Intrathecal liproxstatin-1 delivery inhibits ferroptosis and attenuates mechanical and thermal hypersensitivities in rats with complete Freund's adjuvant-induced inflammatory pain

Previous studies have confirmed the relationship between iron-dependent ferroptosis and a peripheral nerve injury-induced neuropathic pain model.However,the role of fe rroptosis in inflammatory pain remains inconclusive.Therefore,we aimed to explore whether ferroptosis in the spinal cord and do rsal root ganglion contributes to complete Freund's adjuvant(CFA)-induced painful behaviors in rats.Our results revealed that various biochemical and morphological changes were associated with ferroptosis in the spinal cord and dorsal root ganglion tissues of CFA rats.These changes included iron overload,enhanced lipid peroxidation,disorders of anti-acyl-coenzyme A synthetase long-chain family member 4 and glutathione peroxidase 4 levels,and abnormal morphological changes in mitochondria.Intrathecal treatment of liproxstatin-1(a ferroptosis inhibitor)reve rsed these ferroptosis-related changes and alleviated mechanical and thermal hype rsensitivities in CFA rats.Our study demonstrated the occurrence of fe rroptosis in the spinal cord and do rsal root ganglion tissues in a rodent model of inflammatory pain and indicated that intrathecal administration of fe rroptosis inhibitors,such as liproxstatin-1,is a potential therapeutic strategy for treating inflammatory pain.

Tau Accumulation in the Spinal Cord Contributes to Chronic Inflammatory Pain by Upregulation of IL-1βand BDNF

Microtubule-associated protein Tau is responsible for the stabilization of neuronal microtubules under normal physiological conditions.Much attention has been focused on Tau’s contribution to cognition,but little research has explored its role in emotions such as pain,anxiety,and depression.In the current study,we found a significant increase in the levels of p-Tau(Thr231),total Tau,IL-1β,and brain-derived neurotrophic factor(BDNF)on day 7 after complete Freund's adjuvant(CFA)injection;they were present in the vast majority of neurons in the spinal dorsal horn.Microinjection of Mapt-shRNA recombinant adeno-associated virus into the spinal dorsal cord alleviated CFA-induced inflammatory pain and inhibited CFA-induced IL-1βand BDNF upregulation.Importantly,Tau overexpression was sufficient to induce hyperalgesia by increasing the expression of IL-1βand BDNF.Furthermore,the activation of glycogen synthase kinase 3 beta partly contributed to Tau accumulation.These findings suggest that Tau in the dorsal horn could be a promising target for chronic inflammatory pain therapy.

Activation of Dopamine D2 Receptors Alleviates Neuronal Hyperexcitability in the Lateral Entorhinal Cortex via Inhibition of HCN Current in a Rat Model of Chronic Inflammatory Pain

Functional changes in synaptic transmission from the lateral entorhinal cortex to the dentate gyrus(LEC-DG)are considered responsible for the chronification of pain.However,the underlying alterations in fan cells,which are the predominant neurons in the LEC that project to the DG,remain elusive.Here,we investigated possible mechanisms using a rat model of complete Freund’s adjuvant(CFA)-induced inflammatory pain.We found a substantial increase in hyperpolarization-activated/cyclic nucleotide-gated currents(Ih),which led to the hyperexcitability of LEC fan cells of CFA slices.This phenomenon was attenuated in CFA slices by activating dopamine D2,but not D1,receptors.Chemogenetic activation of the ventral tegmental area-LEC projection had a D2 receptor-dependent analgesic effect.Intra-LEC microinjection of a D2 receptor agonist also suppressed CFA-induced behavioral hypersensitivity,and this effect was attenuated by pre-activation of the Ih.Our findings suggest that down-regulating the excitability of LEC fan cells through activation of the dopamine D2 receptor may be a strategy for treating chronic inflammatory pain.

Failure of Placebo Analgesia Model in Rats with Inflammatory Pain

With the shifting role of placebos,there is a need to develop animal models of placebo analgesia and elucidate the mechanisms underlying the effect.In the present study,male Sprague-Dawley rats with chronic inflammatory pain caused by complete Freund’s adjuvant(CFA)underwent a series of conditioning procedures,in which morphine was associated with different cues,but they failed to induce placebo analgesia.Then,conditioning with the conditioned place preference apparatus successfully induced analgesic expectancy and placebo analgesia in naive rats but only induced analgesic expectancy and no analgesic effect in CFA rats.Subsequently,we found enhanced c-fos expression in the nucleus accumbens and reduced expression in the anterior cingulate cortex in naive rats while c-fos expression in the anterior cingulate cortex in CFA rats was not altered.In summary,the behavioral conditioning model demonstrated the difficulty of establishing a placebo analgesia model in rats with a pathological condition.

Targeting the nitric oxide/cGMP signaling pathway to treat chronic pain

Nitric oxide(NO)/cyclic guanosine 3′,5′-monophosphate(cGMP) signaling has been shown to act as a mediator involved in pain transmission and processing. In this review, we summarize and discuss the mechanisms of the NO/cGMP signaling pathway involved in chronic pain, including neuropathic pain, bone cancer pain, inflammatory pain, and morphine tolerance. The main process in the NO/cGMP signaling pathway in cells involves NO activating soluble guanylate cyclase, which leads to subsequent production of cGMP. cGMP then activates cGMP-dependent protein kinase(PKG), resulting in the activation of multiple targets such as the opening of ATP-sensitive K+ channels. The activation of NO/cGMP signaling in the spinal cord evidently induces upregulation of downstream molecules, as well as reactive astrogliosis and microglial polarization which participate in the process of chronic pain. In dorsal root ganglion neurons, natriuretic peptide binds to particulate guanylyl cyclase, generating and further activating the cGMP/PKG pathway, and it also contributes to the development of chronic pain. Upregulation of multiple receptors is involved in activation of the NO/cGMP signaling pathway in various pain models. Notably the NO/cGMP signaling pathway induces expression of downstream effectors, exerting both algesic and analgesic effects in neuropathic pain and inflammatory pain. These findings suggest that activation of NO/cGMP signaling plays a constituent role in the development of chronic pain, and this signaling pathway with dual effects is an interesting and promising target for chronic pain therapy.

Neurochemical mechanism of the gastrointestinal interdigestive migrating motor complex in rats with acute inflammatory stomach ache

The normal gastrointestinal interdigestive migrating motor complex cycle was interrupted, and paroxysmal contraction appeared after formaldehyde-induced stomach ache. Activities of nitric oxide synthase, acetylcholinesterase and vasoactive intestinal peptide neurons were significantly reduced, whereas activities of calcitonin gene-related peptide neurons were significantly increased in the pyloric sphincter muscular layer, myenteric nerve plexus and submucous nerve plexus. Electroacupuncture at Zusanfi （ST36） suppressed paroxysmal contraction in rats with formaldehyde-induced stomach ache, and neurons in the enteric nervous system were normal. These results indicated that nitrergic neurons, cholinergic neurons, vasoactive intestinal peptide neurons and calcitonin gene-related peptide neurons in the enteric nervous system may be involved in changes to the gastrointestinal interdigestive migrating motor complex following stomach ache, and that electroacupuncture can regulate this process.

Diabetic Patients Develop Greater Intensity of Postoperative Pain than Non-Diabetics after Open Cholecystectomy: A Pilot Study

Background: Pre-clinical and clinical studies have shown that inflammatory pain intensity is increased under diabetes condition. Open cholecystectomy (OC) is a surgical procedure with predictable postoperative pain. However, the comparison of postoperative pain due to open cholecystectomy in diabetic and non-diabetic patients remains unknown. The research question to answer was whether diabetic patients undergoing OC development greater intensity of postoperative pain than non-diabetic patients. Methods: The study was conducted between June 2016 and February 2018 at the Regional Hospital of High Specialty “Dr. Juan Graham Casasús” of Villahermosa, Tabasco, Mexico. Seventy patients in two groups of 35 patients each scheduled for OC under general anesthesia were studied. Pain was assessed using the 11-point numerical rating scale (NRS). The primary endpoint was to know NRS pain scores after awaking of general anesthesia. Secondary outcomes included the time of onset of pain and comparing NRS scores between diabetic and non-diabetic patients undergoing OC. Results: Diabetic patients reported significantly greater intensity pain than non-diabetic patients. The mean overall pain score in the diabetic and non-diabetic patients was 7.2 ± 0.3 and 5.3 ± 0.3 (P = 0.0002), respectively. Furthermore, 60% of diabetic patients had severe pain (NRS ≥ 8) compared to 20% of non-diabetics (P = 0.006). The time to onset postoperative pain was about 35 minutes in both groups (P = 0.876). Conclusions: Diabetic patients undergoing OC have greater intensity postoperative pain and also more frequency of patients with severe pain scores compared with non-diabetic patients. Therefore, analgesic treatment in those patients should consider this point in order to provide a satisfactory postoperative analgesia.

Formalin-induced astrocyte glutamate-glutamine cycle involved in rat spinal cord central sensitization

BACKGROUND： Central sensitization, a state of increased excitability of nociceptive neurons in the spinal dorsal horn following peripheral tissue injury and/or inflammation, is an important mechanism underlying hyperalgesia and neuropathic pain. Participation of the glutamate-glutamine cycle in central sensitization of the spinal cord remains poorly understood. OBJECTIVE： To determine whether the astrocyte-neuronal glutamate-glutamine cycle is involved in formalin-induced central sensitization in the spinal cord. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment was performed at the Institute of Orthopedics, Second Hospital, Lanzhou University, China from September 2007 to August 2008. MATERIALS： Methionine sulfoximine （MSO, 0.1 mmol/L）, glutamine （0.25 mmol/L）, and formalin were used for this study. METHODS： A total of 43 male, Sprague Dawley rats, aged 4 months, were randomly assigned to a sham operation group （n = 6） and a model group （n = 37）. Rats in the model group received intrathecal infusion in the spinal cord. 7 days later, 37 model rats were randomly divided into PBS, MSO, glutamine, MSO ＋ glutamine and formalin subcutaneous injection alone groups. The PBS, MSO, glutamine, MSO ＋ glutamine groups were respectively intrathecally injected with PBS, MSO, glutamine, MSO ＋ glutamine （50 μL each）, and then infused with 10 μL of saline. Rats from the sham operation group were not subjected to intrathecal infusion in the spinal cord. At 15 minutes after intrathecal injection, a rat model of formalin-induced inflammatory pain was established by subcutaneous injection of 5% formalin （50 μL） in the left hindpaw. MAIN OUTCOME MEASURES： Changes in spontaneous nociceptive behavior （licking/biting or flinching） were observed following formalin injection into the rat hindpaw. RESULTS： Compared with the PBS group, duration of licking/biting was significantly shortened, and flinching frequency was significantly diminished in the MSO group （P 〈 0.05）. Compared with the MSO group, duration of licking/biting was significantly prolonged, and flinching frequency was significantly increased in the MSO ＋ glutamine group （P 〈 0.05）. There was no significant difference in inflammatory pain behaviors among the sham operation, PBS, glutamine, MSO ＋ glutamine, and formalin subcutaneous injection alone groups （P 〉 0.05）. CONCLUSION： The astrocyte-neuronal glutamate-glutamine cycle in the spinal cord was shown to be involved in central sensitization induced by formalin subcutaneous injection into the hindpaw.

NO-cGMP-K channel-dependent anti-nociceptive activities of methanol stem bark extract of Piptadeniastrum africanum(Mimosaceae) on rats

Objective: To explore anti-hyperalgesic properties of methanol extract of Piptadeniastrum africanum stem bark(PAME) and it possible action mechanism. Methods: PAME was tested on carrageenan ?induced hyperalgesia using plantar test(thermal) and analgesymeter(mechanical) in rats, on prostaglandin E_2(PGE_2) induced mechanical hyperalgesia and vincristine induced neuropathic pain in rat, both with analgesymeter. Modulators of NO/cG MP/K^+ channel pathway and endogenous opioids receptor antagonists and/or agonists were used to determine the possible action mechanism of PAME. Results: PAME significantly decreased carrageenan induced thermal and mechanical hyperalgesia, as well as PGE_2 induced mechanical hyperalgesia. PAME significantly protected the animals against the installation of neuropathic pain. Anti-nociception activity produced by PAME was significantly blocked in animals pre?treated with all the antagonists(naloxone, NW-nitro-L-arginine methyl ester(L-NAME), methylene blue and glibenclamide). Conclusions: Results of this study reveal that, PAME administrate orally, can induce anti-hyperalgesic action against installation of inflammatory pain as well as neuropathic pain. The mechanism underlying PAME antihyperalgesic effect could probably be associated with an activation of opioid receptors and NO/cG MP/K^+ channel pathway.

Inhibition of Muscular Nociceptive Afferents via the Activation of Cutaneous Nociceptors in a Rat Model of Inflammatory Muscle Pain

Topical irritants such as capsaicin(CAP),peppermint oil(PO),and mustard oil(MO)are effective in relieving inflammatory muscle pain.We investigated the effects of topical irritants in a rat model of inflammatory muscle pain produced by injecting complete Freund's adjuvant(CFA)into the tibialis anterior muscle.CFAinduced mechanical hypersensitivity and the spontaneous activity of muscular nociceptive afferents,and decreased weight-bearing of the hindlimb were relieved by topical application of CAP,PO,or MO on the skin overlying the inflamed muscle.The effects of topical irritants were abolished when applied to the skin on the ipsilateral plantar region or on the contralateral leg,or when the relevant cutaneous nerve or dorsal root was transected.Our results demonstrated that topical irritants may alleviate inflammatory muscle pain via activating cutaneous nociceptors and subsequently inhibiting the abnormal activity of muscular nociceptive neurons.

Role of the Anterior Cingulate Cortex in Translational Pain Research

As the most common symptomatic reason to seek medical consultation,pain is a complex experience that has been classified into different categories and stages.In pain processing,noxious stimuli may activate the anterior cingulate cortex(ACC).But the function of ACC in the different pain conditions is not well discussed.In this review,we elaborate the commonalities and differences from accumulated evidence by a variety of pain assays for physiological pain and pathological pain including inflammatory pain,neuropathic pain,and cancer pain in the ACC,and discuss the cellular receptors and signaling molecules from animal studies.We further summarize the ACC as a new central neuromodulation target for invasive and non-invasive stimulation techniques in clinical pain management.The comprehensive understanding of pain processing in the ACC may lead to bridging the gap in translational research between basic and clinical studies and to develop new therapies.

Gait Assessment of Pain and Analgesics: Comparison of the DigiGait^(TM) and CatWalk^(TM） Gait Imaging Systems

Investigation of pain requires measurements of nociceptive sensitivity and other pain-related behaviors.Recent studies have indicated the superiority of gait analysis over traditional evaluations(e.g., skin sensitivity and sciatic function index [SFI]) in detecting subtle improvements and deteriorations in animal models. Here,pain-related gait parameters, whose criteria include(1)alteration in pain models,(2) correlation with nociceptive threshold, and(3) normalization by analgesics, were identified in representative models of neuropathic pain(spared nerve injury: coordination data) and inflammatory pain(intraplantar complete Freund’s adjuvant: both coordination and intensity data) in the DigiGait^TM and CatWalk^TM systems. DigiGait^TM had advantages in fixed speed(controlled by treadmill) and dynamic SFI, while CatWalk^TM excelled in intrinsic velocity, intensity data,and high-quality 3 D images. Insights into the applicability of each system may provide guidance for selecting the appropriate gait imaging system for different animal models and optimization for future pain research.

Tweety-Homolog 1 Facilitates Pain via Enhancement of Nociceptor Excitability and Spinal Synaptic Transmission

Tweety-homolog 1(Ttyh1)is expressed in neural tissue and has been implicated in the generation of several brain diseases.However,its functional significance in pain processing is not understood.By disrupting the gene encoding Ttyh1,we found a loss of Ttyh1 in nociceptors and their central terminals in Ttyh1-deficient mice,along with a reduction in nociceptor excitability and synaptic transmission at identified synapses between nociceptors and spinal neurons projecting to the periaqueductal grey(PAG)in the basal state.More importantly,the peripheral inflammationevoked nociceptor hyperexcitability and spinal synaptic potentiation recorded in spinal-PAG projection neurons were compromised in Ttyh1-deficient mice.Analysis of the paired-pulse ratio and miniature excitatory postsynaptic currents indicated a role of presynaptic Ttyh1 from spinal nociceptor terminals in the regulation of neurotransmitter release.Interfering with Ttyh1 specifically in nociceptors produces a comparable pain relief.Thus,in this study we demonstrated that Ttyh1 is a critical determinant of acute nociception and pain sensitization caused by peripheral inflammation.

电针镇痛及其与刺激参数的量效关系研究:近三十年研究新进展

In this review,we explored the dose-effect relationship of electroacupuncture(EA)analgesia and its stimulus parameters by searching articles on clinical and experimental research of EA analgesia in the past30 years.The impacts on the analgesic effects are discussed in terms of the pulse waveform,frequency,amplitude,wave width,and time effect,as well as parameter combinations,and the optimization of the EA parameters are summarized for the treatment of neuropathic,inflammatory,and cancer pains.It was initially discovered that the distant-dense(D-D)wave(2/15 Hz or 2/100 Hz)and stimulus for 30-45 min were appropriate in the treatment of chronic inflammatory pain,the strong stimulus was applicable to the acute stage of pain(twice a week),while the weak stimulus was for the stable stage(once weekly).The continuous(2 Hz)/D-D wave(2/100 Hz)and moderate/low intensity of stimulus for 30-45 min are preferred in the treatment of neuropathic pain with EA,once daily or every 2 days.Regarding the treatment of cancer pain,EA with continuous(2 Hz or 100 Hz)or D-D wave(2/100 Hz),moderate and low intensity of stimulus for 30-45 min is adopted,once every 2 days.There is a certain rule for the correlation of each parameter and the combination of parameters to ensure an analgesic effect of EA,which should be further explored in future studies.

Far infrared-emitting ceramics decrease Freund's adjuvant-induced inflammatory hyperalgesia in mice through cytokine modulation and activation of peripheral inhibitory neuroreceptors

Objective： The present study aimed to evaluate the analgesic and anti-inflammatory effects of far infrared-emitting ceramics （cFIRs） in a model of persistent inflammatory hyperalgesia and to elucidate the possible mechanisms of these effects. Methods： Mice were injected with complete Freund＇s adjuvant （CFA） and treated with cFIRs via place- ment on a pad impregnated with cFIRs on the bottom of the housing unit for different periods of time. Mice underwent mechanical hyperalgesia and edema assessments, and tumor necrosis factor-α （TNF-α）, interleukin-1β （IL-1β） and IL-10 levels were measured. Twenty-four hours after CFA injection and 30 min before cFIR treatment, mice were pretreated with a nonselective adenosinergic antagonist, caffeine, the selective adenosine receptor A antagonist, 1,3-dipropyl-8-cyclopentylxanthine （DPCPX）, the selective cannabinoid receptor type 1 antagonist, AM281, the selective cannabinoid receptor type 2 antagonist, AM630, or the nonselective opioid receptor antagonist, naloxone, and mechanical hyperalge- sia was assessed. Results： cFIRs statistically （P 〈 0.05） decreased CFA-induced mechanical hyperalgesia （82.86 ±5.21）% in control group vs （56.67±9.54）% in cFIR group） and edema （（1699.0 ± 77.8） gm in control group vs （988.7±107.6） gm in cFIR group）, cFIRs statistically （P 〈 0.05） reduced TNF-α （0.478± 0.072） pg/mg of protein in control group vs （0.273 ±0.055） pg/mg of protein in cFIR group） and IL-113 （（95.81 ± 3.95） pg/mg of protein in control group vs （80.61 ±4.71）pg/mg of protein in cFIR group） levels and statistically （P〈 0.05） increased IL-10 （（18.32 ±0.78） pg/mg of protein in control group vs （25.89 ±1.23） pg/mg of protein in cFIR group） levels in post-CFA-injected paws. Peripheral pre-administration of inhibitory neuroreceptor antagonists （caffeine, DPCPX, AM281, AM630 and naloxone） prevented the analgesic effects of cF1Rs （P 〈 0.05）.Conclusion： These data provide additional support for the use of cFIRs in the treatment of painful inflam- matory conditions and contribute to our understanding of the neurobiological mechanisms of the ther- apeutic effects of cFIRs.

肽能神经元对颈部急性炎性痛模型大鼠穴位敏化的调节作用

Objective:Pain is a health problem frequently reported in the clinical and general populations.Acupoint therapy is one of the most effective ways to treat pain;however,its mechanism remains unclear.Therefore,this study aimed to explore the relationship between distal acupoints,dorsal root ganglion(DRG)neurons,and spinal cord dorsal horn(SDH) neurons in neck acute inflammatory pain(NAIP).Methods:NAIP model rats were used to explore the relationship between acupoint sensitization and pain.Out of fourteen rats,ten rats were grouped into control and NAIP groups,five rats in control and five rats in NAIP.Mustard oil was subcutaneously injected on one side between the C4 and C7 vertebrae of the neck to establish an NAIP model.Evans blue(EB) was injected through the tail vein to detect sensitized acupoints after NAIP modeling.EB exudation in the body,Liè qūe(列缺 LU7),and Língdào(灵道 HT4) were evaluated.An immunofluorescence assay was conducted to detect the expression of calcitonin generelated peptide(CGRP),isolectin B4(IB4),and c-Fos in the dorsal root ganglion(DRG)and spinal cord dorsal medullae spinalis(CDMS).Four rats were used for the retrograde labeling of neurons of the LU7 region to the DRG and CDMS using CTB-488 and CTB-555 microinjection.Results:NAIP was shown to lead to oozing and pain sensitization in the LU7 and HT4 and increased the proportion of c-Fos^(+)/CGRP^(+)and c-Fos^(+)/IB4^(+)cells in both the DRG and CDMS.CTB-488 and CTB-555injected into the LU7 and sensitization point areas were observed in the DRG and CDMS regions of NAIP rats.Conclusions:Our study revealed that NAIP could lead to oozing and pain sensitization in the LU7 and HT4regions and that the pain point around LU7 might result from the transfer of peptidergic(CGRP-positive)and non-peptidergic(IB4-positive) neurons in the DRG and CDMS.

Fibroblast growth factor 7 is a nociceptive modulator secreted via large dense-core vesicles

Fibroblast growth factor(FGF)7,a member of FGF family,is initially found to be secreted from mesenchymal cells to repair epithelial tissues.However,its functions in the nervous system are largely unknown.The present study showed that FGF7 was a neuromodulator localized in the large dense-core vesicles(LDCVs)in nociceptive neurons.FGF7 was mainly expressed in small-diameter neurons of the dorsal root ganglion and could be transported to the dorsal spinal cord.Interestingly,FGF7 was mostly stored in LDCVs that did not contain neuropeptide substance P.Electrophysiological recordings in the spinal cord slice showed that buffer-applied FGF7 increased the amplitude of excitatory post-synaptic current evoked by stimulating the sensory afferent fibers.Behavior tests showed that intrathecally applied FGF7 potentiated the formalin-induced acute nociceptive response.Moreover,both acute and inflammatory nociceptive responses were significantly reduced in Fgf7-deficient mice.These results suggest that FGF7 exerts an excitatory modulation of nociceptive afferent transmission.