Oligodendrocyte precursor cell-derived exosomes combined with cell therapy promote clinical recovery by immunomodulation and gliosis attenuation.

Multiple sclerosis is a chronic inflammatory disease of the central nervous system characterized by autoimmune destruction of the myelin sheath, leading to irreversible and progressive functional deficits in patients. Pre-clinical studies involving the use of neural stem cells (NSCs) have already demonstrated their potential in neuronal regeneration and remyelination. However, the exclusive application of cell therapy has not proved sufficient to achieve satisfactory therapeutic levels. Recognizing these limitations, there is a need to combine cell therapy with other adjuvant protocols. In this context, extracellular vesicles (EVs) can contribute to intercellular communication, stimulating the production of proteins and lipids associated with remyelination and providing trophic support to axons. This study aimed to evaluate the therapeutic efficacy of the combination of NSCs and EVs derived from oligodendrocyte precursor cells (OPCs) in an animal model of multiple sclerosis. OPCs were differentiated from NSCs and had their identity confirmed by gene expression analysis and immunocytochemistry. Exosomes were isolated by differential ultracentrifugation and characterized by Western, transmission electron microscopy and nanoparticle tracking analysis. Experimental therapy of C57BL/6 mice induced with experimental autoimmune encephalomyelitis (EAE) were grouped in control, treated with NSCs, treated with OPC-derived EVs and treated with a combination of both. The treatments were evaluated clinically using scores and body weight, microscopically using immunohistochemistry and immunological profile by flow cytometry. The animals showed significant clinical improvement and weight gain with the treatments. However, only the treatments involving EVs led to immune modulation, changing the profile from Th1 to Th2 lymphocytes. Fifteen days after treatment revealed a reduction in reactive microgliosis and astrogliosis in the groups treated with EVs. However, there was no reduction in demyelination. The results indicate the potential therapeutic use of OPC-derived EVs to attenuate inflammation and promote recovery in EAE, especially when combined with cell therapy.

Effects of local and systemic treatment with human natural killer-1 mimetic peptide (HNK-1) after ventral root avulsion and reimplantation in mice.

Background: Spinal ventral root injuries generate significant motoneuron degeneration, which hinders full functional recovery. The poor prognosis of functional recovery can be attributed to the use or combination of different therapeutic approaches. Several molecules have been screened as potential treatments in combination with surgical reimplantation of the avulsed roots, the gold standard approach for such injuries. Among the studied molecules, human natural killer-1 (HNK-1) stands out as it is related to the stimulation of motor axon outgrowth. Therefore, we aimed to comparatively investigate the effects of local administration of an HNK-1 mimetic peptide (mp-HNK-1) and systemic treatment with ursolic acid (UA), another HNK-1 mimetic, after ventral root avulsion and reimplantation with heterologous fibrin biopolymer (HFB). Methods: Female mice of the isogenic strain C57BL/6JUnib were divided into five experimental groups: Avulsion, Reimplantation, mp-HNK-1 (in situ), and UA (systemic treatment). Mice were evaluated 2 and 12 weeks after surgery. Functional assessment was performed every four days using the Catwalk platform. Neuronal survival was analyzed by cytochemistry, and glial reactions and synaptic coverage were evaluated by immunofluorescence. Results: Treatment with UA elicited long-term neuroprotection, accompanied by a decrease in microglial reactions, and reactive astrogliosis. The neuroprotective effects of UA were preceded by increased glutamatergic and GABAergic inputs in the ventral spinal cord two weeks after injury. However, a single application of mp-HNK-1 had no significant effects. Functional analysis showed that UA treatment led to an improvement in motor and sensory recovery. Conclusion: Overall, the results indicate that UA is neuroprotective, acting on glial cells and synaptic maintenance, and the combination of these findings led to a better functional recovery.

Ultrastructural Evidence of Synapse Preservation and Axonal Regeneration Following Spinal Root Repair with Fibrin Biopolymer and Therapy with Dimethyl Fumarate.

Spinal cord injury causes critical loss in motor and sensory function. Ventral root avulsion is an experimental model in which there is the tearing of the ventral (motor) roots from the surface of the spinal cord, resulting in several morphological changes, including motoneuron degeneration and local spinal cord circuitry rearrangements. Therefore, our goal was to test the combination of surgical repair of lesioned roots with a fibrin biopolymer and the pharmacological treatment with dimethyl fumarate, an immunomodulatory drug. Thus, adult female Lewis rats were subjected to unilateral ventral root avulsion of L4-L6 roots followed by repair with fibrin biopolymer and daily treatment with dimethyl fumarate (15 mg/Kg; gavage) for 4 weeks, the survival time post-surgery being 12 weeks; n = 5/group/technique. Treatments were evaluated by immunofluorescence and transmission electron microscopy, morphometry of the sciatic nerve, and motor function recovery. Our results indicate that the combination between fibrin biopolymer and dimethyl fumarate is neuroprotective since most of the synapses apposed to alfa motoneurons were preserved in clusters. Also, nerve sprouting occurred, and the restoration of the 'g' ratio and large axon diameter was achieved with the combined treatment. Such parameters were combined with up to 50% of gait recovery, observed by the walking track test. Altogether, our results indicate that combining root restoration with fibrin biopolymer and dimethyl fumarate administration can enhance motoneuron survival and regeneration after proximal lesions.

Anti-hyperalgesic effects of photobiomodulation therapy (904 nm) on streptozotocin-induced diabetic neuropathy imply MAPK pathway and calcium dynamics modulation.

Several recent studies have established the efficacy of photobiomodulation therapy (PBMT) in painful clinical conditions. Diabetic neuropathy (DN) can be related to activating mitogen-activated protein kinases (MAPK), such as p38, in the peripheral nerve. MAPK pathway is activated in response to extracellular stimuli, including interleukins TNF-α and IL-1ß. We verified the pain relief potential of PBMT in streptozotocin (STZ)-induced diabetic neuropathic rats and its influence on the MAPK pathway regulation and calcium (Ca2+) dynamics. We then observed that PBMT applied to the L4-L5 dorsal root ganglion (DRG) region reduced the intensity of hyperalgesia, decreased TNF-α and IL-1ß levels, and p38-MAPK mRNA expression in DRG of diabetic neuropathic rats. DN induced the activation of phosphorylated p38 (p-38) MAPK co-localized with TRPV1+ neurons; PBMT partially prevented p-38 activation. DN was related to an increase of p38-MAPK expression due to proinflammatory interleukins, and the PBMT (904 nm) treatment counteracted this condition. Also, the sensitization of DRG neurons by the hyperglycemic condition demonstrated during the Ca2+ dynamics was reduced by PBMT, contributing to its anti-hyperalgesic effects.

Gliosis attenuation in experimental autoimmune encephalomyelitis by a combination of dimethyl fumarate and pregabalin.

Dysregulated microglia and astrocytes have been associated with progressive neurodegeneration in multiple sclerosis (MS), highlighting the need for strategies that additionally target intrinsic inflammation in the central nervous system (CNS). The objective of the present study was to investigate the glial response in experimental autoimmune encephalomyelitis (EAE)-induced mice treated with a combination of dimethyl fumarate (DMF) and pregabalin (PGB). For that, 28 C57BL/6J mice were randomly assigned to the five experimental groups: naïve, EAE, EAE-DMF, EAE-PGB, and EAE-DMF + PGB. Pharmacological treatments were initiated with the beginning of clinical signs, and all animals were euthanized at 28 dpi for the lumbar spinal cord evaluation. The results demonstrated a stronger attenuation of the clinical presentation by the combined approach. DMF alone promoted the downregulation of Iba-1 (microglia/macrophages marker) in the ventral horn compared with the non-treated EAE animals (P < 0.05). PGB treatment was associated with reduced Iba-1 immunofluorescence in both the dorsal (P < 0.05) and ventral horn (P < 0.05) compared to EAE vehicle-treated counterparts. However, the combined approach reduced the Iba-1 marker in the dorsal (P < 0.05) and ventral (P < 0.01) horns compared to non-treated EAE animals and further reduced Iba-1 in the ventral horn compared to each drug-alone approach (P < 0.05). In addition, the combination of DMF and PGB reduced activated astrocytes (GFAP) in both the dorsal and ventral horns of the spinal cord to a naïve-like level and upregulated Nrf-2 expression. Taken together, the data herein suggest robust attenuation of the glial response in EAE mice treated with DMF and PGB.

Neuroprotection and gliosis attenuation by intravenous application of human mesenchymal stem cells (hMSC) following ventral root crush in mice.

Rupture and stretching of spinal roots are common incidents that take place in high-energy accidents. The proximal axotomy of motoneurons by crushing of ventral roots is directly related to the degeneration of half of the lesioned population within the first two weeks. Moreover, only a small percentage of surviving motoneurons can successfully achieve regeneration after such a proximal lesion, and new treatments are necessary to improve this scenario. In this sense, mesenchymal stem cells (MSC) are of great interest once they secrete a broad spectrum of bioactive molecules that are immunomodulatory and can restore the environment after a lesion. The present work aimed at studying the effects of human mesenchymal stem cells (hMSC) therapy after ventral root crush (VRC) in mice. We evaluated motoneuron survival, glial reaction, and synapse preservation at the ventral horn. For this purpose, C57BL/6 J were submitted to a crush procedure of L4 to L6 ventral roots and treated with a single intravenous injection of adipose-derived hMSC. Evaluation of the results was carried out at 7, 14, and 28 days after injury. Analysis of motoneuron survival and astrogliosis showed that hMSC treatment resulted in higher motoneuron preservation (motoneuron survival ipsi/contralateral ratio: VRC group = 53%, VRC + hMSC group = 66%; p < 0.01), combined with reduction of astrogliosis (ipsi/contralateral GFAP immunolabeling: VRC group = 470%, VRC + hMSC group = 250%; p < 0.001). The morphological classification and Sholl analysis of microglial activation revealed that hMSC treatment reduced type V and increased type II profiles, indicating an enhancement of surveying over activated microglial cells. The glial reactivity modulation directly influenced synaptic inputs in apposition to axotomized motoneurons. In the hMSC-treated group, synaptic maintenance was increased (ipsi/contralateral synaptophysin immunolabeling: VRC group = 53%, VRC + hMSC group = 64%; p < 0.05). Overall, the present data show that intravenous injection of hMSC has neuroprotective and anti-inflammatory effects, decreasing reactive astrogliosis, and microglial reaction. Also, such cell therapy results in motoneuron preservation, combined with significant maintenance of spinal cord circuits, in particular those related to the ventral horn.

Leucine-Rich Diet Improved Muscle Function in Cachectic Walker 256 Tumour-Bearing Wistar Rats.

Skeletal muscle atrophy occurs in several pathological conditions, such as cancer, especially during cancer-induced cachexia. This condition is associated with increased morbidity and poor treatment response, decreased quality of life, and increased mortality in cancer patients. A leucine-rich diet could be used as a coadjutant therapy to prevent muscle atrophy in patients suffering from cancer cachexia. Besides muscle atrophy, muscle function loss is even more important to patient quality of life. Therefore, this study aimed to investigate the potential beneficial effects of leucine supplementation on whole-body functional/movement properties, as well as some markers of muscle breakdown and inflammatory status. Adult Wistar rats were randomly distributed into four experimental groups. Two groups were fed with a control diet (18% protein): Control (C) and Walker 256 tumour-bearing (W), and two other groups were fed with a leucine-rich diet (18% protein + 3% leucine): Leucine Control (L) and Leucine Walker 256 tumour-bearing (LW). A functional analysis (walking, behaviour, and strength tests) was performed before and after tumour inoculation. Cachexia parameters such as body weight loss, muscle and fat mass, pro-inflammatory cytokine profile, and molecular and morphological aspects of skeletal muscle were also determined. As expected, Walker 256 tumour growth led to muscle function decline, cachexia manifestation symptoms, muscle fibre cross-section area reduction, and classical muscle protein degradation pathway activation, with upregulation of FoxO1, MuRF-1, and 20S proteins. On the other hand, despite having no effect on the walking test, inflammation status or muscle oxidative capacity, the leucine-rich diet improved muscle strength and behaviour performance, maintained body weight, fat and muscle mass and decreased some protein degradation markers in Walker 256 tumour-bearing rats. Indeed, a leucine-rich diet alone could not completely revert cachexia but could potentially diminish muscle protein degradation, leading to better muscle functional performance in cancer cachexia.

A Novel Multisystem Proteinopathy Caused by a Missense ANXA11 Variant.

OBJECTIVE: Protein misfolding plays a central role not only in amyotrophic lateral sclerosis (ALS), but also in other conditions, such as frontotemporal dementia (FTD), inclusion body myopathy (hIBM) or Paget's disease of bone. The concept of multisystem proteinopathies (MSP) was created to account for those rare families that segregate at least 2 out of these 4 conditions in the same pedigree. The calcium-dependent phospholipid-binding protein annexin A11 was recently associated to ALS in European pedigrees. Herein, we describe in detail 3 Brazilian families presenting hIBM (isolated or in combination with ALS/FTD) caused by the novel p.D40Y change in the gene encoding annexin A11 (ANXA11). METHODS: We collected clinical, genetic, pathological and skeletal muscle imaging from 11 affected subjects. Neuroimaging was also obtained from 8 patients and 8 matched controls. RESULTS: Clinico-radiological phenotype of this novel hIBM reveals a slowly progressive predominant limb-girdle syndrome, but with frequent axial (ptosis/dropped head) and distal (medial gastrocnemius) involvement as well. Muscle pathology identified numerous rimmed vacuoles with positive annexin A11, TDP-43 and p62 inclusions, but no inflammation. Central nervous system was also involved: two patients had FTD, but diffusion tensor imaging uncovered multiple areas of cerebral white matter damage in the whole group (including the corticospinal tracts and frontal subcortical regions). INTERPRETATION: These findings expand the phenotypic spectrum related to ANXA11. This gene should be considered the cause of a novel multisystem proteinopathy (MSP type 6), rather than just ALS. ANN NEUROL 2021;90:239-252.

3D-printed nerve guidance conduits multi-functionalized with canine multipotent mesenchymal stromal cells promote neuroregeneration after sciatic nerve injury in rats.

BACKGROUND: Nerve injuries are debilitating, leading to long-term motor deficits. Remyelination and axonal growth are supported and enhanced by growth factor and cytokines. Combination of nerve guidance conduits (NGCs) with adipose-tissue-derived multipotent mesenchymal stromal cells (AdMSCs) has been performing promising strategy for nerve regeneration. METHODS: 3D-printed polycaprolactone (PCL)-NGCs were fabricated. Wistar rats subjected to critical sciatic nerve damage (12-mm gap) were divided into sham, autograft, PCL (empty NGC), and PCL + MSCs (NGC multi-functionalized with 106 canine AdMSCs embedded in heterologous fibrin biopolymer) groups. In vitro, the cells were characterized and directly stimulated with interferon-gamma to evaluate their neuroregeneration potential. In vivo, the sciatic and tibial functional indices were evaluated for 12 weeks. Gait analysis and nerve conduction velocity were analyzed after 8 and 12 weeks. Morphometric analysis was performed after 8 and 12 weeks following lesion development. Real-time PCR was performed to evaluate the neurotrophic factors BDNF, GDNF, and HGF, and the cytokine and IL-10. Immunohistochemical analysis for the p75NTR neurotrophic receptor, S100, and neurofilament was performed with the sciatic nerve. RESULTS: The inflammatory environment in vitro have increased the expression of neurotrophins BDNF, GDNF, HGF, and IL-10 in canine AdMSCs. Nerve guidance conduits multi-functionalized with canine AdMSCs embedded in HFB improved functional motor and electrophysiological recovery compared with PCL group after 12 weeks. However, the results were not significantly different than those obtained using autografts. These findings were associated with a shift in the regeneration process towards the formation of myelinated fibers. Increased immunostaining of BDNF, GDNF, and growth factor receptor p75NTR was associated with the upregulation of BDNF, GDNF, and HGF in the spinal cord of the PCL + MSCs group. A trend demonstrating higher reactivity of Schwann cells and axonal branching in the sciatic nerve was observed, and canine AdMSCs were engrafted at 30 days following repair. CONCLUSIONS: 3D-printed NGCs multi-functionalized with canine AdMSCs embedded in heterologous fibrin biopolymer as cell scaffold exerted neuroregenerative effects. Our multimodal approach supports the trophic microenvironment, resulting in a pro-regenerative state after critical sciatic nerve injury in rats.

Influence of microcurrent on the modulation of remodelling genes in a wound healing assay.

The literature has shown the beneficial effects of microcurrent (MC) therapy on tissue repair. We investigated if the application of MC at 10 µA/90 s could modulate the expression of remodeling genes transforming growth factor beta (Tgfb), connective tissue growth factor (Ctgf), insulin-like growth factor 1 (Igf1), tenascin C (Tnc), Fibronectin (Fn1), Scleraxis (Scx), Fibromodulin (Fmod) and tenomodulin in NIH/3T3 fibroblasts in a wound healing assay. The cell migration was analyzed between days 0 and 4 in both fibroblasts (F) and fibroblasts + MC (F+MC) groups. On the 4th day, cell viability and gene expression were also analyzed after daily MC application. Higher expression of Ctgf and lower expression of Tnc and Fmod, respectively, were observed in the F+MC group in relation to F group (p < 0.05), and no difference was observed between the groups for the genes Tgfb, Fn1 and Scx. In cell migration, a higher number of cells in the scratch region was observed in group F+MC (p < 0.05) compared to group F on the 4th day, and the cell viability assay showed no difference between the groups. In conclusion, MC therapy at an intensity/time of 10 µA/90 s with 4 daily applications did not affect cell viability, stimulated fibroblasts migration with the involvement of Ctgf, and reduced the Tnc and Fmod expression.

Spinal Reflex Recovery after Dorsal Rhizotomy and Repair with Platelet-Rich Plasma (PRP) Gel Combined with Bioengineered Human Embryonic Stem Cells (hESCs).

Dorsal root rhizotomy (DRZ) is currently considered an untreatable injury, resulting in the loss of sensitive function and usually leading to neuropathic pain. In this context, we recently proposed a new surgical approach to treat DRZ that uses platelet-rich plasma (PRP) gel to restore the spinal reflex. Success was correlated with the reentry of primary afferents into the spinal cord. Here, aiming to enhance previous results, cell therapy with bioengineered human embryonic stem cells (hESCs) to overexpress fibroblast growth factor 2 (FGF2) was combined with PRP. For these experiments, adult female rats were submitted to a unilateral rhizotomy of the lumbar spinal dorsal roots, which was followed by root repair with PRP gel with or without bioengineered hESCs. One week after DRZ, the spinal cords were processed to evaluate changes in the glial response (GFAP and Iba-1) and excitatory synaptic circuits (VGLUT1) by immunofluorescence. Eight weeks postsurgery, the lumbar intumescences were processed for analysis of the repaired microenvironment by transmission electron microscopy. Spinal reflex recovery was evaluated by the electronic Von Frey method for eight weeks. The transcript levels for human FGF2 were over 37-fold higher in the induced hESCs than in the noninduced and the wildtype counterparts. Altogether, the results indicate that the combination of hESCs with PRP gel promoted substantial and prominent axonal regeneration processes after DRZ. Thus, the repair of dorsal roots, if done appropriately, may be considered an approach to regain sensory-motor function after dorsal root axotomy.

Inhibitory effect of red LED irradiation on fibroblasts and co-culture of adipose-derived mesenchymal stem cells.

The objective of this study was to evaluate the effects of red Light Emiting Diode (red LED) irradiation on fibroblasts in adipose-derived mesenchymal stem cells (ASC) co-culture on the scratch assay. We hypothesized that red LED irradiation could stimulate paracrine secretion of ASC, contributing to the activation of genes and molecules involved in cell migration and tissue repair. ASC were co-cultured with NIH/3T3 fibroblasts through direct contact and subjected to red LED irradiation (1.45 J/cm2/5min6s) after the scratch assay, during 4 days. Four groups were established: fibroblasts (F), fibroblasts + LED (FL), fibroblasts + ASC (FC) and fibroblasts + LED + ASC (FLC). The analyzes were based on Ctgf and Reck expression, quantification of collagen types I and III, tenomodulin, VEGF, TGF-ß1, MMP-2 and MMP-9, as well as viability analysis and cell migration. Higher Ctgf expression was observed in FC compared to F. Group FC presented higher amount of tenomodulin and VEGF in relation to the other groups. In the cell migration analysis, a higher number of cells was observed in the scratched area of the FC group on the 4th day. There were no differences between groups considering cell viability, Reck expression, amount of collagen types I and III, MMP-2 and TGF-ß1, whereas TGF-ß1 was not detected in the FC group and the MMP-9 in none of the groups. Our hypothesis was not supported by the results because the red LED irradiation decreased the healing response of ASC. An inhibitory effect of the LED irradiation associated with ASC co-culture was observed with reduction of the amount of TGF-ß1, VEGF and tenomodulin, possibly involved in the reduced cell migration. In turn, the ASC alone seem to have modulated fibroblast behavior by increasing Ctgf, VEGF and tenomodulin, leading to greater cell migration. In conclusion, red LED and ASC therapy can have independent effects on fibroblast wound healing, but the combination of both does not have a synergistic effect. Therefore, future studies with other parameters of red LED associated with ASC should be tested aiming clinical application for tissue repair.

Fibrin biopolymer as scaffold candidate to treat bone defects in rats.

BACKGROUND: Bone tissue repair remains a challenge in tissue engineering. Currently, new materials are being applied and often integrated with live cells and biological scaffolds. The fibrin biopolymer (FBP) proposed in this study has hemostatic, sealant, adhesive, scaffolding and drug-delivery properties. The regenerative potential of an association of FBP, biphasic calcium phosphate (BCP) and mesenchymal stem cells (MSCs) was evaluated in defects of rat femurs. METHODS: Adult male Wistar rats were submitted to a 5-mm defect in the femur. This was filled with the following materials and/or associations: BPC; FBP and BCP; FBP and MSCs; and BCP, FBP and MSCs. Bone defect without filling was defined as the control group. Thirty and sixty days after the procedure, animals were euthanatized and subjected to computed tomography, scanning electron microscopy and qualitative and quantitative histological analysis. RESULTS: It was shown that FBP is a suitable scaffold for bone defects due to the formation of a stable clot that facilitates the handling and optimizes the surgical procedures, allowing also cell adhesion and proliferation. The association between the materials was biocompatible. Progressive deposition of bone matrix was higher in the group treated with FBP and MSCs. Differentiation of mesenchymal stem cells into osteogenic lineage was not necessary to stimulate bone formation. CONCLUSIONS: FBP proved to be an excellent scaffold candidate for bone repair therapies due to application ease and biocompatibility with synthetic calcium-based materials. The satisfactory results obtained by the association of FBP with MSCs may provide a more effective and less costly new approach for bone tissue engineering.

Tempol improves neuroinflammation and delays motor dysfunction in a mouse model (SOD1G93A) of ALS.

BACKGROUND: The development of new therapeutic strategies to treat amyotrophic lateral sclerosis (ALS) is of utmost importance. The use of cyclic nitroxides such as tempol may provide neuroprotection and improve lifespan. We investigated whether tempol (50 mg/kg) presents therapeutic potential in SOD1G93A transgenic mice. METHODS: Tempol treatment began at the asymptomatic phase of the disease (10th week) and was administered every other day until week 14, after which it was administered twice a week until the final stage of the disease. The animals were sacrificed at week 14 (initial stage of symptoms-ISS) and at the end stage (ES) of the disease. The lumbar spinal cord of the animals was dissected and processed for use in the following techniques: Nissl staining to evaluate neuronal survival; immunohistochemistry to evaluate astrogliosis and microgliosis (ISS and ES); qRT-PCR to evaluate the expression of neurotrophic factors and pro-inflammatory cytokines (ISS); and transmission electron microscopy to evaluate the alpha-motoneurons (ES). Behavioral analyses considering the survival of animals, bodyweight loss, and Rotarod motor performance test started on week 10 and were performed every 3 days until the end-stage of the disease. RESULTS: The results revealed that treatment with tempol promoted greater neuronal survival (23%) at ISS compared to untreated animals, which was maintained until ES. The intense reactivity of astrocytes and microglia observed in vehicle animals was reduced in the lumbar spinal cords of the animals treated with tempol. In addition, the groups treated with tempol showed reduced expression of proinflammatory cytokines (IL1ß and TNFα) and a three-fold decrease in the expression of TGFß1 at ISS compared with the group treated with vehicle. CONCLUSIONS: Altogether, our results indicate that treatment with tempol has beneficial effects, delaying the onset of the disease by enhancing neuronal survival and decreasing glial cell reactivity during ALS progression in SOD1G93A mice.

Regular Exercise Modifies Histopathological Outcomes of Pharmacological Treatment in Experimental Autoimmune Encephalomyelitis.

Background: Although it has been suggested that healthier lifestyle may optimize effects of the immunomodulation drugs for treating multiple sclerosis (MS), the knowledge regarding this kind of interactions is limited. Objective: The aim of the present study was to investigate the effects of treadmill exercise in combination with pharmacological treatment in an animal model for MS. Methods: C57BL/6J female mice were subjected to daily treadmill exercise for 4 weeks before immunization and 6 weeks before clinical presentation of disease. Dimethyl fumarate (DMF) or glatiramer acetate (GA) were administered after the first clinical relapse. Histopathological analyses were carried out in the lumbar spinal cord at peak disease and at 1 or 14 days post-treatment (dpt). Results: Exercised-GA treated animals demonstrated decreased astrocytic response in the spinal dorsal horn with an improvement in the paw print pressure. Exercised-DMF treated animals showed an increased microglial/macrophage response on both ventral and dorsal horn that were associated with clinical improvement and synaptic motoneuron inputs density. Conclusion: The present data suggest that prior regular exercise can modify the effects of pharmacological treatment administered after the first relapse in a murine model for MS.

Granulocyte-macrophage colony-stimulating factor improves mouse peripheral nerve regeneration following sciatic nerve crush.

Peripheral nerve injuries severely impair patients' quality of life as full recovery is seldom achieved. Upon axonal disruption, the distal nerve stump undergoes fragmentation, and myelin breaks down; the subsequent regeneration progression is dependent on cell debris removal. In addition to tissue clearance, macrophages release angiogenic and neurotrophic factors that contribute to axon growth. Based on the importance of macrophages for nerve regeneration, especially during the initial response to injury, we treated mice with granulocyte-macrophage colony-stimulating factor (GM-CSF) at various intervals after sciatic nerve crushing. Sciatic nerves were histologically analyzed at different time intervals after injury for the presence of macrophages and indicators of regeneration. Functional recovery was followed by an automated walking track test. We found that GM-CSF potentiated early axon growth, as indicated by the enhanced expression of growth-associated protein at 7 days postinjury. Inducible nitric oxide synthase expression increased at the beginning and at the end of the regenerative process, suggesting that nitric oxide is involved in axon growth and pruning. As expected, GM-CSF treatment stimulated macrophage infiltration, which increased at 7 and 14 days; however, it did not improve myelin clearance. Instead, GM-CSF stimulated early brain-derived neurotrophic factor (BDNF) production, which peaked at 7 days. Locomotor recovery pattern was not improved by GM-CSF treatment. The present results suggest that GM-CSF may have beneficial effects on early axonal regeneration.

Artesunate Ameliorates Experimental Autoimmune Encephalomyelitis by Inhibiting Leukocyte Migration to the Central Nervous System.

BACKGROUND AND AIMS: Experimental autoimmune encephalomyelitis (EAE) is T-cell-dependent disease of the central nervous system (CNS) of mice. This model resembles multiple sclerosis (MS) in many aspects. Therapies that focus in the modulation of the immune response and cellular infiltration in the CNS present best effects in the clinics. Artesunate (Art) is a semi-synthetic sesquiterpene derivative from artemisinin and has been shown to reduce the clinical signs of autoimmune disease models through mechanisms not yet understood. In this study, we aimed to evaluate whether administration of Art would ameliorate EAE. METHODS AND RESULTS: C57BL6 mice were immunized with MOG35-55 peptide to induce EAE. At the same time, Art treatment started (3 mg/kg/day via i.p.) for five consecutive days. We found that Art treatment reduced the clinical signs of EAE and that correlated with a reduced infiltration of cells in the CNS. Disease amelioration did not correlate with immunomodulation as recall responses, leukocyte subpopulations, and gene expression analysis were similar among treated and untreated mice. Ultimately, further analysis provided data indicating that a possible mechanism of action for Art is dependent on the cellular migration to the CNS. CONCLUSIONS: Artesunate reduces the severity of EAE by inhibiting migration of pathogenic T cells to the CNS.

Protection against Paracoccidioides brasiliensis infection in mice treated with modulated dendritic cells relies on inhibition of interleukin-10 production by CD8+ T cells.

Paracoccidioidomycosis is a systemic infection prevalent in Latin American countries. Disease develops after inhalation of Paracoccidioides brasiliensis conidia followed by an improper immune activation by the host leucocytes. Dendritic cells (DCs) are antigen-presenting cells with the unique ability to direct the adaptive immune response by the time of activation of naive T cells. This study was conducted to test whether extracts of P. brasiliensis would induce maturation of DCs. We found that DCs treated with extracts acquired an inflammatory phenotype and upon adoptive transfer conferred protection to infection. Interestingly, interleukin-10 production by CD8(+) T cells was ablated following DC transfer. Further analyses showed that lymphocytes from infected mice were high producers of interleukin-10, with CD8(+) T cells being the main source. Blockage of cross-presentation to CD8(+) T cells by modulated DCs abolished the protective effect of adoptive transfer. Collectively, our data show that adoptive transfer of P. brasiliensis-modulated DCs is an interesting approach for the control of infection in paracoccidioidomycosis.

Triggering of protection mechanism against Phoneutria nigriventer spider venom in the brain.

Severe accidents caused by the "armed" spider Phoneutria nigriventer cause neurotoxic manifestations in victims. In experiments with rats, P. nigriventer venom (PNV) temporarily disrupts the properties of the BBB by affecting both the transcellular and the paracellular route. However, it is unclear how cells and/or proteins participate in the transient opening of the BBB. The present study demonstrates that PNV is a substrate for the multidrug resistance protein-1 (MRP1) in cultured astrocyte and endothelial cells (HUVEC) and increases mrp1 and cx43 and down-regulates glut1 mRNA transcripts in cultured astrocytes. The inhibition of nNOS by 7-nitroindazole suggests that NO derived from nNOS mediates some of these effects by either accentuating or opposing the effects of PNV. In vivo, MRP1, GLUT1 and Cx43 protein expression is increased differentially in the hippocampus and cerebellum, indicating region-related modulation of effects. PNV contains a plethora of Ca(2+), K(+) and Na(+) channel-acting neurotoxins that interfere with glutamate handling. It is suggested that the findings of the present study are the result of a complex interaction of signaling pathways, one of which is the NO, which regulates BBB-associated proteins in response to PNV interference on ions physiology. The present study provides additional insight into PNV-induced BBB dysfunction and shows that a protective mechanism is activated against the venom. The data shows that PNV has qualities for potential use in drug permeability studies across the BBB.