BACKGROUND:The ketamine metabolite (2R,6R)-hydroxynorketamine ([2R,6R]-HNK) has analgesic efficacy in murine models of acute, neuropathic, and chronic pain. The purpose of this study was to evaluate the α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) dependence of (2R,6R)-HNK analgesia and protein changes in the hippocampus in murine pain models administered (2R,6R)-HNK or saline. METHODS:All mice were CD-1 IGS outbred mice. Male and female mice underwent plantar incision (PI) (n = 60), spared nerve injury (SNI) (n = 64), or tibial fracture (TF) (n = 40) surgery on the left hind limb. Mechanical allodynia was assessed using calibrated von Frey filaments. Mice were randomized to receive saline, naloxone, or the brain-penetrating AMPA blocker (1,2,3,4-Tetrahydro-6-nitro-2,3-dioxobenzo [f]quinoxaline-7-sulfonamide [NBQX]) before (2R,6R)-HNK 10 mg/kg, and this was repeated for 3 consecutive days. The area under the paw withdrawal threshold by time curve for days 0 to 3 (AUC 0-3d ) was calculated using trapezoidal integration. The AUC 0-3d was converted to percent antiallodynic effect using the baseline and pretreatment values as 0% and 100%. In separate experiments, a single dose of (2R,6R)-HNK 10 mg/kg or saline was administered to naive mice (n = 20) and 2 doses to PI (n = 40), SNI injury (n = 40), or TF (n = 40) mice. Naive mice were tested for ambulation, rearing, and motor strength. Immunoblot studies of the right hippocampal tissue were performed to evaluate the ratios of glutamate ionotropic receptor (AMPA) type subunit 1 (GluA1), glutamate ionotropic receptor (AMPA) type subunit 2 (GluA2), phosphorylated voltage-gated potassium channel 2.1 (p-Kv2.1), phosphorylated-calcium/calmodulin-dependent protein kinase II (p-CaMKII), brain-derived neurotrophic factor (BDNF), phosphorylated protein kinase B (p-AKT), phosphorylated extracellular signal-regulated kinase (p-ERK), CXC chemokine receptor 4 (CXCR4), phosphorylated eukaryotic translation initiation factor 2 subunit 1 (p-EIF2SI), and phosphorylated eukaryotic translation initiation factor 4E (p-EIF4E) to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). RESULTS:No model-specific gender difference in antiallodynic responses before (2R,6R)-HNK administration was observed. The antiallodynic AUC 0-3d of (2R,6R)-HNK was decreased by NBQX but not with pretreatment with naloxone or saline. The adjusted mean (95% confidence interval [CI]) antiallodynic effect of (2R,6R)-HNK in the PI, SNI, and TF models was 40.7% (34.1%-47.3%), 55.1% (48.7%-61.5%), and 54.7% (46.5%-63.0%), greater in the SNI, difference 14.3% (95% CI, 3.1-25.6; P = .007) and TF, difference 13.9% (95% CI, 1.9-26.0; P = .019) compared to the PI model. No effect of (2R,6R)-HNK on ambulation, rearing, or motor coordination was observed. Administration of (2R,6R)-HNK was associated with increased GluA1, GluA2, p-Kv2.1, and p-CaMKII and decreased BDNF ratios in the hippocampus, with model-specific variations in proteins involved in other pain pathways. CONCLUSIONS:(2R,6R)-HNK analgesia is AMPA-dependent, and (2R,6R)-HNK affected glutamate, potassium, calcium, and BDNF pathways in the hippocampus. At 10 mg/kg, (2R,6R)-HNK demonstrated a greater antiallodynic effect in models of chronic compared with acute pain. Protein analysis in the hippocampus suggests that AMPA-dependent alterations in BDNF-TrkB and Kv2.1 pathways may be involved in the antiallodynic effect of (2R,6R)-HNK.

Tropomyosin receptor kinase A (TrkA/NTRK1) is a high-affinity receptor for nerve growth factor (NGF), a potent pain mediator. NGF/TrkA signaling elevates synovial sensory neuronal distributions in the joints and causes osteoarthritis (OA) pain. We investigated the mechanisms of pain transmission as to whether peripheral sensory neurons are linked to the cellular plasticity in the dorsal root ganglia (DRG) and are critical for OA hyperalgesia. Sensory neuron-specific deletion of TrkA was achieved by tamoxifen injection in 4-week-old TrkAfl/fl;NaV1.8CreERT2 (Ntrk1 fl/fl;Scn10aCreERT2) mice. OA was induced by partial medial meniscectomy (PMM) in 12-week-old mice, and OA-pain-related behavior was analyzed for 12 weeks followed by comprehensive histopathological examinations. OA-associated joint pain was markedly improved without cartilage protection in sensory-neuron-specific conditional TrkA knock-out (cKO) mice. Alleviated hyperalgesia was associated with suppression of the NGF/TrkA pathway and reduced angiogenesis in fibroblast-like synovial cells. Elevated pain transmitters in the DRG of OA-induced mice were significantly diminished in sensory-neuron-specific TrkA cKO and global TrkA cKO mice. Spinal glial activity and brain-derived neurotropic factor (BDNF) were significantly increased in OA-induced mice but were substantially eliminated by sensory-neuron-specific deletion. Our results suggest that augmentation of NGF/TrkA signaling in the joint synovium and the peripheral sensory neurons facilitate pro-nociception and centralized pain sensitization.

BACKGROUND: Disk herniation is a primary cause of radicular back pain. The purpose of this study was to evaluate the antiallodynic effective dose in 50% of the sample (ED50) and dorsal root ganglion (DRG) protein modulation of a peripheral direct adenosine monophosphate kinase alpha (AMPKα) activator (O304) in a murine model of lumbar disk puncture. METHODS: Male (n = 28) and female (n = 28) mice (C57BL6/J) were assessed for hind paw withdrawal threshold (PWT) and burrowing. Abdominal surgery was performed on all mice, and 48 received a lumbar disk puncture (27-G needle), with 8 serving as nondisk puncture controls. Assessments were repeated at day 7, and mice were then randomized into 5 groups of equal numbers of males and females: O304 at 100 mg/kg (n = 10), 150 mg/kg (n = 10), 200 mg/kg (n = 10), and 250 mg/kg (n = 10) or drug vehicle (n = 8). Starting on day 7, mice received daily gavages of O304 or vehicle for 7 days. On days 14 and 21 PWT and on day 14 burrowing were assessed. The area under the PWT by time curve (AUC) from day 7 to 21 was determined by trapezoidal integration. DRG protein modulation was evaluated in male (n = 10) and female (n = 10) mice (C57BL6/J). Following disk puncture, mice were randomized to receive O304 200 mg/kg or vehicle for 7 days starting on day 7. On day 14, mice were euthanized; the DRG harvested and immunoblot performed for mammalian target of rapamycin (mTOR), transient receptor potential ankyrin 1 (TRPA1), phosphorylated adenosine monophosphate kinase (p-AMPK), phosphorylated extracellular signal-regulated kinase (p-ERK), phosphorylated eukaryotic translation initiation factor 2 subunit 1 (p-EIF2S1), phosphorylated eukaryotic translation initiation factor 4e (p-EIF4E), and glyceraldehyde 3-phosphate dehydrogenase (GADPH). RESULTS: Disk puncture decreased PWT greater in female mice compared with male mice and decreased burrowing at 7 days. PWTs were increased with increasing doses of O304 from 150 to 250 mg/g on day 14 and sustained through day 21. The ED50 (95% confidence interval [CI]) for reducing mechanical allodynia was 140 (118–164) mg/kg. Burrowing was not increased at day 14 compared to day 7 by O304 administration. Compared to vehicle-treated animals, O304 increased (95% CI) the p-AMPK/GADPH ratio, difference 0.27 (0.08–0.45; P = .004) and decreased (95% CI) the ratios of p-TRPA1, p-ERK1/2, pEIF4E, and p-EIF2S1 to GADPH by −0.49 (−0.61 to −0.37; P < .001), −0.53 (−0.76 to −0.29; P < .001), −0.27 (−0.42 to 0.11; P = .001), and −0.21 (−0.32 to −0.08; P = .003) in the DRG, respectively. CONCLUSIONS: The direct peripheral AMPK activator O304 reduced allodynia in a dose-dependent manner, and immunoblot studies of the DRG showed that O304 increased p-AMPK and decreased TRPA1, p-ERK1/2, as well as translation factors involved in neuroplasticity. Our findings confirm the role of peripheral AMPKα activation in modulating nociceptive pain.

Background and objectives AMP-activated protein kinase (AMPK) activator drugs decrease hypersensitivity in mice with pain. This study examines if postsurgery treatment with the prototype AMPK activator metformin and a new mechanism-specific AMPK activator, O304, after plantar hindpaw incision in mice, would reduce mechanical hypersensitivity and produce changes in the AMPK pathway in the dorsal root ganglion (DRG). Methods To create postoperative pain, an incision was made in the left plantar hindpaw. Animals were randomized into four oral gavage drug treatment groups (n=8/group): (1) vehicle, (2) metformin 200 mg/kg, (3) O304 200 mg/kg and (4) O304 200 mg/kg plus metformin 200 mg/kg. Drug gavages were performed 4 hours postsurgery and were repeated for 3 days. Mechanical hypersensitivity was measured with von Frey filaments. Changes in phosphorylated AMP-activated protein kinase alpha subunit, phosphorylated mechanistic target of rapamycin and phosphorylated eukaryotic initiation factor 2 alpha in DRG neurons were examined by immunohistochemistry. Results O304 or metformin increased von Frey thresholds (reduced mechanical hypersensitivity) in plantar incision mice versus vehicle-treated incision mice between days 1 and 4 (difference of mean area under the curve, O304: 2.24 g* day; 95% CI of the difference 0.28 to 4.21, p=0.011; metformin: 2.56 g* day; 95% CI of the difference 1.71 to 3.41, p<0.001). The drug combination further elevated von Frey thresholds. In the vehicle-treated group, the AMP-activated protein kinase alpha subunit was downregulated and mechanistic target of rapamycin and eukaryotic initiation factor 2 alpha were upregulated in DRG neurons; these deficits were reversed by the AMPK activator treatments. Conclusions Early treatment with the mechanism-specific AMPK activator O304 or the prototype AMPK activator metformin reduces mechanical hypersensitivity in a postoperative pain model in mice. These drugs also normalize the AMPK pathway in the DRG.

Background and objectives Ketamine has been shown to reduce chronic pain; however, the adverse events associated with ketamine makes it challenging for use outside of the perioperative setting. The ketamine metabolite (2R,6R)-hydroxynorketamine ((2R,6R)-HNK) has a therapeutic effect in mice models of depression, with minimal side effects. The objective of this study is to determine if (2R,6R)-HNK has efficacy in both acute and chronic mouse pain models. Methods Mice were tested in three pain models: nerve-injury neuropathic pain, tibia fracture complex regional pain syndrome type-1 (CRPS1) pain, and plantar incision postoperative pain. Once mechanical allodynia had developed, systemic (2R,6R)-HNK or ketamine was administered as a bolus injection and compared with saline control in relieving allodynia. Results In all three models, 10 mg/kg ketamine failed to produce sustained analgesia. In the neuropathic pain model, a single intraperitoneal injection of 10 mg/kg (2R,6R)-HNK elevated von Frey thresholds over a time period of 1–24hours compared with saline (F=121.6, p<0.0001), and three daily (2R,6R)-HNK injections elevated von Frey thresholds for 3 days compared with saline (F=33.4, p=0.0002). In the CRPS1 model, three (2R,6R)-HNK injections elevated von Frey thresholds for 3 days and then an additional 4 days compared with saline (F=116.1, p<0.0001). In the postoperative pain model, three (2R,6R)-HNK injections elevated von Frey thresholds for 3 days and then an additional 5 days compared with saline (F=60.6, p<0.0001). Conclusions This study demonstrates that (2R,6R)-HNK is superior to ketamine in reducing mechanical allodynia in acute and chronic pain models and suggests it may be a new non-opioid drug for future therapeutic studies.

Background and objectivesIntervertebral disc herniation is one of the common causes of low back pain. Adenosine monophosphate (AMP)-activated protein kinase (AMPK) activator drugs have been shown to reduce pain in several animal models. The present study examines if early treatment with the drug metformin, an indirect AMPK activator, and/or O304, a new direct AMPK activator, can reduce the mechanical hypersensitivity that develops after lumbar disc puncture in mice.MethodsThe L4/L5 and L5/L6 discs in male and female mice were exposed via a retroperitoneal approach and a single puncture was made at the midline of each disc. Mice were randomized into four drug treatment groups: (1) vehicle; (2) metformin 200 mg/kg; (3) O304 200 mg/kg; (4) metformin 100 mg/kg plus O304 100 mg/kg; plus one untreated sham surgery group. Drugs were administered by oral gavage starting 7 days after disc puncture and repeated for six more days. Mechanical allodynia in the plantar hindpaw was measured presurgery and up to day 28.Results7 days after disc puncture, female mice had lower von Frey thresholds than male mice, difference −0.46 g, 95% CI −0.34 to −0.60, p<0.001. Gender adjusted von Frey area under the curve’s (AUC’s) between days 7 and 28 for metformin and/or O304 were greater (reduced allodynia) compared with vehicle-treated mice. The difference of mean AUC’s was: metformin, 41.1 g*d, 95% CI of the difference 26.4 to 54.5, O304, 44.7 g*d, 95% CI of the difference 31.0 to 57.4, drug combination: 33.4 g*d; 95% CI of the difference 18.1 to 46.9. No gender by treatment interactions were observed.ConclusionsLumbar disc puncture in mice produces consistent mechanical hypersensitivity, and postinjury treatment with AMPK activator drugs (indirect and direct) reduces the mechanical hypersensitivity.

BACKGROUND: Metformin, an adenosine monophosphate (AMP)–activated protein kinase activator, as well as a common drug for type 2 diabetes, has previously been shown to decrease mechanical allodynia in mice with neuropathic pain. The objective of this study is to determine if treatment with metformin during the first 3 weeks after fracture would produce a long-term decrease in mechanical allodynia and improve a complex behavioral task (burrowing) in a mouse tibia fracture model with signs of complex regional pain syndrome. METHODS: Mice were allocated into distal tibia fracture or nonfracture groups (n = 12 per group). The fracture was stabilized with intramedullary pinning and external casting for 21 days. Animals were then randomized into 4 groups (n = 6 per group): (1) fracture, metformin treated, (2) fracture, saline treated, (3) nonfracture, metformin treated, and (4) nonfracture, saline treated. Mice received daily intraperitoneal injections of metformin 200 mg/kg or saline between days 14 and 21. After cast removal, von Frey force withdrawal (every 3 days) and burrowing (every 7 days) were tested between 25 and 56 days. Paw width was measured for 14 days after cast removal. AMP-activated protein kinase downregulation at 4 weeks after tibia fracture in the dorsal root ganglia was examined by immunohistochemistry for changes in the AMP-activated protein kinase pathway. RESULTS: Metformin injections elevated von Frey thresholds (reduced mechanical allodynia) in complex regional pain syndrome mice versus saline-treated fracture mice between days 25 and 56 (difference of mean area under the curve, 42.5 g·d; 95% CI of the difference, 21.0–63.9; P < .001). Metformin also reversed burrowing deficits compared to saline-treated tibial fracture mice (difference of mean area under the curve, 546 g·d; 95% CI of the difference, 68–1024; P < .022). Paw width (edema) was reduced in metformin-treated fracture mice. After tibia fracture, AMP-activated protein kinase was downregulated in dorsal root ganglia neurons, and mechanistic target of rapamycin, ribosomal S6 protein, and eukaryotic initiation factor 2α were upregulated. CONCLUSIONS: The important finding of this study was that early treatment with metformin reduces mechanical allodynia in a complex regional pain syndrome model in mice. Our findings suggest that AMP-activated protein kinase activators may be a viable therapeutic target for the treatment of pain associated with complex regional pain syndrome.

Although degenerative disc disease (DDD) and related low back pain (LBP) are growing public health problems, the underlying disease mechanisms remain unclear. An increase in the vascular endothelial growth factor (VEGF) levels in DDD has been reported. This study aimed to examine the role of VEGF receptors (VEGFRs) in DDD, using a mouse model of DDD. Progressive DDD was induced by anterior stabbing of lumbar intervertebral discs in wild type (WT) and VEGFR-1 tyrosine-kinase deficient mice (vegfr-1(TK-/-)). Pain assessments were performed weekly for 12 weeks. Histological and immunohistochemical assessments were made for discs, dorsal root ganglions, and spinal cord. Both vegfr-1(TK-/-) and WT mice presented with similar pathological changes in discs with an increased expression of inflammatory cytokines and matrix-degrading enzymes. Despite the similar pathological patterns, vegfr-1(TK-/-) mice showed insensitivity to pain compared with WT mice. This insensitivity to discogenic pain was related to lower levels of pain factors in the discs and peripheral sensory neurons and lower spinal glial activation in the vegfr-1(TK-)/(-) mice than in the WT mice. Exogenous stimulation of bovine disc cells with VEGF increased inflammatory and cartilage degrading enzyme. Silencing vegfr-1 by small-interfering-RNA decreased VEGF-induced expression of pain markers, while silencing vegfr-2 decreased VEGF-induced expression of inflammatory and metabolic markers without changing pain markers. This suggests the involvement of VEGFR-1 signaling specifically in pain transmission. Collectively, our results indicate that the VEGF signaling is involved in DDD. Particularly, VEGFR-1 is critical for discogenic LBP transmission independent of the degree of disc pathology.

Osteoarthritis (OA) is a painful and debilitating disease. A striking feature of OA is the dramatic increase in vascular endothelial growth factor (VEGF) levels and in new blood vessel formation in the joints, both of which correlate with the severity of OA pain. Our aim was to determine whether anti-VEGF monoclonal antibodies (mAbs) -MF-1 (mAb to VEGFR1) and DC101 (mAb to VEGFR2) - can reduce OA pain and can do so by targeting VEGF signaling pathways such as Flt-1 (VEGFR1) and Flk-1 (VEGFR2). After IACUC approval, OA was induced by partial medial meniscectomy (PMM) in C57/BL6 mice (20 g). ln the first experiment, for validation of VEGFR1 in DRG, the mouse dorsal root ganglion (DRG) was stimulated with NGF for 48 h to find the relative gene induction for VEGFR1 vs. 18S by RT-PCR. In the second experiment, Biotin-conjugated VEGFA (1 mu g/knee joint) was administered in the left knee joint of mice with advanced OA in order to characterization of VEGFR1 and VEGFR2. pVEGFR1/VEGFR2 was detected by immunostaining in DRGs. Finally, MF-1 and DC101 were administered in OA mice by both intrathecal (IT) and intraarticular (IA) injections, and the change in paw withdrawal threshold (PWT) was measured. Retrograde transport of VEGF was confirmed for detection of pVEGFR1/VEGFR2 in the DRG. PMM surgery led to development of OA and mechanical allodynia, with reduced paw withdrawal thresholds (PWT) (P < 0.0001). IT injection of MF-1 led to a reduction of allodynia in advanced OA, but injection of DC101 did not. IA injection of MF-1 or DC101 at one week after PMM injury did not reduce allodynia, but when injected in advanced OA mice joints at 12 weeks, both Mabs increased PWT an indicator of analgesia. Our data show that MF-1 (VEGR1 inhibition) decreases pain in advanced OA after IT or IA injection. Activation of MF-1 or DC101 may ameliorate OA-related joint pain.

Environmental disruption of the circadian rhythm is linked with increased pain due to osteoarthritis (OA). We aimed to characterize the role of the clock gene in OA-induced pain more systemically using both genetic and pharmacological approaches. Genetically modified mice, (bmal1f/fNav1.8CreERT mice), generated by deleting the critical clock gene, bmal1, from Nav1.8 sensory neurons, were resistant to the development of mechanical hyperalgesia associated with OA induced by partial medial meniscectomy (PMM) of the knee. In wild-type mice, induction of OA by PMM surgery led to a substantial increase in BMAL1 expression in DRG neurons. Interestingly, pharmacological activation of the REV-ERB (a negative regulator of bmal1 transcription) with SR9009 resulted in reduction of BMAL1 expression, and a significant decrease in mechanical hyperalgesia associated with OA. Cartilage degeneration was also significantly reduced in mice treated with the REV-ERB agonist SR9009. Based on these data, we also assessed the effect of pharmacological activation of REV-ERB using a model of environmental circadian disruption with its associated mechanical hyperalgesia, and noted that SR9009 was an effective analgesic in this model as well. Our data clearly demonstrate that genetic disruption of the molecular clock, via deletion of bmal1 in the sensory neurons of the DRG, decreases pain in a model of OA. Furthermore, pharmacological activation of REV-ERB leading to suppression of BMAL1 expression may be an effective method for treating OA-related pain, as well as to reduce joint damage associated with this disease.

Discogenic low back pain (DLBP) is extremely common and costly. Effective treatments are lacking due to DLBP's unknown pathogenesis. Currently, there are no in vivo mouse models of DLBP, which restricts research in this field. The aim of this study was to establish a reliable DLBP model in mouse that captures the pathological changes in the disc and allows longitudinal pain testing. The model was generated by puncturing the mouse lumbar discs (L4/5, L5/6, and L6/S1) and removing the nucleus pulposus using a microscalpel under the microscope. Histology, molecular pathways, and pain-related behaviors were examined. Over 12 weeks post-surgery, animals displayed the mechanical, heat, and cold hyperalgesia along with decreased burrowing and rearing. Histology showed progressive disc degeneration with loss of disc height, nucleus pulposus reduction, proteoglycan depletion, and annular fibrotic disorganization. Immunohistochemistry revealed a substantial increase in inflammatory mediators at 2 and 4 weeks. Nerve growth factor was upregulated from 2 weeks to the end of the experiment. Nerve fiber ingrowth was induced in the injured discs after 4 weeks. Disc-puncture also produced an upregulation of neuropeptides in dorsal root ganglia neurons and an activation of glial cells in the spinal cord dorsal horn. These findings indicate that the cellular and structural changes in discs, as well as peripheral and central nervous system plasticity, paralleled persistent, and robust behavioral pain responses. Therefore, this mouse DLBP model could be used to investigate mechanisms underlying discogenic pain, thereby facilitating effective drug screening and development of treatments for DLBP.

Chronic low back pain is a major cause of disability and health care costs. Effective treatments are inadequate for many patients. Animal models are essential to further understanding of the pain mechanism and testing potential therapies. Currently, a number of preclinical models have been developed attempting to mimic aspects of clinical conditions that contribute to low back pain (LBP). This review focused on describing these animal models and the main behavioral tests for assessing pain in each model. Animal models of LBP can be divided into the following five categories: Discogenic LBP, radicular back pain, facet joint osteoarthritis back pain, muscle-induced LBP, and spontaneous occurring LBP models. These models are important not only for enhancing our knowledge of how LBP is generated, but also for the development of novel therapeutic regimens to treat LBP in patients. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1305-1312, 2018.

Background and Objectives Complex regional pain syndrome is a challenging disease to treat. Recently, a mouse fracture model of complex regional pain syndrome has been developed that has many signs of the clinical syndrome. However, many aspects of the sensory neuron biochemistry and behavioral and pharmacological characterization of this model remain to be clarified. Methods Mice were randomly assigned to fracture/cast or control (naive) groups. Fracture/cast mice underwent a closed distal tibia facture, with hindlimb wrapped in casting tape for 3 weeks. After cast removal, mice were tested for mechanical allodynia, burrowing behavior, and motor ability over a 12-week period. Protein immunohistochemistry was performed for substance P, calcitonin gene-related peptide, tropomyosin receptor kinase A, nerve growth factor, Nav1.7, and transient receptor potential cation-channel V1, colocalized in neurons, in the ipsilateral lumbar dorsal root ganglia (DRGs). Analgesic drugs were tested for pain-relieving efficacy. Results Mechanical allodynia was greater in the ipsilateral hindpaw (P = 0.0002) in the fracture/cast group versus the control group, over the 3- to 12-week period. The amount of burrowing material removed was decreased (P = 0.0026), and there were deficits in spontaneous motor-rearing behavior (P = 0.018). Immunostaining of substance P, calcitonin gene-related peptide, Trk A receptor, nerve growth factor, Nav1.7, and transient receptor potential cation-channel V1 all demonstrated up-regulation in the DRGs of fracture mice versus controls (all P < 0.05). Morphine, pregabalin, ketamine, acetaminophen, and dexamethasone transiently increased force withdrawal thresholds on the ipsilateral (fracture) side and improved burrowing activity after injection (all P < 0.05). Ketorolac improved only burrowing. Conclusions Persistent pain-related behavior was demonstrated in this mouse fracture/cast model with wide-scale DRG up-regulation of pain mediators. Antihyperalgesic drugs reduced mechanical allodynia and improved burrowing.

Pathological pain is an enormous medical problem that places a significant burden on patients and can result from an injury that has long since healed or be due to an unidentifiable cause. Although treatments exist, they often either lack efficacy or have intolerable side effects. More importantly, they do not reverse the changes in the nervous system mediating pathological pain, and thus symptoms often return when therapies are discontinued. Consequently, novel therapies are urgently needed that have both improved efficacy and disease-modifying properties. Here we highlight an emerging target for novel pain therapies, adenosine monophosphate-activated protein kinase (AMPK). AMPK is capable of regulating a variety of cellular processes including protein translation, activity of other kinases, and mitochondrial metabolism, many of which are thought to contribute to pathological pain. Consistent with these properties, preclinical studies show positive, and in some cases disease-modifying effects of either pharmacological activation or genetic regulation of AMPK in models of nerve injury, chemotherapy-induced peripheral neuropathy (CIPN), postsurgical pain, inflammatory pain, and diabetic neuropathy. Given the AMPK-activating ability of metformin, a widely prescribed and well-tolerated drug, these preclinical studies provide a strong rationale for both retrospective and prospective human pain trials with this drug. They also argue for the development of novel AMPK activators, whether orthosteric, allosteric, or modulators of events upstream of the kinase. Together, this review will present the case for AMPK as a novel therapeutic target for pain and will discuss future challenges in the path toward development of AMPK-based pain therapeutics.

Intraarticular steroid injection has been the mainstay of short-term treatment of knee osteoarthritis (OA) pain. However, the duration of therapeutic effect from a single injection is not as long as desired. In this study we use a viscous formulation of triamcinolone acetate (TCA) in hyaluronic acid to prolong the anti-allodynia effect of that steroid. OA was induced in mice by a partial medial meniscectomy. Over time the animals' developed a mechanical allodynia in the injected leg. Mice were then given a single intraarticular injection of TCA in a short-acting DMSO formulation, or a standard commercial suspension, or the drug formulated in 5% hyaluronic acid for slow-release. Control injections in OA mice were PBS or 5% hyaluronic acid vehicle. Mechanical allodynia was then monitored over the therapeutic period. Organotypic spinal cord slices and DRG culture were performed to assess whether TCA attenuates expressions of pain mediators induced by interleukin 1β. TCA 40μg in a fast-releasing DMSO formulation produced relief from mechanical allodynia for a few days compared to PBS control injections (P=0.007). Similarly, the commercial suspension of TCA 40μg also produced relief from mechanical allodynia for a few days compared to PBS control injections (P=0.001). However, TCA 100μg in 5% hyaluronic acid produced relief from mechanical allodynia for at least 28days compared to PBS control or 5% hyaluronic acid vehicle injections (P=0.0005). Furthermore, TCA significantly suppressed expression of pain mediators induced by interleukin 1β in spinal cord and DRG organotypic culture. Intraarticular TCA in a sustained release formulation of viscous 5% hyaluronic acid will produce a long-term attenuation of mechanical allodynia in the OA knees of mice.

The purpose of this chapter is to provide a brief introduction to the anatomy and physiology of pain pathways from peripheral nociceptors to central nervous system areas involved in the perception and modulation of pain. This chapter also provides a short introduction to major types of persistent pain: neuropathic and inflammatory persistent pain, and gives an overview of some important molecular targets that are thought to mediate these types of pain. These targets, which include ion channels, receptors, and some neurotransmitters, are further discussed in the context of their relevance as potential drug targets for the better treatment of pain in patients with persistent pain. Finally, this chapter introduces several important concepts in pain research that will be primary topics for chapters that come later in the book.

Post-herpetic neuralgia (PHN) is an extremely painful condition that may develop as a long-term consequence of the shingles that occurs due to reactivation of latent varicella zoster virus (VZV) residing in the dorsal root ganglia after a bout of chicken pox, usually in childhood. PHN is defined as pain that persists for more than 3-months after crusting of the last shingles lesion. PHN is often poorly alleviated by clinically available analgesic and adjuvant drugs. Hence, there is a large unmet medical need for new analgesics to improve relief of this chronic pain condition. Although rodent models of VZV-induced neuropathic pain have been described, there is considerable between-laboratory variability. Hence, there is a need for an optimised rat model of VZV-induced neuropathic pain for use in in vivo efficacy profiling of novel molecules as potential new analgesic agents for the improved relief of PHN. Initially, I assessed the in vitro infectivity of the Schenke, Ellen and AV-92:3L strains of VZV propagated in cultured human foreskin fibroblasts (HFFs) and/or MRC-5 cells in order to identify VZV strains for progression to in vivo establishment of a rat model of PHN. As the Schenke and Ellen strains of VZV exhibited similar infectivity in HFFs and MRC-5 cells, the latter was selected for VZV propagation. VZV-infection in MRC-5 cells was confirmed by the detection of viral proteins IE-62 and VZVgE. Collectively, my in vitro data showed that MRC-5 cells infected with either the Schenke or Ellen strain of VZV were suitable for progression to in vivo establishment of a rat model of PHN. Briefly, groups of adult male Wistar rats received unilateral intraplantar (i.pl.) injections (50 mL) of (i) phosphate buffered saline (pH 7.4, 1 mM, n=3-8; control-group), (ii) uninfected MRC-5 cells (7x105 cells, n=3-8; sham-group) or (iii) VZV-infected MRC-5 cells containing 2x105, 1x105, 2x104, 1x104, 2x103 cells of the Schenke strain of VZV. Following hindpaw inoculation, temporal development of hindpaw hypersensitivity was assessed by measuring paw withdrawal thresholds (PWTs), paw pressure thresholds (PPTs) and paw thermal (noxious heat) thresholds (PTTs). For rats administered unilateral i.pl. injections of 2x105 to 2x103 Schenke-infected MRC-5 cells, bilateral mechanical allodynia developed by ~day 7 and was maintained until day 21, with spontaneous resolution by ~day 56. For rats administered Schenke-infected MRC-5 cells, persistent mechanical hyperalgesia or persistent thermal hyperalgesia did not develop in either hindpaw. For rats administered 2x104 Ellen-infected MRC-5 cells, bilateral mechanical allodynia was fully developed by 2-3 weeks and maintained until at least day 56. Hence, as both MRC-5 cells and the Ellen strain of VZV are available commercially, I standardized my rat model of VZV-induced neuropathic pain using Ellen-infected MRC-5 cells. To pharmacologically characterize my afore-mentioned rat model, I administered clinically available analgesic and adjuvant drugs to VZV-rats with fully developed mechanical allodynia in the bilateral hindpaws. Briefly, VZV-rats received single bolus doses of gabapentin (10-60 mg/kg; s.c.), amitriptyline (5-30 mg/kg; i.p.), morphine, (0.1-3 mg/kg; s.c.), meloxicam (5-20 mg/kg; i.p.) or vehicle, according to a lwashoutr protocol with 2-3 days between successive doses. PWTs in the hindpaws were measured immediately pre-dose and at regular intervals for up to 3 h post-dosing to generate mean ( SEM) PWT versus time curves. Dose-response curves were constructed by plotting mean ( SEM) area under the change in PWT versus time curve (PWT AUC) values versus log dose. The ED50 for single s.c. bolus doses of gabapentin and morphine for the relief of mechanical allodynia in the ipsilateral hindpaws were 44.9 mg/kg and 1.6 mg/kg respectively. Amitriptyline and meloxicam lacked efficacy in the doses tested. Finally, I assessed the pain-relieving efficacy of several novel small molecule angiotensin II type 2 receptor (AT2R) antagonists as previous work by our laboratory had shown that these compounds were efficacious in rat models of peripheral neuropathic pain induced by mechanically and chemically-induced peripheral nerve injury. Hence, VZV-rats exhibiting fully developed mechanical allodynia in the ipsilateral hindpaws (PWTsl6g) received single bolus doses of intravenous (i.v.) PD123319 (0.03-3 mg/kg), subcutaneous (s.c.) EMA300 (0.3-5 mg/kg), oral (p.o.) EMA401 (0.03-1.0 mg/kg) or vehicle, according to a lwashoutr protocol. PWTs measurement in the hindpaws and dose-response curves were performed as described above. The mean ED50rs for i.v. PD123319, s.c. EMA300 and p.o. EMA401 were 1 mg/kg, 2.9 mg/kg and 0.8 mg/kg respectively. In summary, I have established and pharmacologically characterized an optimized rat model of VZV-induced neuropathic pain. I used this model to show that novel small molecule AT2R antagonists produce dose-dependent pain relief mirroring the recent clinical trial success of EMA401 for producing significant analgesia above placebo in patients with PHN.