Transcription factor EB (TFEB) interaction with RagC is disrupted during enterovirus D68 infection.

Enterovirus D68 (EV-D68) is a picornavirus associated with severe respiratory illness and a paralytic disease called acute flaccid myelitis in infants. Currently, no protective vaccines or antivirals are available to combat this virus. Like other enteroviruses, EV-D68 uses components of the cellular autophagy pathway to rewire membranes for its replication. Here, we show that transcription factor EB (TFEB), the master transcriptional regulator of autophagy and lysosomal biogenesis, is crucial for EV-D68 infection. Knockdown of TFEB attenuated EV-D68 genomic RNA replication but did not impact viral binding or entry into host cells. The 3C protease of EV-D68 cleaves TFEB at the N-terminus at glutamine 60 (Q60) immediately post-peak viral RNA replication, disrupting TFEB-RagC interaction and restricting TFEB transport to the surface of the lysosome. Despite this, TFEB remained mostly cytosolic during EV-D68 infection. Overexpression of a TFEB mutant construct lacking the RagC-binding domain, but not the wild-type construct, blocks autophagy and increases EV-D68 nonlytic release in H1HeLa cells but not in autophagy-defective ATG7 KO H1HeLa cells. Our results identify TFEB as a vital host factor regulating multiple stages of the EV-D68 lifecycle and suggest that TFEB could be a promising target for antiviral development against EV-D68. IMPORTANCE: Enteroviruses are among the most significant causes of human disease. Some enteroviruses are responsible for severe paralytic diseases such as poliomyelitis or acute flaccid myelitis. The latter disease is associated with multiple non-polio enterovirus species, including enterovirus D68 (EV-D68), enterovirus 71, and coxsackievirus B3 (CVB3). Here, we demonstrate that EV-D68 interacts with a host transcription factor, transcription factor EB (TFEB), to promote viral RNA(vRNA) replication and regulate the egress of virions from cells. TFEB was previously implicated in the viral egress of CVB3, and the viral protease 3C cleaves TFEB during infection. Here, we show that EV-D68 3C protease also cleaves TFEB after the peak of vRNA replication. This cleavage disrupts TFEB interaction with the host protein RagC, which changes the localization and regulation of TFEB. TFEB lacking a RagC-binding domain inhibits autophagic flux and promotes virus egress. These mechanistic insights highlight how common host factors affect closely related, medically important viruses differently.

Involvement of RIG-I Pathway in Neurotropic Virus-Induced Acute Flaccid Paralysis and Subsequent Spinal Motor Neuron Death.

Poliomyelitis-like illness is a common clinical manifestation of neurotropic viral infections. Functional loss and death of motor neurons often lead to reduced muscle tone and paralysis, causing persistent motor sequelae among disease survivors. Despite several reports demonstrating the molecular basis of encephalopathy, the pathogenesis behind virus-induced flaccid paralysis remained largely unknown. The present study for the first time aims to elucidate the mechanism responsible for limb paralysis by studying clinical isolates of Japanese encephalitis virus (JEV) and Chandipura virus (CHPV) responsible for causing acute flaccid paralysis (AFP) in vast regions of Southeast Asia and the Indian subcontinent. An experimental model for studying virus-induced AFP was generated by intraperitoneal injection of 10-day-old BALB/c mice. Progressive decline in motor performance of infected animals was observed, with paralysis being correlated with death of motor neurons (MNs). Furthermore, we demonstrated that upon infection, MNs undergo an extrinsic apoptotic pathway in a RIG-I-dependent fashion via transcription factors pIRF-3 and pIRF-7. Both gene-silencing experiments using specific RIG-I-short interfering RNA and in vivo morpholino abrogated cellular apoptosis, validating the important role of pattern recognition receptor (PRR) RIG-I in MN death. Hence, from our experimental observations, we hypothesize that host innate response plays a significant role in deterioration of motor functioning upon neurotropic virus infections. IMPORTANCE Neurotropic viral infections are an increasingly common cause of immediate or delayed neuropsychiatric sequelae, cognitive impairment, and movement disorders or, in severe cases, death. Given the highest reported disability-adjusted life years and mortality rate worldwide, a better understanding of molecular mechanisms for underlying clinical manifestations like AFP will help in development of more effective tools for therapeutic solutions.

Slice Culture Modeling of CNS Viral Infection.

The complexity of the central nervous system (CNS) is not recapitulated in cell culture models. Thin slicing and subsequent culture of CNS tissue has become a valued means to study neuronal and glial biology within the context of the physiologically relevant tissue milieu. Modern membrane-interface slice culturing methodology allows for straightforward access to both CNS tissue and feeding medium, enabling experimental manipulations and analyses that would otherwise be impossible in vivo. CNS slices can be successfully maintained in culture for up to several weeks for investigation of evolving pathology and long-term intervention in models of chronic neurologic disease.Herein, membrane-interface slice culture models for studying viral encephalitis and myelitis are detailed, with emphasis on the use of these models for investigation of pathogenesis and evaluation of novel treatment strategies. We describe techniques to (1) generate brain and spinal cord slices from rodent donors, (2) virally infect slices, (3) monitor viral replication, (4) assess virally induced injury/apoptosis, (5) characterize "CNS-specific" cytokine production, and, (6) treat slices with cytokines/pharmaceuticals. Although our focus is on CNS viral infection, we anticipate that the described methods can be adapted to address a wide range of investigations within the fields of neuropathology, neuroimmunology, and neuropharmacology.

Neuregulin-1 beta 1 is implicated in pathogenesis of multiple sclerosis.

Multiple sclerosis is characterized by immune mediated neurodegeneration that results in progressive, life-long neurological and cognitive impairments. Yet, the endogenous mechanisms underlying multiple sclerosis pathophysiology are not fully understood. Here, we provide compelling evidence that associates dysregulation of neuregulin-1 beta 1 (Nrg-1ß1) with multiple sclerosis pathogenesis and progression. In the experimental autoimmune encephalomyelitis model of multiple sclerosis, we demonstrate that Nrg-1ß1 levels are abated within spinal cord lesions and peripherally in the plasma and spleen during presymptomatic, onset and progressive course of the disease. We demonstrate that plasma levels of Nrg-1ß1 are also significantly reduced in individuals with early multiple sclerosis and is positively associated with progression to relapsing-remitting multiple sclerosis. The functional impact of Nrg-1ß1 downregulation preceded disease onset and progression, and its systemic restoration was sufficient to delay experimental autoimmune encephalomyelitis symptoms and alleviate disease burden. Intriguingly, Nrg-1ß1 therapy exhibited a desirable and extended therapeutic time window of efficacy when administered prophylactically, symptomatically, acutely or chronically. Using in vivo and in vitro assessments, we identified that Nrg-1ß1 treatment mediates its beneficial effects in EAE by providing a more balanced immune response. Mechanistically, Nrg-1ß1 moderated monocyte infiltration at the blood-CNS interface by attenuating chondroitin sulphate proteoglycans and MMP9. Moreover, Nrg-1ß1 fostered a regulatory and reparative phenotype in macrophages, T helper type 1 (Th1) cells and microglia in the spinal cord lesions of EAE mice. Taken together, our new findings in multiple sclerosis and experimental autoimmune encephalomyelitis have uncovered a novel regulatory role for Nrg-1ß1 early in the disease course and suggest its potential as a specific therapeutic target to ameliorate disease progression and severity.

FBW7 protects against spinal cord injury by mitigating inflammation-associated neuronal apoptosis in mice.

Spinal cord injury (SCI) leads to severe and long-lasting neurological disability. Presently, the lack of effective therapies for SCI is largely attributable to an incomplete understanding of its pathogenesis. F-box and WD repeat domain-containing protein 7 (FBW7, also known as FBXW7) is a type of E3 ubiquitin ligase complex, and plays essential roles in regulating different pathological and physiological processes. In this study, we attempted to explore the effects of FBW7 on SCI progression by the in vivo and in vitro experiments. SCI mice showed significantly reduced expression of FBW7 in spinal cord tissues. Promoting FBW7 expression via intrathecal injection of AAV9/FBW7 effectively improved locomotor function in SCI mice. Neuronal death in spinal cords of SCI mice was obviously ameliorated by FBW7 over-expression, along with greatly decreased expression of cleaved Caspase-3. In addition, microglial activation in spinal cord specimens was detected in SCI mice through increasing Iba-1 expression levels, which was, however, attenuated in SCI mice injected with AAV9/FBW7. Additionally, FBW7 over-expression dramatically restrained inflammatory response in spinal cord tissues of SCI mice, as evidenced by the down-regulated expression of tumor necrosis factor-α (TNF-α) and interleukin 1ß (IL-1ß) through blocking the activation of nuclear factor-κB (NF-κB) signaling. These anti-inflammatory effects of FBW7 were confirmed in LPS-stimulated mouse microglial BV2 cells. Finally, our in vitro studies showed that conditional medium (CM) collected from LPS-incubated BV2 cells markedly induced apoptosis in the isolated primary spinal neurons; However, this effect was overtly ameliorated by CM from LPS-exposed BV2 cells over-expressing FBW7. Thus, FBW7-regulated inflammation in microglial cells was involved in the amelioration of neuronal apoptosis during SCI development. Collectively, these findings illustrated that FBW7 expression was down-regulated in spinal cords of SCI mice, and promoting its expression could effectively mitigate SCI progression by repressing microglial inflammation and neuronal death.

Contemporary Circulating Enterovirus D68 Strains Infect and Undergo Retrograde Axonal Transport in Spinal Motor Neurons Independent of Sialic Acid.

Enterovirus D68 (EV-D68) is an emerging virus that has been identified as a cause of recent outbreaks of acute flaccid myelitis (AFM), a poliomyelitis-like spinal cord syndrome that can result in permanent paralysis and disability. In experimental mouse models, EV-D68 spreads to, infects, and kills spinal motor neurons following infection by various routes of inoculation. The topography of virus-induced motor neuron loss correlates with the pattern of paralysis. The mechanism(s) by which EV-D68 spreads to target motor neurons remains unclear. We sought to determine the capacity of EV-D68 to spread by the neuronal route and to determine the role of known EV-D68 receptors, sialic acid and intracellular adhesion molecule 5 (ICAM-5), in neuronal infection. To do this, we utilized a microfluidic chamber culture system in which human induced pluripotent stem cell (iPSC) motor neuron cell bodies and axons can be compartmentalized for independent experimental manipulation. We found that EV-D68 can infect motor neurons via their distal axons and spread by retrograde axonal transport to the neuronal cell bodies. Virus was not released from the axons via anterograde axonal transport after infection of the cell bodies. Prototypic strains of EV-D68 depended on sialic acid for axonal infection and transport, while contemporary circulating strains isolated during the 2014 EV-D68 outbreak did not. The pattern of infection did not correspond with the ICAM-5 distribution and expression in either human tissue, the mouse model, or the iPSC motor neurons.IMPORTANCE Enterovirus D68 (EV-D68) infections are on the rise worldwide. Since 2014, the United States has experienced biennial spikes in EV-D68-associated acute flaccid myelitis (AFM) that have left hundreds of children paralyzed. Much remains to be learned about the pathogenesis of EV-D68 in the central nervous system (CNS). Herein we investigated the mechanisms of EV-D68 CNS invasion through neuronal pathways. A better understanding of EV-D68 infection in experimental models may allow for better prevention and treatment strategies of EV-D68 CNS disease.

3D printing collagen/chitosan scaffold ameliorated axon regeneration and neurological recovery after spinal cord injury.

Spinal cord injury (SCI) is a disaster that can cause severe motor, sensory, and functional disorders. Implanting biomaterials have been regarded as hopeful strategies to restore neurological function. However, no optimized scaffold has been available. In this study, a novel 3D printing technology was used to fabricate the scaffold with designed structure. The composite biomaterials of collagen and chitosan were also adopted to balance both compatibility and strength. Female Sprague-Dawley rats were subjected to a T8 complete-transection SCI model. Scaffolds of C/C (collagen/chitosan scaffold with freeze-drying technology) or 3D-C/C (collagen/chitosan scaffold with 3D printing technology) were implanted into the lesion. Compared with SCI or C/C group, 3D-C/C implants significantly promoted locomotor function with the elevation in Basso-Beattie-Bresnahan (BBB) score and angle of inclined plane. Decreased latency and increased amplitude were observed both in motor-evoked potential and somatosensory-evoked potential in 3D-C/C group compared with SCI or C/C group, which further demonstrated the improvement of neurological recovery. Fiber tracking of diffusion tensor imaging (DTI) showed the most fibers traversing the lesion in 3D-C/C group. Meanwhile, we observed that the correlations between the locomotor (BBB score or angle of inclined plane) and the DTI parameters (fractional anisotropy values) were positive. Although C/C implants markedly enhanced biotin dextran amine (BDA)-positive neural profiles compared with SCI group, rats implanted with 3D-C/C scaffold displayed the largest degree of BDA profiles regeneration. Collectively, our 3D-C/C scaffolds demonstrated significant therapeutic effects on rat complete-transected spinal cord model, which provides a promising and innovative therapeutic approach for SCI. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1898-1908, 2019.

Aspirin suppresses neuronal apoptosis, reduces tissue inflammation, and restrains astrocyte activation by activating the Nrf2/HO-1 signaling pathway.

The nuclear factor E2-related factor 2 (Nrf2)/antioxidant response element signaling pathway plays a substantial role in preventing oxidative stress-related diseases. Aspirin has been shown to exert several pharmacological effects by inducing the expression of the heme oxygenase-1 (HO-1) protein. However, the effects of aspirin on spinal cord injury (SCI) have rarely been studied. Therefore, we sought to investigate the neuroprotective effects of aspirin after SCI. We employed a spinal cord contusion model in Sprague-Dawley rats, and aspirin was administered intraperitoneally for 7 days. Nissl staining showed that the aspirin treatment significantly reduced the loss of motor neurons after SCI compared with vehicle-treated animals. The expression of Nrf2, quinine oxidoreductase 1, and HO-1 proteins was increased in aspirin-treated animals after SCI compared with the vehicle group. In addition, aspirin simultaneously decreased the expression of inflammation-related proteins, such as tumor necrosis factor-α and interleukin-6 after SCI. Moreover, the ratio of apoptotic neurons in the anterior horn and the levels of the apoptosis-related proteins caspase-3, cleaved caspase-3, and Bax were significantly decreased in the aspirin group compared with the vehicle group. Immunofluorescence staining was used to detect the colocalization of NeuN and HO-1, and the results showed that aspirin significantly increased expression of the HO-1 protein in neurons. In addition, western blots and immunofluorescence staining showed aspirin restrained astrocyte activation. In conclusion, aspirin induces neuroprotective effects by inhibiting astrocyte activation and apoptosis after SCI through the activation of the Nrf2/HO-1 signaling pathway.

Spinal CCL2 Promotes Central Sensitization, Long-Term Potentiation, and Inflammatory Pain via CCR2: Further Insights into Molecular, Synaptic, and Cellular Mechanisms.

Mounting evidence supports an important role of chemokines, produced by spinal cord astrocytes, in promoting central sensitization and chronic pain. In particular, CCL2 (C-C motif chemokine ligand 2) has been shown to enhance N-methyl-D-aspartate (NMDA)-induced currents in spinal outer lamina II (IIo) neurons. However, the exact molecular, synaptic, and cellular mechanisms by which CCL2 modulates central sensitization are still unclear. We found that spinal injection of the CCR2 antagonist RS504393 attenuated CCL2- and inflammation-induced hyperalgesia. Single-cell RT-PCR revealed CCR2 expression in excitatory vesicular glutamate transporter subtype 2-positive (VGLUT2+) neurons. CCL2 increased NMDA-induced currents in CCR2+/VGLUT2+ neurons in lamina IIo; it also enhanced the synaptic NMDA currents evoked by dorsal root stimulation; and furthermore, it increased the total and synaptic NMDA currents in somatostatin-expressing excitatory neurons. Finally, intrathecal RS504393 reversed the long-term potentiation evoked in the spinal cord by C-fiber stimulation. Our findings suggest that CCL2 directly modulates synaptic plasticity in CCR2-expressing excitatory neurons in spinal lamina IIo, and this underlies the generation of central sensitization in pathological pain.

MiR-30a-5p ameliorates spinal cord injury-induced inflammatory responses and oxidative stress by targeting Neurod 1 through MAPK/ERK signalling.

Spinal cord injury (SCI) is a major disability requiring more effective treatment than is currently available. MicroRNAs have been shown to effectively regulate gene expression at the translational level. The aim of the present study was to explore the potential role of miR-30-5p and possible mechanism in SCI. We found that miR-30-5p was notably down-regulated, while Neurod 1 expression was highly elevated in microglia from the mouse model of SCI. Additionally, overexpression of miR-30a-5p significantly suppressed inflammatory responses as reflected by a decrease in the secretion of the cytokines TNF-α, IL-1ß and IL-10 triggered by SCI. Furthermore, introduction of miR-30a-5p strengthened the scavenging of oxygen free radicals accompanied by an increase in the expression of SEPN1, TXNL1 and GPX1. More importantly, our study explored that Neurod 1 was a direct and functional target of miR-30a-5p, which was validated by the dual luciferase reporter assay. qRT-PCR and western blot analysis further validated that miR-30a-5p negatively regulated the expression of Neurod 1. Mechanistically, overexpression of miR-30a-5p or silencing of the Neurod 1 gene prevented the MAPK/ERK signalling and inhibited inflammatory responses, meanwhile activated SEPN1, TXNL1 and GPX1. These findings indicate that miR-30a-5p ameliorates inflammatory responses and oxidative stress by targeting Neurod 1 through MAPK/ERK signalling.

A green tea polyphenol epigallocatechin-3-gallate enhances neuroregeneration after spinal cord injury by altering levels of inflammatory cytokines.

Spinal cord injury (SCI) is a debilitating condition which is characterized by an extended secondary injury due to the presence of inflammatory local milieu. Epigallocatechin gallate (EGCG) appears to possess strong neuroprotective properties. Here, we evaluated the beneficial effect of EGCG on recovery from SCI. Male Wistar rats were given either EGCG or saline directly to the injured spinal cord and thereafter a daily IP injection. Behavior recovery was monitored by BBB, plantar, rotarod and flat-beam tests. The levels of inflammatory cytokines were determined on days 1, 3, 7, 10 and 14 after SCI. Additionally, NF-κB pathway activity was evaluated. The results demonstrated that EGCG-treated rats displayed a superior behavioral performance in a flat beam test, higher axonal sprouting and positive remodelation of glial scar. Cytokine analysis revealed a reduction in IL-6, IL2, MIP1α and RANTES levels on days 1 and 3, and an upregulation of IL-4, IL-12p70 and TNFα 1 day following SCI in EGCG-treated rats. Treatment with EGCG was effective in decreasing the nuclear translocation of subunit p65 (RelA) of the NF-κB dimer, and therefore canonical NF-κB pathway attenuation. A significant increase in the gene expression of growth factors (FGF2 and VEGF), was noted in the spinal cord of EGCG-treated rats. Further, EGCG influenced expression of M1 and M2 macrophage markers. Our results have demonstrated a therapeutic value of EGCG in SCI, as observed by better behavioral performance measured by flat beam test, modulation of inflammatory cytokines and induction of higher axonal sprouting.

The effects of dexmedetomidine pretreatment on the pro- and anti-inflammation systems after spinal cord injury in rats.

Excessive inflammatory responses play important roles in the aggravation of secondary damage to an injured spinal cord. Dexmedetomidine (DEX), a selective α2-adrenoceptor agonist, has recently been implied to be neuroprotective in clinical anesthesia, but the underlying mechanism is elusive. As signaling through Toll-like receptor 4 (TLR4) and nicotinic receptors (nAChRs, notably α7nAChR) play important roles in the pro- and anti-inflammation systems in the central nervous system, respectively, this study investigated whether and how they were modulated by DEX pretreatment in a rat model of spinal cord compression. The model was used to mimic perioperative compressive spinal cord injury (SCI) during spinal correction. DEX preconditioning improved locomotor scores after SCI, which was accompanied by increased α7nAChR and acetylcholine (Ach, an endogenous ligand of α7nAChR) expression as well as PI3K/Akt activation. However, there was a decrease in Ly6h (a negative regulator for α7nAChR trafficking), TLR4, PU.1 (a critical transcriptional regulator of TLR4), HMGB1 (an endogenous ligand of TLR4), and caspase 3-positive cells, which was prevented by intrathecal preconditioning with antagonists of either α2R, α7nAChR or PI3K/Akt. In addition, application of an α7nAChR agonist produced effects similar to those of DEX after SCI, while application of an α7nAChR antagonist reversed these effects. Furthermore, both α7nAChR and TLR4 were mainly co-expressed in NeuN-positive cells of the spinal ventral horn, but not in microglia or astrocytes after SCI. These findings imply that the α2R/PI3K/Akt/Ly6h and α7nAChR/PI3K/Akt/PU.1 cascades are required for upregulated α7nAChR and downregulated TLR4 expression by DEX pretreatment, respectively, which provided a unique insight into understanding DEX-mediated neuroprotection.

An Agonist of the Protective Factor SIRT1 Improves Functional Recovery and Promotes Neuronal Survival by Attenuating Inflammation after Spinal Cord Injury.

Targeting posttraumatic inflammation is crucial for improving locomotor function. SIRT1 has been shown to play a critical role in disease processes such as hepatic inflammation, rheumatoid arthritis, and acute lung inflammation by regulating inflammation. However, the role of SIRT1 in spinal cord injury (SCI) is unknown. We hypothesized that SIRT1 plays an important role in improving locomotor function after SCI by regulating neuroinflammation. In this study, we investigate the effect of SIRT1 in SCI using pharmacological intervention (SRT1720) and the Mx1-Cre/loxP recombination system to knock out target genes. First, we found that SIRT1 expression at the injured lesion site of wild-type (WT) mice (C57BL/6) decreased 4 h after SCI and lasted for 3 d. Moreover, administration of SRT1720, an agonist of SIRT1, to WT mice significantly improved functional recovery for up to 28 d after injury by reducing the levels of proinflammatory cytokines, the number of M1 macrophages, the number of macrophages/microglia, and the accumulation of perivascular macrophages. In contrast, administration of SRT1720 to SIRT1 knock-out (KO) mice did not improve locomotor recovery or attenuate inflammation. Furthermore, SIRT1 KO mice exhibited worse locomotor recovery, increased levels of inflammatory cytokines, and more M1 macrophages and perivascular macrophages than those of WT mice after SCI. Together, these findings indicate that SRT1720, an SIRT1 agonist, can improve functional recovery by attenuating inflammation after SCI. Therefore, SIRT1 is not only a protective factor but also an anti-inflammatory molecule that exerts beneficial effects on locomotor function after SCI.SIGNIFICANCE STATEMENT Posttraumatic inflammation plays a central role in regulating the pathogenesis of spinal cord injury (SCI). Here, new data show that administration of SRT1720, an SIRT1 agonist, to wild-type (WT) mice significantly improved outcomes after SCI, most likely by reducing the levels of inflammatory cytokines, the number of macrophages/microglia, perivascular macrophages, and M1 macrophages. In contrast, SIRT1 KO mice exhibited worse locomotor recovery than that of WT mice due to aggravated inflammation. Taken together, the results of this study expand upon the previous understanding of the functions and mechanisms of SIRT1 in neuroinflammation following injury to the CNS, suggesting that SIRT1 plays a critical role in regulating neuroinflammation following CNS injury and may be a novel therapeutic target for post-SCI intervention.

Is the Wnt/ß-catenin pathway involved in the anti-inflammatory activity of glucocorticoids in spinal cord injury?

The Wnt canonical or the Wnt/ß-catenin pathway has been implicated in the regulation of several physiopathological pathways such as inflammation. Glucocorticoids (GCs) are administered widely to treat inflammation in several diseases, including spinal cord injury (SCI). The aim of this study was to evaluate whether the Wnt canonical pathway is involved in experimental SCI and whether it is implicated in the anti-inflammatory activity of two different GCs: the methylprednisolone sodium succinate (MPSS), considered the standard treatment for acute SCI, and mometasone furoate (MF), mainly administered for the treatment of airway and skin diseases. Experimental SCI was induced in mice by surgical spinal cord compression at the T6-T7 level. Then, mice were treated with MPSS (6 mg/kg) or MF (0.1 mg/kg) for 7 days until they were killed. Both GCs were found to modulate the Wnt canonical pathway, but in particular, the MF treatment was shown to restore completely the downregulated pathway in SCI. The MF treatment also significantly increased peroxisome proliferator-activated receptor-γ, a Wnt target gene with anti-inflammatory properties, compared with MPSS, and it also inhibited the levels of the proinflammatory cytokines interleukin 1ß and tumor necrosis factor-α. Here, we suggest that MF has more efficacy than MPSS in inhibiting inflammation in an SCI experimental model and we propose the ß-catenin/peroxisome proliferator-activated receptor-γ axis as the mechanism by which MF exerts these beneficial effects.

Effect of exercise on neurogenic inflammation in spinal cord of Type 1 diabetic rats.

Neuropathy is a long-standing and hard to treat complication of diabetes that interferes almost 25-30% of diabetic patients and impacts the quality of life of the patients. Unforeseen side effects, dependency and addiction made the existing medical treatments comparatively ineffective. A number of studies indicate that moderate physical activity provides health-related advantages. However, existing data do not confirm whether regular physical activity would reduce the amount of inflammation in the nervous system of the subjects with Type 1 diabetes. This study reveals the significance of exercise to alleviate inflammation in the spinal cord of the nervous system and preserve sensory nerve function in animals with Type 1 diabetes after 6 weeks of exercise paradigm. Streptozotocin-diabetic animals were placed in motorized running wheels for sixty minutes per day, for five days a week for 6 weeks starting at one week after diabetes. Emerging evidence suggests that the increases in inflammatory mediators play an important role in the development of sensory neuropathy. This study shows that moderate exercise can reduce the release of a number of proinflammatory cytokines in the dorsal horn (DH) of spinal cord, subsequently delaying the development of neuropathy along with an increase in the anti-inflammatory mediator IL10 in the DH. In general, this study indicates that exercise may provide an alternative to the treatment for sensory neuropathy in Type 1 diabetic subjects via reducing the use of medication and providing an easier way to manage neuropathy.

Angelica Sinensis attenuates inflammatory reaction in experimental rat models having spinal cord injury.

This study was aimed to evaluate the effect of Angelica Sinensis on experimental rat models in which spinal cord injury was induced by studying different factors. Different factors causing inflammation play a key role in pathophysiology of SCI. Here three groups of rats (n=15, each was used). These included a sham control group where only laminectomy was performed, SCI group where SCI was induced and AS/SCI group where although SCI was induced but Angelica Sinensis was also administered to study its effect and draw a comparison with control. The expression of I-kBα and NF-kB p65 was also studied using western blotting and after recording optical density (OD) values of western blots. MPO activity was used to measure the effect of 20 mg/kg Angelica Sinensis. The levels of proinflammatory cytokines TNF-α, IL-1ß and IL-6 were also studied. As compared with SCI group and sham control it was observed that Angelica Sinensis significantly reduced the expression of I-kBα and NF-kB p65, (P<0.05), while MPO activity was also significantly reduced. Proinflammatory cytokine level was also reduced in treated group as compared to both other groups. On the basis of this study we concluded that the use of 20 mg/kg Angelica Sinensis in rat models can attenuate the secondary damage caused by SCI and thus help in controlling the pathology of SCI in rats.

Genetic targeting of protease activated receptor 2 reduces inflammatory astrogliosis and improves recovery of function after spinal cord injury.

Inflammatory-astrogliosis exacerbates damage in the injured spinal cord and limits repair. Here we identify Protease Activated Receptor 2 (PAR2) as an essential regulator of these events with mice lacking the PAR2 gene showing greater improvements in motor coordination and strength after compression-spinal cord injury (SCI) compared to wild type littermates. Molecular profiling of the injury epicenter, and spinal segments above and below, demonstrated that mice lacking PAR2 had significantly attenuated elevations in key hallmarks of astrogliosis (glial fibrillary acidic protein (GFAP), vimentin and neurocan) and in expression of pro-inflammatory cytokines (interleukin-6 (IL-6), tumor necrosis factor (TNF) and interleukin-1 beta (IL-1ß)). SCI in PAR2-/- mice was also accompanied by improved preservation of protein kinase C gamma (PKCγ)-immunopositive corticospinal axons and reductions in GFAP-immunoreactivity, expression of the pro-apoptotic marker BCL2-interacting mediator of cell death (BIM), and in signal transducer and activator of transcription 3 (STAT3). The potential mechanistic link between PAR2, STAT3 and astrogliosis was further investigated in primary astrocytes to reveal that the SCI-related serine protease, neurosin (kallikrein 6) promotes IL-6 secretion in a PAR2 and STAT3-dependent manner. Data point to a signaling circuit in primary astrocytes in which neurosin signaling at PAR2 promotes IL-6 secretion and canonical STAT3 signaling. IL-6 promotes expression of GFAP, vimentin, additional IL-6 and robust increases in both neurosin and PAR2, thereby driving the PAR2-signaling circuit forward. Given the significant reductions in astrogliosis and inflammation as well as superior neuromotor recovery observed in PAR2 knockout mice after SCI, we suggest that this receptor and its agonists represent new drug targets to foster neuromotor recovery.

Effect of Tanshinone IIA intrathecal injections on pain and spinal inflammation in mice with bone tumors.

The study aimed to investigate the effect of intrathecal injections of Tanshinone IIA on thermal hyperalgesia in a mouse model of bone cancer-pain. Spinal IL-1ß, IL-6, TNF-α expression levels were analyzed. C3H/HeNCrlVr male mice were assigned to groups that either received dose-dependent injections of Tanshinone IIA, or the DMSO + Sham, Tanshinone IIA + Sham, DMSO + Tumor, and Control groups. Paw withdrawal thermal latency (PWTL) was measured with a radiant heat stimulus and mRNA expression levels were determined using real-time PCR. Fourteen days post-injection, PWTL in the DMSO + Tumor group was lower than that in the controls (P < 0.05). Twenty-one days post-injection, compared with the Control group, there was no significant difference in PWTL and IL-1ß, IL-6, and TNF-α expression levels between the Tanshinone IIA + Sham and DMSO + Sham groups (P > 0.05). PWTL in the DMSO + Tumor group was significantly lower than the Control group (P < 0.05), while the expression levels of IL-1ß, IL-6, and TNF-α were significantly higher than controls. Compared with the DMSO + Tumor group, PWTLs were higher in the Tanshinone IIA - 20-µg and 40-µg groups, while expression levels of IL-1ß, IL-6, and TNF-α were significantly lower (P < 0.05). These measures were not significantly different between the Tanshinone IIA 10 µg and the DMSO + Tumor groups (P > 0.05). In conclusion, Tanshinone IIA may inhibit the release of inflammatory cytokines, such as, IL-1 ß, IL-6 α, TNF-α.

Cdk5 contributes to inflammation-induced thermal hyperalgesia mediated by the p38 MAPK pathway in microglia.

BACKGROUND: The mechanisms underlying cyclin-dependent kinase 5 (Cdk5)-mediated thermal hyperalgesia induced by inflammation remain poorly understood. In the present study, we examined thermal hyperalgesia provoked by peripheral injection of complete Freund׳s adjuvant (CFA) to test for Cdk5 signaling in the spinal dorsal horns of rats through the p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway, which is known to function in mediating inflammatory pain. METHODS: We induced the inflammatory pain model by plantar injection of CFA and compared the inhibitory effects of roscovitine and SB203580 on thermal hyperalgesia. We measured localization of Cdk5, p35, OX-42, and glial fibrillary acidic protein (GFAP) in the dorsal horn at 1 and 3 days after CFA injection using immunohistochemistry, and we measured protein levels of OX-42 and phosphorylated-p38 (p-p38) using Western blot analysis. Tumor necrosis factor-a (TNF-a) was measured by ELISA. RESULTS: The maximum thermal hyperalgesia induced by CFA occurred at 1d following injection and decreased until 5 d. We found colocalization of the Cdk5 activator p35, the microglial marker OX-42 and p-p38 in the same microglial cells and neurons of the spinal cord at day 1 after CFA injection; however, we saw no colocalization of p35 and GFAP, a marker of activated astrocytes. The thermal hyperalgesia induced by CFA was inhibited by intrathecal administration of the Cdk5 inhibitor roscovitine and by the p38 inhibitor SB203580. Furthermore, the expression of OX-42, p-p38, and TNF-a was remarkably increased from days 1 to 5 post-CFA injection and were significantly reversed by roscovitine between 1 and 3 days. CONCLUSIONS: Cdk5, an upstream regulator of p38 and TNF-a, mediates CFA-induced thermal hyperalgesia. As such, pharmacological blocking of the generation of p-p38 mediated by Cdk5 may present a novel approach for diminishing inflammatory pain. This article is part of a Special Issue entitled SI: Spinal cord injury.