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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]

Age-dependent accumulation of amyloid plaques in patients with sporadic Alzheimer's disease (AD) is associated with reduced amyloid clearance. Older microglia have a reduced ability to phagocytose amyloid, so phagocytosis of amyloid plaques by microglia could be regulated to prevent amyloid accumulation. Furthermore, considering the aging-related disruption of cell cycle machinery in old microglia, we hypothesize that regulating their cell cycle could rejuvenate them and enhance their ability to promote more efficient amyloid clearance. First, we used gene ontology analysis of microglia from young and old mice to identify differential expression of cyclin-dependent kinase inhibitor 2A (p16ink4a), a cell cycle factor related to aging. We found that p16ink4a expression was increased in microglia near amyloid plaques in brain tissue from patients with AD and 5XFAD mice, a model of AD. In BV2 microglia, small interfering RNA (siRNA)-mediated p16ink4a downregulation transformed microglia with enhanced amyloid phagocytic capacity through regulated the cell cycle and increased cell proliferation. To regulate microglial phagocytosis by gene transduction, we used poly (D,L-lactic-co-glycolic acid) (PLGA) nanoparticles, which predominantly target microglia, to deliver the siRNA and to control microglial reactivity. Nanoparticle-based delivery of p16ink4a siRNA reduced amyloid plaque formation and the number of aged microglia surrounding the plaque and reversed learning deterioration and spatial memory deficits. We propose that downregulation of p16ink4a in microglia is a promising strategy for the treatment of Alzheimer's disease. [ABSTRACT FROM AUTHOR]

Background and Purpose: There is a scarcity of information regarding the role of prothrombin kringle‐2 (pKr‐2), which can be generated by active thrombin, in hippocampal neurodegeneration and Alzheimer's disease (AD). Experimental Approach To assess the role of pKr‐2 in association with the neurotoxic symptoms of AD, we determined pKr‐2 protein levels in post‐mortem hippocampal tissues of patients with AD and the hippocampi of five familial AD (5XFAD) mice compared with those of age‐matched controls and wild‐type (WT) mice, respectively. In addition, we investigated whether the hippocampal neurodegeneration and object memory impairments shown in 5XFAD mice were mediated by changes to pKr‐2 up‐regulation. Key Results: Our results demonstrated that pKr‐2 was up‐regulated in the hippocampi of patients with AD and 5XFAD mice, but was not associated with amyloid‐β aggregation in 5XFAD mice. The up‐regulation of pKr‐2 expression was inhibited by preservation of the blood–brain barrier (BBB) via addition of caffeine to their water supply or by treatment with rivaroxaban, an inhibitor of factor Xa that is associated with thrombin production. Moreover, the prevention of up‐regulation of pKr‐2 expression reduced neurotoxic symptoms, such as hippocampal neurodegeneration and object recognition decline due to neurotoxic inflammatory responses in 5XFAD mice. Conclusion and Implications: We identified a novel pathological mechanism of AD mediated by abnormal accumulation of pKr‐2, which functions as an important pathogenic factor in the adult brain via blood brain barrier (BBB) breakdown. Thus, pKr‐2 represents a novel target for AD therapeutic strategies and those for related conditions. [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]

An amendment to this paper has been published and can be accessed via a link at the top of the paper. [ABSTRACT FROM AUTHOR]

Activation of nuclear factor-kappa B (NF-κB) in microglia plays a decisive role in the progress of neuropathic pain, and the inhibitor of kappa B (IκB) is a protein that blocks the activation of NF-κB and is degraded by the inhibitor of NF-κB kinase subunit beta (IKBKB). The role of IKBKB is to break down IκB, which blocks the activity of NF-kB. Therefore, it prevents the activity of NK-kB. This study investigated whether neuropathic pain can be reduced in spinal nerve ligation (SNL) rats by reducing the activity of microglia by delivering IKBKB small interfering RNA (siRNA)-encapsulated poly (lactic-co-glycolic acid) (PLGA) nanoparticles. PLGA nanoparticles, as a carrier for the delivery of IKBKB genes silencer, were used because they have shown potential to enhance microglial targeting. SNL rats were injected with IKBKB siRNA-encapsulated PLGA nanoparticles intrathecally for behavioral tests on pain response. IKBKB siRNA was delivered for suppressing the expression of IKBKB. In rats injected with IKBKB siRNA-encapsulated PLGA nanoparticles, allodynia caused by mechanical stimulation was reduced, and the secretion of pro-inflammatory mediators due to NF-κB was reduced. Delivering IKBKB siRNA through PLGA nanoparticles can effectively control the inflammatory response and is worth studying as a treatment for neuropathic pain. [ABSTRACT FROM AUTHOR]

Activation of CX3CR1 in microglia plays an important role in the development of neuropathic pain. Here, we investigated whether neuropathic pain could be attenuated in spinal nerve ligation (SNL)-induced rats by reducing microglial activation through the use of poly(D,L-lactic-co-glycolic acid) (PLGA)-encapsulated CX3CR1 small-interfering RNA (siRNA) nanoparticles. After confirming the efficacy and specificity of CX3CR1 siRNA, as evidenced by its anti-inflammatory effects in lipopolysaccharide-stimulated BV2 cells in vitro, PLGA-encapsulated CX3CR1 siRNA nanoparticles were synthesized by sonication using the conventional double emulsion (W/O/W) method and administered intrathecally into SNL rats. CX3CR1 siRNA-treated rats exhibited significant reductions in the activation of microglia in the spinal dorsal horn and a downregulation of proinflammatory mediators, as well as a significant attenuation of mechanical allodynia. These data indicate that the PLGA-encapsulated CX3CR1 siRNA nanoparticles effectively reduce neuropathic pain in SNL-induced rats by reducing microglial activity and the expression of proinflammatory mediators. Therefore, we believe that PLGA-encapsulated CX3CR1 siRNA nanoparticles represent a valuable new treatment option for neuropathic pain. [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]

Parkinson's disease (PD) and Alzheimer's disease exhibit common features of neurodegenerative diseases and can be caused by numerous factors. A common feature of these diseases is neurotoxic inflammation by activated microglia, indicating that regulation of microglial activation is a potential mechanism for preserving neurons in the adult brain. Recently, we reported that upregulation of prothrombin kringle-2 (pKr-2), one of the domains that make up prothrombin and which is cleaved and generated by active thrombin, induces nigral dopaminergic (DA) neuronal death through neurotoxic microglial activation in the adult brain. In this study, we show that silibinin, a flavonoid found in milk thistle, can suppress the production of inducible nitric oxide synthase and neurotoxic inflammatory cytokines, such as interleukin-1β and tumor necrosis factor-α, after pKr-2 treatment by downregulating the extracellular signal-regulated kinase signaling pathway in the mouse substantia nigra. Moreover, as demonstrated by immunohistochemical staining, measurements of the dopamine and metabolite levels, and open-field behavioral tests, silibinin treatment protected the nigrostriatal DA system resulting from the occurrence of pKr-2-triggered neurotoxic inflammation in vivo. Thus, we conclude that silibinin may be beneficial as a natural compound with anti-inflammatory effects against pKr-2-triggered neurotoxicity to protect the nigrostriatal DA pathway and its properties, and thus, may be applicable for PD therapy. [ABSTRACT FROM AUTHOR]

Abstract: Iron is an essential, but potentially harmful, metal in the brain. In normal brain, iron has been reported to accumulate mainly in glial cells and occasionally in neurons in some particular nuclei. However, the majority of investigations have targeted the adult brain. Here, we investigated spatiotemporal localization of iron in developing and adult chicken cerebellum using iron histochemistry. Iron reactivity was not detected in the chick cerebellum until embryonic day 12. Iron accumulation was first found in mature myelinating oligodendrocytes located in the inner part of the cerebellar folium at embryonic day 14. From embryonic day 20, iron-positive mature myelinating oligodendrocytes were localized in the white matter and the granular layer. From post-hatching day 2, iron accumulation was observed in Bergmann glia in the Purkinje cell layer as well as in mature myelinating oligodendrocytes. Iron accumulation in microglia was observed in the granular and molecular layers at post-hatching month 12. Our data indicate that during cerebellar development iron is accumulated in a unique sequence according to individual requirements or microenvironmental demands. [Copyright &y& Elsevier]

Abstract: CD200 is a glycoprotein that is expressed on the surfaces of neurons and other cells. It interacts with its receptor, CD200R, which is expressed on cells of the myeloid lineage, including microglia. The interaction of CD200 with its receptor plays a significant role in maintaining microglia in a quiescent state; thus, a decrease in CD200 expression in the brain is associated with evidence of microglial activation. However, their roles in pathological progression remain unclear. We examined the expression of CD200 in kainic acid (KA)-induced neurodegeneration of the mouse hippocampus. Our quantitative analysis revealed that CD200 was constitutively expressed in the normal brain and transiently upregulated by KA treatment. At the cellular level, CD200 was expressed in neurons in control, and was upregulated primarily in the microglia of KA-treated mouse hippocampi. We examined the contribution of CD200 to both the classical and alternative activation of microglia in vitro using an adult microglia culture, which was exposed to interleukin-4 (IL-4) with and without lipopolysaccharide (LPS). CD200 expression was increased after exposure to IL-4, but not to LPS. These in vivo experiments demonstrated that CD200 was transiently expressed in microglia in a process mediated by the inflammatory response. Based on CD200R expression in microglia, it suggests that microglia is maintained in an activated state with autocrine signaling by interactions between microglial CD200 and its CD200R. Moreover, we suggest that CD200 may be expressed in the alternative activation of microglia and play a beneficial role in neuroinflammation. [Copyright &y& Elsevier]

This study was undertaken to investigate microglial responses in the avascular central nervous system using the quail retina that is known to be devoid of blood vessels. Following intraorbital optic nerve transection (ONT), the quail retina was examined immunohistochemically at various times up to 6 months. A few days after transection, microglia in the inner retinal layers revealed features of activation. Activated cells displayed an amoeboid shape and enhanced QH1-immunoreactivity. The numbers of these amoeboid cells were rapidly increased, first in the inner plexiform layer (IPL), and then in the ganglion cell/nerve fiber layer (GCL/NFL) of the retina where retrograde degenerating ganglion cell processes and perikarya were located. By 6 months after transection, microglia regained their resting morphology, and their cell counts returned to control levels. At early time points of microglial activation, numerous QH1+ amoeboid cells were observed along the vitreal surface of the pecten and retinal region adjacent to the insertion of the pecten, where some amoeboid cells were attached underneath the internal limiting membrane, and appeared to squeeze through the optic nerve fiber bundles. A considerable number of these amoeboid cells in the GCL/NFL and the IPL were labeled with PCNA, suggesting that active exogenous migration (from the pecten) and in situ proliferation of precursor cells contribute to the increase in microglial population of the degenerating retina. On the other hand, TUNEL-positive microglia appeared in the GCL/NFL at later time points indicate that the decrease of microglial numbers is in part due to apoptosis in these layers. Although some aspects of microglial activation in the avascular retina appear unique, their consequences were similar to those described in vascular retinae of mammals, a finding indicates that blood vessels are not a prerequisite for microglial activation, and microglial precursors could migrate long distance to reach the lesioned site, which is not accessible via blood vessels. Our data provide the first analysis of microglial activation in the avascular central nervous system (CNS), and suggest that the quail retina is a useful model for studies of microglial behavior in CNS. [Copyright &y& Elsevier]