Intraarticular monomethyl fumarate as a perspective therapy for osteoarthritis by macrophage polarization.

BACKGROUND: Osteoarthritis (OA) is a chronic disease that may lead to joint structure degeneration, cartilage destruction, osteophyte formation, subchondral bone disruption, and pain. In this scenario, a higher proportion of the proinflammatory macrophage type 1 (M1) than the anti-inflammatory macrophage type 2 (M2) could be highlighted as a hallmark of OA progression. The balance between these two macrophage types emerges as a new therapeutic target in OA. This study aimed to evaluate the analgesia and macrophage profile in the treatment of experimental osteoarthritis (EOA) with systemic dimethyl fumarate (DMF) or local intra-articular monomethyl fumarate (MMF). RESULTS: DMF via gavage or MMF via intra-articular in the right knee of EOA rats showed improvements in gait parameters and the nociceptive recovery of the mechanical threshold assessment by adapted electronic von Frey treatment on the twenty-first day (long-lasting phase). DMF treatment decreased proinflammatory TNF-α while increasing anti-inflammatory IL-10 cytokines from the macerated capsule on the fifth day (inflammatory phase). MMF treatment showed joint capsule mRNA extraction downregulating iNOS and TNF-α gene expression while upregulating IL-10 and MCP-1. However, CD206 was not significant but higher than untreated EOA rats' joints on the seventh day (inflammatory phase). CONCLUSIONS: Our studies with EOA model induced by MIA suggest a new perspective for human treatment committed with OA based on macrophage polarization as a therapeutic target, switching the proinflammatory profile M1 to the anti-inflammatory profile M2 with DMF systematic or by MMF locally treatment according to the OA severity.

Understanding ayahuasca effects in major depressive disorder treatment through in vitro metabolomics and bioinformatics.

Emerging insights from metabolomic-based studies of major depression disorder (MDD) are mainly related to biochemical processes such as energy or oxidative stress, in addition to neurotransmission linked to specific metabolite intermediates. Hub metabolites represent nodes in the biochemical network playing a critical role in integrating the information flow in cells between metabolism and signaling pathways. Limited technical-scientific studies have been conducted to understand the effects of ayahuasca (Aya) administration in the metabolism considering MDD molecular context. Therefore, this work aims to investigate an in vitro primary astrocyte model by untargeted metabolomics of two cellular subfractions: secretome and intracellular content after pre-defined Aya treatments, based on DMT concentration. Mass spectrometry (MS)-based metabolomics data revealed significant hub metabolites, which were used to predict biochemical pathway alterations. Branched-chain amino acid (BCAA) metabolism, and vitamin B6 and B3 metabolism were associated to Aya treatment, as "housekeeping" pathways. Dopamine synthesis was overrepresented in the network results when considering the lowest tested DMT concentration (1 µmol L-1). Building reaction networks containing significant and differential metabolites, such as nicotinamide, L-DOPA, and L-leucine, is a useful approach to guide on dose decision and pathway selection in further analytical and molecular studies.

Long-term spinal ventral root reimplantation, but not bone marrow mononuclear cell treatment, positively influences ultrastructural synapse recovery and motor axonal regrowth.

We recently proposed a new surgical approach to treat ventral root avulsion, resulting in motoneuron protection. The present work combined such a surgical approach with bone marrow mononuclear cells (MC) therapy. Therefore, MC were added to the site of reimplantation. Female Lewis rats (seven weeks old) were subjected to unilateral ventral root avulsion (VRA) at L4, L5 and L6 levels and divided into the following groups (n = 5 for each group): Avulsion, sealant reimplanted roots and sealant reimplanted roots plus MC. After four weeks and 12 weeks post-surgery, the lumbar intumescences were processed by transmission electron microscopy, to analyze synaptic inputs to the repaired α motoneurons. Also, the ipsi and contralateral sciatic nerves were processed for axon counting and morphometry. The ultrastructural results indicated a significant preservation of inhibitory pre-synaptic boutons in the groups repaired with sealant alone and associated with MC therapy. Moreover, the average number of axons was higher in treated groups when compared to avulsion only. Complementary to the fiber counting, the morphometric analysis of axonal diameter and "g" ratio demonstrated that root reimplantation improved the motor component recovery. In conclusion, the data herein demonstrate that root reimplantation at the lesion site may be considered a therapeutic approach, following proximal lesions in the interface of central nervous system (CNS) and peripheral nervous system (PNS), and that MC therapy does not further improve the regenerative recovery, up to 12 weeks post lesion.

Embryonic stem cells overexpressing high molecular weight FGF2 isoform enhance recovery of pre-ganglionic spinal root lesion in combination with fibrin biopolymer mediated root repair.

BACKGROUND: Spinal ventral root avulsion results in massive motoneuron degeneration with poor prognosis and high costs. In this study, we compared different isoforms of basic fibroblast growth factor 2 (FGF2), overexpressed in stably transfected Human embryonic stem cells (hESCs), following motor root avulsion and repair with a heterologous fibrin biopolymer (HFB). METHODS: In the present work, hESCs bioengineered to overexpress 18, 23, and 31 kD isoforms of FGF2, were used in combination with reimplantation of the avulsed roots using HFB. Statistical analysis was conducted using GraphPad Prism software with one-way or two-way ANOVA, followed by Tukey's or Dunnett's multiple comparison tests. Significance was set at *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. RESULTS: For the first set of experiments, rats underwent avulsion of the ventral roots with local administration of HFB and engraftment of hESCs expressing the above-mentioned FGF2 isoforms. Analysis of motoneuron survival, glial reaction, and synaptic coverage, two weeks after the lesion, indicated that therapy with hESCs overexpressing 31 kD FGF2 was the most effective. Consequently, the second set of experiments was performed with that isoform, so that ventral root avulsion was followed by direct spinal cord reimplantation. Motoneuron survival, glial reaction, synaptic coverage, and gene expression were analyzed 2 weeks post-lesion; while the functional recovery was evaluated by the walking track test and von Frey test for 12 weeks. We showed that engraftment of hESCs led to significant neuroprotection, coupled with immunomodulation, attenuation of astrogliosis, and preservation of inputs to the rescued motoneurons. Behaviorally, the 31 kD FGF2 - hESC therapy enhanced both motor and sensory recovery. CONCLUSION: Transgenic hESCs were an effective delivery platform for neurotrophic factors, rescuing axotomized motoneurons and modulating glial response after proximal spinal cord root injury, while the 31 kD isoform of FGF2 showed superior regenerative properties over other isoforms in addition to the significant functional recovery.

Neuroprotection and reduction of glial reaction by cannabidiol treatment after sciatic nerve transection in neonatal rats.

In neonatal rats, the transection of a peripheral nerve leads to an intense retrograde degeneration of both motor and sensory neurons. Most of the axotomy-induced neuronal loss is a result of apoptotic processes. The clinical use of neurotrophic factors is difficult due to side effects and elevated costs, but other molecules might be effective and more easily obtained. Among them, some are derived from Cannabis sativa. Cannabidiol (CBD) is the major non-psychotropic component found on the surface of such plant leaves. The present study aimed to investigate the neuroprotective potential of CBD. Thus, 2-day-old Wistar rats were divided into the following experimental groups: sciatic nerve axotomy + CBD treatment (CBD group), axotomy + vehicle treatment (phosphate buffer group) and a control group (no-treatment group). The results were analysed by Nissl staining, immunohistochemistry and terminal deoxynucleotidyl transferase dUTP nick end labeling at 5 days post-lesion. Neuronal counting revealed both motor and sensory neuron rescue following treatment with CBD (15 and 30 mg/kg). Immunohistochemical analysis (obtained by synaptophysin staining) revealed 30% greater synaptic preservation within the spinal cord in the CBD-treated group. CBD administration decreased the astroglial and microglial reaction by 30 and 27%, respectively, as seen by glial fibrillary acidic protein and ionised calcium binding adaptor molecule 1 immunolabeling quantification. In line with such results, the terminal deoxynucleotidyl transferase dUTP nick end labeling reaction revealed a reduction of apoptotic cells, mostly located in the spinal cord intermediate zone, where interneurons promote sensory-motor integration. The present results show that CBD possesses neuroprotective characteristics that may, in turn, be promising for future clinical use.

Effect of low-level laser therapy (LLLT) on acute neural recovery and inflammation-related gene expression after crush injury in rat sciatic nerve.

BACKGROUND AND OBJECTIVES: Peripheral nerve function can be debilitated by different kinds of injury. Low-level laser therapy (LLLT) has been used successfully during rehabilitation to stimulate recovery. The aim of this study was to evaluate the effects of LLLT (660 nm, 60 J/cm(2) , 40 mW/cm(2) ) on acute sciatic nerve injury. MATERIALS AND METHODS: Thirty Wistar male rats were divided into three groups: (1) Normal, intact nerves; (2) I3d, crushed nerves evaluated on Day-3 post-injury; (3) I + L3d, crushed nerves submitted to two sessions of LLLT and investigated at 3 days post-injury. Sciatic nerves were removed and processed for gene expression analysis (real-time PCR) of the pro-inflammatory factors TWEAK, Fn14 and TNF-α and extracellular matrix remodeling and axonal growth markers, such as TIMP-1, MMP-2, and MMP-9. Zymography was used to determine levels of MMP-2 and MMP-9 activity and Western blotting was used to evaluate TNF-α protein content. Shapiro-Wilk and Levene's tests were applied to evaluate data normality and homogeneity, respectively. One-way ANOVA followed by Tukey test was used for statistical analysis with a significance level set at 5%. RESULTS: An increase in TNF-α protein level was found in I + L3 compared to Normal and I3d (P < 0.05). Zymography showed an increase in proMMP-9 activity, in both I3d and I + L3d groups (P < 0.05). The increase was more evident in I + L3d (P = 0.02 compared to I3d). Active-MMP-9 isoform activity was increased in I + L3d compared to Normal and I3d groups (P < 0.05). Furthermore, the activity of active-MMP-2 isoform was increased in I3d and I + L3 (P < 0.05). An increase in TIMP-1 expression was observed in both I3d and I + L3d groups (P < 0.05). CONCLUSIONS: The current study showed that LLLT increased MMPs activity, mainly MMP-9, and TNF-α protein level during the acute phase of nerve injury, modulating inflammation. Based on these results, it is recommended that LLLT should be started as soon as possible after peripheral nerve injury.

Neuroprotection by upregulation of the major histocompatibility complex class I (MHC I) in SOD1G93A mice.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that progressively affects motoneurons, causing muscle atrophy and evolving to death. Astrocytes inhibit the expression of MHC-I by neurons, contributing to a degenerative outcome. The present study verified the influence of interferon ß (IFN ß) treatment, a proinflammatory cytokine that upregulates MHC-I expression, in SOD1G93A transgenic mice. For that, 17 days old presymptomatic female mice were subjected to subcutaneous application of IFN ß (250, 1,000, and 10,000 IU) every other day for 20 days. Rotarod motor test, clinical score, and body weight assessment were conducted every third day throughout the treatment period. No significant intergroup variations were observed in such parameters during the pre-symptomatic phase. All mice were then euthanized, and the spinal cords collected for comparative analysis of motoneuron survival, reactive gliosis, synapse coverage, microglia morphology classification, cytokine analysis by flow cytometry, and RT-qPCR quantification of gene transcripts. Additionally, mice underwent Rotarod motor assessment, weight monitoring, and neurological scoring. The results show that IFN ß treatment led to an increase in the expression of MHC-I, which, even at the lowest dose (250 IU), resulted in a significant increase in neuronal survival in the ALS presymptomatic period which lasted until the onset of the disease. The treatment also influenced synaptic preservation by decreasing excitatory inputs and upregulating the expression of AMPA receptors by astrocytes. Microglial reactivity quantified by the integrated density of pixels did not decrease with treatment but showed a less activated morphology, coupled with polarization to an M1 profile. Disease progression upregulated gene transcripts for pro- and anti-inflammatory cytokines, and IFN ß treatment significantly decreased mRNA expression for IL4. Overall, the present results demonstrate that a low dosage of IFN ß shows therapeutic potential by increasing MHC-I expression, resulting in neuroprotection and immunomodulation.

Decreased MHC I expression in IFN γ mutant mice alters synaptic elimination in the spinal cord after peripheral injury.

BACKGROUND: The histocompatibility complex (MHC) class I expression in the central nervous system (CNS) regulates synaptic plasticity events during development and adult life. Its upregulation may be associated with events such as axotomy, cytokine exposition and changes in neuron electrical activity. Since IFNγ is a potent inducer of the MHC I expression, the present work investigated the importance of this pro-inflammatory cytokine in the synaptic elimination process in the spinal cord, as well as the motor recovery of IFN⁻/⁻, following peripheral injury. METHODS: The lumbar spinal cords of C57BL/6J (wild type) and IFNγ⁻/⁻ (mutant) mice, subjected to unilateral sciatic nerve transection, were removed and processed for immunohistochemistry and real time RT-PCR, while the sciatic nerves from animals subjected to unilateral crush, were submitted to immunohistochemistry and electron microscopy for counting of the axons. Gait recovery was monitored using the Cat Walk system. Newborn mice astrocyte primary cultures were established in order to study the astrocytic respose in the absence of the IFNγ expression. RESULTS: IFNγ⁻/⁻ mutant mice showed a decreased expression of MHC I and ß2-microglobulin mRNA coupled with reduced synaptophysin immunolabelling in the lesioned spinal cord segment. Following unilateral nerve transection, the Iba-1 (ionized calcium binding adaptor molecule 1) and glial fibrillary acid protein (GFAP) reactivities increased equally in both strains. In vitro, the astrocytes demonstrated similar GFAP levels, but the proliferation rate was higher in the wild type mice. In the crushed nerves (distal stump), neurofilaments and p75NTR immunolabeling were upregulated in the mutant mice as compared to the wild type and an improvement in locomotor recovery was observed. CONCLUSION: The present results show that a lack of IFNγ affects the MHC I expression and the synaptic elimination process in the spinal cord. Such changes, however, do not delay peripheral nerve regeneration after nerve injury.

Effect of Phoneutria nigriventer venom on the expression of junctional protein and P-gp efflux pump function in the blood-brain barrier.

Phoneutria nigriventer spider venom (PNV) contains Ca(2+), K(+) and Na(+) channel-acting peptides that affect neurotransmitter release and causes excitotoxicity in PNS and CNS. It has been demonstrated that PNV causes blood-brain barrier (BBB) breakdown of hippocampal microvessels time-dependently through enhanced microtubule-mediated vesicular transport. Herein, it is hypothesized that PNV can cause BBB breakdown in the hippocampus and cerebellum time-dependently through other molecular mechanisms. The BBB integrity was assessed through the analysis of expression of Poly-glycoprotein (P-gp) efflux transporter protein, laminin from basement membrane and endothelial tight junctional and adhesion junctional (TJ/AJ) proteins. Phosphatase and tensin homolog (PTEN) and protein phosphatase 2A (PP2A) expression, which are known to have a role in the phosphorylation of junctional proteins and BBB opening, were also investigated. Astrocytes P-gp activity was determined by flow cytometry. The study demonstrated temporary decreased expression of laminin, TJ and AJ proteins (ZO1//occludin//claudin-5//beta-catenin) and P-gp (more prominently in hippocampus), which was completely or partially resolved between 2 and 5 h (and more quickly for cerebellum). PNV inhibited P-gp activity in astrocytes. PP2A phosphorylation, which inhibits the enzyme activity, was increased in both regions (15-45 min); however the phosphorylation level returned to baseline after 2 h. In conclusion, PNV disrupts paracellular transport in the BBB and possesses substrates for the active P-gp efflux transporter located in the BBB complex. Further studies into cellular mechanisms of astrocyte/endothelial interactions, using PNV as tool, may identify how astrocytes regulate the BBB, a characteristic that may be useful for the temporary opening of the BBB.

Impact of acute inflammation on spinal motoneuron synaptic plasticity following ventral root avulsion.

BACKGROUND: Ventral root avulsion is a proximal nerve root lesion in which ventral motor nerve rootlets are torn from surface of the spinal cord, resulting in extensive death of motoneurons. It has been previously shown that if such lesioning is performed in an animal with experimental autoimmune encephalomyelitis (EAE), a significant number of motoneurons can be rescued despite an intense inflammatory reaction. This rescue effect has been attributed to production of a number of neurotrophic factors by invading T cells. Synaptological changes may be involved in neuronal degeneration, and a better understanding of the role of these changes may be of importance for developing new strategies to promote neuronal survival. The objective of the present work was to evaluate neuronal survival, astroglial reaction and synaptic input changes in spinal cord anterior horn motor nuclei after ventral root avulsion in animals with EAE, both during peak disease and after remission. METHODS: Lewis rats were subjected to unilateral avulsion of lumbar ventral roots (VRA) and divided into three groups: VRA control, VRA at peak of EAE, and VRA during EAE remission. The animals were sacrificed and their lumbar spinal cords processed for immunohistochemistry, transmission electron microscopy, and motoneuron counting. RESULTS: The results indicate a reduction in astroglial reaction, a maintenance of microglial reactivity, and increases in synaptic covering of, and survival of, motoneurons in the VRA+EAE group as compared to VRA alone. CONCLUSION: The present findings indicate that CNS inflammation may directly influence synaptic plasticity as well as the stability of neuronal networks, positively influencing the survival of lesioned neurons.

Absence of IFNγ expression induces neuronal degeneration in the spinal cord of adult mice.

BACKGROUND: Interferon gamma (IFNγ) is a pro-inflammatory cytokine, which may be up-regulated after trauma to the peripheral or central nervous system. Such changes include reactive gliosis and synaptic plasticity that are considered important responses to the proper regenerative response after injury. Also, IFNγ is involved in the upregulation of the major histocompatibility complex class I (MHC class I), which has recently been shown to play an important role in the synaptic plasticity process following axotomy. There is also evidence that IFNγ may interfere in the differentiation and survival of neuronal cells. However, little is known about the effects of IFNγ absence on spinal cord neurons after injury. METHODS: We performed a unilateral sciatic nerve transection injury in C57BL/6J (wild type) and IFNγ-KO (mutant) mice and studied motoneuron morphology using light and electron microscopy. One week after the lesion, mice from both strains were sacrificed and had their lumbar spinal cords processed for histochemistry (n = 5 each group) and transmission electron microscopy (TEM, n = 5 each group). Spinal cord sections from non-lesioned animals were also used to investigate neuronal survival and the presence of apoptosis with TUNEL and immunohistochemistry. RESULTS: We find that presumed motoneurons in the lower lumbar ventral horn exhibited a smaller soma size in the IFNγ-KO series, regardless of nerve lesion. In plastic embedded sections stained with toluidine blue, the IFNγ-KO mice demonstrated a greater proportion of degenerating neurons in the ventral horn when compared to the control series (p < 0.05). Apoptotic death is suggested based on TUNEL and caspase 3 immunostaining. A sciatic nerve axotomy did not further aggravate the neuronal loss. The cellular changes were supported by electron microscopy, which demonstrated ventral horn neurons exhibiting intracellular vacuoles as well as degenerating nuclei and cytoplasm in the IFNγ-KO mice. Adjacent glial cells showed features suggestive of phagocytosis. Additional ultrastructural studies showed a decreased number of pre-synaptic terminals apposing to motoneurons in mutant mice. Nevertheless, no statistical difference regarding the input covering could be detected among the studied strains. CONCLUSION: Altogether, these results suggest that IFNγ may be neuroprotective and its absence results in neuronal death, which is not further increased by peripheral axotomy.

Spinal motoneuron synaptic plasticity after axotomy in the absence of inducible nitric oxide synthase.

BACKGROUND: Astrocytes play a major role in preserving and restoring structural and physiological integrity following injury to the nervous system. After peripheral axotomy, reactive gliosis propagates within adjacent spinal segments, influenced by the local synthesis of nitric oxide (NO). The present work investigated the importance of inducible nitric oxide synthase (iNOS) activity in acute and late glial responses after injury and in major histocompatibility complex class I (MHC I) expression and synaptic plasticity of inputs to lesioned alpha motoneurons. METHODS: In vivo analyses were carried out using C57BL/6J-iNOS knockout (iNOS(-/-)) and C57BL/6J mice. Glial response after axotomy, glial MHC I expression, and the effects of axotomy on synaptic contacts were measured using immunohistochemistry and transmission electron microscopy. For this purpose, 2-month-old animals were sacrificed and fixed one or two weeks after unilateral sciatic nerve transection, and spinal cord sections were incubated with antibodies against classical MHC I, GFAP (glial fibrillary acidic protein - an astroglial marker), Iba-1 (an ionized calcium binding adaptor protein and a microglial marker) or synaptophysin (a presynaptic terminal marker). Western blotting analysis of MHC I and nNOS expression one week after lesion were also performed. The data were analyzed using a two-tailed Student's t test for parametric data or a two-tailed Mann-Whitney U test for nonparametric data. RESULTS: A statistical difference was shown with respect to astrogliosis between strains at the different time points studied. Also, MHC I expression by iNOS(-/-) microglial cells did not increase at one or two weeks after unilateral axotomy. There was a difference in synaptophysin expression reflecting synaptic elimination, in which iNOS(-/-) mice displayed a decreased number of the inputs to alpha motoneurons, in comparison to that of C57BL/6J. CONCLUSION: The findings herein indicate that iNOS isoform activity influences MHC I expression by microglial cells one and two weeks after axotomy. This finding was associated with differences in astrogliosis, number of presynaptic terminals and synaptic covering of alpha motoneurons after lesioning in the mutant mice.

Neuroprotection and immunomodulation by dimethyl fumarate and a heterologous fibrin biopolymer after ventral root avulsion and reimplantation.

BACKGROUND: Ventral root avulsion (VRA) is an experimental approach in which there is an abrupt separation of the motor roots from the surface of the spinal cord. As a result, most of the axotomized motoneurons degenerate by the second week after injury, and the significant loss of synapses and increased glial reaction triggers a chronic inflammatory state. Pharmacological treatment associated with root reimplantation is thought to overcome the degenerative effects of VRA. Therefore, treatment with dimethyl fumarate (DMF), a drug with neuroprotective and immunomodulatory effects, in combination with a heterologous fibrin sealant/biopolymer (FS), a biological glue, may improve the regenerative response. METHODS: Adult female Lewis rats were subjected to VRA of L4-L6 roots followed by reimplantation and daily treatment with DMF for four weeks. Survival times were evaluated 1, 4 or 12 weeks after surgery. Neuronal survival assessed by Nissl staining, glial reactivity (anti-GFAP for astrocytes and anti-Iba-1 for microglia) and synapse preservation (anti-VGLUT1 for glutamatergic inputs and anti-GAD65 for GABAergic inputs) evaluated by immunofluorescence, gene expression (pro- and anti-inflammatory molecules) and motor function recovery were measured. RESULTS: Treatment with DMF at a dose of 15 mg/kg was found to be neuroprotective and immunomodulatory because it preserved motoneurons and synapses and decreased astrogliosis and microglial reactions, as well as downregulated the expression of pro-inflammatory gene transcripts. CONCLUSION: The pharmacological benefit was further enhanced when associated with root reimplantation with FS, in which animals recovered at least 50% of motor function, showing the efficacy of employing multiple regenerative approaches following spinal cord root injury.

Toll-like receptor 4 (TLR4) influences the glial reaction in the spinal cord and the neural response to injury following peripheral nerve crush.

After peripheral axotomy, there is a selective retraction of synaptic terminals in contact with injured motoneurons. This process, which actively involves glial cells, is influenced by the expression of immune-related molecules. Since toll-like receptors (TLRs) are upregulated by astrocytes and microglia following lesions, they might be involved in synaptic plasticity processes. Therefore, we administered lipopolysaccharide (LPS) to enhance TLR4 expression in mice and studied retrograde changes in the spinal cord ventral horn following sciatic nerve crush. To this end, adult C57BL/6J male mice were subjected to unilateral sciatic nerve crush at the mid-thigh level and, after a survival time of seven and forty days (acute and chronic phases, respectively), the spinal cords were paraformaldehyde-fixed and dissected out for immunolabeling for synaptophysin, glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter molecule 1 (Iba1). The results show that TLR4 upregulation leads to synaptophysin downregulation close to spinal motoneuron cell bodies, indicating increased synaptic elimination. LPS exposure also further increases astrogliosis and microglial reactions in the both ventral and dorsal horns, especially ipsilateral to nerve axotomy, compared to those in untreated mice. Notably, LPS administration to TLR4-/- mice produces results similar to those observed in untreated wild-type counterparts, reinforcing the role of this receptor in the glial response to injury. Therefore, our results suggest that the overexpression of the TLR4 receptor results in augmented astrogliosis/microglial reactions and the excessive loss of synapses postinjury, which may, in turn, affect the motoneuronal regenerative response and functionality. Additionally, treatment with LPS increases the expression of ß2-microglobulin, a subcomponent of MHC I. Importantly, the absence of TLR4 results in imbalanced axonal regeneration, inducing subsequent improvements and setbacks. In conclusion, our results show the involvement of TLR4 in the process of synaptic remodeling, indicating a new target for future research aimed at developing therapies for CNS and PNS repair.

Local injection of BDNF producing mesenchymal stem cells increases neuronal survival and synaptic stability following ventral root avulsion.

The present study proposed to graft mesenchymal stem cells (MSCs), which continuously produce BDNF, into the spinal cord ventral horn, after ventral root avulsion. Neurotrophin expression was naturally achieved by culturing MSCs in an undifferentiated state for at least 10 weeks. Lewis rats were subjected to unilateral avulsion of lumbar ventral roots, receiving 3 x 10(5) cells injected through the lateral funiculus. Two weeks after surgery, the animals were sacrificed and neuronal survival, astroglial reaction and synaptic inputs within the motor nucleus analyzed. The results indicated that the MSCs treatment significantly rescued avulsed motoneurons. Such neuronal survival was related to in vivo mRNA up regulation as well as expression of BDNF and GDNF. Such increase was correlated to the preservation of synaptophysin- positive nerve terminals. Thus it was proposed that when maintained undifferentiated for a period of 10 weeks, MSCs may be used as a continuous source of BDNF, positively influencing neuronal survival and synaptic plasticity.

Zika virus infection causes temporary paralysis in adult mice with motor neuron synaptic retraction and evidence for proximal peripheral neuropathy.

Clinical evidence is mounting that Zika virus can contribute to Guillain-Barré syndrome which causes temporary paralysis, yet the mechanism is unknown. We investigated the mechanism of temporary acute flaccid paralysis caused by Zika virus infection in aged interferon αß-receptor knockout mice used for their susceptibility to infection. Twenty-five to thirty-five percent of mice infected subcutaneously with Zika virus developed motor deficits including acute flaccid paralysis that peaked 8-10 days after viral challenge. These mice recovered within a week. Despite Zika virus infection in the spinal cord, motor neurons were not destroyed. We examined ultrastructures of motor neurons and synapses by transmission electron microscopy. The percent coverage of motor neurons by boutons was reduced by 20%; more specifically, flattened-vesicle boutons were reduced by 46%, and were normalized in recovering mice. Using electromyographic procedures employed in people to help diagnose Guillain-Barré syndrome, we determined that nerve conduction velocities between the sciatic notch and the gastrocnemius muscle were unchanged in paralyzed mice. However, F-wave latencies were increased in paralyzed mice, which suggests that neuropathy may exist between the sciatic notch to the nerve rootlets. Reversible synaptic retraction may be a previously unrecognized cofactor along with peripheral neuropathy for the development of Guillain-Barré syndrome during Zika virus outbreaks.

Low-level laser and adipose-derived stem cells altered remodelling genes expression and improved collagen reorganization during tendon repair.

OBJECTIVES: The cellular therapy using adipose-derived mesenchymal stem cells (ASCs) aims to improve tendon healing, considering that repaired tendons often result in a less resistant tissue. Our objective was to evaluate the effects of the ASCs combination with a low-level laser (LLL), an effective photobiostimulation for the healing processes. MATERIALS AND METHODS: Rats calcaneal tendons were divided into five groups: normal (NT), transected (T), transected and ASCs (SC) or LLL (L), or with ASCs and LLL (SCL). RESULTS: All treated groups presented higher expression of Dcn and greater organization of collagen fibres. In comparison with T, LLL also up-regulated Gdf5 gene expression, ASCs up-regulated the expression of Tnmd, and the association of LLL and ASCs down-regulated the expression of Scx. No differences were observed for the expression of Il1b, Timp2, Tgfb1, Lox, Mmp2, Mmp8 and Mmp9, neither in the quantification of hydroxyproline, TNF-α, PCNA and in the protein level of Tnmd. A higher amount of IL-10 was detected in SC, L and SCL compared to T, and higher amount of collagen I and III was observed in SC compared to SCL. CONCLUSIONS: Transplanted ASCs migrated to the transected region, and all treatments altered the remodelling genes expression. The LLL was the most effective in the collagen reorganization, followed by its combination with ASCs. Further investigations are needed to elucidate the molecular mechanisms involved in the LLL and ASCs combination during initial phases of tendon repair.

Study of mesenchymal stem cells cultured on a poly(lactic-co-glycolic acid) scaffold containing simvastatin for bone healing.

BACKGROUND: Tissue engineering is a promising alternative for the development of bone substitutes; for this purpose, three things are necessary: stem cells, a scaffold to allow tissue growth and factors that induce tissue regeneration. METHODS: To congregate such efforts, we used the bioresorbable and biocompatible polymer poly(lactic-co-glycolic acid) (PLGA) as scaffold. For the osteoinductive factor, we used simvastatin (SIM), a drug with a pleiotropic effect on bone growth. Mesenchymal stem cells (MSCs) were cultured in PLGA containing SIM, and the bone substitute of PLGA/SIM/MSC was grafted into critical defects of rat calvaria. RESULTS: The in vitro results showed that SIM directly interfered with the proliferation of MSC promoting cell death, while in the pure PLGA scaffold the MSC grew continuously. Scaffolds were implanted in the calvaria of rats and separated into groups: control (empty defect), PLGA pure, PLGA/SIM, PLGA/MSC and PLGA/SIM/MSC. The increase in bone growth was higher in the PLGA/SIM group. CONCLUSIONS: We observed no improvement in the growth of bone tissue after implantation of the PLGA/SIM/MSC scaffold. As compared with in vitro results, our main hypothesis is that the microarchitecture of PLGA associated with low SIM release would have created an in vivo microenvironment of concentrated SIM that might have induced MSC death. However, our findings indicate that once implanted, both PLGA/SIM and PLGA/MSC contributed to bone formation. We suggest that strategies to maintain the viability of MSCs after cultivation in PLGA/SIM will contribute to improvement of bone regeneration.

Enhanced Immune Response in Immunodeficient Mice Improves Peripheral Nerve Regeneration Following Axotomy.

Injuries to peripheral nerves cause loss of motor and sensory function, greatly affecting life quality. Successful repair of the lesioned nerve requires efficient cell debris removal, followed by axon regeneration and reinnervation of target organs. Such process is orchestrated by several cellular and molecular events in which glial and immune cells actively participate. It is known that tissue clearance is largely improved by macrophages, which activation is potentiated by cells and molecules of the acquired immune system, such as T helper lymphocytes and antibodies, respectively. In the present work, we evaluated the contribution of lymphocytes in the regenerative process of crushed sciatic nerves of immunocompetent (wild-type, WT) and T and B-deficient (RAG-KO) mice. In Knockout animals, we found increased amount of macrophages under basal conditions and during the initial phase of the regenerative process, that was evaluated at 2, 4, and 8 weeks after lesion (wal). That parallels with faster axonal regeneration evidenced by the quantification of neurofilament and a growth associated protein immunolabeling. The motor function, evaluated by the sciatic function index, was fully recovered in both mouse strains within 4 wal, either in a progressive fashion, as observed for RAG-KO mice, or presenting a subtle regression, as seen in WT mice between 2 and 3 wal. Interestingly, boosting the immune response by early adoptive transference of activated WT lymphocytes at 3 days after lesion improved motor recovery in WT and RAG-KO mice, which was not ameliorated when cells were transferred at 2 wal. When monitoring lymphocytes by in vivo imaging, in both mouse strains, cells migrated to the lesion site shortly after transference, remaining in the injured limb up to its complete motor recovery. Moreover, a first peak of hyperalgesia, determined by von-Frey test, was coincident with increased lymphocyte infiltration in the damaged paw. Overall, the present results suggest that a wave of immune cell infiltration takes place during subacute phase of axonal regeneration, resulting in transient set back of motor recovery following peripheral axonal injury. Moreover, modulation of the immune response can be an efficient approach to speed up nerve regeneration.

Synaptic rearrangement following axonal injury: Old and new players.

Following axotomy, the contact between motoneurons and muscle fibers is disrupted, triggering a retrograde reaction at the neuron cell body within the spinal cord. Together with chromatolysis, a hallmark of such response to injury is the elimination of presynaptic terminals apposing to the soma and proximal dendrites of the injured neuron. Excitatory inputs are preferentially eliminated, leaving the cells under an inhibitory influence during the repair process. This is particularly important to avoid glutamate excitotoxicity. Such shift from transmission to a regeneration state is also reflected by deep metabolic changes, seen by the regulation of several genes related to cell survival and axonal growth. It is unclear, however, how exactly synaptic stripping occurs, but there is substantial evidence that glial cells play an active role in this process. In one hand, immune molecules, such as the major histocompatibility complex (MHC) class I, members of the complement family and Toll-like receptors are actively involved in the elimination/reapposition of presynaptic boutons. On the other hand, plastic changes that involve sprouting might be negatively regulated by extracellular matrix proteins such as Nogo-A, MAG and scar-related chondroitin sulfate proteoglycans. Also, neurotrophins, stem cells, physical exercise and several drugs seem to improve synaptic stability, leading to functional recovery after lesion. This article is part of a Special Issue entitled 'Neuroimmunology and Synaptic Function'.