sirolimus and Neuralgia

In neuropathic pain following peripheral nerve injury, microglia are rapidly activated and accumulated in the spinal cord. Physical exercise can alleviate neuropathic pain. However, the exact mechanism underlying this analgesic effect is not fully understood.. We aimed to investigate the molecular mechanisms by which exercise alleviates neuropathic pain in relation to brain-derived neurotrophic factor (BDNF), microglia polarization, and autophagy.. A randomized controlled animal study divided into 2 stages. The first stage comprised 4 groups each with 6 mice, and the second stage comprised 6 groups, 3 with 18 mice and 3 with 12 mice.. Department of Anesthesiology, Lanzhou University Second Hospital, Orthopaedics Key Laboratory of Gansu Province, Lanzhou University.. Von Frey filaments, Western blotting, immunofluorescence, and transmission electron microscopy analyses were conducted to detect relevant markers.. After peripheral nerve injury, exercise training downregulated BDNF expression and reversed microglial activation, as indicated by the increased expression of the M2 marker CD206 and decreased expression of the M1 marker CD86 in the spinal dorsal horn of mice. Autophagy flux was enhanced after exercise training, as suggested by the increased expression of the autophagy markers LC3-II/LC3-I and Beclin1 and decreased expression of the autophagy adaptor protein p62. Furthermore, autophagy inhibition by 3-methyladenine aggravated M1 polarization and hyperalgesia, whereas autophagy induced by rapamycin promoted M2 polarization and reduced hyperalgesia. Intrathecal injection of BDNF significantly upregulated BDNF expression, inhibited autophagy, triggered M1 polarization of spinal microglia, and aggravated hyperalgesia. Furthermore, BDNF regulated autophagy through the AKT/mTOR pathway, thereby participating in exercise training-mediated polarization of microglia after nerve injury.. The effect of exercise on autophagy and pain cannot be assessed in an in vitro model. The influence of intrathecal injection of BDNF on the metabolic changes in other neuronal cells and the subsequent effects on pain should be investigated. Further studies on how exercise training modulates microglial autophagy to alleviate neuropathic pain are needed.. Exercise training promoted the recovery of sciatic nerve injury in mice, possibly by regulating microglial polarization through BDNF/AKT/mTOR signaling-mediated autophagy flux. We confirmed the efficacy of exercise training in alleviating neuropathic pain and suggest a new therapeutic target for neuropathic pain.

Topics: Analgesics; Animals; Autophagy; Beclin-1; Brain-Derived Neurotrophic Factor; Hyperalgesia; Mice; Microglia; Neuralgia; Peripheral Nerve Injuries; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Sirolimus; Spinal Cord Dorsal Horn; TOR Serine-Threonine Kinases

Activation of mammalian target of rapamycin (mTOR) has been known as one of the contributing factors in nociceptive sensitization after peripheral injury. Its activation followed by the phosphorylation of downstream effectors causes hyperexcitability of primary sensory neurons in the dorsal root ganglion. We investigated whether a single injection of rAAV-shmTOR would effectively downregulate both complexes of mTOR in the long-term and glial activation as well. Male SD rats were categorized into shmTOR (

Topics: Animals; Ganglia, Spinal; Hyperalgesia; Male; Mammals; Neuralgia; Rats; Rats, Sprague-Dawley; Sciatic Nerve; Sirolimus; TOR Serine-Threonine Kinases; Trauma, Nervous System

Unveiling the etiology and the underlying mechanism of neuropathic pain, a poorly treated disease, is essential for the development of effective therapies. This study aimed to explore the role of mammalian target of rapamycin (mTOR) signaling in autophagy-mediated neuropathic pain. We established a spared nerve injury (SNI) model in adult male SD rats by ligating the common peroneal nerve and tibial, with the distal end cutoff. The paw withdrawal threshold (PWT) and C/A-fiber evoked field potentials were determined by electrophysiologic tests at day 0 (before operation), day 7 and day 14 postoperation, and SNI significantly increased field potentials (P < 0.05). Immunohistochemistry and western blots using spinal cord tissues showed that the expressions of GluR1, GluR2, Beclin-1, p62, mTOR and 4EBP1 were significantly increased after SNI (all P < 0.05), whereas the expressions of LC3 and LAMP2 were significantly decreased after SNI (all P < 0.05). Rapamycin efficiently counteracted the effect of SNI and restored the phenotypes to the level comparable to the sham control. In conclusion, rapamycin inhibits C/A-fiber-mediated long-term potentiation in the SNI rat model of neuropathic pain, which might be mediated by activation of autophagy signaling and downregulation of GluRs expression.

Topics: Animals; Autophagy; Immunosuppressive Agents; Male; Neuralgia; Neuronal Plasticity; Rats; Rats, Sprague-Dawley; Signal Transduction; Sirolimus; Spinal Cord Dorsal Horn; TOR Serine-Threonine Kinases

Neuropathic pain is a chronic condition with little specific treatment. Insulin-like growth factor 1 (IGF1), interacting with its receptor, IGF1R, serves a vital role in neuronal and brain functions such as autophagy and neuroinflammation. Yet, the function of spinal IGF1/IGF1R in neuropathic pain is unclear. Here, we examined whether and how spinal IGF1 signaling affects pain-like behaviors in mice with chronic constriction injury (CCI) of the sciatic nerve. To corroborate the role of IGF1, we injected intrathecally IGF1R inhibitor (nvp-aew541) or anti-IGF1 neutralizing antibodies. We found that IGF1 (derived from astrocytes) in the lumbar cord increased along with the neuropathic pain induced by CCI. IGF1R was predominantly expressed on neurons. IGF1R antagonism or IGF1 neutralization attenuated pain behaviors induced by CCI, relieved mTOR-related suppression of autophagy, and mitigated neuroinflammation in the spinal cord. These findings reveal that the abnormal IGF1/IGF1R signaling contributes to neuropathic pain by exacerbating autophagy dysfunction and neuroinflammation.

Topics: Animals; Autophagy; Insulin-Like Growth Factor I; Mice; Neuralgia; Signal Transduction; Sirolimus; Spinal Cord

The anterior cingulate cortex (ACC) is a well-known brain area that is associated with pain perception. Previous studies reported that the ACC has a specific role in the emotional processing of pain. Chronic pain is characterized by long-term potentiation that is induced in pain pathways and contributes to hyperalgesia caused by peripheral nerve injury. The mammalian target of rapamycin (mTOR) signaling, which is involved in synaptic protein synthesis, could be a key factor controlling long-term potentiation in neuropathic pain conditions. Until now, there have been no reports that studied the role of mTOR signaling in the ACC involved in neuropathic pain. Therefore, this study was conducted to determine the relationship of mTOR signaling in the ACC and neuropathic pain. Male Sprague-Dawley rats were subjected to cannula implantation and nerve injury under pentobarbital anesthesia. Microinjection with rapamycin into the ACC was conducted under isoflurane anesthesia on postoperative day (POD) 7. A behavioral test was performed to evaluate mechanical allodynia, and optical imaging was conducted to observe the neuronal responses of the ACC to peripheral stimulation. Inhibition of mTOR by rapamycin reduced mechanical allodynia, down-regulated mTOR signaling in the ACC, and diminished the expressions of synaptic proteins which are involved in excitatory signaling, thereby reducing neuropathic pain-induced synaptic plasticity. These results suggest that inhibiting mTOR activity by rapamycin in the ACC could serve as a new strategy for treating or managing neuropathic pain before it develops into chronic pain.

Topics: Analgesics; Animals; Electric Stimulation; Gyrus Cinguli; Hyperalgesia; Male; Microinjections; Nerve Tissue; Neuralgia; Rats, Sprague-Dawley; Signal Transduction; Sirolimus; Synapses; TOR Serine-Threonine Kinases

Bortezomib (BTZ) is largely used as a chemotherapeutic agent for the treatment of cancer. However, one of the significant limiting complications of BTZ is painful peripheral neuropathy during BTZ therapy. Drugs preventing and/or treating the painful symptoms induced by BTZ are lacking since the underlying mechanisms leading to neuropathic pain remain largely unclear. The purposes of this study were to examine 1) the effects of blocking mammalian target of rapamycin (mTOR) on mechanical pain and cold hypersensitivity evoked by BTZ and 2) the underlying mechanisms responsible for the role of mTOR in regulating BTZ-induced neuropathic pain.. Behavioral test was performed to determine mechanical pain and cold sensitivity in a rat model. Western blot analysis and ELISA were used to examine expression of mTOR and phosphatidylinositide 3-kinase (p-PI3K) signals, and the levels of substance P and calcitonin gene-related peptide (CGRP).. Systemic injection of BTZ significantly increased mechanical pain and cold sensitivity as compared with control animals (P< 0.05 vs. control rats). The expression of p-mTOR, mTOR-mediated phosphorylation of p70 ribosomal S6 protein kinase 1 (p-S6K1), 4E-binding protein 4 (p-4E-BP1) as well as p-PI3K was amplified in the dorsal horn of spinal cord of BTZ rats as compared with control rats. Blocking mTOR by intrathecal infusion of rapamycin attenuated mechanical pain and cold hypersensitivity. Blocking PI3K signal also attenuated activities of mTOR, which was accompanied with decreasing neuropathic pain. Inhibition of either mTOR or PI3K blunted enhancement of the spinal substance P and CGRP in BTZ rats.. The data for the first time revealed specific signaling pathways leading to BTZ-induced peripheral neuropathic pain, including the activation of mTOR and PI3K. Inhibition of these signal pathways alleviates pain. Targeting one or more of these signaling molecules may present new opportunities for treatment and management of peripheral painful neuropathy observed during chemotherapeutic application of BTZ.

Topics: Animals; Behavior, Animal; Bortezomib; Calcitonin Gene-Related Peptide; Carrier Proteins; Chromones; Disease Models, Animal; Intracellular Signaling Peptides and Proteins; Male; Morpholines; Neuralgia; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphoproteins; Phosphorylation; Rats; Rats, Sprague-Dawley; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; Spinal Cord; Substance P; TOR Serine-Threonine Kinases; Up-Regulation

Nucleoside reverse transcriptase inhibitors (NRTIs) are the earliest and most commonly used anti-human immunodeficiency virus drugs and play an important role in high active antiretroviral therapy. However, NRTI drug therapy can cause peripheral neuropathic pain. In this study, we aimed to investigate the mechanisms of rapamycin on the pain sensitization of model mice by in vivo experiments to explore the effect of mammalian target of rapamycin (mTOR) in the pathogenesis of neuropathic pain caused by NRTIs.. Male Kun Ming (KM) mice weighing 20-22 g were divided into control, 2 mg/kg rapamycin, 12 mg/kg stavudine, and CMC-Na groups. Drugs were orally administered to mice for 42 consecutive days. The von Frey filament detection and thermal pain tests were conducted on day 7, 14, 21, 28, 35, and 42 after drug administration. After the last behavioral tests, immunohistochemistry and western blotting assay were used for the measurement of mTOR and other biomarkers. Multivariate analysis of variance was used.. The beneficial effects of rapamycin on neuropathic pain were attributed to a reduction in mammalian target of rapamycin sensitive complex 1 (mTORC1)-positive cells (70.80 ± 2.41 vs. 112.30 ± 5.66, F = 34.36, P < 0.01) and mTORC1 activity in the mouse spinal cord. Mechanistic studies revealed that Protein Kinase B (Akt)/mTOR signaling pathway blockade with rapamycin prevented the phosphorylation of mTORC1 in stavudine-intoxicated mice (0.72 ± 0.04 vs. 0.86 ± 0.03, F = 4.24, P = 0.045), as well as decreased the expression of phospho-p70S6K (0.47 ± 0.01 vs. 0.68 ± 0.03, F = 6.01, P = 0.022) and phospho-4EBP1 (0.90 ± 0.04 vs. 0.94 ± 0.06, F = 0.28, P = 0.646).. Taken together, these results suggest that stavudine elevates the expression and activity of mTORC1 in the spinal cord through activating the Akt/mTOR signaling pathway. The data also provide evidence that rapamycin might be useful for the treatment of peripheral neuropathic pain.

Topics: Animals; HIV Infections; Humans; Male; Mice; Neuralgia; Phosphatidylinositol 3-Kinase; Phosphatidylinositols; Proto-Oncogene Proteins c-akt; Reverse Transcriptase Inhibitors; Sirolimus; TOR Serine-Threonine Kinases

To investigate the role of autophagy in hydrogen-rich saline attenuating post-herpetic neuralgia( PHN) in rats.. A total of 100 male SD rats were randomly divided into the five groups( n = 20) : control group,PHN group,PHN group treated with hydrogen-rich saline( PHN-H2group),PHN group treated with hydrogen-rich saline and3-MA( PHN-H2-3-MA group),PHN group treated with hydrogen-rich saline and rapamycin( PHN-H2-Rap group). PHN models were established by varicella-zoster virus( VZV) inoculation. After modeling,15 mg / kg 3-MA or 10 mg / kg rapamycin were intraperitoneally injected in corresponding rats with PHN once two days for 3 times. Hydrogen-rich saline( 10 m L / kg)was injected intraperitoneally twice a day for 7 consecutive days in PHN-H2 group,PHN-H2-3-MA group and PHN-H2-Rap group after VZV injection. The paw withdrawal thresholds( PWT) of 50 rats were detected at 3,7,14 and 21 days after modeling. Spinal cord enlargements of the other 50 rats were collected to examine tumor necrosis factor α( TNF-α),interleukine 1β( IL-1β) and IL-6 by ELISA and autophagy protein microtubule-associated protein 1 light chain 3( LC3),beclin 1and P62 by Western blotting.. Compared with the control group,the rats in the PHN group presented with decreased PWT,increased levels of TNF-α,IL-1β,IL-6,LC3Ⅱ and beclin 1,and down-regulated P62 expression. Compared with PHN group,the rats in the PHN-H2 group and PHN-H2-Rap group showed increased PWT,decreased levels of TNF-α,IL-1β and IL-6,further up-regulated expressions of LC3 and beclin 1 as wel as P62 expression. Compared with PHN-H2 group,the rats in the PHN-H2-3-MA group had reduced PWT,elevated expressions of TNF-α,IL-1β and IL-6,suppressed expressions of LC3 and beclin 1,and enhanced p62 expression.. Hydrogen-rich saline attenuated PWT and inhibited the release of cytokines TNF-α,IL-1β,IL-6 in rats with PHN via activating autophagy.

Topics: Animals; Autophagy; Hydrogen; Hyperalgesia; Interleukin-1beta; Interleukin-6; Male; Microtubule-Associated Proteins; Neuralgia; Pain Threshold; Random Allocation; Rats; Rats, Sprague-Dawley; Sirolimus; Sodium Chloride; Spinal Cord; Time Factors; Tumor Necrosis Factor-alpha; Up-Regulation; Varicella Zoster Virus Infection

Rapamycin is an inhibitor of the mammalian target of rapamycin (mTOR) signaling pathway, plays an important role in multiple cellular functions. Our previous study showed rapamycin treatment in acute phase reduced the neural tissue damage and locomotor impairment after spinal cord injury (SCI). However, there has been no study to investigate the therapeutic effect of rapamycin on neuropathic pain after SCI. In this study, we examined whether rapamycin reduces neuropathic pain following SCI in mice. We used a mouse model of thoracic spinal cord contusion injury, and divided the mice into the rapamycin-treated and the vehicle-treated groups. The rapamycin-treated mice were intraperitoneally injected with rapamycin (1 mg/kg) 4 h after SCI. The rapamycin treatment suppressed phosphorylated-p70S6K in the injured spinal cord that indicated inhibition of mTOR. The rapamycin treatment significantly improved not only locomotor function, but also mechanical and thermal hypersensitivity in the hindpaws after SCI. In an immunohistochemical analysis, Iba-1-stained microglia in the lumbar spinal cord was significantly decreased in the rapamycin-treated mice. In addition, the activity of p38 MAPK in the lumbar spinal cord was significantly attenuated by rapamycin treatment. Furthermore, phosphorylated-p38 MAPK-positive microglia was relatively decreased in the rapamycin-treated mice. These results indicated rapamycin administration in acute phase to reduce secondary neural tissue damage can contribute to the suppression of the microglial activation in the lumbar spinal cord and attenuate the development of neuropathic pain after SCI. The present study first demonstrated that rapamycin has significant therapeutic potential to reduce the development of neuropathic pain following SCI. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:93-103, 2017.

Topics: Animals; Calcium-Binding Proteins; Drug Evaluation, Preclinical; Female; Glial Fibrillary Acidic Protein; Hyperalgesia; Locomotion; Mice, Inbred C57BL; Microfilament Proteins; Neuralgia; Neuroglia; p38 Mitogen-Activated Protein Kinases; Ribosomal Protein S6 Kinases, 70-kDa; Sirolimus; Spinal Cord Injuries; TOR Serine-Threonine Kinases

The cerebrospinal fluid-contacting nucleus (CSF-CN) has been demonstrated to be involved in neuropathic pain, but the underlying molecular mechanisms remain unclear. Previous work has shown that mTOR and ERK1/2 are important signaling pathways regulating neuropathic pain. However, studies on the interactions between these major pathways in neuropathic pain are very rare. Therefore, the purpose of this study is to determine whether mTOR and ERK1/2 exist in the CSF-CN and elucidate their alterations in neuropathic pain, especially, the crosstalk between them. Our results showed that mTOR and ERK1/2 were distributed in the CSF-CN, and their expression levels were increased in chronic constriction injury (CCI)-induced neuropathic pain. Furthermore, the injection of both the mTOR antagonist rapamycin and the ERK1/2 antagonist U0126 into the lateral ventricle of the brain attenuated CCI-induced neuropathic pain. Inhibition of the ERK1/2 pathway had little impact on mTOR signaling, but inhibition of the mTOR pathway significantly increased ERK/2 signaling. The coadministration of rapamycin and U0126 inhibited the rapamycin-induced upregulation of ERK, and had a greater effect on pain behaviors than did the single-drug administrations. These data extend our understanding of the relationship between mTOR and ERK in the supraspinal site and demonstrate that the CSF-CN participates in neuropathic pain via the regulation of mTOR and ERK1/2.

Topics: Animals; Cell Nucleus; Cerebrospinal Fluid; Male; MAP Kinase Signaling System; Neuralgia; Pain Measurement; Rats; Rats, Sprague-Dawley; Sirolimus; TOR Serine-Threonine Kinases

Activated mammalian target of rapamycin (P-mTOR) has been shown to maintain the sensitivity of subsets of small-diameter primary afferent A-nociceptors. Local or systemic inhibition of the mTOR complex 1 (mTORC1) pathway reduced punctate mechanical and cold sensitivity in neuropathic pain and therefore offered a new approach to chronic pain control. In this study, we have investigated the effects of the rapamycin analog temsirolimus (CCI-779) on itch. Bouts of scratching induced by the histamine-dependent pruritogenic compound 48/80 and histamine-independent pruritogens, chloroquine and SLIGRL-NH2, injected intradermally were significantly reduced by local (intradermal) or systemic (intraperitoneal, i.p.) pretreatment with CCI-779. We also investigated the action of metformin, a drug taken to control type 2 diabetes and recently shown to inhibit mTORC1 in vivo. Although the response to nonhistaminergic stimuli was reduced at all of the time points tested, scratching to compound 48/80 was modified by metformin only when the drug was injected 24 hours before this pruritogen. We also examined the colocalization of P-mTOR with gastrin-releasing peptide, a putative marker for some itch-sensitive primary afferents, and found that P-mTOR was coexpressed in less than 5% of gastrin-releasing peptide-positive fibers in the mouse skin. Taken together, the data highlight the role that P-mTOR-positive A-fibers play in itch signaling and underline the importance of the mTORC1 pathway in the regulation of homeostatic primary afferent functions such as pain and itch. The actions of the antidiabetic drug metformin in ameliorating nonhistamine-mediated itch also suggest a new therapeutic route for the control of this category of pruritus.

Topics: Animals; Disease Models, Animal; Gastrin-Releasing Peptide; Histamine; Hypoglycemic Agents; Male; Mechanistic Target of Rapamycin Complex 1; Metformin; Mice; Mice, Inbred C57BL; Multiprotein Complexes; Neuralgia; Phosphoproteins; Protein Kinase Inhibitors; Pruritus; Signal Transduction; Sirolimus; Skin; TOR Serine-Threonine Kinases; Treatment Outcome

To evaluate the effects of intrathecal injection of rapamycin on pain threshold and spinal cord glial activation in rats with neuropathic pain.. Healthy 30 male Sprague Dawley (SD) rats were randomly divided into six groups (n = 5 in each group): (1) control group without any treatments; (2) chronic constriction injury (CCI) group; (3) Early-rapamycin group with intrathecal injection of rapamycin 4 hours after CCI days; (4) Early-vehicle group with intrathecal injection of DMSO; (5) Late-rapamycin group with intrathecal injection of rapamycin 7 days after CCI; (6) Late-vehicle group with intrathecal injection of DMSO 7 days after CCI. Rapamycin or DMSO was injected for 3 consecutive days. Mechanical and thermal threshold were tested before and after the CCI operation. Lumbar segment of spinal cords was tested for glial fibrillary acidic protein (GFAP) by immunohistochemistry on 14th day after operation.. Mechanical and thermal hyperalgesia emerged on fourth day were maintained till fourteenth day after operation. After intrathecal injection of rapamycin 4 hours or 7 days after CCI, mechanical and thermal threshold significantly increased compared to injection of DMSO. The area of GFAP positive and the mean density of GFAP positive area in the dorsal horn of the ipsilateral side greatly increased in rapamycin-treated groups.. Intrathecal injection of rapamycin may attenuate CCI-induced hyperalgesia and inhibit the activation of astrocyte.

Topics: Analgesics; Animals; Astrocytes; Chronic Disease; Constriction, Pathologic; Disease Models, Animal; Glial Fibrillary Acidic Protein; Hot Temperature; Hyperalgesia; Injections, Spinal; Male; Neuralgia; Pain Threshold; Random Allocation; Rats, Sprague-Dawley; Sciatic Nerve; Sirolimus; Spinal Cord; Touch

The mammalian target of rapamycin (mTOR), a serine-threonine protein kinase, integrates extracellular signals, thereby modulating several physiological and pathological processes, including pain. Previous studies have suggested that rapamycin (an mTOR inhibitor) can attenuate nociceptive behaviors in many pain models, most likely at the spinal cord level. However, the mechanisms of mTOR at the supraspinal level, particularly at the level of the rostral ventromedial medulla (RVM), remain unclear. Thus, the aim of this study was to elucidate the role of mTOR in the RVM, a key relay region for the descending pain control pathway, under neuropathic pain conditions. Phosphorylated mTOR was mainly expressed in serotonergic spinally projecting neurons and was significantly increased in the RVM after spared nerve injury- (SNI-) induced neuropathic pain. Moreover, in SNI rat brain slices, rapamycin infusion both decreased the amplitude instead of the frequency of spontaneous excitatory postsynaptic currents and reduced the numbers of action potentials in serotonergic neurons. Finally, intra-RVM microinjection of rapamycin effectively alleviated established mechanical allodynia but failed to affect the development of neuropathic pain. In conclusion, our data provide strong evidence for the role of mTOR in the RVM in nerve injury-induced neuropathic pain, indicating a novel mechanism of mTOR inhibitor-induced analgesia.

Topics: Action Potentials; Animals; Hyperalgesia; Male; Medulla Oblongata; Neuralgia; Pain Threshold; Phosphorylation; Rats; Rats, Sprague-Dawley; Sciatic Nerve; Serotonergic Neurons; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

To investigate the effect of locally slow-released rapamycin (RAPA) from bionic peripheral nerve stent to reduce the incidence of neuropathic pain or mitigate the degree of pain after nerve injury.. We constructed a neural tissue engineering scaffold with sustained release of RAPA to repair 20mm defects in rat sciatic nerves. Four presurgical and postsurgical time windows were selected to monitor the changes in the expression of pain-related dorsal root ganglion (DRG) voltage-gated sodium channels 1.3 (Nav1.3), 1.7 (Nav1.7), and 1.8 (Nav1.8) through immunohistochemistry (IHC) and Western Blot, along with the observation of postsurgical pathological pain in rats by pain-related behavior approaches.. Relatively small upregulation of DRG sodium channels was observed in the experimental group (RAPA+poly(lactic-co-glycolic acid) (PLGA)+stent) after surgery, along with low degrees of neuropathic pain and anxiety, which were similar to those in the Autologous nerve graft group.. Autoimmune inflammatory response plays a leading role in the occurrence of post-traumatic neuropathic pain, and that RAPA significantly inhibits the abnormal upregulation of sodium channels to reduce pain by alleviating inflammatory response.

Topics: Animals; Delayed-Action Preparations; Drug-Eluting Stents; Ganglia, Spinal; Gene Expression; Immunosuppressive Agents; Lactic Acid; Male; NAV1.3 Voltage-Gated Sodium Channel; NAV1.7 Voltage-Gated Sodium Channel; NAV1.8 Voltage-Gated Sodium Channel; Neuralgia; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Rats; Rats, Sprague-Dawley; Sciatic Nerve; Sirolimus; Tissue Engineering; Tissue Scaffolds

Autophagy acts as an important homoeostatic mechanism by degradation of cytosolic constituents and plays roles in many physiological processes. Recent studies demonstrated that autophagy can also regulate the production and secretion of the proinflammatory cytokine interleukin-1β (IL-1β), which plays a critical role in the development and maintenance of neuropathic pain. In the present study, the paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were significantly decreased after spinal nerve ligation (SNL), and the changes were accompanied by inhibited autophagy in the spinal microglia and increased mRNA and protein levels of IL-1β in the ipsilateral spinal cord. We then investigated the antinociceptive effect of rapamycin, a widely used autopahgy inducer, on SNL-induced neuropathic pain in rats and found that treatment with intrathecal rapamycin significantly attenuated the mechanical allodynia and thermal hyperalgesia. Moreover, rapamycin significantly enhanced autophagy in the spinal microglia, whereas it reduced the mRNA and protein levels of IL-1β in the ipsilateral spinal cord. Our results showed that rapamycin could ameliorate neuropathic pain by activating autophagy and inhibiting IL-1β in the spinal cord.

Topics: Animals; Autophagy; Immunosuppressive Agents; Interleukin-1beta; Male; Neuralgia; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sirolimus; Spine

Protein translation controlled through activation of mammalian target of rapamycin (mTOR) participates in many physiological and pathological processes. However, whether such activation is required for chronic pain is still unknown. Here, we examined activation of the mTOR signaling pathway during complete Freund's adjuvant (CFA)-induced chronic inflammatory pain and L5 spinal nerve ligation (SNL)-induced neuropathic pain in rats. Western blot analysis showed significantly increased levels of phosphorylated mTOR (p-mTOR) and phosphorylated p70S6 kinase 1 (p-S6K1, a downstream effector of mTOR) in the ipsilateral L4/5 spinal cord 2h, 1 day, 3 days, and 7 days after intraplantar CFA injection and in the ipsilateral L4/5 dorsal root ganglions (DRGs) 1 and 3 days after CFA injection. Immunohistochemistry also demonstrated increases in number of p-mTOR-labeled neurons in the ipsilateral L4/5 DRGs and in density of p-mTOR-labeled immunoreactivity in the ipsilateral L4/5 superficial dorsal horn 1 day after CFA injection. Moreover, intrathecal administration of rapamycin, a selective inhibitor of mTOR, significantly blocked CFA-induced mechanical allodynia and thermal hyperalgesia 1 day post-CFA injection. Interestingly, expression of neither p-mTOR nor p-S6K1 was markedly altered on days 3, 7, or 14 after L5 SNL in L5 spinal cord or DRG. These findings indicate that in DRG and spinal cord, mTOR and S6K1 are activated during chronic inflammatory pain, but not during neuropathic pain. Our results strongly suggest that mTOR and its downstream pathway contribute to the development of chronic inflammatory pain.

Topics: Animals; Disease Models, Animal; Freund's Adjuvant; Functional Laterality; Ganglia, Spinal; Gene Expression Regulation; Hyperalgesia; Immunosuppressive Agents; Male; Neuralgia; Neurogenic Inflammation; Pain Measurement; Pain Threshold; Rats; Rats, Sprague-Dawley; Ribosomal Protein S6 Kinases; Signal Transduction; Sirolimus; Spinal Cord; Spinal Nerves; Statistics, Nonparametric; Time Factors; TOR Serine-Threonine Kinases

Current treatments used in Multiple Sclerosis (MS) are partly effective in the early stages of the disease but display very limited benefits in patients affected by progressive MS. One possible explanation is that these therapies are unable to target the inflammatory component most active during the progressive phase of the disease, and compartmentalized behind the blood-brain barrier. Our findings show that Rapamycin ameliorates clinical and histological signs of chronic EAE when administered during ongoing disease. Moreover, Rapamycin significantly reduced the hyperalgesia observed before clinical development of EAE which, in turn, is completely abolished by the administration of the drug.

Topics: Analysis of Variance; Animals; Cytokines; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Glycoproteins; Hyperalgesia; Immunosuppressive Agents; Mice; Mice, Inbred C57BL; Myelin Basic Protein; Myelin Sheath; Myelin-Oligodendrocyte Glycoprotein; Neuralgia; Pain Threshold; Peptide Fragments; Pertussis Toxin; RNA, Messenger; Sirolimus; T-Lymphocytes; Time Factors

The management of neuropathic pain is unsatisfactory, and new treatments are required. Because the sensitivity of a subset of fast-conducting primary afferent nociceptors is thought to be regulated by the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway, selectively targeting mTORC1 represents a new strategy for the control of chronic pain. Here we show that activated mTOR was expressed largely in myelinated sensory fibers in mouse and that inhibiting the mTORC1 pathway systemically alleviated mechanical hypersensitivity in mouse models of inflammatory and neuropathic pain. Specifically, systemic administration of mTORC1 inhibitor temsirolimus (CCI-779), both acutely (25 mg/kg i.p.) and chronically (4 daily 25 mg/kg i.p.), inhibited the mTORC1 pathway in sensory axons and the spinal dorsal horn and reduced mechanical and cold hypersensitivity induced by nerve injury. Moreover, systemic treatment with CCI-779 also reduced mechanical but not heat hypersensitivity in an inflammatory pain state. This treatment did not influence nociceptive thresholds in naive or sham-treated control animals. Also, there was no evidence for neuronal toxicity after repeated systemic treatment with CCI-779. Additionally, we show that acute and chronic i.p. administration of Torin1 (20 mg/kg), a novel ATP-competitive inhibitor targeting both mTORC1 and mTORC2 pathways, reduced the response to mechanical and cold stimuli in neuropathic mice. Our findings emphasize the importance of the mTORC1 pathway as a regulator of nociceptor sensitivity and therefore as a potential target for therapeutic intervention, particularly in chronic pain.

Topics: Animals; Hyperalgesia; Male; Mice; Mice, Inbred C57BL; Neural Inhibition; Neuralgia; Nociceptors; Posterior Horn Cells; Protein Kinase Inhibitors; Sirolimus; TOR Serine-Threonine Kinases

The protein kinase mammalian target of rapamycin (mTOR) regulates mRNA translation and is inhibited by rapamycin. Signaling pathways involving mTOR are implicated in physiological and pathophysiological processes. We determined the spinal effects of the rapamycin analogue cell cycle inhibitor (CCI)-779 on neuronal responses and behavioral hypersensitivity in a model of persistent neuropathic pain. We also assessed the anatomical distribution of spinal mTOR signaling pathways. Specifically, we ligated rat spinal nerves L5 and L6 to produce a model of neuropathic pain. After confirming neuropathy with behavioral testing, we obtained in vivo single-unit extracellular stimulus-evoked recordings from deep dorsal horn spinal neurons. We applied CCI-779 spinally in electrophysiological and behavioral studies and assessed its effects accordingly. We also used immunohistochemistry to probe for mTOR signaling pathways in dorsal root ganglia (DRG) and the spinal cord. We found that spinally administered CCI-779 rapidly attenuated calibrated mechanically but not thermally evoked neuronal responses and mechanically evoked behavioral responses. Immunohistochemistry showed presence of mTOR signaling pathways in nociceptive-specific C-fiber DRG and in neurons of inner lamina II of the spinal cord. We conclude that alterations in the activity of spinal mTOR signaling pathways are crucial to the full establishment of spinal neuronal plasticity and behavioral hypersensitivity associated with nerve injury.. This study is consistent with growing evidence implicating mTOR signaling pathways as important modulators of persistent pain, providing novel insights into the molecular mechanisms of pain maintenance and potential for novel approaches into treating chronic pain.

Topics: Animals; Behavior, Animal; Electrophysiology; Hyperalgesia; Immunohistochemistry; Male; Neuralgia; Neuronal Plasticity; Peripheral Nervous System Diseases; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Signal Transduction; Sirolimus; Spinal Cord; Spinal Nerves; TOR Serine-Threonine Kinases

Misexpression and cytosolic retention of peripheral myelin protein 22 (PMP22) within Schwann cells (SCs) is associated with a genetically heterogeneous group of demyelinating peripheral neuropathies. PMP22 overproducer C22 and spontaneous mutant Trembler J (TrJ) mice display neuropathic phenotypes and affected nerves contain abnormally localized PMP22. Nutrient deprivation-induced autophagy is able to suppress the formation of PMP22 aggregates in a toxin-induced cellular model, and improve locomotor performance and myelination in TrJ mice. As a step toward therapies, we assessed whether pharmacological activation of autophagy by rapamycin (RM) could facilitate the processing of PMP22 within neuropathic SCs and enhance their capacity to myelinate peripheral axons. Exposure of mouse SCs to RM induced autophagy in a dose- and time-dependent manner and decreased the accumulation of poly-ubiquitinated substrates. The treatment of myelinating dorsal root ganglion (DRG) explant cultures from neuropathic mice with RM (25 nm) improved the processing of PMP22 and increased the abundance and length of myelin internodes, as well as the expression of myelin proteins. Notably, RM is similarly effective in both the C22 and TrJ model, signifying that the benefit overlaps among distinct genetic models of PMP22 neuropathies. Furthermore, lentivirus-mediated shRNA knockdown of the autophagy-related gene 12 (Atg12) abolished the activation of autophagy and the increase in myelin proteins, demonstrating that autophagy is critical for the observed improvement. Together, these results support the potential use of RM and other autophagy-enhancing compounds as therapeutic agents for PMP22-associated demyelinating neuropathies.

Topics: Animals; Autophagy; Demyelinating Diseases; Female; Gene Knockdown Techniques; Male; Mice; Mice, Neurologic Mutants; Myelin Sheath; Nerve Fibers, Myelinated; Neuralgia; Organ Culture Techniques; Sirolimus