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.

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

Abstract 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.

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.

The Time Course of MHC-I Expression in C57BL/6J and A/J Mice Correlates with the Degree of Retrograde Gliosis in the Spinal Cord following Sciatic Nerve Crush.

The pleiotropic role of the major histocompatibility complex class I (MHC-I) reflects the close association between the nervous and immune systems. In turn, MHC-I upregulation postinjury is associated with a better regenerative outcome in isogenic mice following peripheral nerve damage. In the present work, we compared the time course of neuronal, glial, and sensorimotor recovery (1, 3, 5, 7, and 28 days after lesion­dal) following unilateral sciatic nerve crush in A/J and C57BL/6J mice. The A/J strain showed higher expression of MHC-I (7 dal, ** p < 0.01), Iba-1 (microglial reaction, 7 dal, *** p < 0.001), and GFAP (astrogliosis, 5 dal, * p < 0.05) than the C57BL/6J counterpart. Synaptic coverage (synaptophysin) was equivalent in both strains over time. In addition, mRNA expression of microdissected spinal motoneurons revealed an increase in cytoskeleton-associated molecules (cofilin, shp2, and crmp2, * p < 0.05), but not trkB, in C57BL/6J mice. Gait recovery, studied by the sciatic functional index, was faster in the A/J strain, despite the equivalent results of C57BL/6J at 28 days after injury. A similar recovery was also seen for the nociceptive threshold (von Frey test). Interestingly, when evaluating proprioceptive recovery, C57BL/6J animals showed an enlarged base of support, indicating abnormal ambulation postinjury. Overall, the present results reinforce the role of MHC-I expression in the plasticity of the nervous system following axotomy, which in turn correlates with the variable recovery capacity among strains of mice.

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.

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.

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.

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.

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.

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.

Role of MHC-I Expression on Spinal Motoneuron Survival and Glial Reactions Following Ventral Root Crush in Mice.

Lesions to the CNS/PNS interface are especially severe, leading to elevated neuronal degeneration. In the present work, we establish the ventral root crush model for mice, and demonstrate the potential of such an approach, by analyzing injury evoked motoneuron loss, changes of synaptic coverage and concomitant glial responses in ß2-microglobulin knockout mice (ß2m KO). Young adult (8-12 weeks old) C57BL/6J (WT) and ß2m KO mice were submitted to a L4-L6 ventral roots crush. Neuronal survival revealed a time-dependent motoneuron-like cell loss, both in WT and ß2m KO mice. Along with neuronal loss, astrogliosis increased in WT mice, which was not observed in ß2m KO mice. Microglial responses were more pronounced during the acute phase after lesion and decreased over time, in WT and KO mice. At 7 days after lesion ß2m KO mice showed stronger Iba-1+ cell reaction. The synaptic inputs were reduced over time, but in ß2m KO, the synaptic loss was more prominent between 7 and 28 days after lesion. Taken together, the results herein demonstrate that ventral root crushing in mice provides robust data regarding neuronal loss and glial reaction. The retrograde reactions after injury were altered in the absence of functional MHC-I surface expression.

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.

During Development NG2 Glial Cells of the Spinal Cord are Restricted to the Oligodendrocyte Lineage, but Generate Astrocytes upon Acute Injury.

NG2 glia are self-renewal cells widely populating the entire central nervous system (CNS). The differentiation potential of NG2 glia in the brain has been systematically studied. However, the fate of NG2 glia in the spinal cord during development and after injury is still unclear. Here, we took advantage of faithful expression of Cre in NG2-CreERT2 knock-in mice to demonstrate that spinal NG2 glia remain committed to the oligodendrocyte (OL) lineage and generate OLs, but not astrocytes or neurons, during development. However, we found significant age- and region dependent differences in differentiation into OLs. Embryonic or neonatal NG2 glia generated more than 90% of the white matter OLs, but only 50% (embryonic) or 75% (neonatal) of gray matter OLs. Such differences disappeared after myelin completion coinciding with a decrease in the differentiation rate. While we never detected the generation of astrocytes from NG2 glia during spinal cord development, we found a small portion of NG2 glia could generate astrocytes in adult spinal cord upon acute traumatic injury.

In vivo two-photon imaging of motoneurons and adjacent glia in the ventral spinal cord.

BACKGROUND: Interactions between motoneurons and glial cells are pivotal to regulate and maintain functional states and synaptic connectivity in the spinal cord. In vivo two-photon imaging of the nervous system provided novel and unexpected knowledge about structural and physiological changes in the grey matter of the forebrain and in the dorsal white matter of the spinal cord. NEW METHOD: Here, we describe a novel experimental strategy to investigate the spinal grey matter, i.e. the ventral horn motoneurons and their adjacent glial cells by employing in vivo two-photon laser-scanning microscopy (2P-LSM) in anesthetized transgenic mice. RESULTS: After retrograde tracer labelling in transgenic mice with cell-specific expression of fluorescent proteins and surgical exposure of the lumbar intumescence groups of motoneurons could be visualized deeply localized in the ventral horn. In this region, morphological responses of microglial cells to ATP could be recorded for an hour. In addition, using in mice with expression of GCaMP3 in astrocytes, physiological Ca2+ signals could be recorded after local noradrenalin application. COMPARISON WITH EXISTING METHODS: Previous in vivo imaging protocols were restricted to the superficial dorsal white matter or upper layers of the dorsal horn. Here, we modified a multi-step procedure originally established for a root-crush injury. We adapted it to simultaneously visualize motoneurons and adjacent glial cells in living animals. CONCLUSION: A modified surgery approach is presented to visualize fluorescently labelled motoneurons and glial cells at a depth of more than 200 µm in the grey matter ventral horn of the mouse spinal cord.

Multiple uses of fibrin sealant for nervous system treatment following injury and disease.

Lesions to the nervous system often produce hemorrhage and tissue loss that are difficult, if not impossible, to repair. Therefore, scar formation, inflammation and cavitation take place, expanding the lesion epicenter. This significantly worsens the patient conditions and impairment, increasing neuronal loss and glial reaction, which in turn further decreases the chances of a positive outcome. The possibility of using hemostatic substances that also function as a scaffold, such as the fibrin sealant, reduces surgical time and improve postoperative recovery. To date, several studies have demonstrated that human blood derived fibrin sealant produces positive effects in different interventions, becoming an efficient alternative to suturing. To provide an alternative to homologous fibrin sealants, the Center for the Study of Venoms and Venomous Animals (CEVAP, Brazil) has proposed a new bioproduct composed of certified animal components, including a thrombin-like enzyme obtained from snake venom and bubaline fibrinogen. Thus, the present review brings up to date literature assessment on the use of fibrin sealant for nervous system repair and positions the new heterologous bioproduct from CEVAP as an alternative to the commercial counterparts. In this way, clinical and pre-clinical data are discussed in different topics, ranging from central nervous system to peripheral nervous system applications, specifying positive results as well as future enhancements that are necessary for improving the use of fibrin sealant therapy.

Multiple uses of fibrin sealant for nervous system treatment following injury and disease

Lesions to the nervous system often produce hemorrhage and tissue loss that are difficult, if not impossible, to repair. Therefore, scar formation, inflammation and cavitation take place, expanding the lesion epicenter. This significantly worsens the patient conditions and impairment, increasing neuronal loss and glial reaction, which in turn further decreases the chances of a positive outcome. The possibility of using hemostatic substances that also function as a scaffold, such as the fibrin sealant, reduces surgical time and improve postoperative recovery. To date, several studies have demonstrated that human blood derived fibrin sealant produces positive effects in different interventions, becoming an efficient alternative to suturing. To provide an alternative to homologous fibrin sealants, the Center for the Study of Venoms and Venomous Animals (CEVAP, Brazil) has proposed a new bioproduct composed of certified animal components, including a thrombin-like enzyme obtained from snake venom and bubaline fibrinogen. Thus, the present review brings up to date literature assessment on the use of fibrin sealant for nervous system repair and positions the new heterologous bioproduct from CEVAP as an alternative to the commercial counterparts. In this way, clinical and pre-clinical data are discussed in different topics, ranging from central nervous system to peripheral nervous system applications, specifying positive results as well as future enhancements that are necessary for improving the use of fibrin sealant therapy.(AU)

Multiple uses of fibrin sealant for nervous system treatment following injury and disease

Abstract Lesions to the nervous system often produce hemorrhage and tissue loss that are difficult, if not impossible, to repair. Therefore, scar formation, inflammation and cavitation take place, expanding the lesion epicenter. This significantly worsens the patient conditions and impairment, increasing neuronal loss and glial reaction, which in turn further decreases the chances of a positive outcome. The possibility of using hemostatic substances that also function as a scaffold, such as the fibrin sealant, reduces surgical time and improve postoperative recovery. To date, several studies have demonstrated that human blood derived fibrin sealant produces positive effects in different interventions, becoming an efficient alternative to suturing. To provide an alternative to homologous fibrin sealants, the Center for the Study of Venoms and Venomous Animals (CEVAP, Brazil) has proposed a new bioproduct composed of certified animal components, including a thrombin-like enzyme obtained from snake venom and bubaline fibrinogen. Thus, the present review brings up to date literature assessment on the use of fibrin sealant for nervous system repair and positions the new heterologous bioproduct from CEVAP as an alternative to the commercial counterparts. In this way, clinical and pre-clinical data are discussed in different topics, ranging from central nervous system to peripheral nervous system applications, specifying positive results as well as future enhancements that are necessary for improving the use of fibrin sealant therapy.

Expressão do complexo de histocompatilidade principal de classe I (MHC I) no sistema nervoso central: plasticidade sináptica e regeneração / Expression of class I major histocompatibility complex (MHC I) in the central nervous system: role in synaptic plasticity and regeneration / Expresión del complejo principal de histocompatibilidad de clase I (MHC I) en el sistema nervioso central: plasticidad sináptica y regeneración

Foi demonstrado recentemente que o complexo de histocompatibilidade principal de classe I (MHC I), expresso no sistema nervoso central (SNC), não funciona somente como molécula com papel imunológico, mas também como parte de um mecanismo envolvido na plasticidade sináptica. A expressão de MHC I interfere na intensidade e seletividade da retração de sinapses em contato com neurônios que sofreram lesão e também influencia a reatividade das células gliais próximas a esses neurônios. A intensidade do rearranjo sináptico e resposta glial após lesão, ligadas à expressão de MHC I no SNC, repercute em diferenças na capacidade regenerativa e recuperação funcional em linhagens de camundongos isogênicos. Dessa forma, os novos aspectos sobre a função do MHC I no SNC direcionam futuras pesquisas no sentido de buscar o envolvimento do MHC I em doenças neurológicas e também o desenvolvimento de novas estratégias terapêuticas.

It has been recently demonstrated that the major histocompatibility complex of class I (MHC I) expressed in the central nervous system (CNS) does not only function as a molecule of the immune system, but also plays a role in the synaptic plasticity. The expression of MHC I influences the intensity and selectivity of elimination of synapses apposed to neurons that were subjected to lesion, besides influencing the reactivity of neighboring glial cells. MHC I expression and the degree of synaptic rearrangement and glial response after injury correlate with differences in the regenerative potential and functional recovery of isogenic mice strains. In this way, the new aspects regarding MHC I functions in the CNS may guide further studies aiming at searching the involvement of MCH I in neurologic disorders, as well as the development of new therapeutic strategies.

El complejo mayor de histocompatibilidad de clase I (MHC I), expresado en el sistema nervioso central (SNC), no sólo funciona como una molécula con función inmunológica, sino que es crucial para las respuestas del tejido nervioso en casos de lesiones. El MHC I está involucrado con los procesos de plasticidad sináptica y las células gliales en el microambiente de la médula espinal después de realizada axotomía periférica. La expresión de MHC I interfiere con la intensidad y la forma en que se producen la contracción y la eliminación de sinapsis con relación a las neuronas, cuyos axones se han comprometido, y también influye en la reactividad de las células gliales, cerca de estas neuronas. La intensidad de estos cambios, que responden a la expresión de MHC I en el SNC, implica diferencias en la capacidad de regeneración axonal de las células dañadas por axotomía, por lo que el nivel de expresión de las moléculas MHC I se relaciona con el proceso de regeneración de los axones y, en consecuencia, con la recuperación funcional. Por consiguiente, estos nuevos aspectos sobre la función del MHC I en el SNC orientan nuevas investigaciones con miras a entender el papel del MHC I en las enfermedades neurológicas y a desarrollar nuevas estrategias terapéuticas.