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 β2-microglobulin knockout mice (β2m KO). Young adult (8–12 weeks old) C57BL/6J (WT) and β2m 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 β2m KO mice. Along with neuronal loss, astrogliosis increased in WT mice, which was not observed in β2m 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 β2m KO mice showed stronger Iba-1+ cell reaction. The synaptic inputs were reduced over time, but in β2m 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

Long-standing motor and sensory recovery following acute fibrin sealant based neonatal sciatic nerve repair

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOCAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIORBrachial plexus lesion results in loss of motor and sensory function, being more harmful in the neonate. Therefore, this study evaluated neuroprotection and regeneration after neonatal peripheral nerve coaptation with fibrin sealant. Thus, P2 neonatal Lewis rats were divided into three groups: AX: sciatic nerve axotomy (SNA) without treatment; AX+FS: SNA followed by end-to-end coaptation with fibrin sealant derived from snake venom; AX+CFS: SNA followed by end-to-end coaptation with commercial fibrin sealant. Results were analyzed 4, 8, and 12 weeks after lesion. Astrogliosis, microglial reaction, and synapse preservation were evaluated by immunohistochemistry. Neuronal survival, axonal regeneration, and ultrastructural changes at ventral spinal cord were also investigated. Sensory-motor recovery was behaviorally studied. Coaptation preserved synaptic covering on lesioned motoneurons and led to neuronal survival. Reactive gliosis and microglial reaction decreased in the same groups (AX+FS, AX+CFS) at 4 weeks. Regarding axonal regeneration, coaptation allowed recovery of greater number of myelinated fibers, with improved morphometric parameters. Preservation of inhibitory synaptic terminals was accompanied by significant improvement in the motor as well as in the nociceptive recovery. Overall, the present data suggest that acute repair of neonatal peripheral nerves with fibrin sealant results in neuroprotection and regeneration of motor and sensory axons.Brachial plexus lesion results in loss of motor and sensory function, being more harmful in the neonate. Therefore, this study evaluated neuroprotection and regeneration after neonatal peripheral nerve coaptation with fibrin sealant. Thus, P2 neonatal Lew2016119FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOCAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIORFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOCAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR2014/06892-3; 2012/19646-6; 2012/08101-8; 2011/23236-4; 2009/53846-9; 2011/23377-7563582/2010-3; 300552/2013-9; 310207/2011-823038.000823/2011-21; 23038.005536/2012-3

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

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. 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. 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 (IL1β and TNFα) and a three-fold decrease in the expression of TGFβ1 at ISS compared with the group treated with vehicle. 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 mice161CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP300553/2013-9; 303085/2017-72013/16168-8; 2014/06892-3; 2017/02895-6; 2018/05006-

Expression of class I major histocompatibility complex (MHC I) in the central nervous system: role in synaptic plasticity and regeneration

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.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.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.19319

Estudo dos mecanismos imunomoduladores exercidos pelas células tronco mesenquimais sobre a reatividade das células gliais e correlação com a capacidade regenerativa após axotomia de raízes lombares

Orientadores: Alexandre Leite Rodrigues de Oliveira, Frank KirchhoffTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Lesões na interface CNS/PNS são especialmente severas, levando a até 80% de degeneração neuronal nas primeiras duas semanas. Evidências recentes apontam para o envolvimento das moléculas de MHC-I na interação entre neurônios pré ¿ e pós - sinápticos, bem como entre neurônios axotomizados e células gliais, tendo papel importante na manutenção sináptica seletiva após lesão. O presente trabalho tem por objetivo a padronização do esmagamento de raízes ventrais (VRC) em camundongos e posterior tratamento com células tronco mesenquimais humanas (hMSC), avaliando ainda a potencial interferência da ausência de MHC-I na sobrevivência neuronal, reação glial e cobertura sináptica com e sem a terapia celular. Para isto, camundongos C57BL/6J WT e beta2mKO foram submetidos ao esmagamento das raízes ventrais espinais L4 ¿ L6, tratados ou não com uma injeção intravenosa de hMSC e mantidos por 7, 14 ou 28 dias após a lesão. As análises da sobrevivência neuronal e da astrogliose mostraram padrões parecidos no que se refere ao aumento da perda neuronal e aumento da astrogliose reativa com o tempo em animais WT, sendo que a ausência de MHC-I, aumentou a susceptibilidade dos motoneurônios no período mais agudo. O tratamento com hMSC resultou na maior preservação dos motoneurônios e controle da astrogliose independente da expressão de MHC-I. A reação microglial foi mais intensa 7 dias após a lesão em animais WT, sendo reduzida no 28º dia. Na ausência de MHC-I, padrão semelhante foi detectado, porém com uma reação microglial 33% mais intensa no período agudo após a lesão. Os inputs sinápticos foram reduzidos ao redor dos neurônios axotomizados a partir de 7 dias após a lesão, sendo agudamente mais intensa nos beta2mKO, alcançando uma redução de até 50% no 28º dia após a lesão. Após o tratamento com hMSC, tanto em animais WT quando nos beta2mKO, aproximadamente 65% das sinapses foram mantidas. Os resultados aqui descritos, demonstram que após esmagamento de raízes ventrais em camundongos, MHC I possui um papel no controle da reação microglial aguda afetando temporariamente a perda sináptica e que o tratamento com hMSC reduziu a astrogliose reativa e reação microglial, culminando na neuroproteção de motoneurônios e manutenção da cobertura sináptica independente da expressão de MHC-IAbstract: Lesions on CNS/PNS interface are especially severe, leading up to 80% of neuronal degeneration within the first two weeks. Recent data point out to the involvement of MHC-I in the interactions between pre- and post-synaptic neurons, as well as between axotomized neurons and glial cells, having an important role in selective synaptic maintenance after lesion. The present work objectives the stabilization of ventral root crush (VRC) in mice and further treatment with human mesenchymal stem cells (hMSC), evaluating the potential effect of lack of MHC-I on motoneuron survival, glial reaction, and synaptic covering, with and without cell therapy. For this purpose, C57BL/6J WT e beta2mKO mice were submitted to the crush of L4 to L6 ventral roots, treated or not with one intravenous injection of hMSC and kept for 7, 14 and 28 days after injury. Analysis of motoneuron survival and astrogliosis showed similar patterns regarding the increasing loss of motoneurons and astrogliosis over time on WT animals, while the lack of MHC-I increased the motoneuron susceptibility in the acute phase. hMSC treatment resulted in higher motoneuron preservation and astrogliosis control independent of MHC-I. Microglial reaction was more intense 7 days after lesion in WT animals, becoming reduced over time. In the lack of MHC-I, an analogous pattern was detected, only with a microglial reaction 33% more intense in the acute time point after lesion. Synaptic inputs were reduced around axotomized motoneurons from the 7th day after lesion, being acutely more intense on beta2mKO mice, reaching up to 50% reduction 28 days after injury. After hMSC treatment, both in WT and beta2mKO mice, around 65% of synapses were maintained. Results described herein show that after ventral root crush in mice, MHC I plays a role on acute microglial reaction control, affecting temporarily synaptic loss and that hMSC treatment reduced reactive astrogliosis and microglial reaction, causing motoneuron neuroprotection and synaptic covering maintenance independent of the presence of MHC-IDoutoradoBiologia CelularDoutora em Biologia Celular e Estrutural2013/16134-6FAPES

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 2-microglobulin knockout mice (2m KO). Young adult (8-12 weeks old) C57BL/6J (WT) and 2m 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 2m KO mice. Along with neuronal loss, astrogliosis increased in WT mice, which was not observed in 2m 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 2m KO mice showed stronger Iba-1(+) cell reaction. The synaptic inputs were reduced over time, but in 2m 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 expression85CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP303085/2017-7sem informação2013/16134-6; 2014/06892-3Sao Paulo Research Foundation (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [2013/16134-6, 2014/06892-3]; National Council for Scientific and Technological Development (CNPq)National Council for Scientific and Technological Development (CNPq) [303085/2017-7]; Coordination for the Improvement of Higher Education Personnel (CAPES)CAPES; Deutsche ForschungsgemeinschaftGerman Research Foundation (DFG) [SPP 1757, FOR 2289

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 β2-microglobulin knockout mice (β2m KO). Young adult (8−12 weeks old) C57BL/6J (WT) and β2m 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 β2m KO mice. Along with neuronal loss, astrogliosis increased in WT mice, which was not observed in β2m 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 β2m KO mice showed stronger Iba-1+ cell reaction. The synaptic inputs were reduced over time, but in β2m 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

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 lesion—dal) 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

Flow Cytometry Characterization and Analysis of Glial and Immune Cells from the Spinal Cord

Several protocols have been developed with the aim of characterizing glial and immune cells from the central and peripheral nervous systems. However, a small number of these protocols have demonstrated the ability to yield satisfactory results following conventional isolation. Considering this necessity and the difficulties encountered in enzymatic and bead isolation, our work proposes a method for the isolation of glial and immune cells from the spinal cord utilizing a Percoll gradient. For this purpose, C57BL/6J spinal cords were dissected, and the lumbar intumescence was dissociated and subjected to a Percoll gradient centrifugation (70%, 50%, 37%, and 10%). Each layer was then separated and labeled for astrocytes (anti-GFAP, TNF-α, IFN-γ, IL-10, IL-4), microglia (anti-CD45, CD11b, CD206, CD68, TNF-α, IFN-γ), and lymphocytes (anti-CD3, CD4, IFN-γ, IL-4). The gate detections were mathematically performed by computational analysis utilizing the K-means clustering algorithm. The results demonstrated that astrocytes were concentrated at the Percoll 10/37 interface, microglia at the Percoll 37/50 layer, and lymphocytes at the Percoll 50/70 layer. Our findings indicate that astrocytes in healthy animals are putative of the A1 profile, while microglia and lymphocytes are more frequently labeled with M1 and Th1 markers, suggesting a propensity towards inflammatory responses. The computational method enabled the semi-autonomous gate detection of flow cytometry data, which might facilitate and expedite the processing of large amounts of data