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(327) Preemptive local bupivacaine attenuates mechanical and cold allodynia in a rat model of neuropathic pain
Abstracts
E22 Models of CNS Stimulation: Brain and Spinal Cord
The Journal of Pain
S57
E23 Neuropathic Pain
(324) A novel optogenetic spinal cord stimulator for wireless, bi-directional control of nociceptive circuitry in freely moving mice
(326) Supraspinal adenosine A3 receptor (A3AR) activation reverses chemotherapy-induced neuropathic pain through an IL-10 dependent mechanism
C Morgan, V Samineni, S Sundaram, J Golden, G Shin, S Oh, J Rogers, and R Gereau, IV; Washington University School of Medicine, St. Louis, MO
J Little, K Janes, Z Chen, C Wahlman, D Tosh, K Jacobson, and D Salvemini; Saint Louis University School of Medicine, St. Louis, MO
Optogenetics has dramatically expanded the tools available for studying peripheral and central circuitry involved in pain. In order to realize the full potential of optogenetics for the study of pain, devices are needed which allow integration of miniaturized light sources that are minimally invasive, provide sufficient light, and allow chronic, free movement of rodents during the study of spontaneous and evoked pain behaviors. Here, we designed a highly flexible, wirelessly-powered, micro-LED spinal implant which allows optogenetic stimulation and inhibition of neurons in the spinal cord during free movement over a relatively large area. We characterized two transgenic mouse lines where light-sensitive ChR2-eYFP or Arch-eGFP is specifically expressed exclusively in Nav1.8+ sensory neurons. Implantation of the optogenetic spinal stimulator produces no gross motor or sensory deficits. Optogenetic spinal activation of ChR2 in these mice produce robust, reproducible, frequencydependent, spontaneous pain behaviors which are not present in control mice. Further studies utilizing this novel device will allow for specific, bi-directional, and wireless targeting of discrete spinal nociceptive populations.
We have recently reported that activation of the adenosine subtype 3 receptor (A3AR) potently attenuates evoked and non-evoked pain behaviors associated with preclinical models of nerve injury- and chemotherapy-induced neuropathies. In nerve injury models, we have demonstrated that these antinociceptive effects are a result of modulation of spinal nociceptive processing, in part, through the induction of descending inhibitory bulbospinal pathways from the rostral ventromedial medulla (RVM). The role of the RVM in chemotherapy-induced neuropathic pain and the mechanisms by which these effects occur are unknown. As A3AR activation attenuates inflammation via the release of anti-inflammatory mediators such as interleukin (IL)-10 and RVM neuroinflammation is associated with nerve injury- and chemotherapyinduced behavioral responses (e.g. allodynia and hyperalgesia), here we examined if A3AR activation in the RVM during chemotherapy (oxaliplatin)-induced neuropathic pain is antinociceptive through an IL-10 dependent mechanism. Systemic injection of MRS5698 (1 mg/kg), a recently characterized selective A3AR agonist, reversed established mechanical allodynia and hyperalgesia (n=4 per group). These effects were blocked by intra-RVM delivery of a selective A3AR antagonist, MRS1523 (1 nmol), but not its vehicle, suggesting supraspinal mechanisms are critical for the actions of MRS5698. Further examination demonstrated that A3AR activation following intra-RVM injection of MRS5698 (3 nmol; n=4 per group) reversed mechanical allodynia and hyperalgesia; this effect was blocked by RVM injection of an anti-IL10 antibody (0.2 mg). There were no observed effects in oxaliplatin control animals following administration of MRS5698, MRS1523, or anti-IL10. Collectively, these experiments suggest that the RVM contributes to the protective effects of A3AR activation through the IL-10 pathway during chemotherapy-induced neuropathic pain. This research was supported by NIH/NCI 1RO1CA169519-01, the Saint Louis University President Research Fund, and the Saint Louis Cancer Center.
(325) DREADD activation of dopaminergic and glutamatergic neurons in the periaquaductal gray produces differing analgesic responses
(327) Preemptive local bupivacaine attenuates mechanical and cold allodynia in a rat model of neuropathic pain
N Taylor, J Pei, C Van Dort, E Brown, and K Solt; Massachusetts General Hospital, Boston, MA Despite years of study, current treatments do not sufficiently treat chronic pain syndromes. Electrical brain stimulation of the periaqueductal gray (PAG) produces profound antinociception, and shows promise in treating these difficult conditions. However, this treatment can also produce intense anxiety which has limited its use. The goal of this study was to better define the complex neural circuitry in the PAG specifically responsible for the analgesic effect and hypothesized that in targeting specific neural circuits we would be able to produce analgesia without anxiety. DREADDs (Designer Receptors Exclusively Activated by Designer Drugs) are G-protein coupled receptors engineered to be selectively activated by the ligand Clozapine N-Oxide (CNO). We targeted DREADDs to dopamine, GABA and glutamate neurons within the ventral lateral PAG (vlPAG) using viral-mediated delivery into different Cre-mice lines. Anesthetized dopamine transporter promoter (DAT-cre) and vesicular glutamate tranporter 2 (Vglut-2-cre) mice received targeted injections of adeno-associated virus carrying excitatory DREADDs (AAV5-hM3Dq), while vesicular GABA transporter (Vgat-ires-cre) mice were injected with an inhibitory DREADDs construct (AAV8-hM4Di). After a 3 week recovery, analgesia was assessed using the Hargreaves method before and after i.p. CNO injection. Viral expression and localization were confirmed using immunohistochemistry upon completion of the study. At baseline, paw withdrawal occurred at 2.7060.27s in DAT-cre mice (n=3), 2.6260.10s in Vgat-cre mice (n=6) and 2.9360.10s in Vglut-2-cre mice (n=6). CNO was injected ip and paw withdrawal times were measured hourly for 6 hours. No statistically significant difference in withdrawal times were seen in GABA targeted animals. Significant analgesia was accompanied with anxiety behaviors in glutamate targeted mice, while dopamine targeted animals showed only an analgesic response. In summary, only activation of dopamine neurons in the vlPAG produced profound analgesia without signs of anxiety. vlPAG dopamine neurons represent a novel target for producing analgesia that may lead to new treatments for pain.
J Clifford, A Mares, J Hansen, and D Averitt; US Army Institute of Surgical Research, Fort Sam Houston, TX Neuropathic pain is evasive to treat once developed, however evidence suggests that local administration of anesthetics near the time of injury reduces the development of neuropathic pain. As abnormal electrical signaling in the damaged nerve contributes to the initiation and maintenance of neuropathic pain, local administration of anesthetics prior to injury may prevent neuropathic pain. We hypothesized that local preemptive treatment with bupivacaine prior to nerve injury in a rat model of spinal nerve ligation (SNL) would reduce the initiation and/or maintenance of neuropathic pain behaviors. On the day prior to SNL, baseline measures of thermal hyperalgesia, mechanical allodynia and acetone-induced cold alloynia were recorded in male SpragueDawley rats. During surgery immediately prior to ligation, the nerve was perineurally bathed in either bupivacaine (0.4% bupivacaine in 0.2 mL) or sterile saline (0.2 mL) for 30 minutes. The nerve was then ligated, the wound sutured and the rats were allowed to recover for 3 days prior to behavioral testing. A separate group of rats received the same surgical manipulations without ligation (sham controls). All rats were then examined for the development of thermal hyperalgesia, mechanical allodynia, and cold allodynia on 3, 7, 10, 14 and 21 days following surgery. All ligated rats exhibited both mechanical and cold allodynia, but not thermal hyperalgesia, within 3 days post-SNL and lasting for 21 days. No pain behaviors were observed in sham controls. While there was no effect of bupivacaine on mechanical allodynia 3 days post-SNL, mechanical allodynia was significantly diminished by day 7 and similar to sham controls by 2 weeks post-SNL. Bupivacaine significantly attenuated cold allodynia at all time points measured. These data indicate that local treatment with bupivacaine prior to surgical manipulations that are known to cause nerve damage, such as amputation, may protect against the initiation and maintenance of neuropathic pain.
E22 Models of CNS Stimulation: Brain and Spinal Cord
The Journal of Pain
S57
E23 Neuropathic Pain
(324) A novel optogenetic spinal cord stimulator for wireless, bi-directional control of nociceptive circuitry in freely moving mice
(326) Supraspinal adenosine A3 receptor (A3AR) activation reverses chemotherapy-induced neuropathic pain through an IL-10 dependent mechanism
C Morgan, V Samineni, S Sundaram, J Golden, G Shin, S Oh, J Rogers, and R Gereau, IV; Washington University School of Medicine, St. Louis, MO
J Little, K Janes, Z Chen, C Wahlman, D Tosh, K Jacobson, and D Salvemini; Saint Louis University School of Medicine, St. Louis, MO
Optogenetics has dramatically expanded the tools available for studying peripheral and central circuitry involved in pain. In order to realize the full potential of optogenetics for the study of pain, devices are needed which allow integration of miniaturized light sources that are minimally invasive, provide sufficient light, and allow chronic, free movement of rodents during the study of spontaneous and evoked pain behaviors. Here, we designed a highly flexible, wirelessly-powered, micro-LED spinal implant which allows optogenetic stimulation and inhibition of neurons in the spinal cord during free movement over a relatively large area. We characterized two transgenic mouse lines where light-sensitive ChR2-eYFP or Arch-eGFP is specifically expressed exclusively in Nav1.8+ sensory neurons. Implantation of the optogenetic spinal stimulator produces no gross motor or sensory deficits. Optogenetic spinal activation of ChR2 in these mice produce robust, reproducible, frequencydependent, spontaneous pain behaviors which are not present in control mice. Further studies utilizing this novel device will allow for specific, bi-directional, and wireless targeting of discrete spinal nociceptive populations.
We have recently reported that activation of the adenosine subtype 3 receptor (A3AR) potently attenuates evoked and non-evoked pain behaviors associated with preclinical models of nerve injury- and chemotherapy-induced neuropathies. In nerve injury models, we have demonstrated that these antinociceptive effects are a result of modulation of spinal nociceptive processing, in part, through the induction of descending inhibitory bulbospinal pathways from the rostral ventromedial medulla (RVM). The role of the RVM in chemotherapy-induced neuropathic pain and the mechanisms by which these effects occur are unknown. As A3AR activation attenuates inflammation via the release of anti-inflammatory mediators such as interleukin (IL)-10 and RVM neuroinflammation is associated with nerve injury- and chemotherapyinduced behavioral responses (e.g. allodynia and hyperalgesia), here we examined if A3AR activation in the RVM during chemotherapy (oxaliplatin)-induced neuropathic pain is antinociceptive through an IL-10 dependent mechanism. Systemic injection of MRS5698 (1 mg/kg), a recently characterized selective A3AR agonist, reversed established mechanical allodynia and hyperalgesia (n=4 per group). These effects were blocked by intra-RVM delivery of a selective A3AR antagonist, MRS1523 (1 nmol), but not its vehicle, suggesting supraspinal mechanisms are critical for the actions of MRS5698. Further examination demonstrated that A3AR activation following intra-RVM injection of MRS5698 (3 nmol; n=4 per group) reversed mechanical allodynia and hyperalgesia; this effect was blocked by RVM injection of an anti-IL10 antibody (0.2 mg). There were no observed effects in oxaliplatin control animals following administration of MRS5698, MRS1523, or anti-IL10. Collectively, these experiments suggest that the RVM contributes to the protective effects of A3AR activation through the IL-10 pathway during chemotherapy-induced neuropathic pain. This research was supported by NIH/NCI 1RO1CA169519-01, the Saint Louis University President Research Fund, and the Saint Louis Cancer Center.
(325) DREADD activation of dopaminergic and glutamatergic neurons in the periaquaductal gray produces differing analgesic responses
(327) Preemptive local bupivacaine attenuates mechanical and cold allodynia in a rat model of neuropathic pain
N Taylor, J Pei, C Van Dort, E Brown, and K Solt; Massachusetts General Hospital, Boston, MA Despite years of study, current treatments do not sufficiently treat chronic pain syndromes. Electrical brain stimulation of the periaqueductal gray (PAG) produces profound antinociception, and shows promise in treating these difficult conditions. However, this treatment can also produce intense anxiety which has limited its use. The goal of this study was to better define the complex neural circuitry in the PAG specifically responsible for the analgesic effect and hypothesized that in targeting specific neural circuits we would be able to produce analgesia without anxiety. DREADDs (Designer Receptors Exclusively Activated by Designer Drugs) are G-protein coupled receptors engineered to be selectively activated by the ligand Clozapine N-Oxide (CNO). We targeted DREADDs to dopamine, GABA and glutamate neurons within the ventral lateral PAG (vlPAG) using viral-mediated delivery into different Cre-mice lines. Anesthetized dopamine transporter promoter (DAT-cre) and vesicular glutamate tranporter 2 (Vglut-2-cre) mice received targeted injections of adeno-associated virus carrying excitatory DREADDs (AAV5-hM3Dq), while vesicular GABA transporter (Vgat-ires-cre) mice were injected with an inhibitory DREADDs construct (AAV8-hM4Di). After a 3 week recovery, analgesia was assessed using the Hargreaves method before and after i.p. CNO injection. Viral expression and localization were confirmed using immunohistochemistry upon completion of the study. At baseline, paw withdrawal occurred at 2.7060.27s in DAT-cre mice (n=3), 2.6260.10s in Vgat-cre mice (n=6) and 2.9360.10s in Vglut-2-cre mice (n=6). CNO was injected ip and paw withdrawal times were measured hourly for 6 hours. No statistically significant difference in withdrawal times were seen in GABA targeted animals. Significant analgesia was accompanied with anxiety behaviors in glutamate targeted mice, while dopamine targeted animals showed only an analgesic response. In summary, only activation of dopamine neurons in the vlPAG produced profound analgesia without signs of anxiety. vlPAG dopamine neurons represent a novel target for producing analgesia that may lead to new treatments for pain.
J Clifford, A Mares, J Hansen, and D Averitt; US Army Institute of Surgical Research, Fort Sam Houston, TX Neuropathic pain is evasive to treat once developed, however evidence suggests that local administration of anesthetics near the time of injury reduces the development of neuropathic pain. As abnormal electrical signaling in the damaged nerve contributes to the initiation and maintenance of neuropathic pain, local administration of anesthetics prior to injury may prevent neuropathic pain. We hypothesized that local preemptive treatment with bupivacaine prior to nerve injury in a rat model of spinal nerve ligation (SNL) would reduce the initiation and/or maintenance of neuropathic pain behaviors. On the day prior to SNL, baseline measures of thermal hyperalgesia, mechanical allodynia and acetone-induced cold alloynia were recorded in male SpragueDawley rats. During surgery immediately prior to ligation, the nerve was perineurally bathed in either bupivacaine (0.4% bupivacaine in 0.2 mL) or sterile saline (0.2 mL) for 30 minutes. The nerve was then ligated, the wound sutured and the rats were allowed to recover for 3 days prior to behavioral testing. A separate group of rats received the same surgical manipulations without ligation (sham controls). All rats were then examined for the development of thermal hyperalgesia, mechanical allodynia, and cold allodynia on 3, 7, 10, 14 and 21 days following surgery. All ligated rats exhibited both mechanical and cold allodynia, but not thermal hyperalgesia, within 3 days post-SNL and lasting for 21 days. No pain behaviors were observed in sham controls. While there was no effect of bupivacaine on mechanical allodynia 3 days post-SNL, mechanical allodynia was significantly diminished by day 7 and similar to sham controls by 2 weeks post-SNL. Bupivacaine significantly attenuated cold allodynia at all time points measured. These data indicate that local treatment with bupivacaine prior to surgical manipulations that are known to cause nerve damage, such as amputation, may protect against the initiation and maintenance of neuropathic pain.