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Aim: Amitriptyline (AMI) has been used to treat neuropathic pain. However, the clinical outcomes remain unsatisfactory, presumably due to a limited understanding of the underlying molecular mechanisms. Here, we investigated a drug repositioning strategy using a low-dose of AMI encapsulated in poly (D, L lactic-co-glycolic acid) (PLGA) nanoparticles (AMI NPs) for neuropathic pain, since PLGA nanoparticles are known to enhance delivery to microglia. Methods: We evaluated the anti-allodynic effects of AMI and AMI NPs on neuropathic pain by assessing behaviors and inflammatory responses in a rat model of spinal nerve ligation (SNL). While the anti-allodynic effect of AMI (30 μg) drug injection on SNL-induced neuropathic pain persisted for 12 h, AMI NPs significantly alleviated mechanical allodynia for 3 days. Results: Histological and cytokine analyses showed AMI NPs facilitated the reduction of microglial activation and pro-inflammatory mediators in the spinal dorsal horn. This study suggests that AMI NPs can provide a sustained anti-allodynic effect by enhancing the targeting of microglia and regulating the release of pro-inflammatory cytokines from activated microglia. Conclusion: Our findings suggest that the use of microglial-targeted NPs continuously releasing AMI (2 μg) as a drug repositioning strategy offers long-term anti-allodynic effects. Graphical Abstract Article highlights This study investigated a drug repositioning strategy using a low-dose of AMI encapsulated in poly (D, L lactic-co-glycolic acid) (PLGA) nanoparticles (AMI NPs) for neuropathic pain, since PLGA nanoparticles are known to enhance delivery to microglia. While the anti-allodynic effect of AMI (30 μg) drug injection on SNL-induced neuropathic pain persisted for 12 h, AMI NPs significantly alleviated mechanical allodynia for 3 days. This study suggests that AMI NPs can provide a sustained anti-allodynic effect by enhancing the targeting of microglia and regulating the release of pro-inflammatory cytokines from activated microglia. [ABSTRACT FROM AUTHOR]

Neuropathic pain is caused by injury or disease of the somatosensory system, and its course is usually chronic. Several studies have been dedicated to investigating neuropathic pain-related targets; however, little attention has been paid to the persistent alterations that these targets, some of which may be crucial to the pathophysiology of neuropathic pain. The present study aimed to identify potential targets that may play a crucial role in neuropathic pain and validate their long-term impact. Through bioinformatics analysis of RNA sequencing results, we identified Slc9a1 and validated the reduced expression of sodium-hydrogen exchanger 1 (NHE1), the protein that Slc9a1 encodes, in the spinal nerve ligation (SNL) model. Colocalization analysis revealed that NHE1 is primarily co-localized with vesicular glutamate transporter 2-positive neurons. In vitro experiments confirmed that poly(lactic-co-glycolic acid) nanoparticles loaded with siRNA successfully inhibited NHE1 in SH-SY5Y cells, lowered intracellular pH, and increased intracellular calcium concentrations. In vivo experiments showed that sustained suppression of spinal NHE1 expression by siRNA-loaded nanoparticles resulted in delayed hyperalgesia in naïve and SNL model rats, whereas amiloride-induced transient suppression of NHE1 expression yielded no significant changes in pain sensitivity. We identified Slc9a1 , which encodes NHE1, as a key gene in neuropathic pain. Utilizing the sustained release properties of nanoparticles enabled us to elucidate the chronic role of decreased NHE1 expression, establishing its significance in the mechanisms of neuropathic pain. [ABSTRACT FROM AUTHOR]

Aims: We investigated whether miRNA (miR) 146a-5p-loaded nanoparticles (NPs) can attenuate neuropathic pain behaviors in the rat spinal nerve ligation-induced neuropathic pain model by inhibiting activation of the NF-κB and p38 MAPK pathways in spinal microglia. Materials & methods: After NP preparation, miR NPs were assessed for their physical characteristics and then injected intrathecally into the spinal cords of rat spinal nerve ligation rats to test their analgesic effects. Results: miR NPs reduced pain behaviors for 11 days by negatively regulating the inflammatory response in spinal microglia. Conclusion: The anti-inflammatory effects of miR 146a-5p along with nanoparticle-based materials make miR NPs promising tools for treating neuropathic pain. [ABSTRACT FROM AUTHOR]

Microglia play a critical role in neuropathic pain. Since upregulated Foxp3 in microglia enhances tissue repair by resolving neuroinflammation in excitotoxin-induced neuronal death, it may attenuate neuropathic pain in a similar manner. Therefore, this study tests whether Foxp3 introduced with poly (D, L-lactic-co-glycolic acid) (PLGA) nanoparticles (Foxp3 NPs) can alleviate neuropathic pain by inhibiting microglia activity. The prepared Foxp3 NPs had an anti-inflammatory effect on lipopolysaccharide-stimulated BV2 cells in vitro , and localized to spinal microglia in vivo. Further, the Foxp3 NPs significantly attenuated pain behavior induced by spinal nerve ligation in rats for 7 days by suppressing microglial activity, followed by the downregulation of pro-nociceptive genes and the upregulation of anti-nociceptive genes in the spinal dorsal horn. Collectively, these data suggest that Foxp3 NPs effectively relieve neuropathic pain in animals by reducing microglia activity and subsequent modulation of neuroinflammation, and may be of therapeutic value in the treatment of neuropathic pain. Neuropathic pain was induced by lumbar 5 spinal nerve ligation in rats. NC or Foxp3 plasmids-encapsulated PLGA nanoparticles (NC NPs or Foxp3 NPs) were then administered intrathecally into the spinal subarachnoid space of rats with neuropathic pain. Pain behavior in von Frey filaments tests was significantly attenuated in rats with the Foxp3 NPs compared with the NC NPs. Unlabelled Image [ABSTRACT FROM AUTHOR]

Aim: To investigate whether p38 small-interfering RNA-loaded nanoparticles (p38 siRNA NPs) attenuate spinal nerve ligation (SNL)-induced neuropathic pain in rats by suppressing spinal microglia activation via p38 targeting. Materials & methods: After synthesizing p38 siRNA NPs with sonication, physical characteristics were measured for size and zeta potential. p38 siRNA NPs were then administrated intrathecally into SNL rats if they could reduce pain behavior excellently. Results: p38 siRNA NPs significantly reduced mechanical allodynia as well as microgliosis in the spinal dorsal horns of SNL rats, consistent with a downregulation of p38-related proinflammatory mediators. Conclusion: As p38 in the spinal microglia plays a critical role in neuropathic pain, we expect that p38 siRNA NPs could be a promising tool for the treatment of neuropathic pain. [ABSTRACT FROM AUTHOR]

Targeting microglial activation is emerging as a clinically promising drug target for neuropathic pain treatment. Fexofenadine, a histamine receptor 1 antagonist, is a clinical drug for the management of allergic reactions as well as pain and inflammation. However, the effect of fexofenadine on microglial activation and pain behaviors remains elucidated. Here, we investigated nanomedicinal approach that targets more preferentially microglia and long-term analgesics. Fexofenadine significantly abolished histamine-induced microglial activation. The fexofenadine-encapsulated poly(lactic-co-glycolic acid) nanoparticles (Fexo NPs) injection reduced the pain sensitivity of spinal nerve ligation rats in a dose-dependent manner. This alleviation was sustained for 4 days, whereas the effective period by direct fexofenadine injection was 3 h. Moreover, Fexo NPs inhibited microglial activation, inflammatory signaling, cytokine release, and a macrophage phenotype shift towards the alternative activated state in the spinal cord. These results show that Fexo NPs exhibit drug repositioning promise as a long-term treatment modality for neuropathic pain. Fexofenadine-encapsulated PLGA nanoparticles (Fexo NPs) and negative control nanoparticles (NC NPs) were generated by sonication with the typical double emulsion (W/O/W) method. As a neuropathic pain model, lumbar 5 spinal nerve ligated rats were administered intrathecally into the spinal subarachnoid space with/without fexofenadine alone, NC NPs, and Fexo NPs. Then, the thermal hyperalgesia was assessed as paw withdrawal latency (PWL) by using Hargreaves Apparatus. The results highlighted that fexofenadine-loaded nanoparticles prolong pain relief in SNL rats. Fexo NPs also reduced M1/M2 macrophage ratio and inhibited cytokine release. [Display omitted] [ABSTRACT FROM AUTHOR]

Microglia, the resident macrophages, act as the first and main form of active immune defense in the central nervous system. Arginase 2 (Arg2) is an enzyme involved in L-arginine metabolism and is expressed in macrophages and nervous tissue. In this study, we determined whether the absence of Arg2 plays a beneficial or detrimental role in the neuroinflammatory process. We then investigated whether the loss of Arg2 potentiated microglia activation and pain behaviors following nerve injury-induced neuropathic pain. A spinal nerve transection (SNT) experimental model was used to induce neuropathic pain in mice. As a result of the peripheral nerve injury, SNT induced microgliosis and astrogliosis in the spinal cord, and upregulated inflammatory signals in both wild-type (WT) and Arg2 knockout (KO) mice. Notably, inflammation increased significantly in the Arg2 KO group compared to the WT group. We also observed a more robust microgliosis and a lower mechanical threshold in the Arg2 KO group than those in the WT group. Furthermore, our data revealed a stronger upregulation of M1 pro-inflammatory cytokines, such as interleukin (IL)-1β, and a stronger downregulation of M2 anti-inflammatory cytokines, including IL4 and IL-10, in Arg2 KO mice. Additionally, stronger formation of enzyme-inducible nitric oxide synthase, oxidative stress, and decreased expression of CD206 were detected in the Arg2 KO group compared to the WT group. These results suggest that Arg2 deficiency contributes to inflammatory response. The reduction or the loss of Arg2 results in the stronger neuroinflammation in the spinal dorsal horn, followed by more severe pain behaviors arising from nerve injury-induced neuropathic pain. [ABSTRACT FROM AUTHOR]

Upon peripheral nerve injury, vesicular ATP is released from damaged primary afferent neurons. This extracellular ATP subsequently activates purinergic receptors of the spinal cord, which play a critical role in neuropathic pain. As an inhibitor of the vesicular nucleotide transporter (VNUT), Evans blue (EB) inhibits the vesicular storage and release of ATP in neurons. Thus, we tested whether EB could attenuate neuropathic pain behavior induced by spinal nerve ligation (SNL) in rats by targeting VNUT. An intrathecal injection of EB efficiently attenuated mechanical allodynia for five days in a dose-dependent manner and enhanced locomotive activity in an SNL rat model. Immunohistochemical analysis showed that EB was found in VNUT immunoreactivity on neurons in the dorsal root ganglion and the spinal dorsal horn. The level of ATP in cerebrospinal fluid in rats with SNL-induced neuropathic pain decreased upon administration of EB. Interestingly, EB blocked ATP release from neurons, but not glial cells in vitro. Eventually, the loss of ATP decreased microglial activity in the ipsilateral dorsal horn of the spinal cord, followed by a reduction in reactive oxygen species and proinflammatory mediators, such as interleukin (IL)-1β and IL-6. Finally, a similar analgesic effect of EB was demonstrated in rats with monoiodoacetate-induced osteoarthritis (OA) pain. Taken together, these data demonstrate that EB prevents ATP release in the spinal dorsal horn and reduces the ATP/purinergic receptor-induced activation of spinal microglia followed by a decline in algogenic substances, thereby relieving neuropathic pain in rats with SNL. [ABSTRACT FROM AUTHOR]