Involvement of GDNF/PI3K/AKT pathway in promoting facial nerve regeneration using electro acupuncture in a rabbit model of facial nerve crush injury / 中国组织工程研究

BACKGROUND: The mechanism of electroacupuncture on multi-acupoints in the treatment of peripheral facial paralysis is still unknown. Glial cell-derived neurotrophic factor (GDNF) is currently the most effective factor in promoting the survival of motoneurons in vitro, and the PI3K/AKT pathway plays an important role in protecting damaged motoneurons. There is yet no research on GDNF/PI3K/AKT pathway involved in electroacupuncture promoting facial nerve regeneration in rabbits. OBJECTIVE: To observe the effect of electroacupuncture on regeneration after peripheral facial nerve crush injury, and to explore the protective mechanism of electroacupuncture on facial motoneurons through the GDNF/PI3K/AKT signaling pathway. METHODS: Sixty-six adult healthy New Zealand white rabbits provided by the Animal Experimental Center of Southwest Medical University were randomly divided into a normal group and a model group. The facial nerves on the right side in the model group were subjected to a crush injury. Then the animal models were randomly divided into a model control group and an electroacupuncture group. Animals in the model control group recovered naturally, while those in the electroacupuncture group underwent electroacupuncture at Yifeng, Jiache, Sibai, Dicang, Yangbai, and Quanliao acupoints daily for 30 minutes. The improvement of facial paralysis symptoms in experimental animals were observed and scored. Tissue samples were directly taken form the normal group, and pons tissues with facial neurons were taken in the model group at 1, 4, 7, 14, and 28 days postoperatively. The morphologies of facial motoneurons and Nissl bodies were observed by hematoxylin-eosin staining and Nissl staining, respectively. Immunohistochemical techniques and western blot assay were used to detect the protein expression of GDNF, PI3K, AKT, and p-AKT in the facial motoneurons. The study protocol was approved by the Animal Ethics Committee of Southwest Medical University with approval No. 20170120001. RESULTS AND CONCLUSION: The symptoms of facial paralysis were that the animal’s mouth was drooped at the affected side, with lodging tentacles and the movement being weakened, and the eyelids that could not be lifted, which recovered faster and more completely in the electroacupuncture group than the model control group. The morphological changes of facial neurons and changes of Nissl bodies in the electroacupuncture group were lighter than those in the model control group. At each time point postoperatively, the stronger GDNF immune response could be seen in the electroacupuncture group, and the number of GDNF-positive cells was higher than that of the model control group except 1 day postoperatively (P < 0.001). The expressions of GDNF, PI3K, p-AKT proteins in the facial motoneurons were significantly increased in the electroacupuncture group compared with the model control group (P < 0.05; P < 0.01; P < 0.001). To conclude, electroacupuncture can effectively treat the peripheral facial paralysis caused by the crushed injury of facial nerve and promote the recovery of facial neurons. The up-regulation of GDNF expression in the facial motoneurons and the activation of PI3K/AKT signaling pathway may be the underlying protective mechanism of electroacupuncture.

Vesicular Glutamate Transporter 1 (VGLUT1)- and VGLUT2-containing Terminals on the Rat Jaw-closing γ-Motoneurons

Currently, compared to jaw-closing (JC) α-motoneurons, the information on the distribution and morphology of glutamatergic synapses on the jaw-closing (JC) γ-motoneurons, which may help elucidate the mechanism of isometric contraction of the JC muscle, is very limited. This study investigated the distribution and ultrastructural features of vesicular glutamate transporter 1 (VGLUT1)- and VGLUT2-immunopositive (+) axon terminals (boutons) on JC γ-motoneurons by retrograde tracing with horseradish peroxidase, electron microscopic immunocytochemistry, and quantitative analysis. About 35% of the boutons on identified JC γ-motoneurons were VGLUT+, and of those, 99% were VGLUT2+. The fraction of VGLUT1+ boutons of all boutons and the percentage of membrane of JC γ-motoneurons covered by these boutons were significantly lower than those for the JC α-motoneurons, revealed in our previous work. The bouton volume, mitochondrial volume, and active zone area of the VGLUT2+ boutons on the JC γ-motoneurons were uniformly small. These findings suggest that the JC γ-motoneurons, in contrast to the JC α-motoneurons, receive generally weak glutamatergic synaptic input almost exclusively from VGLUT2+ premotoneurons that form direct synapse with motoneurons.

Modelo in vitro para el estudio del papel de la unión mesopontina en la generación del sueño de movimientos oculares rápidos y la vigilia / In vitro model for the study of the role of the mesopontine region in rapid eye movement (REM) sleep and wakefulness / Modelo in vitro para o estudo do papel da união mesopontina na geração de sono de movimentos oculares rápidos e da vigília

El estudio de las estrategias neurales para la organización del comportamiento en vertebrados constituye un desafío mayor para la Neurociencia. El avance del conocimiento en este campo depende de manera crítica de la utilización de modelos experimentales adecuados que admitan múltiples niveles de análisis (p.ej: comportamental, circuital, celular, sináptico, molecular) y abordajes multitécnicos. Nos propusimos analizar in vitro una red neural de la unión mesopontina del tronco encefálico críticamente implicada en el control del sueño de movimientos oculares rápidos (S-REM). Pese al cúmulo de evidencias que apoyan el papel desempeñado por esta red en relación al S-REM, los mecanismos celulares y sinápticos que subyacen a este control son poco conocidos y continúan siendo objeto de intensa investigación. Para avanzar en el conocimiento de estos mecanismos, se llevó a cabo la caracterización morfológica y funcional de una rodaja de tronco encefálico de la rata, en la que las estructuras críticas para el control del S-REM, i.e.: núcleos tegmentales laterodorsal y pedúnculopontino, y su proyección al núcleo reticular pontis oralis (PnO), están presentes y son operativas. La inclusión del núcleo motor del trigémino en la rodaja permitió detectar cambios de la excitabilidad de las motoneuronas ante manipulaciones farmacológicas del PnO, representativos de los cambios del tono muscular asociados a maniobras similares realizadas in vivo. La utilización de este modelo in vitro de S-REM, permitirá aportar a la dilucidación de las estrategias neurales que operan en niveles intermedios de organización del SN en mamíferos para la generación y regulación de un estado comportamental.

The study of the neural basis of behavior is a major challenge in Neuroscience. Advancing our knowledge in this field depends, critically, on the use of experimental paradigms that provide multiple levels of analysis, as well as powerful techniques. We have selected, as a model of a neural plan that organizes a complex behavior, a neural network located in the mesopontine junction. This region is thought to be both necessary and sufficient for the generation of rapid eye movement (REM) sleep, although the cellular and synaptic mechanisms involved in the control of this behavioral state at the mesopontine level are still under debate and remain poorly understood. As part of a long term effort to gain insight into these mechanisms, we carried out the morphological and functional characterization of a slice preparation of rat brainstem and we demonstrate that critical structures for the control of REM sleep - the laterodorsal and pedunculopontine tegmental nuclei and their projection to the oral part of the pontine reticular nucleus (PnO) - are present and are operational. The presence of the trigeminal motor nucleus in the slice sought to include in the experimental model a structure capable of expressing changes of the excitability of the motorneurons caused by pharmacological manipulations of the PnO, representative of changes of muscle tone associated with similar maneuvers performed in vivo. The use of this in vitro model of REM sleep will provide critical information to elucidate neural strategies that operate at intermediate levels of central nervous system organization in mammals to control behavioral states.

O estudo de estratégias neurais para a organização do comportamento em vertebrados constitui um desafio maior para a Neurociencia. O avanço do conhecimento nessa área depende criticamente da utilização de modelos experimentais adequados que suportem múltiplos níveis de análise (por exemplo: comportamental, circuital, celular, sináptico e molecular) e abordagens por múltiplas técnicas. Decidiu-se analisar in vitro uma rede neural da união mesopontina do tronco encefálico criticamente envolvida no controle do sono de movimentos oculares rápidos (S-REM). Apesar da riqueza de provas que sustentam o papel desta rede em relação ao S-REM, os mecanismos celulares e sinápticos subjacentes a este controle são pouco conhecidos e permanecem sob intensa investigação. Para avançar no conhecimento desses mecanismos, caracterizou-se morfológica e funcionalmente uma fatia de tronco encefálico de rato, na qual as estruturas críticas para o controle do S-REM, i.e.: núcleos tegmentais laterodorsal e pedunculopontino, e sua projeção para o núcleo reticular pontis oralis (PnO) estão presentes e operantes. A inclusão do núcleo motor do trigêmeo na fatia permitiu detectar mudanças da excitabilidade das motoneuronas provocadas por manipulações farmacológicas do PnO, representativas das alterações do tônus muscular associados com operações semelhantes quando realizados in vivo. A utlização deste modelo in vitro de S-REM permitirá contribuir para a elucidação de estratégias neurais que operam em níveis intermedios de organização do SN de mamíferos para a geração e regulação de um estado comportamental.

VGCC expression in facial nucleus motoneurons after facial nerve injury in adult rats / 实用口腔医学杂志

Objective:To investigate the expression change of voltage-gated calcium channels(VGCC) in the facial nucleus motoneurons of adult rats after facial nerve injury.Methods:The facial motor nucleus was localized by retrograde labeling with a fluorescent dye,Dil,and identified by Nissl staining.The facial nerve injury model was established by amputation of the main trunk of left facial nerve.Exposure of the right facial nerve without nerve transection was used as the control.Rats were sacrificed at 3,7,14 and 28 days after injury respectively(n =10),the brainstem containing facial nucleus were collected,the expression of P/Q,N,L,R-type calcium channel α1A,α1B,α1C and α1E subunits was examined by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR).Results:Immunohistochemistry results show that whereas α1A,α1E subunits levels did not vary compared with control group 3,7,14,28 days after injury(P ＞0.05),α1B and α1C subunits immunoreactivity decreased in the motoneurons after injury,a sharp decrease was detected at 14 days after injury(P ＜ 0.01),thereafter returned to the control level at 28 days after axotomy(P ＞0.05).The expression of α1B and α1C mRNA was down-regulated,especially 14 days after the injury(P ＜0.01),and then recovered to normal level at 28 days (P ＞ 0.05).In addition,there was no significant difference of α1A and α1E subunits and their correspoding mRNA between operated group and control group at all time points(P ＞ 0.05).Conclusion:VGCC is involved in facial nerve injury and down-regulation of N-type and L-type calcium channels may be one of the role.

Disfuncionalidades neurofisiológicas y alteraciones en la calidad de vida enpoblaciones con esclerosis lateral amiotrófica / Neurophysiological dysfunctions and alterations in the quality of life in populations with amyotrophic lateral sclerosis

La esclerosis lateral amiotrófica (ELA), es una enfermedad neurodegenerativa que deteriora gradualmente las motoneuronas. La corteza cerebral es una de las áreas más afectadas durante la evolución de la ELA, comprometiendo además, regiones del tronco encefálico y los núcleos basales. Los daños provocados por esta enfermedad, ocurren a nivel neuromotor y respiratorio, siendo ésta última, la causa de los decesos en pacientes que la padecen. La Calidad de Vida (CV), en las poblaciones con esta enfermedad, tiende a decrecer significativamente, y los métodos de diagnóstico y previsión son poco efectivos para detectar la ELA y abordarla eficazmente. Es necesaria una labor mancomunada e interdisciplinaria para conseguir mejorar el grado de CV en estospacientes y en sus cuidadores.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that gradually deteriorates motor neurons. The cerebral cortex is one of the most affected areas during the course of ALS committing regions of the brainstem and basal ganglia. The damage caused by this disease occurs on a neuromotor and respiratory level; being this the last cause of deaths in patients who have it. The Quality of Life (QoL) in populations with this disease tends to decrease significantly, and the methods of diagnosis and forecasting are ineffective when detecting ALS and address it effectively. More interdisciplinary studies need to be done in order to improve the degree of QoL in patients that have ALS and their caregivers.

Protective Effect of Erythropoietin on Anoxia of Motoneurons after Spinal Cord Injury in Rats / 中国康复理论与实践

@#Objective To observe the protective effect of erythropoietin (EPO) on anoxia of motoneurons after spinal cord injury in rats.Methods The motoneuron was dissociated from neonate Wistar rats and cultivated in vitro with deprivation of oxygen in the medium. The changes of morphology were observed by phase-contrast microscopy, and the expression of EPO receptor (EPOR) was detected with Western blotting. The survival rate of motoneuron was measured with methyl thiazolyl tetrazolium (MTT) assay, and the level of lactate dehydrogenase (LDH) was observed. Results Compared with the control group, the expression of EPOR and the level of LDH increased (P<0.01),and the survival rate of motoneuron decreased (P<0.01) in the anoxia group. Compared with the anoxia group, the expression of EPOR and the level of LDH decreased (P<0.05), and the survival rate of motoneuron increased (P<0.01) in the EPO group, which was related with the level of EPO. Conclusion EPO could relieve the damage of motoneuron caused by anoxia, especially by upregulating EPOR.

Protective Effect of Erythropoietin on Anoxia of Motoneurons after Spinal Cord Injury in Rats / 中国康复理论与实践

Objective To observe the protective effect of erythropoietin (EPO) on anoxia of motoneurons after spinal cord injury in rats. Methods The motoneuron was dissociated from neonate Wistar rats and cultivated in vitro with deprivation of oxygen in the medium. The changes of morphology were observed by phase-contrast microscopy, and the expression of EPO receptor (EPOR) was detected with West-ern blotting. The survival rate of motoneuron was measured with methyl thiazolyl tetrazolium (MTT) assay, and the level of lactate dehydro-genase (LDH) was observed. Results Compared with the control group, the expression of EPOR and the level of LDH increased (P<0.01), and the survival rate of motoneuron decreased (P<0.01) in the anoxia group. Compared with the anoxia group, the expression of EPOR and the level of LDH decreased (P<0.05), and the survival rate of motoneuron increased (P<0.01) in the EPO group, which was related with the level of EPO. Conclusion EPO could relieve the damage of motoneuron caused by anoxia, especially by upregulating EPOR.

Ghrelin Protects Spinal Cord Motoneurons Against Chronic Glutamate Excitotoxicity by Inhibiting Microglial Activation

Glutamate excitotoxicity is emerging as a contributor to degeneration of spinal cord motoneurons in amyotrophic lateral sclerosis (ALS). Recently, we have reported that ghrelin protects motoneurons against chronic glutamate excitotoxicity through the activation of extracellular signal-regulated kinase 1/2 and phosphatidylinositol-3-kinase/Akt/glycogen synthase kinase-3beta pathways. Previous studies suggest that activated microglia actively participate in the pathogenesis of ALS motoneuron degeneration. However, it is still unknown whether ghrelin exerts its protective effect on motoneurons via inhibition of microglial activation. In this study, we investigate organotypic spinal cord cultures (OSCCs) exposed to threohydroxyaspartate (THA), as a model of excitotoxic motoneuron degeneration, to determine if ghrelin prevents microglial activation. Exposure of OSCCs to THA for 3 weeks produced typical motoneuron death, and treatment of ghrelin significantly attenuated THA-induced motoneuron loss, as previously reported. Ghrelin prevented THA-induced microglial activation in the spinal cord and the expression of pro-inflammatory cytokines tumor necrosis factor-alpha and interleukin-1beta. Our data indicate that ghrelin may act as a survival factor for motoneurons by functioning as a microglia-deactivating factor and suggest that ghrelin may have therapeutic potential for the treatment of ALS and other neurodegenerative disorders where inflammatory responses play a critical role.

Comparative Study of the L5 Spinal Nerve Transection Model and Sciatic Nerve Axotomy Model as a Peripheral Nerve Injury Model in Rat / 체질인류학회지

The aim of this study was to propose new more reliable peripheral nerve transection model to overcome the defect of the traditional sciatic axotomy model by specifically transecting L5 spinal nerve just after emerging from the intervertebral foramen and confining analysis area to the L5 spinal segment. The adult male Sprague-Dawley rats, weighing 300~350 g at the time of surgery, were used for the experiments. Four different experimental groups were used. 1. Sciatic nerve transection (Sc-Tx) group: transect the sciatic nerve in the popliteal fossa where it divided into the common peroneal nerve and tibial nerve. 2. L5 spinal nerve transection (L5-Tx) group: L5 spinal nerve was specifically transected. 3. Suture (Su) group: L5 spinal nerve was transected and immediately sutured. 4. Control group: the same surgical procedure with L5 spinal nerve transection group was performed except for the excision of L5 spinal nerve. To distinguish L5 motoneurons from the other level ones, the animals were received the retrograde tracer, FluoroGold into the axotomized proximal nerve stump. Serial coronal frozen sections at 40 microm thick through the L4 to L6 spinal segment was performed and the resultant total number of sections was about 180. Approximate serial 50 sections (approximately 2 mm) could be considered as the L5 segment based on the number of the fluorescent signals (above 20). L5 spinal segment could be differentiated from L4 and L6 segment based on their morphological characteristics under Cresyl violet stain. In L5-Tx group, at 2 and 4 weeks post-transection, the number of L5 spinal motoneurons was reduced by 8%. Meanwhile, Sc-Tx and Su groups showed no statistically notable changes. In this study, the authors could propose more reliable peripheral nerve axotomy model than the conventional sciatic nerve axotomy model by specifically transecting L5 spinal nerve and confining the investigating area within the L5 spinal segment.

Comparative Study of the L5 Spinal Nerve Transection Model and Sciatic Nerve Axotomy Model as a Peripheral Nerve Injury Model in Rat / 체질인류학회지

The aim of this study was to propose new more reliable peripheral nerve transection model to overcome the defect of the traditional sciatic axotomy model by specifically transecting L5 spinal nerve just after emerging from the intervertebral foramen and confining analysis area to the L5 spinal segment. The adult male Sprague-Dawley rats, weighing 300~350 g at the time of surgery, were used for the experiments. Four different experimental groups were used. 1. Sciatic nerve transection (Sc-Tx) group: transect the sciatic nerve in the popliteal fossa where it divided into the common peroneal nerve and tibial nerve. 2. L5 spinal nerve transection (L5-Tx) group: L5 spinal nerve was specifically transected. 3. Suture (Su) group: L5 spinal nerve was transected and immediately sutured. 4. Control group: the same surgical procedure with L5 spinal nerve transection group was performed except for the excision of L5 spinal nerve. To distinguish L5 motoneurons from the other level ones, the animals were received the retrograde tracer, FluoroGold into the axotomized proximal nerve stump. Serial coronal frozen sections at 40 microm thick through the L4 to L6 spinal segment was performed and the resultant total number of sections was about 180. Approximate serial 50 sections (approximately 2 mm) could be considered as the L5 segment based on the number of the fluorescent signals (above 20). L5 spinal segment could be differentiated from L4 and L6 segment based on their morphological characteristics under Cresyl violet stain. In L5-Tx group, at 2 and 4 weeks post-transection, the number of L5 spinal motoneurons was reduced by 8%. Meanwhile, Sc-Tx and Su groups showed no statistically notable changes. In this study, the authors could propose more reliable peripheral nerve axotomy model than the conventional sciatic nerve axotomy model by specifically transecting L5 spinal nerve and confining the investigating area within the L5 spinal segment.

Interações não lineares na arborização dendrítica de modelos de motoneurônios / Nonlinear interactions in the dendritic tree of motoneuron models

Este trabalho tem como objetivo verificar as diferenças funcionais entre sinapses proximais e distais e investigar as interações não line­ares de ativações sinápticas em modelos de motoneurônios em con­dições passivas. Os modelos utilizados, implementados em C++, são uma versão expandida dos desenvolvidos por Vieira e Kohn (2005), com uma arborização dendrítica mais detalhada: 5 ramos dendríti­cos equivalentes com 17 ou 19 compartimentos dendríticos cada um. Testes verificaram as diferenças funcionais de sinapses que ocorrem próximas ao soma e ao longo do dendrito além de avaliarem o grau de não linearidade de resposta da arborização dendrítica para dife­rentes ativações sinápticas em diferentes sítios. As diferenças funcio­nais entre as sinapses proximais e distais foram avaliadas pela injeção de senoides puras em diferentes compartimentos dendríticos. Os re­sultados mostraram atenuações mais intensas nas altas frequências e frequência de corte mais baixas em compartimentos dendríticos mais distantes do soma. As interações não lineares foram avaliadas apli­cando entradas sinápticas senoidais com frequências primas entre si, em dois ou mais ramos dendríticos equivalentes simultaneamente e em compartimentos dendríticos diferentes. O espectro de frequên­cia da corrente efetiva foi analisado, comparando a amplitude do pico das distorções harmônicas e por intermodulação com o pico da frequência fundamental de menor amplitude: quanto menores estas diferenças maior o grau de não linearidade entre as ativações sinápti­cas em compartimentos dendríticos distintos. Os resultados sugerem expressivo grau de não linearidade entre as entradas sinápticas nos dendritos. Notou­-se que, em todas as situações, quando se variou a condutância sináptica, maior foi o grau de não linearidade em relação aos casos em que se variou a corrente sináptica, bem como entradas sinápticas mais próximas eletrotonicamente apresentaram maior grau de não linearidade em...

The aim of this work was to verify the functional differences between proximal and distal synapses and to investigate the nonlinear interactions of synaptic activation in models of motoneurons in passive conditions. The models, implemented in C++, are an expanded version of that developed by Vieira and Kohn (2005), with a more detailed dendritic arborization: five equivalent dendrites with 17 or 19 dendritic compartments each one. Tests verified the functional differences of synapses that occur close to the soma and along the dendrite in addition to evaluating the degree of nonlinearity response from dendritic arborization for different synaptic activation at different sites. The functional differences between the proximal and distal synapses were evaluated by injection of pure sinusoids in different dendritic compartments. The results showed severe attenuation at high frequencies and a lower cut-off frequency in more distant dendritic compartments of the soma. The nonlinear interactions were evaluated by applying sinusoidal synaptic input at prime frequencies with each other in two or more dendritic branches simultaneously and at different dendritic compartments. The frequency spectrum of the effective current was analyzed by comparing the peak amplitude of harmonics and spurious rays with the peak amplitude of the fundamental frequency of smaller amplitude: the smaller these differences are, the greater the degree of nonlinearity between the synaptic activation of distinct dendritic compartments. The results suggest a high degree of nonlinearity between the dendrites. In all situations was noted that when the synaptic conductance was varied, the nonlinearities was greater in relation to the cases in which the synaptic current was varied, and nonlinear summation is expected for synapses located electrotonically close together in relation for electrotonically separated inputs, suggesting a dependence on membrane potential.

Protection of testosterone on spinal cord anterior horn motor neurons after sciatic nerve injury in mice / 解剖学报

ObjectiveTo investigate the protective effect of testosterone on motor neuron after sciatic nerve injury. MethodsTwelve adult C57 male mice were randomly divided into two groups: the sesame oil control group (n= 6) and the testosterone experimental group (n= 6). Unilateral sciatic nerve cutting severed as the animal model. Following operation, sesame oil or testosterone was administered for the control and experimental animals respectively via subcutaneous injection every other day for two weeks. The number and soma area of the anterior horn motor neurons in the lumbosacral cord of the sciatic nerve injury side were then measured on Nissl stained sections. Results In the testosterone group the motor neurons appeared healthier than that in the sesame control group. The neurons were plumper and showed more processes. Their number and average soma size were significantly larger than that in the sesame control (P<0.01). Conclusion The findings demonstrate that testosterone has a significant protective effect on sciatic nerve motor neurons after nerve injury.

The immunomodulator glatiramer acetate influences spinal motoneuron plasticity during the course of multiple sclerosis in an animal model

The immunomodulador glatiramer acetate (GA) has been shown to significantly reduce the severity of symptoms during the course of multiple sclerosis and in its animal model - experimental autoimmune encephalomyelitis (EAE). Since GA may influence the response of non-neuronal cells in the spinal cord, it is possible that, to some extent, this drug affects the synaptic changes induced during the exacerbation of EAE. In the present study, we investigated whether GA has a positive influence on the loss of inputs to the motoneurons during the course of EAE in rats. Lewis rats were subjected to EAE associated with GA or placebo treatment. The animals were sacrificed after 15 days of treatment and the spinal cords processed for immunohistochemical analysis and transmission electron microscopy. A correlation between the synaptic changes and glial activation was obtained by performing labeling of synaptophysin and glial fibrillary acidic protein using immunohistochemical analysis. Ultrastructural analysis of the terminals apposed to alpha motoneurons was also performed by electron transmission microscopy. Interestingly, although the GA treatment preserved synaptophysin labeling, it did not significantly reduce the glial reaction, indicating that inflammatory activity was still present. Also, ultrastructural analysis showed that GA treatment significantly prevented retraction of both F and S type terminals compared to placebo. The present results indicate that the immunomodulator GA has an influence on the stability of nerve terminals in the spinal cord, which in turn may contribute to its neuroprotective effects during the course of multiple sclerosis.

Effects of systemic administration of ciliary neurotrophic factor on Bax and Bcl-2 proteins in the lumbar spinal cord of neonatal rats after sciatic nerve transection

Ciliary neurotrophic factor (CNTF) is a cytokine that plays a neuroprotective role in relation to axotomized motoneurons. We determined the effect of daily subcutaneous doses of CNTF (1.2 µg/g for 5 days; N = 13) or PBS (N = 13) on the levels of mRNA for Bcl-2 and Bax, as well as the expression and inter-association of Bcl-2 and Bax proteins, and the survival of motoneurons in the spinal cord lumbar enlargement of 2-day-old Wistar rats after sciatic nerve transection. Five days after transection, the effects were evaluated on histological and molecular levels using Nissl staining, immunoprecipitation, Western blot analysis, and reverse transcriptase-polymerase chain reaction. The motoneuron survival ratio, defined as the ratio between the number of motoneurons counted on the lesioned side vs those on the unlesioned side, was calculated. This ratio was 0.77 ± 0.02 for CNTF-treated rats vs 0.53 ± 0.02 for the PBS-treated controls (P < 0.001). Treatment with CNTF modified the level of mRNA, with the expression of Bax RNA decreasing 18 percent (with a consequent decrease in the level of Bax protein), while the expression of Bcl-2 RNA was increased 87 percent, although the level of Bcl-2 protein was unchanged. The amount of Bcl-2/Bax heterodimer increased 91 percent over that found in the PBS-treated controls. These data show, for the first time, that the neuroprotective effect of CNTF on neonatal rat axotomized motoneurons is associated with a reduction in free Bax, due to the inhibition of Bax expression, as well as increased Bcl-2/Bax heterodimerization. Thus, the neuroprotective action of the CNTF on axotomized motoneurons can be related to the inhibition of this apoptotic pathway.

Role of spinal microglial responses in peripheral nerve regeneration in rats / 神经解剖学杂志

To explore the relationship of mieroglia activation,motoneuronal loss in the ventral horn of spinal cord and sciatic nerve regen-eration after the sciatic nerve injury,Sprague-Dawley rats were prepared a modal of the fight sciatic nerve crash injury.The immunoreactiv-ity(-ir)ofmicroglia and number of the motoneurons inthe ventral horn of spinal cord were detected at 3 and7 days,light and electron mi-croscopic detection of sciatic nerve degeneration and regeneration were performed at 4 weeks after the nerve injury.The results showed:(1)At 3 days after the sciatic nerve injury,OX-42-irinthe ventral horn of spinal cord begantoincrease significantly(P<0.05);(2)The number ratio of motoneurons in ventral horn of spinal cord in ipsilateral to contralateral for injury decreased markedly(P<0.05),in-dicating the numbers of ipsilateral motoneuroanl survival decreased;(3)Histological assessment showed the poor regeneration of the in-jured nerves;(4)Simvastatin(an inhibitor of cholesterol biosynthesis,having potential immunomodulatory capacities)facilitated the mi-eroglial activation.the motoneuronal survival and sciatic nerve regeneration were better than non-simvastatin-treated vehicle rats.The pres-ent results suggest that mieroglia activation in the ventral horns ofthe spinal cord may play an important protective role in the nerve regener-ation after peripheral nerve injury of the rat.

An experimental study on protective effect of extracellulsr ATP to motorneurons on spinal cord after brachial plexus avulsion / 中华显微外科杂志

Objective To investigate the protective effect of extracellular ATP on motoneurons of spinal anterior horn from spinal root avulsion induced cell injurey. Methods Thirty-six Wister rats were randomized into 2 groups: single root avulsion and root avulsion with ATP. The left C5-C8 nerve roots were avulsed, the experiment group was gioven extracellular ATP (2 mg/kg) 0.4 ml by intraperitoneal injection and control group physiological saline water was gioven 0.4ml as the same method, all one time daily, contin-ue injection ATP or saline water 2 weeks. At 2,4 and 6 week postoperatively, C5-C8 spinal cord was har-vested after the rats were executed. The change of nitric oxide synthase (NOS) and neurofilament protein (NF-200) were observed after histochemistrical examination. Results At 2、4 and 6 week postoperatively, the survival of motorneurons in the spinal anterior was 80.48%, 73.55% and 53.43% in experiment group, was 68.90%、63.58% and 37.72% in control group;compare experiment group with control group, the le-don-induced motomeurons death in the spinal anterior bern was decreased by 11.58%、9.78% and 15.71% respectively (P < 0.01) ; the activity changes of NOS expression rate was 17.85% ,40.20% and 18.03% in experiment group, was 25.53%,53.88% and 25.58% in control group, compare experiment group with con-trol group, the activity changes of NOS expression rate was decreased by 7.68% (P < 0.01), 13.68% and 7.55% (P < 0.05) respectively. The motoneurons expressed NF-200 in the experiment group was larger than the control group, there was significant difference was found between the control group and the experiment group. Conclusion ATP might play certain protective role in survival of motoneurous after spinal root avulsion.

Morphological analysis of developmental changes in soma area of digastric motoneurons in the rat trigeminal motor nuclei

To analyze the developmental changes in soma diameters of digastric motoneurons, wheat-germ agglutinin conjugated horseradish peroxidase (WGA-HRP) was injected into the digastric muscle and visualized the retrogradely HRP-labeled motoneurons through tungstate/tetramethylbenzidine (TMB) and following diaminobenzidine (DAB) reactions. The results obtained from Sprague-Dawley rats at postnatal days 1 (P1), 10 (P10) and 30 (P30) indicated as follows: firstly, soma diameters of digastric motoneurons showed unimodal distribution in all postnatal days examined; secondly, the period of P1 to P10 (period 1) showed about 2 times faster growth rate than that of P10 to P30 (period 2); thirdly, the smallest soma examined in each postnatal day exhibited slower growth rate with that of the largest one (increase ratio in soma diameters from P1 to P30, smallest vs. largest =1.62 : 1.93); Finally, relative growth rates a day showed again that period 1 had faster growth rate than that of period 2. Consequently, developmental changes in soma diameters of digastric motoneurons resulted in very different growth rates between both periods. This implies that the growth of the soma is almost completing within P10 and thereafter growing slowly. The period 1 and 2 are corresponding to sucking and sucking/masticatory period, respectively. Therefore present study providing morphological changes in soma diameters of digastric motoneurons suggests that both periods and their different growth rates of the motoneurons in each period may closely be related with each other.

NEURAL PATHWAYS OF TRIGEMINAL PROPRIOCEPTIVE AFFERENTS COORDINATE ORAL MOTOR BEHAVIORS / 神经解剖学杂志

Neural pathways and synaptic connections from the trigeminal mesencephalic nucleus (Vme) neurons to the cranial motor nuclei were studied in the rat using double labelling methodologies of intracellular Neurobiotin staining combined with retrograde horseradish peroxidase (HRP) transport, anterograde biotinylated dextran amine (BDA) tracing combined with retrograde HRP transport, and a dual fluorescent labelling of BDA anterograde combined tracing with Cholera Toxin B (CTB) retrograde transport. Direct projections and synapses were demonstrated from Vme neuronal boutons to motoneurons (MNs) of the trigeminal motor nucleus (Vmo), the hypoglossal nucleus (Ⅻ) and the ambiguus nucleus (Amb). Indirect projections and pathways from Vme neurons to the cranial motor nuclei including Vmo, Ⅻ, the facial nucleus (Ⅶ) and the cervical spinal cord (C1～5) were seen to relay on their premotor neurons. The premotor neurons of above cranial motor nuclei were overlapped in bilateral premotor neuronal pool including the parvocellular reticular formation (PCRt) and its alpha division (PCRtA), the dorsomedial part of the spinal trigeminal nucleus oralis (Vodm), and interpolaris (Vidm), the medullary reticular nucleus dorsal division (MdD), the supratrigeminal region (Vsup) and the dorsomedial part of the principal trigeminal sensory nucleus (Vpdm).Synapses between Vme neuronal boutons and Vmo and Ⅻ MNs and Ⅻ premotor neurons were predominantly asymmetric.There were four types of synaptic organizations, i.e. synaptic convergence; synaptic divergence presynaptic inhibition and afferent feedforward inhibition seen between Vme boutons and Vmno, Ⅻ MNs and between Vme boutons and Ⅻ premotor neurons.The results of present studies have demonstrated direct pathways from the trigeminal proprioceptive afferents to Vmo, Ⅻ and Amb MNs, and indirect pathways from the trigeminal proprioceptive afferents to bilateral Vmno, Ⅻ, Ⅶ and C1～s via their premotor neurons. It provides neuroanatomical network to elucidate trigeminal proprioceptive afferents coordinate oral motor behaviors.

EXOGENOUS FGF-2 REGULATES THE METABOLISM OF RAT FACIAL MOTONEURONS AFTER INJURY / 解剖学报

Objective To study the regulatory effect of exogenous fibroblast growth factor(FGF\|2) in expressions of calcitonin gene\|related peptide(CGRP) and FGF\|2 in facial motoneurons after injury. Methods The right facial nerve was transected 6?mm distal to the stylomastoid foraman.A 3?mm\+3 piece of Gelfoam presoaked in 25??l of various test solutions(normal saline,or 16?10 6?IU/L FGF\|2,or 32?10 6?IU/L FGF\|2) was implanted adjacent to the proximal nerve stump.After three days survival,the rats were killed and brainstems were removed.Serial 20??m cryosections were cut through the whole brainstem.The CGRP and FGF\|2 immunoreactivity were studied by immunohistochemisty and image analysis. Results An increase in the CGRP and decrease in the FGF\|2 signals were presented in axotomized motoneurons with saline\|treated animals.In FGF\|2\|treated animals,the lesion\|induced up\|regulation of CGRP and down\|regulation of FGF\|2 were attenuated compairing with normal saline treated animals.Conclusion\ Exogenous FGF\|2 can increase FGF\|2 expression and decrease CGRP expression in rat facial motoneurons after injury.\;[

Ultrastructural Analysis of GABA- and Glycine-Immunoreactive Nerve Terminals on Jaw-Closing and Jaw-Opening Motoneurons in the Rat / 대한해부학회지

Previous studies have shown that inhibitory synaptic inputs are different between in spinal and trigeminal motor systems and activities of jaw closing and opening alpha motoneurons are different during a chewing cycle. This study examined the distribution of inhibitory synapses made on masseter and digastric motoneurons by using retrograde tracing of wheat germ agglutinin conjugated to horseradish peroxides (WGA-HRP) combined with postembedding immunogold labeling on serial ultrathin sections.Many boutons IR (immunoreactive) to GABA and/or glycine were found to appose on two kinds of motoneurons, which were containing pleomorphic vesicles (a mixture of round, oval and flattened vesicles) and exhibited symmetrical synaptic contacts on the somata. Packing density and synaptic covering % were higher in digastric than in masseter motoneurons. Of 703 boutons apposing on 12 masseter motoneurons, 6.08+/-3.51, 29.67+/-8.89 and 17.78+/-5.22% were IR to GABA only, glycine only, and both GABA and glycine, respectively. Of 637 boutons apposing on 11 digastric motoneurons, 6.37+/-4.64, 19.74+/-8.25 and 12.01+/-5.38% were IR to GABA only, glycine only, and both GABA and glycine, respectively. Proportions of glycine IR boutons were higher than that of GABA IR boutons in both masseter and digastric motoneurons. Packing density and proportion of boutons IR to GABA and/or glycine were higher in jaw closing than in jaw opening motoneurons (packing density, 12.03+/-1.58 vs 10.28+/-2.99; proportion of IR boutons, 53.54+/-8.94% vs 38.12+/-9.38% in jaw closing and opening motoneurons, respectively). These results provide ultrastructural evidence that GABA and glycine act as important neurotransmitters for modulation of jaw movement and that proportion of inhibitory synapses is higher in jaw closing than in jaw opening motoneurons.