Upregulation of neuronal kynurenine 3-monooxygenase mediates depression-like behavior in a mouse model of neuropathic pain.

Pain and depression often co-occur, but the underlying mechanisms have not been elucidated. Here, we used the spared nerve injury (SNI) model in mice to induce both neuropathic pain and depression-like behavior. We investigated whether brain interleukin (IL)-1 signaling and activity of kynurenine 3-monoxygenase (KMO), a key enzyme for metabolism of kynurenine into the neurotoxic NMDA receptor agonist quinolinic acid, are necessary for comorbid neuropathic pain and depression-like behavior. SNI mice showed increased expression levels of Il1b and Kmo mRNA in the contralateral side of the brain. The SNI-induced increase of Kmo mRNA was associated with increased KMO protein and elevated quinolinic acid and reduced kynurenic acid in the contralateral hippocampus. The increase in KMO-protein in response to SNI mostly took place in hippocampal NeuN-positive neurons rather than microglia. Inhibition of brain IL-1 signaling by intracerebroventricular administration of IL-1 receptor antagonist after SNI prevented the increase in Kmo mRNA and depression-like behavior measured by forced swim test. However, inhibition of brain IL-1 signaling has no effect on mechanical allodynia. In addition, intracerebroventricular administration of the KMO inhibitor Ro 61-8048 abrogated depression-like behavior without affecting mechanical allodynia after SNI. We show for the first time that the development of depression-like behavior in the SNI model requires brain IL-1 signaling and activation of neuronal KMO, while pain is independent of this pathway. Inhibition of KMO may represent a promising target for treating depression.

Combination strategy of PARP inhibitor with antioxidant prevent bioenergetic deficits and inflammatory changes in CCI-induced neuropathy.

Neuropathic pain, a debilitating pain condition and the underlying pathogenic mechanisms are complex and interwoven amongst each other and still there is scant information available regarding therapies which promise to treat the condition. Evidence indicate that oxidative/nitrosative stress induced poly (ADP-ribose) polymerase (PARP) overactivation initiate neuroinflammation and bioenergetic crisis culminating into neurodegenerative changes following nerve injury. Hence, we investigated the therapeutic effect of combining an antioxidant, quercetin and a PARP inhibitor, 4-amino 1, 8-naphthalimide (4-ANI) on the hallmark deficits induced by chronic constriction injury (CCI) of sciatic nerve in rats. Quercetin (25 mg/kg, p.o.) and 4-ANI (3 mg/kg, p.o.) were administered either alone or in combination for 14 days to examine sciatic functional index, allodynia and hyperalgesia using walking track analysis, Von Frey, acetone spray and hot plate tests respectively. Malondialdehyde, nitrite and glutathione levels were estimated to detect oxidative/nitrosative stress; mitochondrial membrane potential and cytochrome c oxidase activity to assess mitochondrial function; NAD & ATP levels to examine the bioenergetic status and levels of inflammatory markers were evaluated in ipsilateral sciatic nerve. Quercetin and 4-ANI alone improved the pain behaviour and biochemical alterations but the combination therapy demonstrated an appreciable reversal of CCI-induced changes. Nitrotyrosine and Poly ADP-Ribose (PAR) immunopositivity was decreased and nuclear factor erythroid 2-related factor (Nrf-2) levels were increased significantly in micro-sections of the sciatic nerve and dorsal root ganglion (DRG) of treatment group. These results suggest that simultaneous inhibition of oxidative stress-PARP activation cascade may potentially be useful strategies for management of trauma induced neuropathic pain.

Effects of electroacupuncture intervention on expression of cyclooxygenase 2 and microglia in spinal cord in rat model of neuropathic pain.

OBJECTIVE: To investigate the effect of electroacupuncture (EA) treatment on the expression of cyclooxygenase (COX) 2 and microglia in spinal cord by using rat model of neuropathic pain, and to probe into the relationship between COX 2 and microglia. METHODS: The rats were randomly divided into 6 groups, including normal control group, model group, sham group, EA 1 group (distant acupoints + local acupoints), EA 2 group (local acupoints), and EA 3 group (distant acupoints). Thermal withdrawal latencies were evaluated at 1 day preoperatively and 3, 5 and 7 days postoperatively. At 7 days postoperatively, the spinal COX 2 mRNA was detected by reverse-transcription polymerase chain reaction. Double immunofluorescent staining technology was applied to screen and verify the relationship between altered COX 2 and microglia. RESULTS: Compared with the model group, thermal withdrawal latencies increased after EA treatment (P<0.01). The expressions of COX 2 mRNA were up-regulated in spinal cord of rat on day 7 after surgery (P<0.05). Compared with the model group, EA stimulation (EA 1 and EA 2 groups) reversed the up-regulation of COX 2 mRNA expression (P<0.05). EA 1 and EA 2 groups might have better treatment effect compared with the EA 3 group. Fluorescent images displayed COX 2 and microglia expressed at common areas. CONCLUSIONS: EA was effective in analgesic and anti-inflammatory. EA has decreased the expression of spinal COX 2 mRNA in the trend of the therapeutic effect of "distant acupoints + local acupoints", and "local acupoints" intervention may be superior to that of "distant acupoints" intervention. Microglia may be related to the formation of COX 2.

Activation of hippocampal MEK1 contributes to the cumulative antinociceptive effect of electroacupuncture in neuropathic pain rats.

BACKGROUND: Electroacupuncture (EA) intervention can relieve a variety of pain; however, optimal EA protocols have not been clearly determined. In addition, although central mitogen-activated protein kinase kinase (MEK) signaling has been shown to be involved in the antinociceptive effect of acupuncture stimulation, its characteristics at different time-points of EA intervention have not been fully elucidated. Therefore, the present study investigated the relationship between the effects of different numbers of EA intervention sessions and the activation of MEK1 in the hippocampus and hypothalamus in a rat model of neuropathic pain. METHODS: After ligation of the left sciatic nerve, which induces chronic constriction injury (CCI), the acupoints Zusanli (ST36) and Yanglingquan (GB34) were applied. The thermal withdrawal latency of the hind paw was used to evaluate the effect of EA on pain thresholds. Intra-hippocampus microinjection of PD98059, a MEK inhibitor, was performed to validate the involvement of MEK in EA analgesia. The hippocampus and hypothalamus were harvested to examine the phosphorylation levels of MEK (pMEK) by western blotting. RESULTS: In CCI rats, the thermal pain threshold of the affected hind paw decreased significantly relative to the control. Following subsequent daily EA interventions, CCI-induced ipsilateral hyperalgesia was markedly improved from day 4 and the analgesic effect of EA lasted 3 days after cessation of EA. Four sessions of EA markedly suppressed CCI-induced decrease of hippocampal pMEK1 (normalized to the total MEK level). In contrast, successive sessions of EA intervention gradually down-regulated the CCI-induced up-regulation of hypothalamic pMEK1 along with the increase numbers of EA intervention. However, EA did not exert the same analgesic effect after microinjection of PD98059 into the contralateral hippocampus during the first 3 days of EA intervention. CONCLUSIONS: EA intervention can induce time-dependent cumulative analgesia in neuropathic pain rats after 4 successive sessions of daily EA intervention, which is at least in part related to the activation of hippocampal MEK1.

The deubiquitinating enzyme USP5 modulates neuropathic and inflammatory pain by enhancing Cav3.2 channel activity.

T-type calcium channels are essential contributors to the transmission of nociceptive signals in the primary afferent pain pathway. Here, we show that T-type calcium channels are ubiquitinated by WWP1, a plasma-membrane-associated ubiquitin ligase that binds to the intracellular domain III-IV linker region of the Cav3.2 T-type channel and modifies specific lysine residues in this region. A proteomic screen identified the deubiquitinating enzyme USP5 as a Cav3.2 III-IV linker interacting partner. Knockdown of USP5 via shRNA increases Cav3.2 ubiquitination, decreases Cav3.2 protein levels, and reduces Cav3.2 whole-cell currents. In vivo knockdown of USP5 or uncoupling USP5 from native Cav3.2 channels via intrathecal delivery of Tat peptides mediates analgesia in both inflammatory and neuropathic mouse models of mechanical hypersensitivity. Altogether, our experiments reveal a cell signaling pathway that regulates T-type channel activity and their role in nociceptive signaling.

[Effect of mild and strong manual acupuncture stimulation of "Huantiao" (GB 30) on mechanical pain thresholds and extracellular signal-regulated kinase protein expression in spinal dorsal horns in rats with neuropathic mirror-image pain].

OBJECTIVE: To observe the effect of different intensities of manual acupuncture (MA) stimulation on mechanical pain thresholds (PTs) and the expression of phosphorylated extracellular signal-regulated kinases (p-ERK) in lumbar spinal dorsal horn regions in rats with neuropathic mirror-image pain, so as to explore its mechanisms underlying analgesia. METHODS: Forty male SD rats were equally and randomly divided into control, spinal nerve ligation (SNL) model, mild MA-stimulation, and strong MA-stimulation groups. Neuropathological pain model was established by ligature of the spinal nerve (L 5). Three days after the SNL, bilateral "Huantiao" (GB 30) were stimulated by rotating the thin (0.22 mm x 13 mm) or thick (0.3 mm x 13 mm) filiform needles at a frequencies of 60 times/min or 180 times/min and at an angle of 180 degrees or 360 degrees for 2 min for rats in the mild and strong MA-stimulation groups, respectively, followed by remaining the needle in place for 30 min. The mechanical PTs were measured before and after SNL. The expression of p-ERK protein in bilateral dorsal horn regions of the lumbar spinal cord (L4- L 6) was detected by Western blot. RESULTS: In comparison with the control group, the mechanical PTs were significantly decreased beginning from the 3rd day on after SNL on the affected side and from the 7th day on after SNL on the healthy hindpaw (P < 0.05), simultaneously, p-ERK protein expression levels of dorsal horn regions on both sides of the spinal cord were considerably up-regulated on the 12th day (P < 0.05). Compared with the model group, the PTs of the affected hindpaw and the healthy hindpaw were significantly increased on the 7th and 12th day in the strong MA-stimulation group (P < 0.05, P < 0.01), whereas pERK expression levels in the bilateral spinal dorsal horn regions were obviously down-regulated in the strong MA-stimulation group (P < 0.05). No significant differences were found between the model and mild MA-stimulation groups in the PTs of bilateral hindpaws and p-ERK expression levels of the bilateral spinal dorsal horn regions (P > 0.05) except the PTs of the healthy hindpaw on 7th day (P < 0.05). CONCLUSION: Strong MA-stimulation can alleviate neuropathic mirror-image pain in SNL rats, which is closely related to its effect in down-regulating the expression of p-ERK in the bilateral spinal dorsal horn regions.

Effects of hyperbaric oxygen on pain-related behaviors and nitric oxide synthase in a rat model of neuropathic pain.

BACKGROUND: Neuropathic pain is complex, and a satisfactory therapeutic method of treatment has yet to be developed; therefore, finding a new and effective therapeutic method is an important issue in the field of neuropathic pain. OBJECTIVE: To determine the effects of hyperbaric oxygen (HBO) on pain-related behaviours and nitric oxide synthase (NOS) expression in a rat model of neuropathic pain. METHODS: Forty male Sprague Dawley rats were randomly divided into five groups (eight rats per group) including control, sham operation, sciatic nerve with chronic constriction injury (CCI), HBO pretreatment (pre-HBO) and HBO post-treatment (post-HBO) groups. Pain-related behaviours and NOS expression in the spinal cord were compared among the five groups. RESULTS: Compared with the CCI group, the mechanical withdrawal threshold was significantly increased and thermal withdrawal latency was significantly extended in the pre-HBO and post-HBO groups (all P<0.05). After CCI, expression of spinal neuronal NOS and inducible NOS were increased. Expression of spinal neuronal NOS and inducible NOS were significantly decreased in the pre-HBO and post-HBO groups compared with the CCI group (all P<0.05). Spinal eNOS expression changed very little. DISCUSSION: HBO has been used as an effective and noninvasive method for the treatment of spinal cord injuries and high-altitude sickness, and in immunosuppression and stem-cell research; however, it has yet to be applied to the treatment of neuropathic pain. The present study indicated that HBO effectively increased mechanical withdrawal threshold and thermal withdrawal latency, demonstrating that HBO has therapeutic effects on neuropathic pain. CONCLUSION: HBO inhibits pain in rats with CCI through the regulation of spinal NOS expression.

Analysis on interrelation between electroacupuncture-induced cumulative analgesic effect and hypothalamic cholinergic activities in chronic neuropathic pain rats.

OBJECTIVE: To observe the effects of repeated electroacupuncture (EA) of Zusanli (ST36)- Yanglingquan (GB34) on hypothalamic acetylcholinesterase (AchE) and vesicular acetylcholine (ACh) transporter (VAChT) activities and choline acetyltransferase (ChAT) mRNA and muscarinic M1 receptor (M1R) mRNA expression in chronic constrictive injury (CCI) and/or ovariectomy (OVX) rats so as to reveal its underlying mechanism in cumulative analgesia. METHODS: A total of 103 female Wistar rats were randomly divided into normal control (n =15), CCI (n =15), CCI+EA2d (n =15), CCI+EA2W (n =15), OVX+CCI =13), OVX+CCI+EA2d (n =15), and OVX+CCI+EA2W groups (n =15). CCI model was established by ligature of the unilateral sciatic nerve with surgical suture. Memory impairment model was established by removal of the bilateral ovaries. Morris water test was conducted to evaluate the OVX rats' memory learning ability, and the thermal pain threshold (PT) of the bilateral paws was detected the next morning after EA. EA (2/15 Hz, 1 mA) was applied to bilateral ST36-GB34 for 30 min, once daily for 2 days or 2 weeks, respectively. Hypothalamic AChE activity was detected by histochemistry, VAChT immunoactivity was determined by immunohistochemistry, and ChAT mRNA and M1R mRNA expressions were assayed by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: In comparison with the normal control group, the AChE activity in hypothalamic arcuate nucleus (ARC) and supraoptic nucleus (SON) regions of CCI group, AChE activity in paraventricular nucleus (PVN), ARC, and SON regions of OVX+CCI group, and hypothalamic muscarinic M1R mRNA expression levels in both CCI and OVX+CCI groups were down-regulated significantly (P <0.05). Compared with the CCI group, the AChE activities in hypothalamic ARC and SON regions of CCI+EA2d and CCI+EA2W groups and PVN region of CCI+EA2W group and hypothalamic ChAT mRNA and M1R mRNA expression levels in CCI+EA2W group were up-regulated considerably (P <0.05). In comparison with the OVX+CCI group, the AChE activities in PVN, ARC, and SON regions and the expressions of hypothalamic ChAT mRNA and VAChT in ARC region of OVX+CCI+EA2W group were up-regulated remarkably (P <0.05). The effects in rats of CCI+EA2W group were evidently superior to those of OVX+CCI+EA2d group in up-regulating AChE activities in PVN, ARC, and SON regions, VAChT immunoactivity in ARC region, and expression levels of hypothalamic ChAT mRNA and M1R mRNA (P <0.05). Similar situations were found in OVX+CCI rats after EA2W. It suggested a cumulative effect after repeated EA of ST36-GB34. Comparison between CCI+EA2W and OVX+CCI+EA2W groups showed that the effects in rats of the former group were evidently better than those of the latter group in up-regulating AChE activity in ARC and SON regions and the expressions of hypothalamic ChAT mRNA and M1 mRNA (P <0.05), suggesting a reduction of EA2W effects after OVX. CONCLUSION: Repeated EA can significantly up-regulate AChE and VAChT activities and ChAT mRNA and M1R mRNA expressions in the hypothalamus of CCI and OVX+CCI rats, which may contribute to the cumulative analgesic effects of repeated EA and be closely related to the animals' neuromemory ability.

Inhibition of ROS-induced p38MAPK and ERK activation in microglia by acupuncture relieves neuropathic pain after spinal cord injury in rats.

Acupuncture (AP) is currently used worldwide to relieve pain. However, little is known about its mechanisms of action. We found that after spinal cord injury (SCI), AP inhibited the production of superoxide anion (O(2)·), which acted as a modulator for microglial activation, and the analgesic effect of AP was attributed to its anti-microglial activating action. Direct injection of a ROS scavenger inhibited SCI-induced NP. After contusion injury which induces the below-level neuropathic pain (NP), Shuigou and Yanglingquan acupoints were applied. AP relieved mechanical allodynia and thermal hyperalgesia, while vehicle and simulated AP did not. AP also decreased the proportion of activated microglia, and inhibited both p38MAPK and ERK activation in microglia at the L4-5. Also, the level of prostaglandin E(2) (PGE2), which is produced via ERK signaling and mediates the below-level pain through PGE2 receptor, was reduced by AP. Injection of p38MAPK or ERK inhibitors attenuated NP and decreased PGE2 production. Furthermore, ROS produced after injury-induced p38MAPK and ERK activation in microglia, and mediated mechanical allodynia and thermal hyperalgesia, which were inhibited by AP or a ROS scavenger. AP also inhibited the expression of inflammatory mediators. Therefore, our results suggest that the analgesic effect of AP may be partly mediated by inhibiting ROS-induced microglial activation and inflammatory responses after SCI and provide the possibility that AP can be used effectively as a non-pharmacological intervention for SCI-induced chronic NP in patients.

Cyclooxygenase 2 expression in the spared nerve injury model of neuropathic pain.

Cyclooxygenase-2 (COX-2) after induction peripherally, and within the CNS, plays an important role in producing inflammatory pain. However, its role in neuropathic pain models is controversial. Recently a robust and persistent model of partial nerve injury pain, the spared nerve injury (SNI) model, has been developed. The aim of the present study was to examine the regulation of COX-2 in the rat SNI model and to evaluate the effectiveness of the selective COX-2 inhibitor rofecoxib in preventing neuropathic allodynia and hyperalgesia. RNase protection assays revealed only a very small and transient increase in COX-2 mRNA in the dorsal horn of the spinal cord in the SNI model with a maximum change at 24 h. Immunohistochemical analysis showed a small increase in COX-2 protein in the deep layers of the dorsal horn 10 h following SNI surgery. Rofecoxib (100 microM) did not affect spontaneous excitatory postsynaptic currents or alpha-amino-3-hydroxy-5-methyl-4-isoxazole propanoic acid (AMPA) and N-methyl-d-aspartate (NMDA) responses in lamina II neurons from spinal cords of animals with SNI indicating no detectable action on transmitter release or postsynaptic activity. Furthermore, rofecoxib treatment (1 and 3.2 mg/kg for 5 and 3 days respectively starting on the day of surgery) failed to modify the development of allodynia and hyperalgesia in the SNI model. However, rofecoxib significantly reduced inflammatory hypersensitivity evoked by injection of complete Freund's adjuvant into one hindpaw, indicating that the doses used were pharmacologically active. The pain hypersensitivity produced by the SNI model is not COX-2-dependent.