A subset of spinal dorsal horn interneurons crucial for gating touch-evoked pain-like behavior.

A cardinal, intractable symptom of neuropathic pain is mechanical allodynia, pain caused by innocuous stimuli via low-threshold mechanoreceptors such as Aß fibers. However, the mechanism by which Aß fiber-derived signals are converted to pain remains incompletely understood. Here we identify a subset of inhibitory interneurons in the spinal dorsal horn (SDH) operated by adeno-associated viral vectors incorporating a neuropeptide Y promoter (AAV-NpyP+) and show that specific ablation or silencing of AAV-NpyP+ SDH interneurons converted touch-sensing Aß fiber-derived signals to morphine-resistant pain-like behavioral responses. AAV-NpyP+ neurons received excitatory inputs from Aß fibers and transmitted inhibitory GABA signals to lamina I neurons projecting to the brain. In a model of neuropathic pain developed by peripheral nerve injury, AAV-NpyP+ neurons exhibited deeper resting membrane potentials, and their excitation by Aß fibers was impaired. Conversely, chemogenetic activation of AAV-NpyP+ neurons in nerve-injured rats reversed Aß fiber-derived neuropathic pain-like behavior that was shown to be morphine-resistant and reduced pathological neuronal activation of superficial SDH including lamina I. These findings suggest that identified inhibitory SDH interneurons that act as a critical brake on conversion of touch-sensing Aß fiber signals into pain-like behavioral responses. Thus, enhancing activity of these neurons may offer a novel strategy for treating neuropathic allodynia.

The Role of Microglial Purinergic Receptors in Pain Signaling.

Pain is an essential modality of sensation in the body. Purinergic signaling plays an important role in nociceptive pain transmission, under both physiological and pathophysiological conditions, and is important for communication between both neuronal and non-neuronal cells. Microglia and astrocytes express a variety of purinergic effectors, and a variety of receptors play critical roles in the pathogenesis of neuropathic pain. In this review, we discuss our current knowledge of purinergic signaling and of the compounds that modulate purinergic transmission, with the aim of highlighting the importance of purinergic pathways as targets for the treatment of persistent pain.

Direct energy spectrum measurement of X-ray from a clinical linac.

A realistic X-ray energy spectrum is essential for accurate dose calculation using the Monte Carlo (MC) algorithm. An energy spectrum for dose calculation in the radiation treatment planning system is modeled using the MC algorithm and adjusted to obtain acceptable agreement with the measured percent depth dose (PDD) and off-axis ratio. The simulated energy spectrum may not consistently reproduce a realistic energy spectrum. Therefore, direct measurement of the X-ray energy spectrum from a linac is necessary to obtain a realistic spectrum. Previous studies have measured low photon fluence directly, but the measurement was performed with a nonclinical linac with a thick target and a long target-to-detector distance. In this study, an X-ray energy spectrum from a clinical linac was directly measured using a NaI(Tl) scintillator at an ultralow dose rate achieved by adjusting the gun grid voltage. The measured energy spectrum was unfolded by the Gold algorithm and compared with a simulated spectrum using statistical tests. Furthermore, the PDD was calculated using an unfolded energy spectrum and a simulated energy spectrum was compared with the measured PDD to evaluate the validity of the unfolded energy spectrum. Consequently, there was no significant difference between the unfolded and simulated energy spectra by nonparametric, Wilcoxon's rank-sum, chi-square, and two-sample Kolmogorov-Smirnov tests with a significance level of 0.05. However, the PDD calculated from the unfolded energy spectrum better agreed with the measured compared to the calculated PDD results from the simulated energy spectrum. The adjustment of the incident electron parameters using MC simulation is sensitive and takes time. Therefore, it is desirable to obtain the energy spectrum by direct measurement. Thus, a method to obtain the realistic energy spectrum by direct measurement was proposed in this study.

Sensitization of spinal itch transmission neurons in a mouse model of chronic itch requires an astrocytic factor.

BACKGROUND: Chronic itch is a highly debilitating symptom among patients with inflammatory skin diseases. Recent studies have revealed that gastrin-releasing peptide (GRP) and its receptor (gastrin-releasing peptide receptor [GRPR]) in the spinal dorsal horn (SDH) play a central role in itch transmission. OBJECTIVE: We aimed to investigate whether GRP-GRPR signaling is altered in SDH neurons in a mouse model of chronic itch and to determine the potential mechanisms underlying these alterations. METHODS: Patch-clamp recordings from enhanced green fluorescent protein (EGFP)-expressing (GRPR+) SDH neurons were used to examine GRP-GRPR signaling in spinal cord slices obtained from Grpr-EGFP mice. Immunohistochemical, genetic (gene expression and editing through adeno-associated virus vectors), and behavioral approaches were also used for in vivo experiments. RESULTS: We observed potentiation of GRP-evoked excitation in the GRPR+ SDH neurons of mice with contact dermatitis, without concomitant changes in GRPR expression. Interestingly, increases in excitation were attenuated by suppressing the reactive state of SDH astrocytes, which are known to be reactive in patients with chronic itch conditions. Furthermore, CRISPR-Cas9-mediated astrocyte-selective in vivo editing of a gene encoding lipocalin-2 (LCN2), an astrocytic factor implicated in chronic itch, suppressed increases in GRP-induced excitation of GRPR+ neurons, repetitive scratching, and skin damage in mice with contact dermatitis. Moreover, LCN2 potentiated GRP-induced excitation of GRPR+ neurons in normal mice. CONCLUSION: Our findings indicate that, under chronic itch conditions, the GRP-induced excitability of GRPR+ SDH neurons is enhanced through a non-cell-autonomous mechanism involving LCN2 derived from reactive astrocytes.

Mechanical pain of the lower extremity after compression of the upper spinal cord involves signal transducer and activator of transcription 3-dependent reactive astrocytes and interleukin-6.

Chronic pain is one of the main symptoms of spinal disorders such as spinal canal stenosis. A major cause of this pain is related to compression of the spinal cord, and chronic pain can develop at the level of the compressed spinal segment. However, in many patients chronic pain arises in an area that does not correspond to the compressed segment, and the underlying mechanism involved remains unknown. This was investigated in the present study using a mouse model of spinal cord compression in which mechanical pain of the hindpaws develops after compression of the first lumbar segment (L1) of the spinal cord. Compression induced the activation of astrocytes in the L1 spinal dorsal horn (SDH)-but not the L4 SDH that corresponds to the hindpaws-and activated signal transducer and activator of transcription 3 (STAT3). Suppressing reactive astrocytes by expressing a dominant negative form of STAT3 (dnSTAT3) in the compressed SDH prevented mechanical pain. Expression of interleukin (IL)-6 was also upregulated in the compressed SDH, and it was inhibited by astrocytic expression of dnSTAT3. Intrathecal administration of a neutralizing anti-IL-6 antibody reversed the compression-induced mechanical pain. These results suggest that astrocytic STAT3 and IL-6 in the compressed SDH are involved in remote mechanical pain observed in the lower extremity, and may provide a target for treating chronic pain associated with spinal cord compression such as spinal canal stenosis.

Involvement of exchange protein directly activated by cAMP and tumor progression locus 2 in IL-1ß production in microglial cells following activation of ß-adrenergic receptors.

Endogenous noradrenaline (NA) has multiple bioactive functions and, in the central nervous system (CNS), has been implicated in modulating neuroinflammation via ß-adrenergic receptors (ß-ARs). Microglia, resident macrophages in the CNS, have a central role in the brain immune system and have been reported to be activated by NA. However, intracellular signaling mechanisms of the AR-mediated proinflammatory responses of microglia are not fully understood. Using a rapid and stable in vitro reporter assay system to evaluate IL-1ß production in microglial BV2 cells, we found that NA and the ß-AR agonist isoproterenol upregulated the IL-1ß reporter activity. This effect was suppressed by ß-AR antagonists. We further examined the involvement of EPAC (exchange protein directly activated by cAMP) and TPL2 (tumor progression locus 2, MAP3K8) and found that inhibitors for EPAC and TPL2 reduced AR agonist-induced IL-1ß reporter activity. These inhibitors also suppressed NA-induced endogenous Il1b mRNA expression and IL-1ß protein production. Our results suggest that EPAC and TPL2 are involved in ß-AR-mediated IL-1ß production in microglial cells, and extend our understanding of its intracellular signaling mechanism.

Transcription factor MafB contributes to the activation of spinal microglia underlying neuropathic pain development.

Microglia, which are pathological effectors and amplifiers in the central nervous system, undergo various forms of activation. A well-studied microglial-induced pathological paradigm, spinal microglial activation following peripheral nerve injury (PNI), is a key event for the development of neuropathic pain but the transcription factors contributing to microglial activation are less understood. Herein, we demonstrate that MafB, a dominant transcriptional regulator of mature microglia, is involved in the pathology of a mouse model of neuropathic pain. PNI caused a rapid and marked increase of MafB expression selectively in spinal microglia but not in neurons. We also found that the microRNA mir-152 in the spinal cord which targets MafB expression decreased after PNI, and intrathecal administration of mir-152 mimic suppressed the development of neuropathic pain. Reduced MafB expression using heterozygous Mafb deficient mice and by intrathecal administration of siRNA alleviated the development of PNI-induced mechanical hypersensitivity. Furthermore, we found that intrathecal transfer of Mafb deficient microglia did not induce mechanical hypersensitivity and that conditional Mafb knockout mice did not develop neuropathic pain after PNI. We propose that MafB is a key mediator of the PNI-induced phenotypic alteration of spinal microglia and neuropathic pain development.

New pharmacological effect of fulvestrant to prevent oxaliplatin-induced neurodegeneration and mechanical allodynia in rats.

Oxaliplatin, which is widely used as chemotherapy for certain solid cancers, frequently causes peripheral neuropathy. Commonly described neuropathic symptoms include aberrant sensations such as mechanical allodynia (hypersensitivity to normally innocuous stimuli). Although oxaliplatin neuropathy is a dose-limiting toxicity, there are no established preventive strategies available at present. By screening several sets of small-molecule chemical libraries (more than 3,000 compounds in total) using a newly established in vitro high-throughput phenotypic assay, we identified fulvestrant, a clinically approved drug for the treatment of breast cancer in postmenopausal women, as having a protective effect on oxaliplatin-induced neuronal damage. Furthermore, histological and behavioural analyses using a rat model of oxaliplatin neuropathy demonstrated the in vivo efficacy of fulvestrant to prevent oxaliplatin-induced axonal degeneration of the sciatic nerve and mechanical allodynia. Furthermore, fulvestrant did not interfere with oxaliplatin-induced cytotoxicity against cancer cells. Thus, our findings reveal a previously unrecognised pharmacological effect of fulvestrant to prevent oxaliplatin-induced painful peripheral neuropathy without impairing its cytotoxicity against cancer cells and may represent a novel prophylactic option for patients receiving oxaliplatin chemotherapy.

Evidence for detection of rat P2X4 receptor expressed on cells by generating monoclonal antibodies recognizing the native structure.

P2X purinergic receptors are ATP-driven ionic channels expressed as trimers and showing various functions. A subtype, the P2X4 receptor present on microglial cells is highly involved in neuropathic pain. In this study, in order to prepare antibodies recognizing the native structure of rat P2X4 (rP2X4) receptor, we immunized mice with rP2X4's head domain (rHD, Gln111-Val167), which possesses an intact structure stabilized by S-S bond formation (Igawa and Abe et al. FEBS Lett. 2015), as an antigen. We generated five monoclonal antibodies with the ability to recognize the native structure of its head domain, stabilized by S-S bond formation. Site-directed mutagenesis revealed that Asn127 and Asp131 of the rHD, in which combination of these amino acid residues is only conserved in P2X4 receptor among P2X family, were closely involved in the interaction between rHD and these antibodies. We also demonstrated the antibodies obtained here could detect rP2X4 receptor expressed in 1321N1 human astrocytoma cells.

Evaluation of gantry angle during respiratory-gated VMAT using triggered kilovoltage x-ray image.

Respiratory-gated volumetric modulated arc therapy (gated VMAT) involves further complexities to the dose delivery process because the gantry rotation must repeatedly stop and restart according to the gating signals. In previous studies, the gantry rotation performances were evaluated by the difference between the plan and the machine log. However, several reports pointed out that log analysis does not sufficiently replicate the machine performance. In this report, a measurement-based quality assurance of the relation between the gantry angle and gate-on or gate-off using triggered kilovoltage imaging and a cylinder phantom with 16 ball bearings is proposed. For the analysis, an in-house program that estimates and corrects the phantom offset was developed. The gantry angle in static and gated arc delivery was compared between the machine log and the proposed method. The gantry was set every 5 deg through its full motion range in static delivery, and rotated at three speeds (2, 4 and 6 deg s-1 ) with different gating intervals (1.5 or 3.0 s) in gated arc delivery. The mean and standard deviation of the angular differences between the log and the proposed method was -0.05 deg ± 0.12 deg in static delivery. The mean of the angular difference was within ±0.10 deg and the largest difference was 0.41 deg in gated arc delivery. The log records the output of the encoder so that miscalibration and mechanical sagging will be disregarded. However, the proposed method will help the users to detect the mechanical issues due to the repeated gantry stops and restarts in gated VMAT.

Astrocytic Ca2+ responses in the spinal dorsal horn by noxious stimuli to the skin.

The role of astrocytes in the spinal dorsal horn (SDH) for sensory information processing under normal conditions is poorly understood. In this study, we investigated whether SDH astrocytes respond to noxious and innocuous stimuli to the skin of normal mice using in vivo two-photon Ca2+ imaging under anesthesia. We found that noxious stimulation evoked by intraplantar formalin injection provoked an elevation in intracellular Ca2+ levels in SDH astrocytes. By contrast, neither instantaneous noxious pinching nor innocuous stimuli (cooling or brushing) to the hindpaw elicited astrocytic Ca2+ responses. Thus, SDH astrocytes could respond preferentially to a strong and/or sustained noxious stimulus.

Temporal Kinetics of Microgliosis in the Spinal Dorsal Horn after Peripheral Nerve Injury in Rodents.

Neuropathic pain, a highly debilitating chronic pain following nerve damage, is a reflection of the aberrant functioning of a pathologically altered nervous system. Previous studies have implicated activated microglia in the spinal dorsal horn (SDH) as key cellular intermediaries in neuropathic pain. Microgliosis is among the dramatic cellular alterations that occur in the SDH in models of neuropathic pain established by peripheral nerve injury (PNI), but detailed characterization of SDH microgliosis has yet to be realized. In the present study, we performed a short-pulse labeling of proliferating cells with ethynyldeoxyuridine (EdU), a marker of the cell cycle S-phase, and found that EdU+ microglia in the SDH were rarely observed 32 h after PNI, but rapidly increased to the peak level at 40 h post-PNI. Numerous EdU+ microglia persisted for the next 20 h (60 h post-PNI) and decreased to the baseline on day 7. These results demonstrate a narrow time window for rapidly inducing a proliferation burst of SDH microglia after PNI, and these temporally restricted kinetics of microglial proliferation may help identify the molecule that causes microglial activation in the SDH, which is crucial for understanding and managing neuropathic pain.

Density scaling of phantom materials for a 3D dose verification system.

In this study, the optimum density scaling factors of phantom materials for a commercially available three-dimensional (3D) dose verification system (Delta4) were investigated in order to improve the accuracy of the calculated dose distributions in the phantom materials. At field sizes of 10 × 10 and 5 × 5 cm2 with the same geometry, tissue-phantom ratios (TPRs) in water, polymethyl methacrylate (PMMA), and Plastic Water Diagnostic Therapy (PWDT) were measured, and TPRs in various density scaling factors of water were calculated by Monte Carlo simulation, Adaptive Convolve (AdC, Pinnacle3 ), Collapsed Cone Convolution (CCC, RayStation), and AcurosXB (AXB, Eclipse). Effective linear attenuation coefficients (µeff ) were obtained from the TPRs. The ratios of µeff in phantom and water ((µeff )pl,water ) were compared between the measurements and calculations. For each phantom material, the density scaling factor proposed in this study (DSF) was set to be the value providing a match between the calculated and measured (µeff )pl,water . The optimum density scaling factor was verified through the comparison of the dose distributions measured by Delta4 and calculated with three different density scaling factors: the nominal physical density (PD), nominal relative electron density (ED), and DSF. Three plans were used for the verifications: a static field of 10 × 10 cm2 and two intensity modulated radiation therapy (IMRT) treatment plans. DSF were determined to be 1.13 for PMMA and 0.98 for PWDT. DSF for PMMA showed good agreement for AdC and CCC with 6 MV x ray, and AdC for 10 MV x ray. DSF for PWDT showed good agreement regardless of the dose calculation algorithms and x-ray energy. DSF can be considered one of the references for the density scaling factor of Delta4 phantom materials and may help improve the accuracy of the IMRT dose verification using Delta4.

P2Y12 receptors in primary microglia activate nuclear factor of activated T-cell signaling to induce C-C chemokine 3 expression.

Microglia are widely accepted as surveillants in the central nervous system that are continually searching the local environment for signs of injury. Following an inflammatory situation, microglia alter their morphology, extend ramified processes, and undergo cell body hypertrophy. Extracellular nucleotides are recognized as a danger signal by microglia. ADP acting on P2Y12 receptors induce process extension of microglia thereby attracting microglia to the site of adenosine tri-phosphate/ADP leaking or release. However, the question whether ADP/P2Y12 receptor signaling directly stimulates the production or release of inducible factors such as cytokines remains unclear. In this study, we found that CC chemokine ligand 3 (CCL3) is induced by ADP-treated primary microglia. Pharmacological characterization using pertussis toxin, a P2Y12 receptor inhibitor, and a calcium chelator revealed that CCL3 induction was caused by P2Y12 receptor-mediated intracellular calcium elevation. Next, nuclear factor of activated T-cell dephosphorylation and nuclear translocalization were observed. Calcineurin, an inhibitor for nuclear factor of activated T cell, suppressed CCL3 induction. These data indicate that microglial P2Y12 receptors are utilized to trigger an acute inflammatory response in microglia via rapid CCL3 induction after ADP stimulation.

Contrast enhancement for portal images by combination of subtraction and reprojection processes for Compton scattering.

For patient setup of the IGRT technique, various imaging systems are currently available. MV portal imaging is performed in identical geometry with the treatment beam so that the portal image provides accurate geometric information. However, MV imaging suffers from poor image contrast due to larger Compton scatter photons. In this work, an original image processing algorithm is proposed to improve and enhance the image contrast without increasing the imaging dose. Scatter estimation was performed in detail by MC simulation based on patient CT data. In the image processing, scatter photons were eliminated and then they were reprojected as primary photons on the assumption that Compton interaction did not take place. To improve the processing efficiency, the dose spread function within the EPID was investigated and implemented on the developed code. Portal images with and without the proposed image processing were evaluated by the image contrast profile. By the subtraction process, the image contrast was improved but the EPID signal was weakened because 15.2% of the signal was eliminated due to the contribution of scatter photons. Hence, these scatter photons were reprojected in the reprojection process. As a result, the tumor, bronchi, mediastinal space and ribs were observed more clearly than in the original image. It was clarified that image processing with the dose spread functions provides stronger contrast enhancement while maintaining a sufficient signal-to-noise ratio. This work shows the feasibility of improving and enhancing the contrast of portal images.

Neuronal CCL21 up-regulates microglia P2X4 expression and initiates neuropathic pain development.

Up-regulation of P2X4 receptors in spinal cord microglia is crucial for tactile allodynia, an untreatable pathological pain reaction occurring after peripheral nerve injury. How nerve injury in the periphery leads to this microglia reaction in the dorsal horn of the spinal cord is not yet understood. It is shown here that CCL21 was rapidly expressed in injured small-sized primary sensory neurons and transported to their central terminals in the dorsal horn. Intrathecal administration of a CCL21-blocking antibody diminished tactile allodynia development in wild-type animals. Mice deficient for CCL21 did not develop any signs of tactile allodynia and failed to up-regulate microglial P2X4 receptor expression. Microglia P2X4 expression was enhanced by CCL21 application in vitro and in vivo. A single intrathecal injection of CCL21 to nerve-injured CCL21-deficient mice induced long-lasting allodynia that was undistinguishable from the wild-type response. This effect of CCL21 injection was strictly dependent on P2X4 receptor function. Since neuronal CCL21 is the earliest yet identified factor in the cascade leading to tactile allodynia, these findings may lead to a preventive therapy in neuropathic pain.

Transcription factor IRF1 is responsible for IRF8-mediated IL-1ß expression in reactive microglia.

Interferon regulatory factor-8 (IRF8) plays a crucial role in the transformation of microglia to a reactive state by regulating the expression of various genes. In the present study, we show that IRF1 is required for IRF8-induced gene expression in microglia. Peripheral nerve injury induced IRF1 gene upregulation in the spinal microglia in an IRF8-dependent manner. IRF8 transduction in cultured microglia induced de novo gene expression of IRF1. Importantly, knockdown of the IRF1 gene in IRF8-transduced microglia prevented upregulation of interleukin-1ß (IL-1ß). Therefore, our findings suggest that expression of IL-1ß is dependent on IRF1 in IRF8-expressing reactive microglia.

Involvement of the chemokine CCL3 and the purinoceptor P2X7 in the spinal cord in paclitaxel-induced mechanical allodynia.

BACKGROUND: Paclitaxel is an effective chemotherapeutic agent widely used for the treatment of solid tumors. The major dose-limiting toxicity of paclitaxel is peripheral neuropathy. The mechanisms underlying the development and maintenance of paclitaxel-induced peripheral neuropathy are still unclear, and there are no currently established effective treatments. Accumulating evidence in models of neuropathic pain in which peripheral nerves are lesioned has implicated spinal microglia and chemokines in pain hypersensitivity, but little is know about their roles in chemotherapy-induced peripheral neuropathy. In the present study, we investigated the role of CC-chemokine ligand 3 (CCL3) in the spinal cord in the development and maintenance of mechanical allodynia using a rat model of paclitaxel-induced neuropathy. FINDINGS: Repeated intravenous administration of paclitaxel induced a marked decrease in paw withdrawal threshold in response to mechanical stimulation (mechanical allodynia). In these rats, the number of microglia in the spinal dorsal horn (SDH) was significantly increased. Paclitaxel-treated rats showed a significant increase in the expression of mRNAs for CCL3 and its receptor CCR5 in the SDH. Intrathecal administration of a CCL3-neutralizing antibody not only attenuated the development of paclitaxel-induced mechanical allodynia but also reversed its maintenance. Paclitaxel also upregulated expression of purinoceptor P2X7 receptors (P2X7Rs), which have been implicated in the release of CCL3 from microglia, in the SDH. The selective P2X7R antagonist A438079 had preventive and reversal effects on paclitaxel-induced allodynia. CONCLUSIONS: Our findings suggest a contribution of CCL3 and P2X7Rs in the SDH to paclitaxel-induced allodynia and may provide new therapeutic targets for paclitaxel-induced painful neuropathy.

Rice husk-derived graphene with nano-sized domains and clean edges.

A new synthetic method is demonstrated for transforming rice husks into bulk amounts of graphene through its calcination and chemical activation. The bulk sample consists of crystalline nano-sized graphene and corrugated individual graphene sheets; the material generally contains one, two, or a few layers, and corrugated graphene domains are typically observed in monolayers containing topological defects within the hexagonal lattice and edges. Both types of graphenes exhibit atomically smooth surfaces and edges.

Chemokine (C-C motif) receptor 5 is an important pathological regulator in the development and maintenance of neuropathic pain.

BACKGROUND: The chemokine family has been revealed to be involved in the pathogenesis of neuropathic pain. In this study, the authors investigated the role of chemokine (C-C motif) ligand 3 and its receptors chemokine (C-C motif) receptor 1 and chemokine (C-C motif) receptor (CCR) 5 in neuropathic pain. METHODS: A spinal nerve injury model was established in adult male Wistar rats. The von Frey test and hot plate test were performed to evaluate neuropathic pain behavior, and real-time quantitative reverse transcription polymerase chain reaction, in situ hybridization, and immunohistochemistry were performed to understand the molecular mechanisms. RESULTS: The expression levels of chemokine (C-C motif) ligand 3 and CCR5 messenger RNA in the spinal cord were up-regulated after nerve injury, which was possibly due to CD11b-positive microglia. Single intrathecal administration of recombinant chemokine (C-C motif) ligand 3 produced biphasic tactile allodynia; each phase of pain behavior was induced by different receptors. Intrathecal injection of CCR5 antagonist suppressed the development of tactile allodynia (12.81 ± 1.33 g vs. 3.52 ± 0.41 g [mean ± SEM, drug vs. control in paw-withdrawal threshold]; P < 0.05, n = 6 each) and could reverse established tactile allodynia (10.87 ± 0.91 g vs. 3.43 ± 0.28 g; P < 0.05, n = 8 and 7). Furthermore, Oral administration of CCR5 antagonist could reverse established tactile allodynia (8.20 ± 1.27 g vs. 3.18 ± 0.46 g; P < 0.05, n = 4 each). CONCLUSIONS: Pharmacological blockade of CCR5 was effective in the treatment of the development and maintenance phases of neuropathic pain. Thus, CCR5 antagonists may be potential new drugs for the treatment of neuropathic pain.