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.

Caloric restriction prevents inflammation and insulin dysfunction in middle-aged ovariectomized mice.

BACKGROUND: Loss of ovarian function is associated with increased visceral fat. In this study, we aimed to study the effects of caloric restriction (CR) on metabolism in ovariectomized mice. METHODS AND RESULTS: Female, 8-12-month-old mice were divided into three groups: OVX (ovariectomized mice), OVXR (40% CR) and Sham. CR increased insulin sensitivity and glucose tolerance. AMPK phosphorylation was observed in the liver of OVXR mice. CR also increased hepatic cholesterol and triglyceride levels. The reductions in the level of TBARS in the serum and liver and of H2O2 in the liver of OVXR mice suggested alterations in the redox state of the liver. Although expression of catalase protein was reduced by CR, expression of superoxide dismutase was not altered by CR. Although interleukin IL-6 and IL-10 levels in OVXR mice were similar to those in Sham mice, macrophage infiltration was reduced in OVXR mice. OVXR mice had increased sirtuin1 levels and decreased sirtuin3 levels in the liver. CONCLUSIONS: In conclusion, CR improved the condition of ovariectomized mice by reducing adiposity and increasing insulin sensitivity and glucose tolerance through a mechanism that may involve AMPK.

Interferon-beta induces major histocompatibility complex of class I (MHC-I) expression and a proinflammatory phenotype in cultivated human astrocytes.

Major histocompatibility complex class I (MHC-I) has been implicated in several types of neuroplasticity phenomena. Interferon beta-1b (IFN-ß) increases MHC-I expression by motoneurons after sciatic nerve crush in mice, improving axonal growth and functional recovery. Additionally, IFN-ß induces glial hypertrophy associated with upregulation of glial fibrillary acidic protein (GFAP) and MHC-I in murine astrocytes in vitro. As knowledge about MHC-I and its role in synaptic plasticity in human astrocytes (HAs) is scarce, we investigated these aspects in mature HAs obtained from the neocortex of patients undergoing surgery due to hippocampal sclerosis. Cells were exposed to media in the absence (0 IU/ml) or presence of IFN-ß for 5 days (500 IU/ml). Beta-2 microglobulin (ß2m), a component of the MHC-I, GFAP and vimentin proteins, was quantified by flow cytometry (FC) and increased by 100%, 60% and 46%, respectively, after IFN-ß exposure. We also performed qRT-PCR gene expression analyses for ß2m, GFAP, vimentin, and pro- and anti-inflammatory cytokines. Our data showed that IFN-ß-treated astrocytes displayed ß2m and GFAP gene upregulation. Additionally, they presented a proinflammatory profile with increase in the IL-6 and IL-1ß genes and a tendency to upregulate TNF-α. Moreover, we evaluated the effect of HAs conditioned medium (CM) on the formation/maintenance of neurites/synapses by the PC12 lineage. Synaptophysin protein expression was quantified by FC. The CM of IFN-ß-activated astrocytes was not harmful to PC12 neurites, and there was no change in synaptophysin protein expression. Therefore, IFN-ß activated HAs by increasing GFAP, vimentin and MHC-I protein expression. Like MHC-I modulation and astrocyte activation may be protective after peripheral nerve damage and in some neurodegenerative conditions, this study opens perspectives on the pathophysiological roles of astroglial MHC-I in the human CNS.

Pregabalin synchronizes the regeneration of nerve and muscle fibers optimizing the gait recovery of MDX dystrophic mice.

Duchenne muscular dystrophy (DMD) is an X-linked genetic disorder induced by mutations in the dystrophin gene, leading to a degeneration of muscle fibers, triggering retrograde immunomodulatory, and degenerative events in the central nervous system. Thus, neuroprotective drugs such as pregabalin (PGB) can improve motor function by modulating plasticity, together with anti-inflammatory effects. The present work aimed to study the effects of PGB on axonal regeneration after axotomy in dystrophic and non-dystrophic mice. For that, MDX and C57BL/10 mouse strains were subjected to peripheral nerve damage and were treated with PGB (30 mg/kg/day, i.p.) for 28 consecutive days. The treatment was carried out in mice as soon as they completed 5 weeks of life, 1 week before the lesion, corresponding to the peak period of muscle degeneration in the MDX strain. Six-week-old mice were submitted to unilateral sciatic nerve crush and were sacrificed in the 9th week of age. The ipsi and contralateral sciatic nerves were processed for immunohistochemistry and qRT-PCR, evaluating the expression of proteins and gene transcripts related to neuronal and Schwann cell activity. Cranial tibial muscles were dissected for evaluation of neuromuscular junctions using α-bungarotoxin, and the myelinated axons of the sciatic nerve were analyzed by morphometry. The recovery of motor function was monitored throughout the treatment through tests of forced locomotion (rotarod) and spontaneous walking track test (Catwalk system). The results show that treatment with PGB reduced the retrograde cyclic effects of muscle degeneration/regeneration on the nervous system. This fact was confirmed after peripheral nerve injury, showing better adaptation and response of neurons and glia for rapid axonal regeneration, with efficient muscle targeting and regain of function. No side effects of PGB treatment were observed, and the expression of pro-regenerative proteins in neurons and Schwann cells was upregulated. Morphometry of the axons was in line with the preservation of motor endplates, resulting in enhanced performance of dystrophic animals. Overall, the present data indicate that pregabalin is protective and enhances regeneration of the SNP during the development of DMD, improving motor function, which can, in turn, be translated to the clinic.

Delayed onset, immunomodulation, and lifespan improvement of SOD1G93A mice after intravenous injection of human mesenchymal stem cells derived from adipose tissue.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the selective and progressive loss of motor neurons from the spinal cord, brain stem, and motor cortex. Although the hallmark of ALS is motor neuron degeneration, astrocytes, microglia, and T cells actively participate. Pharmacological treatment with riluzole has little effect on the lifespan of the patient. Thus, the development of new therapeutic strategies is of utmost importance. The objective of this study was to verify whether human mesenchymal stem cells (hMSCs) from adipose tissue have therapeutic potential in SOD1G93A transgenic mice. The treatment was carried out in the asymptomatic phase of the disease (10th week) by a single systemic application of ad-hMSCs (1 ×105 cells). The animals were sacrificed at the 14th week (the initial stage of symptoms) or the end-stage (ES) of the disease. The lumbar spinal cords were dissected and processed for Nissl staining (neuronal survival), immunohistochemistry (gliosis and synaptic preservation), and gene transcript expression (qRT-PCR). Behavioral analyses considering the onset of disease and its progression, neurological score, body weight, and motor control (rotarod test) started on the 10th week and were performed every three days until the ES of the disease. The results revealed that treatment with ad-hMSCs promoted greater neuronal survival (44%) than vehicle treatment. However, no effect was seen at the ES of the disease. Better structural preservation of the ventral horn in animals treated with ad-hMSCs was observed, together with decreased gliosis and greater synapse protection. In line with this, we found that the transcript levels of Hgf1 were upregulated in ad-hMSCs-treated mice. These results corroborate the behavioral data showing that ad-hMSCs had delayed motor deficits and reduced weight loss compared to vehicle animals. Additionally, cell therapy delayed the course of the disease and significantly improved survival by 20 days. Overall, our results indicate that treatment with ad-hMSCs has beneficial effects, enhancing neuronal survival and promoting a less degenerative neuronal microenvironment. Thus, this may be a potential therapy to improve the quality of life and to extend the lifespan of ALS patients.

Effects of different physical training protocols on inflammatory markers in Zymosan-induced rheumatoid arthritis in Wistar rats.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by inflammation and involvement of the synovial membrane, causing joint damage and deformities. No effective drug treatment is available, and physical exercise has been utilized to alleviate the inflammatory processes. This study aimed to investigate the effects of different exercise training protocols on Zymosan-induced RA inflammatory markers in the right knee of Wistar rats. The rodents were subjected to aerobic, resisted, and combined physical training protocols with variations in the total training volume (50% or 100% of resistance and aerobic training volume) for 8 weeks. All physical training protocols reduced cachexia and systemic inflammatory processes. The histological results showed an increase in the inflammatory influx to the synovial tissue of the right knee in all physical training protocols. The rats that underwent combined physical training with reduced volume had a lower inflammatory influx compared to the other experimental groups. A reduction in the mRNA expression of inflammatory genes and an increase in anti-inflammatory gene expression were also observed. The physical training protocol associated with volume reduction attenuated systemic and synovial inflammation of the right knee, reducing the impact of Zymosan-induced RA in rats.

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.

Short and long-term neuroprotective effects of cannabidiol after neonatal peripheral nerve axotomy.

Neonatal rat sciatic nerve crush mimics obstetric axonotmesis, leading to extensive loss of motor and sensory neurons. The present study aimed to investigate the neuroprotective potential of cannabidiol (CBD) and the role of cannabinoid receptors after sciatic nerve crush in neonatal rats. For that, two-day-old Wistar rats were used, organized into the following experimental groups: sciatic nerve crush plus CBD treatment (CBD), crush plus vehicle treatment (VE), crush + CBD + AM251 treatment (AM251 - CB1 inverse agonist), crush + CBD + AM630 treatment (AM630 - CB2 antagonist). Spinal motoneuron survival was evaluated by Nissl staining of the lumbar spinal cord, 5- and 56-days following injury. CBD treatment enhanced neuronal survival by ~54 % both 5 days and 8 weeks after injury. However, AM251 and AM630 treatment decreased neuronal rescue by 30 % when compared to the CBD group, suggesting that CBD acts partially through such pathways. However, in the long term, only the CB1 blockade reverted CBD positive results. Synaptic preservation was evaluated by anti-synaptophysin immunolabeling. Five days after the lesion, CBD treatment preserved ~35 % of synapses in the ventral horn, and such effect was partially reversed by CB1 inactivation. Additionally, CBD treatment reduced astroglial reaction both at 5 days (39 %, compared to VE) and 8 weeks (31 %, compared to VE) after lesion. The microglial response was acutely reduced by 62 % after CBD treatment. Overall, the results herein show that CBD is neuroprotective, increasing neuronal survival and reducing glial reaction after neonatal axotomy. Such effects require CB1 and CB2 receptors to be effective, in turn influencing neuroprotection, glial reactivity, and functional recovery.

Pregabalin-induced neuroprotection and gait improvement in dystrophic MDX mice.

Duchenne muscular dystrophy (DMD) is a genetic disease linked to the X chromosome induced by mutations in the dystrophin gene. Neuroprotective drugs, such as pregabalin (PGB), can improve motor function through the modulation of excitatory synapses, together with anti-apoptotic and anti-inflammatory effects. The present work studied the effects of PGB in the preservation of dystrophic peripheral nerves, allowing motor improvements in MDX mice. Five weeks old MDX and C57BL/10 mice were treated with PGB (30 mg/kg/day, i.p.) or vehicle, for 28 consecutive days. The mice were sacrificed on the 9th week, the sciatic nerves were dissected out and processed for immunohistochemistry and qRT-PCR, for evaluating the expression of proteins and gene transcripts related to neuronal activity and Schwann cell function. The lumbar spinal cords were also processed for qRT-PCR to evaluate the expression of neurotrophic factors and pro- and anti-inflammatory cytokines. Cranial tibial muscles were dissected out for endplate evaluation with α-bungarotoxin. The recovery of motor function was monitored throughout the treatment, using a spontaneous walking track test (Catwalk system) and a forced locomotion test (Rotarod). The results showed that treatment with PGB reduced the retrograde effects of muscle degeneration/regeneration on the nervous system from the 5th to the 9th week in MDX mice. Thus, PGB induced protein expression in neurons and Schwann cells, protecting myelinated fibers. In turn, better axonal morphology and close-to-normal motor endplates were observed. Indeed, such effects resulted in improved motor coordination of dystrophic animals. We believe that treatment with PGB improved the balance between excitatory and inhibitory inputs to spinal motoneurons, increasing motor control. In addition, PGB enhanced peripheral nerve homeostasis, by positively affecting Schwann cells. In general, the present results indicate that pregabalin is effective in protecting the PNS during the development of DMD, improving motor coordination, indicating possible translation to the clinic.

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.

In Vivo Investigation of Polymer-Ceramic PCL/HA and PCL/ß-TCP 3D Composite Scaffolds and Electrical Stimulation for Bone Regeneration.

Critical bone defects are a major clinical challenge in reconstructive bone surgery. Polycaprolactone (PCL) mixed with bioceramics, such as hydroxyapatite (HA) and tricalcium phosphate (TCP), create composite scaffolds with improved biological recognition and bioactivity. Electrical stimulation (ES) aims to compensate the compromised endogenous electrical signals and to stimulate cell proliferation and differentiation. We investigated the effects of composite scaffolds (PCL with HA; and PCL with ß-TCP) and the use of ES on critical bone defects in Wistar rats using eight experimental groups: untreated, ES, PCL, PCL/ES, HA, HA/ES, TCP, and TCP/ES. The investigation was based on histomorphometry, immunohistochemistry, and gene expression analysis. The vascular area was greater in the HA/ES group on days 30 and 60. Tissue mineralization was greater in the HA, HA/ES, and TCP groups at day 30, and TCP/ES at day 60. Bmp-2 gene expression was higher in the HA, TCP, and TCP/ES groups at day 30, and in the TCP/ES and PCL/ES groups at day 60. Runx-2, Osterix, and Osteopontin gene expression were also higher in the TCP/ES group at day 60. These results suggest that scaffolds printed with PCL and TCP, when paired with electrical therapy application, improve bone regeneration.

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.

Neuroprotection by dimethyl fumarate following ventral root crush in C57BL/6J mice.

CNS lesions usually result in permanent loss of function and are an important problem in the medical field. In order to investigate neuroprotection/degeneration mechanisms and the synaptic plasticity of motoneurons, in addition to the potential for a variety of treatments, different experimental models of axonal injury have been proposed. Recent studies have tested the immunomodulatory drug dimethyl fumarate (DMF) for the treatment of neurodegenerative diseases and have shown promising outcomes. Therefore, in this work, we investigated the effects of DMF with regard to neuroprotection and its influence on the glial response in C57BL/6J animals subjected to crushing of the motor roots in the lumbar intumescence of the spinal cord. The animals were divided into a vehicle-treated injury group (0.08 % methylcellulose solution control group, n = 7) and injured groups treated with DMF at different doses (15, 30, 45, 90 and 180 mg/kg; n = 6-7 per dose). The 90 mg/kg dose showed the best neuroprotective results, so it was used for treatment over a period of eight weeks. Neuronal survival was assessed through Nissl staining, and functional recovery was evaluated with the CatWalk system (walking track test) and the von Frey test (mechanoreception). Immunohistochemistry was used to assess synaptic coverage and astroglial and microglial reactivity using the primary antibodies anti-synaptophysin (pre-synaptic terminal pan marker), GAD65 (GABAergic pre-synaptic terminations - inhibitory), and VGLUT1 (glutamatergic pre-synaptic terminations - excitatory). Glial reactions were evaluated with anti-IBA1 (microglia) and GFAP (astrocytes). Gene transcript levels of IL-3, IL-4, TNF-α, IL-6, TGF-ß, iNOS-M1, and arginase-M2 were quantified by RT-qPCR. The results indicated that treatment with DMF, at a dose of 90 mg/kg, promoted neuroprotection and immunomodulation towards an anti-inflammatory response. It also resulted in greater preservation of inhibitory synapses and reduced astroglial reactivity, providing a more favorable environment for sensorimotor recovery.

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.

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

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.(AU)

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.

Reflex arc recovery after spinal cord dorsal root repair with platelet rich plasma (PRP).

Spinal dorsal roots can be affected by a wide range of lesions, leading to a significant loss of proprioceptive information transmission and greatly affecting motor behavior. In this context, the reimplantation of lesioned roots with platelet-rich plasma (PRP) may allow nerve regeneration. Therefore, the present study evaluated sensorimotor improvement following dorsal root rhizotomy and repair with PRP. For this purpose, female Lewis rats were subjected to unilateral rhizotomy (RZ) of the L4-L6 dorsal roots and divided into the following groups: (1) the unlesioned control group; (2) the group that underwent rhizotomy (RZ) without repair; and (3) the group that underwent RZ followed by root repair with PRP. PRP was obtained from human blood and characterized regarding platelet concentration, integrity, and viability. Reflex arc recovery was evaluated weekly for eight weeks by the electronic von Frey method. The spinal cords were processed 1 week postlesion to evaluate the in vivo gene expression of TNFα, TGF-ß, BDNF, GDNF, VEGF, NGF, IL-4, IL-6, IL-13 by qRT-PCR and eight weeks postlesion to evaluate changes in the glial response (GFAP and Iba-1) and excitatory synaptic circuits (VGLUT1) by immunofluorescence. The results indicated that PRP therapy partially restores the paw withdrawal reflex over time, indicating the reentry of primary afferents from the dorsal root ganglia into the spinal cord without exacerbating glial reactivity. Additionally, the analysis of mRNA levels showed that PRP therapy has immunomodulatory properties. Overall, the present data suggest that the repair of dorsal roots with PRP may be considered a promising approach to improve sensorimotor recovery following dorsal rhizotomy.

Neuronal preservation and reactive gliosis attenuation following neonatal sciatic nerve axotomy by a fluorinated cannabidiol derivative.

Immature peripheral nervous system damage, such as the transection of a peripheral nerve, results in the extensive degeneration of motoneurons and dorsal root ganglia (DRG) sensory neurons, mostly due to apoptotic events. We have previously shown that cannabidiol (CBD), the most abundant non-psychotropic molecule present in the Cannabis sativa plant, exhibits neuroprotective action when administered daily at a dose of 15 mg/kg. This study shows that use of the fluorinated synthetic version of CBD (4'-fluoro-cannabidiol, HUF-101) significantly improves neuronal survival by 2-fold compared to that achieved with traditional CBD at one-third the dose. Furthermore, we show that HUF-101 administration significantly upregulates anti-apoptotic genes and blocks the expression of pro-apoptotic nuclear factors. Two-day-old Wistar rats were subjected to unilateral sectioning of the sciatic nerve and treated daily with HUF-101 (1, 2.5, 5 mg/kg/day, i.p.) or a vehicle solution for five days. The results were evaluated by Nissl staining, immunohistochemistry, and qRT-PCR. Neuronal counting revealed a 47% rescue of spinal motoneurons and a 79% rescue of DRG neurons (HUF-101, 5 mg/kg). Survival was associated with complete depletion of p53 and a 60-fold elevation in BCL2-like 1 gene expression. Additionally, peroxisome proliferator-activated receptor gamma (PPAR-gamma) gene expression was downregulated by 80%. Neuronal preservation was coupled with a high preservation of synaptic coverage and a reduction in astroglial and microglial reactions that were evaluated in nearby spinal motoneurons present in the ventral horn of the lumbar intumescence. Overall, these data strongly indicate that HUF-101 exerts potent neuroprotective effects that are related to anti-apoptotic protection and the reduction of glial reactivity.

The Emerging Role of the Major Histocompatibility Complex Class I in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting upper and lower motoneurons (MNs). The etiology of the disease is still unknown for most patients with sporadic ALS, while in 5-10% of the familial cases, several gene mutations have been linked to the disease. Mutations in the gene encoding Cu, Zn superoxide dismutase (SOD1), reproducing in animal models a pathological scenario similar to that found in ALS patients, have allowed for the identification of mechanisms relevant to the ALS pathogenesis. Among them, neuroinflammation mediated by glial cells and systemic immune activation play a key role in the progression of the disease, through mechanisms that can be either neuroprotective or neurodetrimental depending on the type of cells and the MN compartment involved. In this review, we will examine and discuss the involvement of major histocompatibility complex class I (MHCI) in ALS concerning its function in the adaptive immunity and its role in modulating the neural plasticity in the central and peripheral nervous system. The evidence indicates that the overexpression of MHCI into MNs protect them from astrocytes' toxicity in the central nervous system (CNS) and promote the removal of degenerating motor axons accelerating collateral reinnervation of muscles.

Neuroprotective effect of tempol (4 hydroxy-tempo) on neuronal death induced by sciatic nerve transection in neonatal rats.

Peripheral nerve injury in newborn rats triggers extensive neuronal death within the spinal cord. Because most neurodegeneration is related to oxidative stress and apoptosis, the use of antioxidants may be of therapeutic interest. Tempol is promising because of its ability to chelate reactive oxygen species and to minimize or even prevent tissue damage. Here, we evaluated neuroprotective effects of tempol following neonatal sciatic nerve transection. Two-day-old pups underwent sciatic nerve axotomy followed by tempol (12, 24 and 48 mg/kg) treatment (i.p.) at 10 min, 6 h, and every 24 h up to 1 week after injury. The rats were then killed for lumbar intumescence analysis. Nissl staining, TUNEL, synaptophysin immunolabeling and qRT-PCR (Caspase 3, Bax and Bcl2) were carried out. The results indicated that tempol treatment, at 24 mg/kg, increased up to 21% spinal cord motoneuron survival (p<0.001), also preserving pre-synaptic terminals in the neuropile. Likewise, the TUNEL-positive cell number decreased in tempol-treated animals. qRT-PCR results indicated differential increase in Caspase 3 (3-fold), Bax (13-fold) and Bcl2 (28-fold) gene expression, after 12 h following axotomy and tempol treatment. In conclusion, tempol administration has proven to be neuroprotective after neonatal nerve injury, leading to improved motoneuron survival, synapse preservation and minimizing apoptosis.