Decrease In Paw Withdrawal Latency Research Articles

Electroacupuncture Alleviates Diabetic Neuropathic Pain and Downregulates p-PKC and TRPV1 in Dorsal Root Ganglions and Spinal Cord Dorsal Horn.

Diabetic neuropathic pain (DNP) is a common complication of diabetes. Streptozotocin (STZ)-induced changes of protein in dorsal root ganglion (DRG) and spinal cord dorsal horn (SCDH) are critical for DNP genesis. However, which proteins change remains elusive. Here, the DNP model was established by a single intraperitoneal injection of STZ, accompanied by increased fasting blood glucose (FBG), decreased body weight (BW), and decreased paw withdrawal latency (PWL). Proteins change in L4-L6 DRGs and SCDH of rats were detected. Western blot and immunofluorescence results showed that expression levels of phosphorylated protein kinase C (p-PKC), transient receptor potential vanilloid-1 (TRPV1), Substance P (SP) and calcitonin gene-related peptide (CGRP) in the DRG and the SCDH of rats were increased after STZ injection. A preliminary study from our previous study showed that 2 Hz electroacupuncture (EA) effectively alleviates DNP. However, the analgesic mechanism of EA needs further elucidation. Here, EA at the bilateral Zusanli (ST36) and KunLun (BL60) acupoints was applied for one week, and to investigate the effect on DNP. EA reversed thermal hyperalgesia in DNP rats and downregulated the expression of p-PKC, TRPV1, SP, and CGRP in DRG and SCDH.

Effect of apple peel extract on diabetes-induced peripheral neuropathy and wound injury.

Diabetic peripheral neuropathy (DPN) affects up to 50 % diabetic patients. Moreover, uncontrolled diabetes associated with impaired wound healing. The present study was aimed at exploring the effect of apple peel extract (APE) on type 2 diabetes (T2D)-induced DPN and delayed wound healing. In adult male Sprague-Dawley rats on high-fat diet, a single low dose streptozotocin (STZ, 35mg/kg) was administeredvia intraperitoneal route to induce T2D. Plantar test using Hargreaves apparatus was used to evaluate the DPN. Six different groups of rats were treated orally with saline (naïve control and DPN control), APE (100, 200 and 400mg/kg) and gabapentin (30mg/kg) daily for 7 consecutive days and thermal paw withdrawal latency (PWL) was measured. To elucidate the underlying antioxidant effect of APE, the catalase (CAT), glutathione (GSH) and malonaldehyde (MDA) levels were measured. To evaluate the wound healing potential of APE, excision ischemic open wound model was used. Six different groups of rats were applied with 2 % gum acacia (naïve control and diabetic control), 1 % silver sulfadiazine (SSD) cream and APE cream (5, 10 and 20 %)twice daily for 28 days. Dry connective tissue parameters like hydroxyproline and hexosamine were also measured to further confirm the wound healing activity. Diabetes produced thermal hyperalgesia in rats with a significant decrease in PWL as compared to naive controls indicating induction of DPN. APE and gabapentin significantly improved PWL in diabetic animals. Biochemical analysis revealed a significant improvement in oxidative stress parameters such as catalase, GSH and MDA. Wound closure was significantly more after day 15 of topical application of APE and SSD as compared to control group. APE significantly increased hydroxyproline and hexosamine levels as compared to standard cream. Moreover, histopathology revealed that, topical application of APE cream showed an enhanced healing process. On the basis of the findings, we conclude that APE has a potential to be used as a therapeutic intervention for the management of DPN and delayed wound healing in the diabetic condition.

Ferula asafoetida Linn. is effective for early functional recovery following mechanically induced insult to the sciatic nerve of a mouse model

Purpose: To evaluate the effect of Ferula asafoetida (oleo gum resin powder) on sensory and motor functions retrieval on an induced sciatic nerve injury in a mouse model.Methods: A mechanical crush was inserted in the sciatic nerve of all the experimental mice after acclimatization. The mice were allocated to four groups; one normal chow group (control, n = 7) and three Ferula asafoetida chow groups (each n = 7) of different doses (50, 100 and 200 mg/kg). Muscle grip strength, muscle mass, and sciatic functional index were measured to evaluate the motor function regain, while sensory function regain was assessed by hot plate test. Oxidative stress and glycemic levels were measured by biochemical assays.Results: The findings of this study indicate that Ferula asafoetida 200 mg/kg has a highly significant (p≤ 0.001) ameliorating effect in terms of improved grip strength (77.7 ± 5.4 % for 200 mg/kg vs. 46 ± 5.1 % for control), reversal of SFI towards normal ( -34 ± 8.1 for 200 mg/kg group vs. –61 ± 6.1 for control), decrease in paw withdrawal latency (7.10 ± 0.06 s for 200 mg/kg group vs. 15 ± 0.5 s for control) on day 12 post-injury, as well as restoration of skeletal muscle mass towards normal. Interestingly, F. asafoetida chow 50 mg/kg and 100 mg/kg groups also impacted significant (p < 0.01) improvement in the ameliorative effect. However, the differences among all treatment groups in ameliorating recovery were not significant (p > 0.05). Moreover, comparatively improved (p < 0.0001) total antioxidant capacity along with reduced total oxidant status (p = 0.01) in the Ferula asafoetida chow (200 mg/kg) group, indicate the antioxidative effect of this plant. Furthermore, the treated mice (200 mg/kg) also expressedan improved glycemic level (p = 0.0005).Conclusion: Ferula asafoetida supplementation helps to accelerate both sensory and motor function retrieval following sciatic nerve injury. This improvement is thought to be correlated with the antioxidant capacity of the plant. However, further investigations are required to identify the therapeutic principles responsible for the observed actions.
 Keywords: Sciatic nerve injury, Ferula asafoetida, Function recovery, Oxidative stress, Biochemical analysis

MicroRNA-146a attenuates the development of morphine analgesic tolerance in a rat model

ABSTRACT Background: Morphine plays an irreplaceable role in relieving severe pain clinically, while long-term medication inevitably leads to drug resistance. MicroRNA (miR) 146a has been reported to be a negative regulator in the process of morphine-tolerance formation. This study aimed to investigate how miR-146a affects the development of morphine analgesic tolerance. Methods: The morphine-tolerance rat model was established by means of one-week continuous morphine administration. Paw withdrawal latency test was performed every day, and spinal cord samples were dissected on the seventh day for Q-PCR and Western blotting to detect the expression level of miR-146a, and IRAK1/TRAF6 participated in TLR4 signaling pathway. Results: The expression of miR-146 was significantly decreased in morphine-tolerant model. Also, overexpression of miR-146a reduced the resistance caused by morphine, followed by the down-regulation of IRAK1/TRAF6 in TLR4 pathway. The inhibition of miR-146a remarkably decreased paw withdrawal latency as well as increased the expression levels of TLR4 signaling pathway-related molecules, IRAK1 and TRAF6. Conclusion: This study suggests that miR-146a attenuates morphine tolerance by inhibiting the expression of IRAK1/TRAF6 in TLR4 pathway, which could provide an essential experimental basis for the settlement of morphine resistance-associated matters.

6-Methoxyflavanone abates cisplatin-induced neuropathic pain apropos anti-inflammatory mechanisms: A behavioral and molecular simulation study

Cisplatin is used as a first line therapy in treating cancers. However, its use is often accompanied with the development of peripheral neuropathy. 6-Methoxyflavanone (6-MeOF) is a positive allosteric modulator at GABAA receptors and is known for attenuating diabetes-induced neuropathic pain. Neuropathy was induced in male Sprague-Dawley rats (150–250 g), via intraperitoneal injection of cisplatin (3 mg/kg) once a week for four consecutive weeks. 6-MeOF (25, 50 and 75 mg/kg, i.p) and gabapentin (75 mg/kg, i.p) were administered 30 min before each cisplatin injection. Static and dynamic allodynia were assessed using von Frey filaments and cotton buds. The anti-inflammatory activity was analyzed with plethysmometer. Body weights were also measured each week. The binding affinity of 6-MeOF with chloride channel, Cyclooxygenase-1 (COX-1) and Cyclooxygenase-2 (COX-2) was studied using docking approach. The in vitro COX-1 and COX-2 inhibitory effect of 6-MeOF was conducted with COX colorimetric assay. Administration of cisplatin for four consecutive weeks induced static (decreased paw withdrawal threshold; PWT) and dynamic allodynia (decreased paw withdrawal latency; PWL). Co-administration of 6-MeOF for four weeks significantly attenuated the cisplatin-induced expression of nocifensive behaviors observed as significant increase in PWT and PWL. Moreover, it also prevented the body weight loss induced by cisplatin administration. In silico studies depicted a good interaction of 6-MeOF with chloride ion channels and COX-1 and COX-2 enzymes. The in vitro study confirmed the inhibitory activity of 6-MeOF for COX-1 and COX-2. 6-MeOF may be effective in attenuating cisplatin-induced allodynia, probably through interaction with GABAergic receptors and reducing inflammation.

Dermorphin [D-Arg2, Lys4] (1-4) amide inhibits below-level heat hypersensitivity in mice after contusive thoracic spinal cord injury.

Opioid use for chronic pain is limited by severe central adverse effects. We examined whether activating mu-opioid receptors (MORs) in the peripheral nervous system attenuates spinal cord injury (SCI) pain-like behavior in mice. We produced a contusive SCI at the T10 vertebral level and examined motor and sensory dysfunction for 6 weeks. At 6 weeks, we tested the effect of subcutaneous (s.c.) injection of dermorphin [D-Arg2, Lys4] (1-4) amide (DALDA), a peripherally acting MOR-preferring agonist, on mechanical and heat hypersensitivity. Basso mouse scale score was significantly decreased after SCI, and mice showed hypersensitivity to mechanical and heat stimulation at the hind paw beginning at 2 weeks, as indicated by increased paw withdrawal frequency to mechanical stimulation and decreased paw withdrawal latency to heat stimulation. In wild-type SCI mice, DALDA (1 mg/kg, s.c.) attenuated heat but not mechanical hypersensitivity. The effect was blocked by pretreatment with an intraperitoneal injection of methylnaltrexone (5 mg/kg), a peripherally restricted opioid receptor antagonist, and was also diminished in Pirt-MOR conditional knockout mice. DALDA did not adversely affect exploratory activity or induced preference to drug treatment in SCI mice. In vivo calcium imaging showed that DALDA (1, 10 mg/kg, s.c.) inhibited responses of small dorsal root ganglion neurons to noxious heat stimulation in Pirt-GCaMP6s mice after SCI. Western blot analysis showed upregulation of MOR in the lumbar spinal cord and sciatic nerves at 6 weeks after SCI. Our findings suggest that peripherally acting MOR agonist may inhibit heat hypersensitivity below the injury level with minimal adverse effects.

Comparisons of motor and sensory abnormalities after lumbar and thoracic contusion spinal cord injury in male rats

Rodent models of contusion spinal cord injury (SCI) are widely studied for the mechanisms underlying functional deficits after SCI. Yet, how does lesion level affect SCI-induced motor and sensory dysfunctions remains unclear. Using a computer-controlled impactor (Impact One™, Leica) and the same parameters (diameter, 2.0 mm; Speed: 4.0 m/s; Depth: 1.5 mm; Dwell time: 0.1 s), we produced contusions at mid-thoracic (T10) and rostral-lumbar (L2) spinal cord in male rats, and compared locomotor and sensory dysfunctions within the same experimental setting. The time courses of locomotor deficit were comparable between thoracic (n = 8) and lumbar (n = 7) SCI rats, but the severity was greater after thoracic SCI especially during the first week post-injury, as indicated by the lower Basso, Beattle and Bresnahan open-field locomotion scores. Both groups showed similar heightened avoiding response (hyper-reactivity) to mechanical stimulation applied at the hindpaws from day 21–56 post-injury, as indicated by decreased paw withdrawal thresholds. Compared to lumbar SCI, thoracic SCI induced a greater decrease of paw withdrawal latency in hot-plate test from day 28–56 post-injury. In contrast, lumbar SCI rats showed a greater reduction of avoidance threshold to mechanical stimulation at the girdle region, and larger overgroomed area than thoracic SCI rats at day 14 post-injury. Thus, thoracic SCI may induce greater motor deficits and hindpaw heat hyper-reactivity than did lumbar SCI. In contrast, lumbar SCI may elicit greater at-level mechanical hyper-reactivity and overgrooming behavior than thoracic SCI. Future study needs to examine the specific pathological changes underlying different dysfunctions in two SCI models.

Music-Enhanced Analgesia and Antiseizure Activities in Animal Models of Pain and Epilepsy: Toward Preclinical Studies Supporting Development of Digital Therapeutics and Their Combinations With Pharmaceutical Drugs.

Digital therapeutics (software as a medical device) and mobile health (mHealth) technologies offer a means to deliver behavioral, psychosocial, disease self-management and music-based interventions to improve therapy outcomes for chronic diseases, including pain and epilepsy. To explore new translational opportunities in developing digital therapeutics for neurological disorders, and their integration with pharmacotherapies, we examined analgesic and antiseizure effects of specific musical compositions in mouse models of pain and epilepsy. The music playlist was created based on the modular progression of Mozart compositions for which reduction of seizures and epileptiform discharges were previously reported in people with epilepsy. Our results indicated that music-treated mice exhibited significant analgesia and reduction of paw edema in the carrageenan model of inflammatory pain. Among analgesic drugs tested (ibuprofen, cannabidiol (CBD), levetiracetam, and the galanin analog NAX 5055), music intervention significantly decreased paw withdrawal latency difference in ibuprofen-treated mice and reduced paw edema in combination with CBD or NAX 5055. To the best of our knowledge, this is the first animal study on music-enhanced antinociceptive activity of analgesic drugs. In the plantar incision model of surgical pain, music-pretreated mice had significant reduction of mechanical allodynia. In the corneal kindling model of epilepsy, the cumulative seizure burden following kindling acquisition was lower in animals exposed to music. The music-treated group also exhibited significantly improved survival, warranting further research on music interventions for preventing Sudden Unexpected Death in Epilepsy (SUDEP). We propose a working model of how musical elements such as rhythm, sequences, phrases and punctuation found in K.448 and K.545 may exert responses via parasympathetic nervous system and the hypothalamic-pituitary-adrenal (HPA) axis. Based on our findings, we discuss: (1) how enriched environment (EE) can serve as a preclinical surrogate for testing combinations of non-pharmacological modalities and drugs for the treatment of pain and other chronic diseases, and (2) a new paradigm for preclinical and clinical development of therapies leading to drug-device combination products for neurological disorders, depression and cancer. In summary, our present results encourage translational research on integrating non-pharmacological and pharmacological interventions for pain and epilepsy using digital therapeutics.

Prostaglandin E2 Glyceryl Ester Contributes to Hyperalgesia in a Humanized Model Pf Sickle Cell Disease through P2Y6 Receptors

Sickle Cell Disease (SCD) is the most common monogenic red blood cell disorder characterized by a remarkable phenotypic complexity. Common complications of SCD are unpredictable episodic and chronic pain. Transgenic homozygous HbSS-BERK mice expressing >99% human sickle hemoglobin and exhibiting both pain and neurochemical changes similar to humans, and HbAA control mice expressing normal human hemoglobin, were used to determine the contribution of the prostaglandin E2 glyceryl ester (PGE2-G), to mechanical and heat hyperalgesia and the mechanism of its pro-nociceptive effect. Mechanical hyperalgesia was defined by a decrease in paw withdrawal threshold, and heat hyperalgesia was defined by a decrease in paw withdrawal latency to radiant heat applied to the plantar hind paw. Considering that prostaglandin E2-G (PGE2-G) is the product of oxidation of 2-arachidonylglycerol (2-AG) by cycloxygenase-2 (COX-2), the expression of COX-2 protein and COX-2 activity was measured in dorsal root ganglia (DRG). The levels of 2-AG and PGE2-G in DRG were determined by HPLC-MS. Mechanical and heat hyperalgesia in HbSS mice were associated with a decreased level of 2-AG and increased level of PGE2-G in DRGs of HbSS mice due to elevated level of COX-2 protein and COX-2 activity. In HbAA control mice, intraplantar (i.pl.) injection of PGE2-G (1 µg/10 µl) produced mechanical hyperalgesia in the injected paw independently from PGE2 receptors. Also, PGE2-G (1 µg/10 µl, i.pl.) produced mechanical and heat hyperalgesia in the injected paw in C3H mice. The hyperalgesia was blocked by co-injection of PGE2-G with MRS 2578, the purinergic P2Y6 receptor antagonist (1.4 mg/10 ml, i.pl.). Importantly, MRS 2578 (4.7 mg/50 ml, i.p.) also attenuated hyperalgesia in HbSS mice. Thus, increased expression of COX-2 in DRG neurons of HbSS mice results in the oxidation of 2-AG and production of PGE2-G, a pronociceptive mediator. PGE2-G produced mechanical and heat hyperalgesia in HbAA and C3H mice, and these effects were mediated by P2Y6 receptors.Acknowledgments: Supported by the National Heart, Lung and Blood Institute (HL135895-16). DisclosuresGupta:Novartis: Honoraria; Tau tona: Consultancy.

ORL1 Activation Mediates a Novel ORL1 Receptor Agonist SCH221510 Analgesia in Neuropathic Pain in Rats.

Opioid receptor like 1 (ORL1) receptor activation displayed an anti-nociceptive effect at spinal level for acute and neuropathic pain. SCH221510, an orally active non-peptide ORL1 agonist, was reported to be effective in treating neuropathic pain. The present study used ORL1 antagonist and siRNA to investigate that ORL1 activation mediates intrathecal SCH221510 analgesia in neuropathic pain induced by chronic constrictive injury (CCI) to rat sciatic nerve. Paw withdrawal latency and 50% mechanical threshold were measured for thermal and mechanical hypersensitivity in rats. CCI significantly decreased paw withdrawal latency and mechanical threshold. SCH221510 (3, 10, 30μg) or ORL1 antagonist ([Nphe1]nociceptin(1-13)NH2, 10μg) was intrathecally injected to test the behavioral effects on neuropathic pain. Intrathecal siRNA was started on 1day before CCI surgery and maintained for 7days. L4-L5 spinal cord ORL1 mRNA and protein were measured by real-time PCR and Western blot. The effect of intrathecal siRNA on SCH2210510 was tested in CCI rats on day 7. Intrathecal SCH221510 dose-dependently reduced thermal and mechanical hypersensitivity induced by CCI. [Nphe1]nociceptin(1-13)NH2 blocked SCH221510 analgesia in CCI rats. Intrathecal siRNA blocked ORL1 mRNA and protein increase induced by CCI. Intrathecal ORL1 siRNA did not change thermal and mechanical hypersensitivity induced by nerve injury. Intrathecal siRNA blocked SCH221510 analgesia in neuropathic pain at spinal level. Conclusively, ORL1 activation mediates SCH221510 analgesia in neuropathic pain at spinal level. The results warrant a potential clinically applicable drug in treating neuropathic pain.

Notoginseng Radix and Rehmanniae Radix Preparata Extract Combination (YH23537) Reduces Pain and Cartilage Degeneration in Rats with Monosodium Iodoacetate-Induced Osteoarthritis

Notoginseng Radix and Rehmanniae Radix Preparata have been widely used traditionally for treating inflammatory diseases. This research studies the therapeutic effects of YH23537, the extracts of Notoginseng Radix and Rehmanniae Radix Preparata, on pain and cartilage degeneration in an experimental osteoarthritis (OA) model. Male Wistar rats were inoculated intra-articularly with 3 mg of monosodium iodoacetate (MIA) in the right intra-articular. Four days later, the animals were administrated orally with YH23537 daily for 24 days. Tactile allodynia and weight bearing were measured. Macroscopic and microscopic observations for articular cartilage were performed at the end of the experiment. Protein expression in the joint was determined by immunohistochemistry. The effects of YH23537 on mRNA levels in chondrocytes stimulated with interleukin (IL)-1β were analyzed using random polymerase chain reaction. OA induction was confirmed by significant decrease of paw withdrawal latency, paw withdrawal threshold, and weight bearing compared with the normal group at 3 days after MIA injection. The YH23537-treated groups displayed significant increases in pain thresholds and weight bearing throughout the observation period. The damage to articular cartilage was significantly lessened visually and histopathologically by YH23537 treatment. YH23537 suppressed the expression of metalloproteinase-3, nitrotyrosine, IL-1β and IL-6 increased in OA joints. YH23537 upregulated tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-3 in IL-1β-stimulated human OA chondrocytes. The protein levels of the NF-κBp65 and HIF-2α in the joint tissues were reduced by YH23537. YH23537 exerted antinociceptive effects and cartilage protective effects in experimental OA rats by suppressing oxidative injury, inflammatory mediators, and inducing anabolic factors. We suggest that YH23537 may have efficacy for treating OA in humans.

Persistent Catechol-O-methyltransferase-dependent Pain Is Initiated by Peripheral β-Adrenergic Receptors.

Patients with chronic pain disorders exhibit increased levels of catecholamines alongside diminished activity of catechol-O-methyltransferase (COMT), an enzyme that metabolizes catecholamines. The authors found that acute pharmacologic inhibition of COMT in rodents produces hypersensitivity to mechanical and thermal stimuli via β-adrenergic receptor (βAR) activation. The contribution of distinct βAR populations to the development of persistent pain linked to abnormalities in catecholamine signaling requires further investigation. Here, the authors sought to determine the contribution of peripheral, spinal, and supraspinal βARs to persistent COMT-dependent pain. They implanted osmotic pumps to deliver the COMT inhibitor OR486 (Tocris, USA) for 2 weeks. Behavioral responses to mechanical and thermal stimuli were evaluated before and every other day after pump implantation. The site of action was evaluated in adrenalectomized rats receiving sustained OR486 or in intact rats receiving sustained βAR antagonists peripherally, spinally, or supraspinally alongside OR486. The authors found that male (N = 6) and female (N = 6) rats receiving sustained OR486 exhibited decreased paw withdrawal thresholds (control 5.74 ± 0.24 vs. OR486 1.54 ± 0.08, mean ± SEM) and increased paw withdrawal frequency to mechanical stimuli (control 4.80 ± 0.22 vs. OR486 8.10 ± 0.13) and decreased paw withdrawal latency to thermal heat (control 9.69 ± 0.23 vs. OR486 5.91 ± 0.11). In contrast, adrenalectomized rats (N = 12) failed to develop OR486-induced hypersensitivity. Furthermore, peripheral (N = 9), but not spinal (N = 4) or supraspinal (N = 4), administration of the nonselective βAR antagonist propranolol, the β2AR antagonist ICI-118,511, or the β3AR antagonist SR59230A blocked the development of OR486-induced hypersensitivity. Peripheral adrenergic input is necessary for the development of persistent COMT-dependent pain, and peripherally-acting βAR antagonists may benefit chronic pain patients.

(312) Peripherally located β2- and β3-adrenergic receptors contribute to the development of COMT-dependent pain in rats, but not the maintenance of pain in COMT-/- mice

Genetic and pharmacologic alterations resulting in reduced catechol-O-methyltransferase (COMT) activity are associated with increased pain sensitivity and onset of chronic pain conditions. COMT-dependent pain is mediated via β2 and β3-adrenergic receptors (ARs), which are located in various regions where they could potentially drive pain transmission. In order to determine the site of action whereby βARs drive COMT-dependent pain, the present study measured evoked pain behaviors in rats receiving βAR antagonists peripherally, spinally or supraspinally alongside administration of COMT inhibitor OR486. Osmotic pumps were utilized to achieve sustained subcutaneous (s.c.), intrathecal and intracerebroventricular delivery of βAR antagonists, and sustained s.c. delivery of OR486, over a 2-week period. Responses to mechanical and thermal stimuli were assessed prior to and following pump implantation. Rats receiving sustained OR486 demonstrated decreased paw withdrawal thresholds and increased paw withdrawal frequency in response to mechanical stimuli and decreased paw withdrawal latency in response to thermal heat. Peripheral, but not spinal or supraspinal, administration of non-selective βAR antagonist propranolol, β2AR antagonist ICI-118,511 or β3AR antagonist SR59230A blocked the development of OR486-induced pain. Next, we examined the ability of peripherally administered β2- and β3AR antagonists to reverse pain in COMT-/- mice. Osmotic pumps were utilized to achieve sustained s.c. delivery of ICI-118,511 together with SR59230A over a 2-week period. Responses to mechanical and thermal stimuli were assessed prior to and following pump implantation. Peripheral delivery of βAR antagonists failed to block endogenous mechanical and thermal pain in COMT-/- mice. In summary, peripherally located β2- and β3-adrenergic receptors contribute to the development of COMT-dependent pain in rats, but not the maintenance of pain in COMT-/- mice. These results suggest that the use of peripherally-acting βAR antagonists may be beneficial in acute clinical and/or surgical settings to prevent the development of chronic pain among susceptible individuals. Funding: NIH/NINDS R01-NS072205 and P01-NS045685.

Neurotropin(®) ameliorates chronic pain via induction of brain-derived neurotrophic factor.

Neurotropin (NTP)(®), a non-protein extract isolated from the inflamed skin of rabbits inoculated with vaccinia virus, is used clinically for the treatment of neuropathic pain. Moreover, NTP may activate the descending pain inhibitory system. Depression-like behavior is often complicated by chronic pain. However, little is known about NTP-mediated prevention of mood disorders in chronic pain and its molecular mechanisms. We aimed to investigate the effects of NTP on brain-derived neurotrophic factor (BDNF)-mediated signaling and gene expression in chronic pain. In addition, these effects of NTP were compared with pregabalin which is an anticonvulsant, anxiolytic analgesic used to treat neuropathic pain and fibromyalgia. A chronic constriction injury model was established in Sprague-Dawley rats. The pain response was assessed using a paw withdrawal latency (PWL) test and depression was assessed by the immobility time in a forced swim test (FST). NTP was orally administered in two doses of 50 NU (Neurotropin Unit) and 100 NU/kg for 7 days from day 7 after injury. To measure the analgesic and anti-depressant effects of NTP, either K252a (a tyrosine kinase inhibitor), or 5,7-dihydroxy tryptamine (5,7-DHT, a selective toxin for 5-HTergic neurons) was administered by intracerebroventricular injection. Changes in pERK1/2 and pCREB (immunohistochemistry), 5-HT, and BDNF protein level (ELISA) and BDNF mRNA (RT-PCR) were measured in the anterior cingulate cortex (ACC) and in the rostral ventromedial medulla (RVM) 14 days after injury. After injury, the rats showed a decrease in PWL associated with the increase in time of immobility in FST. In this injury model, NTP blocked both the decrease in PWL and the increase in the FST, while pregabalin (10 mg/kg, po.) did not affect the increase in the FST. These effects of NTP were reversed by K252a, and 5,7-DHT. The analgesic effects of pregabalin were not reversed by K252a. NTP normalized the injury-induced excessive activation of pERK1/2 associated with decreased pCREB and BDNF mRNA in the ACC and in the RVM, and these changes were reversed by 5,7-DHT. In contrast, pregabalin did not affect either pCREB or BDNF levels in the chronic pain model. NTP ameliorated chronic pain and pain-related depression by normalizing the induction of BDNF associated with the 5-HTergic system. Pregabalin showed the analgesic effects but had no effects on either depression or the BDNF pathway. These results suggest that NTP may represent an additional drug strategy for chronic pain associated with depression.

Dopamine D3 receptor dysfunction prevents anti-nociceptive effects of morphine in the spinal cord

Dopamine (DA) modulates spinal reflexes, including nociceptive reflexes, in part via the D3 receptor subtype. We have previously shown that mice lacking the functional D3 receptor (D3KO) exhibit decreased paw withdrawal latencies from painful thermal stimuli. Altering the DA system in the CNS, including D1 and D3 receptor systems, reduces the ability of opioids to provide analgesia. Here, we tested if the increased pain sensitivity in D3KO might result from a modified μ-opioid receptor (MOR) function at the spinal cord level. As D1 and D3 receptor subtypes have competing cellular effects and can form heterodimers, we tested if the changes in MOR function may be mediated in D3KO through the functionally intact D1 receptor system. We assessed thermal paw withdrawal latencies in D3KO and wild type (WT) mice before and after systemic treatment with morphine, determined MOR and phosphorylated MOR (p-MOR) protein expression levels in lumbar spinal cords, and tested the functional effects of DA and MOR receptor agonists in the isolated spinal cord. In vivo, a single morphine administration (2 mg/kg) increased withdrawal latencies in WT but not D3KO, and these differential effects were mimicked in vitro, where morphine modulated spinal reflex amplitudes (SRAs) in WT but not D3KO. Total MOR protein expression levels were similar between WT and D3KO, but the ratio of pMOR/total MOR was higher in D3KO. Blocking D3 receptors in the isolated WT cord precluded morphine's inhibitory effects observed under control conditions. Lastly, we observed an increase in D1 receptor protein expression in the lumbar spinal cord of D3KO. Our data suggest that the D3 receptor modulates the MOR system in the spinal cord, and that a dysfunction of the D3 receptor can induce a morphine-resistant state. We propose that the D3KO mouse may serve as a model to study the onset of morphine resistance at the spinal cord level, the primary processing site of the nociceptive pathway.

4-Methylcatechol prevents derangements of brain-derived neurotrophic factor and TrkB-related signaling in anterior cingulate cortex in chronic pain with depression-like behavior

Chronic pain with mood disorder, resulting from a peripheral nerve injury, is a serious clinical problem affecting the quality of life. A lack of brain-derived neurotrophic factor (BDNF) and abnormal intercellular signaling in the brain can mediate this symptom. BDNF is induced in cultured neurons by 4-methylcatechol (4-MC), but little is known about its role in pain-emotion. Thus, we characterized the actions of 4-MC on TrkB receptor-related pERK and BDNF mRNA in discreet brain regions related to pain-emotion after chronic pain in rat. Rats implanted with a stainless steel cannula into the lateral ventricular were subjected to chronic constriction injury (CCI). Pain was assessed by changes in paw withdrawal latency (PWL) to heat stimuli after CCI. Immobility time during the forced swimming testing was measured for depression-like behavior. Analgesic and antidepression modulations with 4-MC were examined by an anti-BDNF antibody (K252a, a TrkB receptor inhibitor). The animals were perfused and fixed (4% paraformaldehyde) for immunohistochemistry analysis (c-FOS/pERK). BDNF mRNA expression (anterior cingulate cortex) was determined using reverse transcription-PCR. Rats showed a sustained decrease in PWL, associated with a prolonged immobility time after CCI. 4-MC reduced decreases in PWL and increased immobility time. 4-MC reduced increases in pERK immunoreactivity and decreases in BDNF mRNA expression in regions related to pain and the limbic system. Anti-BDNF blocked effects induced by 4-MC. We suggest that a lack of BDNF associated with activated extracellular signal-regulated kinase in the pain-emotion network may be involved in depression-like behavior during chronic pain. 4-MC ameliorates pain-emotion symptoms by inducing BDNF and normalizing pERK activities.

DAMGO in the central amygdala alleviates the affective dimension of pain in a rat model of inflammatory hyperalgesia

Pain has sensory-discriminative and emotional-affective dimensions. Recent studies show that the affective component can be assessed with a conditioned place avoidance (CPA) test. We hypothesized that systemic morphine before a post-conditioning test would more potently attenuate the affective aspect compared to the sensory component and that [d-Ala2-N-Me-Phe4, Gly-ol5]-enkephalin (DAMGO), a μ-selective opioid receptor agonist, injected into the central nucleus of the amygdala (CeA) would reduce established CPA. A rat model of inflammatory pain, produced by a complete Freund adjuvant (CFA) injection into the hind paw, was combined with a CPA test. Three experiments were performed on adult male Sprague–Dawley rats. Systemic morphine (0.5 or 1.0mg/kg) in Experiment 1, intrathecal (i.t.) morphine (2.5μg/rat) in Experiment 2, and intra-CeA DAMGO (7.7–15.4ng/0.4μl) in Experiment 3 were given to CFA-injected rats (n=6–8/group) prior to a post-conditioning test. Saline-injected rats were used as control. Time spent in a pain-paired compartment was recorded twice, before conditioning and after a post-conditioning test. Paw withdrawal latency (PWL) to a noxious thermal stimulus was measured before experiment at day-1 and after the post-conditioning test; hyperalgesia was defined as a decrease in PWL. The data showed that CFA-injected rats had significantly negative CPA compared to those of saline-injected rats (P<0.05). Low-dosage systemic morphine significantly (P<0.05) reduced CFA-induced CPA but had no effect on PWL. I.t. morphine did not inhibit the display of CPA but significantly increased PWL, suppressing hyperalgesia (P<0.05). Intra-CeA DAMGO significantly inhibited the display of CPA compared to saline (P<0.05) but had no effect on PWL. The data demonstrate that morphine attenuates the affective component more powerfully than it does the sensory and suggests that the sensory and the emotional-affective dimensions are underpinned by different mechanisms.

Electroacupuncture Inhibits Spinal Interleukin-17A to Alleviate Inflammatory Pain in a Rat Model

Although acupuncture analgesia has been reported in clinical trials, its mechanisms have been unclear. It was recently reported that spinal astrocytes-produced interleukin-17A (IL-17A) facilitates inflammatory pain. Hypothesizing that electroacupuncture (EA) would suppress inflammation-enhanced IL-17A synthesis to inhibit pain, we induced hyperalgesia, as measured by decreased paw withdrawal latency (PWL) to a noxious thermal stimulus, by subcutaneously injecting complete Freund's adjuvant (CFA, 0.08 ml, 40 μg Mycobacterium tuberculosis) into the hind paws of rats, or intrathecal (i.t.) IL-17A (400 ng in 10 μl) into the lumbar spinal cord. We then gave EA at acupoint GB30 for two 20-min periods, once immediately after CFA or IL-17A administration and again 2 h post-injection. For sham control, EA needles were inserted into GB30 without stimulation. PWL was measured before and 2.5 and 24 h after injection. Spinal IL-17A, IL-17 receptor A (IL-17RA), and phosphorylated NR1, an essential subunit of the N-methyl D-aspartate receptor (NMDAR), were determined 24 h post-CFA or –IL-17A using immunohistochemistry and western blot. Compared to sham control, EA inhibited CFA-caused thermal hyperalgesia 2.5 and 24 h post-CFA and concurrently suppressed inflammation-enhanced IL-17A and IL-17RA synthesis and NR1 phosphorylation in the ipsilateral spinal cord. EA inhibited IL-17A-produced thermal hyperalgesia, IL-17RA synthesis and NR1 phosphorylation. Our data suggest that EA inhibits inflammatory pain by blocking spinal IL-17A synthesis. Since previous study shows that IL-17A is located in astrocytes and IL-17RA and NR1 are in neurons, the data suggest that EA alleviates pain by modulating glia-neuronal interactions that involve IL-17A, IL-17RA, and NR1 phosphorylation.

Curcumin Attenuates Diabetic Neuropathic Pain by Downregulating TNF-α in a Rat Model

The mechanisms involved in diabetic neuropathic pain are complex and involve peripheral and central pathophysiological phenomena. Proinflammatory tumour necrosis factor α (TNF-α) and TNF-α receptor 1, which are markers of inflammation, contribute to neuropathic pain. The purpose of this experimental study was to evaluate the effect of curcumin on diabetic pain in rats. We tested 24 rats with diabetes induced by a single intraperitoneal injection of streptozotocin and 24 healthy control rats. Twelve rats in each group received 60 mg/kg oral curcumin daily for 28 days, and the other 12 received vehicle. On days 7, 14, 21, and 28, we tested mechanical allodynia with von Frey hairs and thermal hyperalgesia with radiant heat. Markers of inflammation in the spinal cord dorsal horn on day 28 were estimated with a commercial assay and Western blot analysis. Compared to control rats, diabetic rats exhibited increased mean plasma glucose concentration, decreased mean body weight, and significant pain hypersensitivity, as evidenced by decreased paw withdrawal threshold to von Frey hairs and decreased paw withdrawal latency to heat. Curcumin significantly attenuated the diabetes-induced allodynia and hyperalgesia and reduced the expression of both TNF-α and TNF-α receptor 1. Curcumin seems to relieve diabetic hyperalgesia, possibly through an inhibitory action on TNF-α and TNF-α receptor 1.

Intracerebroventricular 4-Methylcatechol (4-MC) Ameliorates Chronic Pain Associated with Depression-Like Behavior via Induction of Brain-Derived Neurotrophic Factor (BDNF)

Neuropathic pain concurrent with mood disorder from peripheral nerve injury is a serious clinical problem that significantly affects quality of life. Recent studies have suggested that a lack of brain-derived neurotrophic factor (BDNF) in the limbic system may cause this pain-emotion. BDNF is induced in cultured neurons by 4-methylcatechol (4-MC), but the role of 4-MC-induced BDNF in pain-emotion is poorly understood. Thus, we assessed the possible involvement of BDNF in brain in depression-like behavior during chronic pain following peripheral nerve injury. In addition, we examined whether intracerebroventricular (i.c.v.) 4-MC prevents chronic pain in rats and produces an antidepressant effect. Sprague-Dawley rats implanted intracerebroventricularly with a PE-10 tube were subjected to chronic constriction injury (CCI). Pain was assessed by a reduction in paw withdrawal latency (PWL) to heat stimuli after CCI. We also used a forced swimming testing (FST; time of immobility, in seconds) from day 14 to day 21 after CCI. Modulation of pain and emotional behavior was performed by injection of PD0325901 (a MEK1/2 inhibitor). 4-MC (100 nM) was continuously administered i.c.v. for 3 days during the period from day 14 to day 21 after CCI. To block analgesic and antidepressant effects, anti-BDNF antibody or K252a (a TrkB receptor inhibitor) was injected in combination with 4-MC. Naloxone was also coadministered to confirm the analgesic effect of 4-MC. During the chronic stage after CCI, the rats showed a sustained decrease in PWL (thermal hyperalgesia) associated with extension of the time of immobility (depression-like behavior). PD0325901 significantly reduced the decrease in PWL and the increased time of immobility after CCI. The decreased PWL and increased time of immobility were also reduced by 4-MC and by treatment with an ERK1/2 inhibitor. These effects of 4-MC i.c.v. were reversed by anti-BDNF and K252a. The analgesic effect of 4-MC i.c.v. was also antagonized by naloxone. Based on these results, we suggest that a lack of BDNF and activation of ERK1/2 in the pain-emotion network in the CNS may be involved in depression-like behavior during chronic pain. 4-MC i.c.v. ameliorates chronic pain and depression-like behavior by producing of BDNF and normalization of ERK1/2 activation. Therefore, enhancement of BDNF may be a new treatment strategy for chronic pain associated with depression.