls. The reduction in these behaviors occurred on postsurgical days 3 and 6 and increased to levels of the control mice by days 10 and 13. Further analysis revealed a positive correlation of time spent digging and the number of marbles buried, (r = 0.84, P < 0.0001, Fig. 1C). Locomotor speed (P = 0.4, Supplemental Digital Content Figure 1A, available online at http://links.lww.com/PAIN/A549) and distance traveled (P = 0.5, Supplemental Digital Content Figure 1B, available online at http://links.lww.com/PAIN/A549) did not differ between sham and CCI groups. To determine whether repeated exposure to the marble-burying assay accounted for the complete resolution of the observed effects, new groups of mice undergoing sham or CCI surgery were prepared and tested on postsurgical days 3 and 13, only. On day 3, mice in the CCI group buried significantly fewer marbles than the sham mice (P < 0.01, Supplemental Digital Content Figure 1C, available online at http://links.lww.com/PAIN/A549) and spent less time digging than mice in the sham group (P < 0.05). However, on postsurgical day 13, both groups buried a similar number of marbles (P = 0.40) and spent a comparable duration of time digging, (P = 0.39, Supplemental Digital Content Figure 1D, available online at http://links.lww.com/PAIN/A549). Thus, CCI surgery depressed marble burying and digging time for the first postsurgical week.
Because previous research using the SNI neuropathic pain model reported increases in marble burying,24,55 we next compared marble burying behavior among SNI, CCI, and sham-operated mice on days 3 and 14 postsurgery. Both the CCI and SNI groups exhibited significant decreases in marble burying compared with sham-operated controls on day 3 (P < 0.05, Supplemental Digital Content Figure 2A, available online at http://links.lww.com/PAIN/A549), no group differences were observed on day 14 (P = 0.7, Supplemental Digital Content Figure 2B, available online at http://links.lww.com/PAIN/A549). However, mice undergoing SNI or CCI surgery exhibited significant ipsilateral mechanical allodynia (P < 0.0001, Supplemental Digital Content Figure 2C, available online at http://links.lww.com/PAIN/A549) and thermal hyperalgesia on day 13 compared with sham mice (P < 0.0001, Supplemental Digital Content Figure 2D, available online at http://links.lww.com/PAIN/A549).
3.2. Evaluation of standard analgesics and diazepam in chronic constriction injury–depressed marble burying and chronic constriction injury–evoked behaviors
We next examined if a panel of drugs (ie, morphine, gabapentin, and valdecoxib) known to reverse CCI-induced mechanical allodynia and thermal hyperalgesia would also ameliorate CCI-depressed marble burying. Morphine significantly reversed CCI-induced decreases in marble burying (P < 0.05, Fig. 2A), thermal hyperalgesia (P < 0.0001, Fig. 2B), and mechanical allodynia (P < 0.0001, Fig. 2C). Similarly, gabapentin significantly reversed CCI-induced decreases in the marble-burying assay (P < 0.001, Fig. 2A), thermal hyperalgesia (P < 0.0001, Fig. 2B), and mechanical allodynia (P < 0.0001, Fig. 2C). Similarly, valdecoxib reversed CCI-depressed marble burying (P < 0.001, Fig. 2A), thermal hyperalgesia (P < 0.001, Fig. 2B), and mechanical allodynia (P < 0.0001, Fig. 2C).
None of the drugs altered marble burying in sham mice (Supplemental Digital Content Figure 3A; morphine: P = 0.7, valdecoxib: P = 0.1, gabapentin: P = 0.7, available online at http://links.lww.com/PAIN/A549). Morphine elicited antinociception in the hot-plate test (P < 0.0001), but neither valdecoxib (P = 0.94) nor gabapentin (P = 0.06) significantly altered basal hot-plate responses (Supplemental Digital Content Figure 3B, available online at http://links.lww.com/PAIN/A549). In sham mice, morphine elevated mechanical stimulus thresholds (P < 0.01), but gabapentin (P = 0.9) and valdecoxib (P = 0.06) did not significantly affect this measure (Supplemental Digital Content Figure 3C, available online at http://links.lww.com/PAIN/A549).
Next, we examined whether the standard analgesic morphine would reverse the depressive effects of the kappa-opioid receptor agonist U69593 on marble burying. Naive mice given U69593 (0.1 mg/kg) buried fewer marbles compared with the vehicle control mice (Fig. 2D). Unlike its effects in reversing CCI-induced depression of marble burying, morphine (10 mg/kg) did not alter marble-burying behavior in either vehicle or U69593-injected mice (main effect of U69593, P < 0.0001).
We next sought to investigate if either the anxiolytic diazepam or U69593 would alter CCI depression of marble burying or CCI-induced allodynia or thermal hyperalgesia. Both diazepam and U69593 produced further decreases in marble burying within the CCI group (P < 0.05, Fig. 3A), but these drugs had differential effects on CCI-stimulated behavior. Although diazepam failed to alter CCI-induced thermal hyperalgesia (P = 0.4, Fig. 3B), or mechanical allodynia (P = 0.6, Fig. 3C), U69593 produced a dose-responsive reversal of CCI-induced thermal hyperalgesia (P < 0.0001, Fig. 3B), and mechanical allodynia (P < 0.001, Fig. 3C).
Both diazepam and U69593 decreased marble burying in sham mice (diazepam: P < 0.0001, U69593: P < 0.01; Supplemental Digital Content Figure 4A, available online at http://links.lww.com/PAIN/A549), but neither drug altered hot-plate latencies (diazepam: P = 0.1; U69593: P = 0.6; Supplemental Digital Content Figure 4B, available online at http://links.lww.com/PAIN/A549) or mechanical stimulus thresholds (U69593: P = 0.4; diazepam: P = 0.9; Supplemental Digital Content Figure 4C, available online at http://links.lww.com/PAIN/A549) in sham control mice.
3.3. Modulation of the endocannabinoid system reverses both pain-evoked and pain-depressed behavior
Finally, because the endogenous cannabinoid system contains multiple targets that show promise for the treatment of sustained pain, we tested cannabinoid receptor agonists as well as inhibitors of FAAH and MAGL in CCI-induced depression of marble burying. In addition, the same mice were tested for mechanical allodynia and thermal hyperalgesia.
The mixed CB1/CB2 receptor agonist CP55,940 dose dependently reversed marble burying (P < 0.001, Fig. 4A), thermal hyperalgesia (P < 0.0001, Fig. 4B), and mechanical allodynia (P < 0.0001, Fig. 4C) in the CCI group. Likewise, the CB2 receptor agonist LEI101 dose dependently reversed CCI-induced decreases in marble burying (P < 0.05, Fig. 4A), thermal hyperalgesia (P < 0.01, Fig. 4B), and mechanical allodynia (P < 0.05, Fig. 4C).
The MAGL inhibitor MJN110 reversed CCI-induced reduction in marble burying (P < 0.01, Fig. 4A), thermal hyperalgesia (P < 0.001, Fig. 4B), and mechanical allodynia (P < 0.0001, Fig. 4C). By contrast, the FAAH inhibitor PF3845 did not reverse marble-burying behavior (P = 0.1591, Fig. 4A), but reversed thermal hyperalgesia (P = 0.001, Fig. 4B) and mechanical allodynia (P = 0.0004, Fig. 4C) in the CCI group. A summary of the drug effects on CCI-induced decreases in marble-burying and pain-evoked behaviors can be found in Table 2.
In sham mice, 2 mg/kg CP55,940 significantly decreased marble burying (P < 0.05, Supplemental Figure 5A, available online at http://links.lww.com/PAIN/A549), as well as elevated hot-plate latencies (P < 0.05, Supplemental Digital Content Figure 5B, available online at http://links.lww.com/PAIN/A549) and withdrawal thresholds in the von Frey assay (P < 0.01, Supplemental Digital Content Figure 5C, available online at http://links.lww.com/PAIN/A549). Meanwhile, 40 mg/kg LEI101 and 1.25 mg/kg MJN110 did not alter the number of marbles buried (LEI101: P = 0.4, MJN110: P = 0.4, Supplemental Digital Content Figure 5A, available online at http://links.lww.com/PAIN/A549), hot-plate latencies (LEI101: P = 0.3, MJN110: P = 0.3, Supplemental Digital Content Figure 5B, available online at http://links.lww.com/PAIN/A549), or paw withdrawal thresholds (LEI101: P = 0.6, MJN110: P = 0.1, Supplemental Digital Content Figure 5C, available online at http://links.lww.com/PAIN/A549) in sham mice. PF3845 produced a small, but significant decrease in the number of marble buried (P < 0.001, Supplemental Digital Content Figure 5A, available online at http://links.lww.com/PAIN/A549) and reduced hot-plate latencies (P < 0.05, Supplemental Digital Content Figure 5B, available online at http://links.lww.com/PAIN/A549), but did not affect von Frey thresholds (P = 0.6, Supplemental Digital Content Figure 5C, available online at http://links.lww.com/PAIN/A549).
Pain disrupts performance of otherwise routine behaviors such as housekeeping, social function, grooming, and can severely impact job performance as well as overall quality of life.41,45,62 Despite pain-depressed normative life-oriented endpoints, most preclinical studies screening new analgesics use a cadre of assays using pain-evoked behaviors, such as lifting or licking hind paws in response to light mechanical touch, cold, or heat.50, 51 Conversely, evaluation of pain-depressed behaviors offers parallel preclinical lines of evidence for screening potential therapeutics and has been hypothesized to predict clinical efficacy better than pain-stimulated pain assays.43,51,53,64 Examples of pain-depressed assays include reductions in voluntary wheel running70 and burrowing behavior3 in rats after CCI or SNI surgery, respectively. We report that CCI or SNI surgery transiently decreases marble-burying behavior and overall time spent digging. Moreover, established antinociceptive agents from distinct drug classes (ie, morphine, gabapentin, and valdecoxib) and a variety of drugs targeting different components of the endocannabinoid system (ie, cannabinoid receptors and MAGL) reversed CCI-induced depression of marble burying, as well as CCI-evoked behaviors of thermal hyperalgesia and mechanical allodynia.
Although CCI and SNI surgery led to mechanical allodynia and thermal hyperalgesia that persisted beyond the second postsurgical week, the decreased marble-burying effect occurred during the first postsurgical week, only. However, these decreases are likely not due to the surgical procedure itself, as sham mice display similar rates of marble burying to surgically naive vehicle–vehicle treated mice (Fig. 2D). Our findings agree with other evidence for robust and sustained expression of many pain-stimulated behaviors in comparison with expression of pain-depressed behaviors.12,43,61 The high correlation between the number of marbles buried and the time spent digging is consistent with work from Gyertyan,27 who concluded that this assay reflects overall digging behavior. Other data suggest that marble burying reflects a repetitive and perseverative behavior,66 rather than a model of anxiety or depression. Given that CCI surgery led to a transient decrease in marble-burying behavior compared with the long duration of hypersensitive withdrawal responses from mechanical and thermal stimuli,36 this may be related to inflammatory and pronociceptive mediators associated with the early-phase after the nerve ligation. Alternatively, the relatively quick resolution of this pain-depressed behavior compared with the pain-stimulated behaviors may reflect an adaptive response in which mice rely on such behaviors such as digging to survive.16 Mechanical allodynia and thermal hyperalgesia are regulated largely through the spinothalamic tract,56, 63 whereas numerous regions within the central nervous system (eg, inputs from the hippocampus, prefrontal cortex, hypothalamus, thalamus, and spinal cord16,40) regulate digging behavior. Thus, clear anatomical distinctions, specifically regarding higher order brain region inputs, may be responsible for the facilitated resolution of CCI-induced depression of marble burying, compared with CCI-induced thermal hyperalgesia or mechanical allodynia.
Morphine, gabapentin, and valdecoxib are reported to elicit antinociceptive effects in preclinical models of pain.26,34,63 In this study, each drug fully reversed both CCI-stimulated nociceptive behaviors and the depressive effects of CCI surgery on marble-burying behavior, despite producing pharmacological effects through distinct mechanisms. Specifically, morphine dampens transmission of the sensory and affective components of nociception through activation of the mu-opioid receptor, gabapentin dampens neuronal excitability, and valdecoxib elicits anti-inflammatory effects through the inhibition of COX-2. Similarly, the selective COX-2 inhibitor celecoxib decreases upregulation of P2X3 receptors in dorsal root ganglia and decreases CCI-stimulated nociceptive behaviors when administered early after CCI.69 Although U69593 reversed CCI-stimulated behaviors, it depressed marble burying irrespective of CCI surgery. Similarly, U69593 reverses lactic acid–induced stretching behavior, but not lactic acid-depressed nest-building behaviors.53 Although morphine reversed CCI-depressed marble burying, which at higher doses might be due to enhanced locomotion rather than digging, it did not reverse drug-induced depression of marble burying by U69593. This pattern of findings suggests a degree of selectivity for morphine in reversing depression of marble burying by a pain stimulus, but not by a nonpain stimulus. It also highlights the sensitivity of the marble-burying assay to drugs that increase locomotion. Diazepam was tested as another negative control, and it did not alter mechanical allodynia or thermal hyperalgesia, and only exacerbated CCI-induced suppression of marble-burying behavior. The inhibitory effects of diazepam on marble-burying behavior are well described.37,60
This study also demonstrates that drugs targeting multiple components of the endocannabinoid system reverse both CCI-stimulated and CCI-depressed behaviors at approximately comparable doses for a given drug. Specifically, the CB1/CB2 receptor agonist CP55,940 or the selective CB2 receptor agonist LEI101, reversed CCI-stimulated and CCI-depressed behaviors. This effect is consistent with a recent report that [INCREMENT]9-tetrahydrocannabinol, another mixed CB1/CB2 receptor agonist, alleviated migraine pain-related depression of wheel running in rats.30 However, [INCREMENT]9-tetrahydrocannabinol and CP55,940 lacked efficacy in other pain-depressed assays, including i.p. acid-induced depression of feeding or wheel running in mice,48 i.p. acid-induced depression of feeding or positively reinforced operant behavior in rats, intraplantar formalin-induced depression of operant responding in rats, or noxious heat-induced depression of operant responding squirrel monkeys.31,39,42,48 The effects of LEI101 are consistent with previous reports showing that CB2 receptor agonists reverse CCI-induced behaviors.36,71 Thus, the effectiveness of cannabinoid receptor agonists in reversing pain-depressed behavior may depend on multiple procedural factors, such as the type of noxious stimulus/injury, the behavioral endpoint, and species.
Indirect modulation of cannabinoid receptors through inhibitors of endocannabinoid-regulating enzymes also holds promise as a potential strategy to treat pain. Specifically, MAGL inhibitors block 2-AG degradation leading to increased levels of this endocannabinoid, and consequently increased signaling at cannabinoid CB1 and CB2 receptors.29,34,36,66 In this study, the MAGL inhibitor MJN110 reversed CCI-induced behaviors in the von Frey, hot-plate, and marble-burying assays. Notably, although MJN110 did not affect marble burying in sham mice, the MAGL inhibitor JZL184 reduced marble burying in naive mice.37 As shown previously, MJN110 and JZL184 differentially alter rates of operant responding for food administration and locomotor behavior, which may be time and dose dependent.29
The FAAH inhibitor PF3845 led to a different pattern of results than the other drugs acting on the endocannabinoid system. Although PF3845 reversed thermal hyperalgesia and allodynia, as reported elsewhere,6,23 it failed to reverse CCI-induced decreases of marble burying. Likewise, the FAAH inhibitor PF-0445784 did not reverse pain-related decreases of burrowing behavior in a rat model of osteoarthritis,9 which is consistent with its failure in a clinical trial for osteoarthritis pain.28 By contrast, another FAAH inhibitor, URB597, reversed acetic acid–depressed feeding and wheel running behaviors48 and partially reversed lactic acid–depressed rates of intracranial self-stimulation.38 Translation of preclinical studies to the clinic may be affected by multiple factors, including the type of noxious stimulus used, the dependent measures of pain-depressed and pain-stimulated behavior, and differential pharmacokinetics and pharmacodynamics of drugs categorized in the same class of drugs.
In contrast to the observation that CCI decreased the number of marbles buried and time spent digging, others reported that SNI increased marble burying in mice beginning at 2-week postsurgery.55,74 To ascertain whether the type of nerve injury model accounted for these disparate findings on marble burying, we compared the consequences of CCI and SNI surgery in the marble burying on days 3 and 14. Both surgeries elicited a similar pattern of effects in which marble burying was reduced on day 3 compared with the sham controls, and this pain-depressed behavior resolved by day 14. Although previous studies concluded that increased marble burying equated to “pain-induced anxiety,” this study revealed a high correlation between marble-burying and digging behavior.4 Similarly, other research suggests that marble burying reflects a nongoal directed digging behavior,66 which can be affected by numerous environmental and pharmacological manipulations. However, this study did not assess digging behavior in experiments testing the various pharmacological agents and did not distinguish between goal-directed and incidental behavioral responses that resulted in the wood chips covering the marbles.
The marble-burying assay offers a straightforward procedure with sensitivity to pain-depressed behavior during the early stages of SNI- and CCI-induced neuropathy, and is readily reversed by known analgesics (ie, morphine, gabapentin, and valdecoxib). In addition, a variety of pharmacological agents targeting distinct components of the endocannabinoid system (ie, cannabinoid receptors and MAGL) reverse both CCI-induced depression of marble-burying behavior and CCI-stimulated nociceptive behavior, which adds credence for potential clinical efficacy. One important caveat of our findings is that this assay is only useful for 1 week after surgery, which may limit its preclinical drug discovery utility. Thus, this assay is not useful for measuring changes in chronic pain. Nonetheless, incorporation of pain-depressed behaviors, such as marble burying, in conjunction with pain-stimulated behaviors is relatively straightforward behavioral assays and may serve to identify new analgesic drugs with increased translational implications for treating patients suffering from pain.
Conflict of interest statement
Z.A. Curry declares that he received personal fees and other from National Institutes of Health, during the conduct of the study. The remaining authors have no conflict of interest to declare.
Research was supported by NIH Grants: DA009789, DA017259, DA032933, DA035864, DA007027, and DA038493-01A1.
The authors thank Mr. Arjun Goyal for his assistance in scoring digging behavior, as well as Dr. Micah Niphakis for the original synthesis of MJN110. The authors are also indebted for the highly critical feedback of a colleague who wished to remain anonymous.
Supplemental digital content
Supplemental digital content associated with this article can be found online at http://links.lww.com/PAIN/A549.
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Cannabinoid; Pain depressed; CCI; SNI
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