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.

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.

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.

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.

Cytokine-regulated neutrophil recruitment is required for brain but not spinal cord inflammation during experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is an autoimmune disease in which inflammatory lesions lead to tissue injury in the brain and/or spinal cord. The specific sites of tissue injury are strong determinants of clinical outcome in MS, but the pathways that determine whether damage occurs in the brain or spinal cord are not understood. Previous studies in mouse models of MS demonstrated that IFN-γ and IL-17 regulate lesion localization within the brain; however, the mechanisms by which these cytokines mediate their effects have not been identified. In the present study, we show that IL-17 promoted, but IFN-γ inhibited, ELR(+) chemokine-mediated neutrophil recruitment to the brain, and that neutrophil infiltration was required for parenchymal tissue damage in the brain. In contrast, IFN-γ promoted ELR(+) chemokine expression and neutrophil recruitment to the spinal cord. Surprisingly, tissue injury in the spinal cord did not exhibit the same dependence on neutrophil recruitment that was observed for the brain. Our results demonstrate that the brain and spinal cord exhibit distinct sensitivities to cellular mediators of tissue damage, and that IL-17 and IFN-γ differentially regulate recruitment of these mediators to each microenvironment. These findings suggest an approach toward tailoring therapies for patients with distinct patterns of neuroinflammation.

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.

Experimental autoimmune prostatitis induces microglial activation in the spinal cord.

BACKGROUND: The pathogenesis of chronic prostatitis/chronic pelvic pain syndrome is unknown and factors including the host's immune response and the nervous system have been attributed to the development of CP/CPPS. We previously demonstrated that mast cells and chemokines such as CCL2 and CCL3 play an important role in mediating prostatitis. Here, we examined the role of neuroinflammation and microglia in the CNS in the development of chronic pelvic pain. METHODS: Experimental autoimmune prostatitis (EAP) was induced using a subcutaneous injection of rat prostate antigen. Sacral spinal cord tissue (segments S14-S5) was isolated and utilized for immunofluorescence or QRT-PCR analysis. Tactile allodynia was measured at baseline and at various points during EAP using Von Frey fibers as a function for pelvic pain. EAP mice were treated with minocycline after 30 days of prostatitis to test the efficacy of microglial inhibition on pelvic pain. RESULTS: Prostatitis induced the expansion and activation of microglia and the development of inflammation in the spinal cord as determined by increased expression levels of CCL3, IL-1ß, Iba1, and ERK1/2 phosphorylation. Microglial activation in mice with prostatitis resulted in increased expression of P2X4R and elevated levels of BDNF, two molecular markers associated with chronic pain. Pharmacological inhibition of microglia alleviated pain in mice with prostatitis and resulted in decreased expression of IL-1ß, P2X4R, and BDNF. CONCLUSION: Our data show that prostatitis leads to inflammation in the spinal cord and the activation and expansion of microglia, mechanisms that may contribute to the development and maintenance of chronic pelvic pain.

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.

Migration and remyelination by oligodendrocyte progenitor cells transplanted adjacent to focal areas of spinal cord inflammation.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system. Exogenous cell replacement in MS lesions has been proposed as a means of achieving remyelination when endogenous remyelination has failed. However, the ability of exogenous cells to remyelinate axons in the presence of inflammation remains uncertain. We have explored the remyelinating capacity of an oligodendrocyte progenitor cell line CG-4 transduced with the GFP gene and transplanted adjacent to a zymosan-induced focal demyelination model in the rat spinal cord. The resulting zymosan-induced lesions were characterized by persistent macrophage/microglia activation, focal demyelination, degeneration of axons, and reactive astrogliosis. GFP(+) CG-4 cells were found to migrate preferentially toward the inflammatory lesion and survive inside the lesion. A proportion of GFP(+) CG-4 cells differentiated into mature oligodendrocytes and remyelinated axons within the lesion. These findings suggest that grafted oligodendrocyte progenitors may migrate toward areas of inflammation in the adult rat spinal cord, where they can survive and differentiate into myelinating oligodendrocytes.

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.

Tolerogenic dendritic cells in the control of autoimmune neuroinflammation: an emerging role of protein-glycan interactions.

During the past decade, a great deal of information has contributed to our understanding of the immunosuppressive pathways that operate during the resolution of autoimmune pathology, including central nervous system (CNS) inflammation. Activation of these pathways is accomplished through the integration of an intricate network of inhibitory signals and immune suppressive cells, including regulatory T cells, myeloid-derived suppressor cells, 'alternatively activated' macrophages and tolerogenic dendritic cells (DCs). During the course of inflammatory diseases, immature or mature DCs may be licensed by different stimuli (e.g. cytokines, neuropeptides and growth factors) to become tolerogenic and suppress pathogenic T cell responses, thus emphasizing the outstanding plasticity of these cells. Recent findings have shed light to an immunoregulatory circuit by which galectin-1, an endogenous glycan-binding protein, favors the differentiation of regulatory DCs which promote T cell tolerance and contribute to resolution of autoimmune pathology through mechanisms involving IL-27 and IL-10. Together with the ability of galectin-1-glycan interactions to selectively blunt T helper (Th)1 and Th17 responses, this effect provides a rational explanation for the broad immunosuppressive effects of this glycan-binding protein in several experimental models of chronic inflammation and cancer. In this mini review, we will summarize the regulatory signals leading to the differentiation of tolerogenic DCs and their participation in CNS inflammation. In addition, we will underscore recent findings on the emerging role of galectin-glycan interactions in the establishment of immunosuppressive networks during the resolution of chronic inflammation.

Neurodegeneration and increased production of nitrotyrosine, nitric oxide synthase, IFN-gamma and S100beta protein in the spinal cord of IL-12p40-deficient mice infected with Trypanosoma cruzi.

BACKGROUND/AIM: Chagas' disease is caused by Trypanosoma cruzi and occurs in most Latin American countries. The protozoan may colonize the central nervous system (CNS) of immune-compromised human hosts, thus causing neuronal disorders. Systemic control of the intracellular forms of the parasite greatly depends on the establishment of a TH1 response and subsequent nitric oxide (NO) release. At the CNS, it is known that low concentrations of NO promote neuronal survival and growth, while high concentrations exert toxic effects and neuron death. Accounting for NO production by astrocytes is the glia-derived factor S100beta, which is overproduced in some neurodegenerative diseases. In the current work, we studied the expression of NO, interferon (IFN)-gamma and S100beta in the spinal cord tissue of IL-12p40KO mice infected with T. cruzi, a model of neurodegenerative process. METHODS: IL-12p40KO and wild-type (WT) female mice infected with T. cruzi Sylvio X10/4 (10(5) trypomastigotes, intraperitoneally) were euthanized when IL-12p40KO individuals presented limb paralysis. Spinal cord sections were submitted to immunohistochemical procedures for localization of neurofilament, laminin, nitrotyrosine, NO synthases (NOS), IFN-gamma and S100beta. The total number of neurons was estimated by stereological analysis and the area and intensity of immunoreactivities were assessed by microdensitometric/morphometric image analysis. RESULTS: No lesion was found in the spinal cord sections of WT mice, while morphological disarrangements, many inflammatory foci, enlarged vessels, amastigote nests and dying neurons were seen at various levels of IL-12p40KO spinal cord. Compared to WT mice, IL-12p40KO mice presented a decrement on total number of neurons (46.4%, p < 0.05) and showed increased values of immunoreactive area for nitrotyrosine (239%, p < 0.01) and NOS (544%, p < 0.001). Moreover, the intensity of nitrotyrosine (16%, p < 0.01), NOS (38%, p < 0.05) and S100beta (21%, p < 0.001) immunoreactivities were also augmented. No IFN-gamma-labeled cells were seen in WT spinal cord tissue, contrary to IL-12p40KO tissue that displayed inflammatory infiltrating cells and also some parenchymal cells positively labeled. CONCLUSION: We suggest that overproduction of NO may account for neuronal death at the spinal cord of T. cruzi-infected IL-12p40KO mice and that IFN-gamma and S100beta may contribute to NOS activation in the absence of IL-12.

[Analysis of morphological manifestations of the inflammatory reaction in wistar rat spinal cord in experimental model].

Morphological changes in the spinal cord of rats with different intensity of pathological symptoms were studied at the peak of the experimental encephalomyelitis development. Light-microscopical and immunohistochemical methods were used. Distribution of proliferating cell nuclear antigen (PCNA), astrocyte marker - glial fibrillar acidic protein (GFAP), and microglia and macrophage marker Iba-1, was studied. Heterogeneity in morphological manifestations of the experimental allergic encephalomyelitis was shown. Four typical patterns of morphological manifestations of the disease were demonstrated depending on the preferential involvement of pia mater, vessels, spinal cell nuclei or conductive tracts in the pathological process.

Erythropoietin in spinal cord injury.

Spinal cord injury (SCI) is a devastating condition for individual patients and costly for health care systems requiring significant long-term expenditures. Cytokine erythropoietin (EPO) is a glycoprotein mediating cytoprotection in a variety of tissues, including spinal cord, through activation of multiple signaling pathways. It has been reported that EPO exerts its beneficial effects by apoptosis blockage, reduction of inflammation, and restoration of vascular integrity. Neuronal regeneration has been also suggested. In the present review, the pathophysiology of SCI and the properties of endogenous or exogenously administered EPO are briefly described. Moreover, an attempt to present the current traumatic, ischemic and inflammatory animal models that mimic SCI is made. Currently, a clearly effective pharmacological treatment is lacking. It is highlighted that administration of EPO or other recently generated EPO analogues such as asialo-EPO and carbamylated-EPO demonstrate exceptional preclinical characteristics, rendering the evaluation of these tissue-protective agents imperative in human clinical trials.

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.

Cytoskeletal protein carbonylation and degradation in experimental autoimmune encephalomyelitis.

Protein carbonylation, the non-enzymatic addition of aldehydes or ketones to specific amino acid residues, has been implicated in the pathophysiology of multiple sclerosis. In this study, we investigated whether protein carbonyls also accumulate in the spinal cord of Lewis rats with acute experimental autoimmune encephalomyelitis (EAE). Western blots analysis after derivatization with dinitrophenyl hydrazine (oxyblot) showed elevated protein carbonylation at the time of maximal clinical disability. During the same period glutathione levels were substantially reduced, suggesting a causal relationship between these two markers. In contrast, lipid peroxidation products accumulated in EAE spinal cord well before the appearance of neurological symptoms. Carbonyl staining was not restricted to inflammatory lesions but present throughout the spinal cord particularly in neuronal cell bodies and axons. By 2-dimensional-oxyblot, we identified several cytoskeletal proteins, including beta-actin, glial acidic fibrillary protein, and the neurofilament proteins as the major targets of carbonylation. These findings were confirmed by pull-down experiments, which also showed an increase in the number of carbonylated beta-actin molecules and a decrease in that of oxidized neurofilament proteins in EAE. These data suggest the possibility that oxidation targets neurofilament proteins for degradation, which may contribute to axonal pathology observed in multiple sclerosis and EAE.

Effects of single administration of morphine on G-protein mRNA level in the presence and absence of inflammation in the rat spinal cord.

Antinociceptive potency of opioids is greater against various noxious stimuli in animals with peripheral inflammation. Opioid agonists stimulate activation of G-protein-coupled receptor. Changes in the resting levels of G-protein subtypes could have an effect on intracellular signalling pathways. The present study was designed to investigate the effects of analgesic morphine treatment on the level G-protein subunits mRNA in the presence and absence of inflammation. Our results showed that the carrageenan administration increased G-protein subunits. Administration of analgesic dose of morphine alone and in the presence of inflammation induced different alterations in the levels of G-protein mRNA. Taken together, the results obtained using real time RT-PCR suggested that G-protein genes expression levels following the acute administration of morphine between animals with and without inflammation could influence, at least in part, analgesic responsiveness.