ABSTRACT
We have surveyed the motor changes in rats subjected to sciatic nerve axotomy. The rats were divided into two groups, each one consisting of ten animals, which underwent the following intervention: The first group (control): healthy rats without any injuries and experimental group: rats with injured sciatic nerve without treatment. at 12 weeks, the L4 and L5 spinal cord segments were removed. We evaluated nerve function using muscle electromyography (EMG) activity and sciatic function index (SFI) simultaneously with histological spinal cord analyses by stereological methods at 12 week. After nerve injury presented gross locomotor deficits at week 12. We also found that the volume of the anterior horn of spinal cord and total number of motor neurons were decreased after nerve axotomy (p<0.05). In conjunction, these results indicate that peripheral nerve injuries have more severe consequences on hind limb motor output.
En este estudio se examinaron los cambios motores en ratas sometidas a axotomía del nervio ciático. Las ratas se dividieron en dos grupos diez animales. El primer grupo (control) eran ratas sanas sin lesiones, y el grupo experimental consistió en ratas con nervio ciático lesionado sin tratamiento. A las 12 semanas, los segmentos de la médula espinal L4 y L5 fueron removidos. Se evaluó la función nerviosa mediante electromiografía muscular (EMG) y el índice de función ciática (IFC), simultáneamente con análisis histológicos de la médula espinal mediante métodos estereológicos. A las 12 semanas de la lesión nerviosa presentó déficit locomotor grueso. Además, se observó que el volumen del asta anterior y el número total de neuronas motoras disminuyeron después de la axotomía nerviosa (P <0,05). En conjunto, estos resultados indican que las lesiones de los nervios periféricos determinan graves consecuencias de la función motora de los miembros posteriores.
Subject(s)
Animals , Male , Rats , Spinal Cord/physiopathology , Spinal Cord/pathology , Sciatic Nerve/physiology , Sciatic Nerve/injuries , Rats, Wistar , Axotomy , Electromyography , Anterior Horn CellsABSTRACT
Resumen Introducción. El grupo de investigación del Laboratorio de Neurofisiología Comportamental de la Universidad Nacional de Colombia ha descrito modificaciones estructurales y electrofisiológicas en neuronas piramidales de la corteza motora producidas por la lesión del nervio facial contralateral en ratas. Sin embargo, poco se sabe sobre la posibilidad de que dichos cambios neuronales se acompañen también de modificaciones en las células gliales circundantes. Objetivo. Caracterizar el efecto de la lesión unilateral del nervio facial sobre la activación y proliferación de las células de la microglía en la corteza motora primaria contralateral en ratas. Materiales y métodos. Se hicieron pruebas de inmunohistoquímica para detectar las células de la microglía en el tejido cerebral de ratas sometidas a lesión del nervio facial, las cuales se sacrificaron en distintos momentos después de la intervención. Se infligieron dos tipos de lesiones: reversible (por compresión, lo cual permite la recuperación de la función) e irreversible (por corte, lo cual provoca parálisis permanente). Los tejidos cerebrales de los animales sin lesión (grupo de control absoluto) y de aquellos sometidos a falsa cirugía se compararon con los de los animales lesionados sacrificados 1, 2, 7, 21 y 35 días después de la lesión. Resultados. Las células de la microglía en la corteza motora de los animales lesionados irreversiblemente mostraron signos de proliferación y activación entre el tercero y séptimo días después de la lesión. La proliferación de las células de la microglía en animales con lesión reversible fue significativa solo a los tres días de infligida la lesión. Conclusiones. La lesión del nervio facial produce modificaciones en las células de la microglía de la corteza motora primaria. Estas modificaciones podrían estar involucradas en los cambios morfológicos y electrofisiológicos descritos en las neuronas piramidales de la corteza motora que comandan los movimientos faciales.
Abstract Introduction: Our research group has described both morphological and electrophysiological changes in motor cortex pyramidal neurons associated with contralateral facial nerve injury in rats. However, little is known about those neural changes, which occur together with changes in surrounding glial cells. Objective: To characterize the effect of the unilateral facial nerve injury on microglial proliferation and activation in the primary motor cortex. Materials and methods: We performed immunohistochemical experiments in order to detect microglial cells in brain tissue of rats with unilateral facial nerve lesion sacrificed at different times after the injury. We caused two types of lesions: reversible (by crushing, which allows functional recovery), and irreversible (by section, which produces permanent paralysis). We compared the brain tissues of control animals (without surgical intervention) and sham-operated animals with animals with lesions sacrificed at 1, 3, 7, 21 or 35 days after the injury. Results: In primary motor cortex, the microglial cells of irreversibly injured animals showed proliferation and activation between three and seven days post-lesion. The proliferation of microglial cells in reversibly injured animals was significant only three days after the lesion. Conclusions: Facial nerve injury causes changes in microglial cells in the primary motor cortex. These modifications could be involved in the generation of morphological and electrophysiological changes previously described in the pyramidal neurons of primary motor cortex that command facial movements.
Subject(s)
Animals , Male , Rats , Microglia/pathology , Facial Nerve Injuries/pathology , Facial Paralysis/physiopathology , Motor Cortex/pathology , Time Factors , Random Allocation , Afferent Pathways , Cell Division , Rats, Wistar , Pyramidal Cells/physiology , Pyramidal Cells/pathology , Axotomy , Facial Nerve Injuries/complications , Facial Nerve Injuries/physiopathology , Facial Muscles/innervation , Facial Paralysis/etiology , Facial Paralysis/pathology , Nerve Crush , Nerve RegenerationABSTRACT
This study reports on changes in the number of somatostatin-like immunoreactive (SOM-LI) endocrine cells in the porcine descending colon, caused by chemically driven inflammation, axotomy and proliferative enteropathy (PE). The distribution pattern of SOM-LI endocrine cells has been studied using the routine single-labelling immunofluorescence technique. Semi-quantitative evaluation of the number of the SOM-immunostained endocrine cells within the mucosal layer of the porcine descending colon has been based on counting of all endocrine cells immunoreactive to SOM per unit area (0,1 mm²). Under physiological conditions the number of SOM-LI endocrine cells has been shown to constitute 3,30±0,22. All applied pathological processes resulted in changes in the SOM-like immunoreactivity, which varied in particular processes studied. The number of SOM-LI endocrine cells increased to 6,28±0,31 and 4,43±0,35 during chemically driven inflammation and proliferative enteropathy, respectively, and decreased to 1,17%±0,16 after axotomy. The obtained results suggest that SOM-LI endocrine cells may participate in various pathological states within porcine descending colon and their functions probably depend on the type of pathological factor.(AU)
Subject(s)
Male , Swine/abnormalities , Somatostatin , Endocrine Cells/pathology , Pathologic Processes , Immunohistochemistry/veterinary , AxotomyABSTRACT
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.
Subject(s)
Adult , Animals , Humans , Male , Rats , Axotomy , Benzoxazines , Frozen Sections , Peripheral Nerve Injuries , Peripheral Nerves , Peroneal Nerve , Rats, Sprague-Dawley , Sciatic Nerve , Spinal Nerves , Sutures , Tibial Nerve , ViolaABSTRACT
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.
Subject(s)
Adult , Animals , Humans , Male , Rats , Axotomy , Benzoxazines , Frozen Sections , Peripheral Nerve Injuries , Peripheral Nerves , Peroneal Nerve , Rats, Sprague-Dawley , Sciatic Nerve , Spinal Nerves , Sutures , Tibial Nerve , ViolaABSTRACT
Este trabalho teve como objetivo o estudo da reprodução e descrição de técnica para colheita digital de imagens durante a marcha de ratos e determinação do Índice Funcional do Ciático (IFC), através de uma passarela de vidro e obtenção de imagens com uma câmera digital. Após lesão por estrangulamento controlado de nervo ciático, na extensão de 3mm, durante 30 segundos, utilizando pinça hemostática, um grupo de 32 ratos foram amostrados 24h antes da lesão que serviu de controle e 24h, 7, 14 e 21 dias após a compressão do nervo ciático. Estes ensaios consistiam na filmagem da marcha de cada animal em vista inferior (através de um espelho a 45º), e posteriormente analisadas através do programa IMAGE-J. As medidas retiradas foram os comprimentos das patas (direita e esquerda posteriores), bem como, a distância entre os artelhos. Na análise do IFC, valores próximos do "zero" (0) sugerem que a função do nervo Ciático mantém-se preservada e valores próximos de "menos cem" (-100) indicam perda total de função. Verificou-se neste estudo que 24horas antes da cirurgia causadora da lesão, a média do IFC foi de -7,07 ± 7,88 e 24 horas após a lesão estes valores passaram para uma média de -77,95 ± 13,81, sendo cerca de 10 vezes maiores, em que 78% dos animais apresentavam de 60 a 100% de perda funcional na atividade motora que demonstrou recuperação gradativa ao longo dos dias analisados, comprovando a acuidade e eficácia da metodologia proposta. Tais resultados sugerem que estudos possam ser conduzidos de forma mais simples e barata com a técnica de obtenção e manipulação de imagens digitais das pegadas, durante a marcha de ratos em laboratório.
This work aimed to study the reproduction and description of technique for digital sampling images during rats gait and determination of the sciatic functional index (SFI), through a glass walkway to obtain shoots with a digital camera. After controlled injury by strangulation of sciatic nerve, with 3mm of length, during 30 seconds, using hemostatic forceps, a group of 32 rats was performed 24 hours before the lesion which served as control and 24 hours, 7, 14 and 21 days after injury. The tests consisted in the filming and shooting each animal in order to observe the view from below (by a mirror to 45°) and subsequently analyzed using the IMAGE-J program. Measures were taken from the lengths of the legs (right and left), and the distance between the ankle. In the analysis of IFC, values close to zero (0) suggest that the function of the sciatic nerve is still preserved and values coming from "less one hundred" (-100) indicate total loss of function. It was verified in this study that 24 hours before surgery the average SFI was -7.07 ± 7.88 and 24 hours after injury these values rose to an average of -77.95 ± 13.81, being about 10 times larger, where 78% of the animals showed 60 to 100% of functional loss in motor activity, demonstrated by the gradual recovery over the days analyzed, confirming the accuracy and effectiveness of the proposed methodology. These results suggest that studies can be conducted to simplify and reduce costs using the technique for digital images of footprints during rats gait in the laboratory.
Subject(s)
Rats , Axotomy , Diagnostic Imaging , Sciatic NerveABSTRACT
Activating transcription factor 3 (ATF3) and c-Jun play key roles in either cell death or cell survival, depending on the cellular background. To evaluate the functional significance of ATF3/c-Jun in the peripheral nervous system, we examined neuronal cell death, activation of ATF3/c-Jun, and microglial responses in facial motor nuclei up to 24 weeks after an extracranial facial nerve axotomy in adult rats. Following the axotomy, neuronal survival rate was progressively but significantly reduced to 79.1% at 16 weeks post-lesion (wpl) and to 65.2% at 24 wpl. ATF3 and phosphorylated c-Jun (pc-Jun) were detected in the majority of ipsilateral facial motoneurons with normal size and morphology during the early stage of degeneration (1-2 wpl). Thereafter, the number of facial motoneurons decreased gradually, and both ATF3 and pc-Jun were identified in degenerating neurons only. ATF3 and pc-Jun were co-localized in most cases. Additionally, a large number of activated microglia, recognized by OX6 (rat MHC II marker) and ED1 (phagocytic marker), gathered in the ipsilateral facial motor nuclei. Importantly, numerous OX6- and ED1-positive, phagocytic microglia closely surrounded and ingested pc-Jun-positive, degenerating neurons. Taken together, our results indicate that long-lasting co-localization of ATF3 and pc-Jun in axotomized facial motoneurons may be related to degenerative cascades provoked by an extracranial facial nerve axotomy.
Subject(s)
Adult , Animals , Humans , Rats , Activating Transcription Factor 3 , Axotomy , Cell Death , Cell Survival , Facial Nerve , Microglia , Neurons , Peripheral Nervous System , Survival RateABSTRACT
We examined the degeneration of post-mitotic ganglion cells in ex-vivo neonatal retinal explants following axon damage. Ultrastructural features of both apoptosis and autophagy were detected. Degenerating cells reacted with antibodies specific for activated caspase-3 or -9, consistent with the presence of caspase activity. Furthermore, peptidic inhibitors of caspase-9, -6 or -3 prevented cell death (100 µM Ac-LEDH-CHO, 50 µM Ac-VEID-CHO and 10 µM Z-DEVD-fmk, respectively). Interestingly, inhibition of autophagy by 7-10 mM 3-methyl-adenine increased the rate of cell death. Immunohistochemistry data, caspase activation and caspase inhibition data suggest that axotomy of neonatal retinal ganglion cells triggers the intrinsic apoptotic pathway, which, in turn, is counteracted by a pro-survival autophagic response, demonstrated by electron microscopy profiles and pharmacological autophagy inhibitor.
Subject(s)
Animals , Rats , Apoptosis/physiology , Autophagy/physiology , Caspase 9/metabolism , /metabolism , Retinal Ganglion Cells/enzymology , Axotomy , Autophagy/drug effects , Immunohistochemistry , Microscopy, Electron, Transmission , Retinal Ganglion Cells/ultrastructureABSTRACT
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.
Subject(s)
Axons , Axotomy , Major Histocompatibility Complex , Neuronal Plasticity , Spinal Cord , SynapsesABSTRACT
<p><b>BACKGROUND</b>By unbiased stereological methods, we have observed preferential dorsal root ganglion (DRG) B-cell loss in rodents after nerve injury, and caspase-3 activation and cell loss were related to the present of p75 receptor (p75(NTR)). We hypothesized that DRG B-cells express higher levels of pro-apoptotic proteins as compared to A-cells and the expressions of pro-apoptotic proteins can be reduced by depletion of p75(NTR). This study aimed to identify the p75(NTR) involved apoptotic pathway in DRG neurons after nerve injury.</p><p><b>METHODS</b>The p75(NTR) knockout mice (p75-/-) and wildtype Balb/C mice (p75+/+) were used in this study. The expressions of pro-apoptotic proteins, c-Jun-N-terminal kinase (JNK), c-jun and p38 in DRG were evaluated with immunohistochemistry 2 and 7 days following unilateral sciatic nerve transection. In addition, extra-cellular related kinase (ERK), a transducer of survival signals, was also tested with immunohistochemistry and Western blotting methods in these animal models.</p><p><b>RESULTS</b>Phosphorylated JNK (P-JNK) and phosphorylated p38 (P-p38) were mainly located in small B-cells, whereas phosphorylated c-jun (P-c-jun) was located in both A- and B-cells. Phosphorylated ERK (P-ERK) was located in both B-cells and satellite cells. Axotomy dramatically increased the expressions of P-JNK and P-c-jun (paired t-test), with no influence on the expressions of P-p38 and P-ERK. Furthermore, the increase of P-JNK in p75+/+ mice 2 days after nerve axotomy was approximately 2.2-folds of that in p75-/- mice (P = 0.001, unpaired t-test).</p><p><b>CONCLUSION</b>p75(NTR)-dependent JNK-caspase-3 pathway is involved in DRG B-cell loss after nerve injury and JNK is not the unique upstream of c-jun activation.</p>
Subject(s)
Animals , Male , Mice , Apoptosis , Genetics , Physiology , Axotomy , Blotting, Western , Ganglia, Spinal , Cell Biology , Immunohistochemistry , Mice, Inbred BALB C , Mice, Knockout , Neurons , Cell Biology , Metabolism , Receptors, Nerve Growth Factor , Genetics , Metabolism , Sciatic Nerve , General SurgeryABSTRACT
<p><b>OBJECTIVE</b>To investigate the effect of exogenous glial cell line-derived neurotrophic factor (GDNF) infused into the cavitas subarachnoidealis on cornu anterius medullae spinalis motor neurons after sciatic nerve axotomy.</p><p><b>METHODS</b>Forty-eight healthy SD rats were divided into 2 groups randomly: GDNF group and NS group. The left sciatic nerve in rats were cut off. And then 0.9% saline (6 microl) and GDNF solution (6 microl) were injected into cavitas subarachnoidealis at L4-L6 in NS group and GDNF group,respectively. The rats were sacrificed on postoperative 1, 2, 4 and 8 weeks respectively. Their specimen of L4-L6 spinal cord were taken at different time and sectioned. The HE staining, Nissl staining and cholinesterase (ChE) staining in motor neurons were used for counting of motor neurons.</p><p><b>RESULTS</b>In GDNF group the number of motor neurons in cornu anterius medullae spinalis and the ChE activity were higher than that of NS group.</p><p><b>CONCLUSION</b>The exogenous GDNF infused into the cavitas subarachnoidealis are supposed to protect the degenerated spinal motor neuron from death after sciatic nerve injury.</p>
Subject(s)
Animals , Male , Rats , Axotomy , Cholinesterases , Metabolism , Glial Cell Line-Derived Neurotrophic Factor , Pharmacology , Motor Neurons , Metabolism , Random Allocation , Rats, Sprague-Dawley , Sciatic Nerve , Cell Biology , Metabolism , Spinal Cord , Cell Biology , Metabolism , General SurgeryABSTRACT
We previously reported that nidogen is an extracellular matrix protein regulating Schwann cell proliferation and migration. Since Schwann cells play a critical role in peripheral nerve regeneration, nidogen may play a role in it via regulation of Schwann cells. Here, we demonstrate direct evidence that nidogen induces elongation of regenerative axon growth of adult sensory neurons, and that the effect is Schwann cell dependent. Continuous infusion of recombinant ectodomain of tumor endothelial marker 7, which specifically blocks nidogen function in Schwann cells, suppressed regenerative neurite growth in a sciatic nerve axotomy model. Taken together, it is likely that nidogen is required for proper regeneration of peripheral nerves after injury.
Subject(s)
Animals , Male , Rats , Axotomy , Cell Movement , Cell Proliferation , Membrane Glycoproteins/physiology , Membrane Proteins/pharmacology , Nerve Regeneration , Nerve Tissue Proteins/pharmacology , Neurites/drug effects , Rats, Sprague-Dawley , Recombinant Proteins/pharmacology , Schwann Cells/cytology , Sensory Receptor Cells/physiologyABSTRACT
BACKGROUND AND OBJECTIVES: The object of this study was to evaluate the effect of platelet rich plasma (PRP) on facial nerve regeneration from an axotomy injury in the guinea pig model. MATERIALS AND METHOD: Experiments involved the transection and repair of right facial nerve. The right facial nerve of 14 albino guinea pigs were completely transected and immediately sutured, followed by fibrin glue only (control group) or fibrin glue +PRP (PRP group). Western blot assay was used to detect neurotrophic factors secreted by PRP. Nerve regeneration was assessed by motor function, electrophysiology, and histology studies. RESULTS: High levels of neurotrophin-3, angiopoietin-1, glial cell line derived neurotrophic factors, nerve growth factors and brain derived neurotrophic factors were demonstrated in PRP. Motor function recovery, compound motor action potentials, and axon count showed significant improvement in guinea pig treated with PRP. CONCLUSION: There was an improved functional outcome with the use of PRP in comparison with control. The increased nerve regeneration found in this study may be due to the neurotrophic factors secreted by PRP.
Subject(s)
Animals , Action Potentials , Angiopoietin-1 , Axons , Axotomy , Blood Platelets , Blotting, Western , Brain-Derived Neurotrophic Factor , Electrophysiology , Facial Nerve , Fibrin Tissue Adhesive , Glial Cell Line-Derived Neurotrophic Factor , Glial Cell Line-Derived Neurotrophic Factors , Guinea Pigs , Nerve Growth Factor , Nerve Growth Factors , Nerve Regeneration , Platelet-Rich Plasma , Recovery of Function , RegenerationABSTRACT
PURPOSE: The neurotrophic factor fibroblast growth factor-2 (FGF-2, bFGF) and Ca++ binding protein S100ß are expressed by the Schwann cells of the peripheral nerves and by the satellite cells of the dorsal root ganglia (DRG). Recent studies have pointed out the importance of the molecules in the paracrine mechanisms related to neuronal maintenance and plasticity of lesioned motor and sensory peripheral neurons. Moreover, cultured Schwann cells have been employed experimentally in the treatment of central nervous system lesions, in special the spinal cord injury, a procedure that triggers an enhanced sensorymotor function. Those cells have been proposed to repair long gap nerve injury. METHODS: Here we used double labeling immunohistochemistry and Western blot to better characterize in vitro and in vivo the presence of the proteins in the Schwann cells and in the satellite cells of the DRG as well as their regulation in those cells after a crush of the rat sciatic nerve. RESULTS: FGF-2 and S100ß are present in the Schwann cells of the sciatic nerve and in the satellite cells of the DRG. S100ß positive satellite cells showed increased size of the axotomized DRG and possessed elevated amount of FGF-2 immunoreactivity. Reactive satellite cells with increased FGF-2 labeling formed a ring-like structure surrounding DRG neuronal cell bodies.Reactive S100ß positive Schwann cells of proximal stump of axotomized sciatic nerve also expressed higher amounts of FGF-2. CONCLUSION: Reactive peripheral glial cells synthesizing FGF-2 and S100ß may be important in wound repair and restorative events in the lesioned peripheral nerves.
OBJETIVO: O fator neurotrófico fator de crescimento de fibroblastos-2 (FGF-2, bFGF) e a proteína ligante de Ca++ S100ß são expressos pelas células de Schwann dos nervos e por células satélites do gânglio da raiz dorsal (GRD). Estudos recentes indicam a importância das moléculas nos mecanismos parácrinos relacionados à manutenção neuronal e à plasticidade de neurônios periféricos motores e sensoriais. Além disso, células de Schwann cultivadas têm sido empregadas experimentalmente no tratamento de lesões no sistema nervo central, especialmente na lesão da medula espinal, a qual mostrou uma melhora da função sensoriomotora. Estas células são ainda propostas no reparo do nervo lesado com perda de tecido. MÉTODOS: Usamos a dupla marcação imunohistoquímica e o Western blot para caracterizar melhor in vitro e in vivo a presença das proteínas nas células de Schwann e nas células satélites do GRD assim como sua regulação nessas células após a compressão do nervo ciático de ratos. RESULTADOS: FGF-2 e S100ß estão presentes nas células de Schwann do nervo ciático e nas células satélites do GRD. Células satélites do GRD axotomizado positivas para S100ß possuíam quantidade aumentada de imurreatividade da FGF-2. Células satélites reativas apresentando maior quantidade de FGF-2 formaram um anel ao redor dos corpos neuronais do GRD. Células de Schwann do coto proximal à axotomia do nervo ciático e positivas para S100ß também expressaram quantidades aumentadas de FGF-2. CONCLUSÃO: As células gliais periféricas ao sintetizar FGF-2 e S100ß podem ser importantes no reparo de cicatrização e em eventos restaurativos nas lesões do nervo.
Subject(s)
Animals , Male , Rats , /metabolism , Ganglia, Spinal/metabolism , Nerve Growth Factors/metabolism , Peripheral Nerves/injuries , /metabolism , Schwann Cells/metabolism , Axotomy , Blotting, Western , Cells, Cultured , /analysis , Ganglia, Spinal/chemistry , Ganglia, Spinal/cytology , Immunohistochemistry , Nerve Crush , Nerve Growth Factors/analysis , Paracrine Communication , Peripheral Nerves/physiology , Peripheral Nerves/surgery , Rats, Wistar , /analysis , Satellite Cells, Perineuronal/metabolism , Schwann Cells/cytology , Sciatic Nerve/cytology , Sciatic Nerve/injuries , Sciatic Nerve/metabolismABSTRACT
Traumatic injury or inflammatory irritation of the peripheral nervous system often leads to persistent pathophysiological pain states. It has been well-documented that, after peripheral nerve injury or inflammation, functional and anatomical alterations sweep over the entire peripheral nervous system including the peripheral nerve endings, the injured or inflamed afferent fibers, the dorsal root ganglion (DRG), and the central afferent terminals in the spinal cord. Among all the changes, ectopic discharge or spontaneous activity of primary sensory neurons is of great clinical interest, as such discharges doubtless contribute to the development of pathological pain states such as neuropathic pain. Two key sources of abnormal spontaneous activity have been identified following peripheral nerve injury: the injured afferent fibers (neuroma) leading to the DRG, and the DRG somata. The purpose of this review is to provide a global account of the abnormal spontaneous activity in various animal models of pain. Particular attention is focused on the consequence of peripheral nerve injury and localized inflammation. Further, mechanisms involved in the generation of spontaneous activity are also reviewed; evidence of spontaneous activity in contributing to abnormal sympathetic sprouting in the axotomized DRG and to the initiation of neuropathic pain based on new findings from our research group are discussed. An improved understanding of the causes of spontaneous activity and the origins of neuropathic pain should facilitate the development of novel strategies for effective treatment of pathological pain.
Subject(s)
Animals , Humans , Axotomy , Ganglia, Spinal , Cell Biology , Neuralgia , Neurons, Afferent , Cell Biology , Peripheral Nerve Injuries , Spinal Cord , Cell BiologyABSTRACT
Objective A calcium-activated chloride current (IClCa) has been observed in medium-sized sensory neurons of the dorsal root ganglion (DRG). Axotomy of the sciatic nerve induces a similar current in the majority of medium and large diameter neurons. Our aim is to identify the molecule(s) underlying this current. Methods Using conventional and quantitative RT-PCR, we examined the expression in DRG of members of three families of genes, which have been shown to have IClCa current inducing properties. Results We showed the detection of transcripts representing several members of these families, i.e. chloride channel calcium-activated (CLCA), Bestrophin and Tweety gene families in adult DRG, in the normal state and 3 d after sciatic nerve section, a model for peripheral nerve injury. Conclusion Our analysis revealed that that mBest1 and Tweety2 appear as the best candidates to play a role in the injury-induced IClCa in DRG neurons.
Subject(s)
Animals , Mice , Axotomy , Chloride Channels , Genetics , DNA Primers , Ganglia, Spinal , Metabolism , Gene Expression , Neurons, Afferent , Metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sciatic Nerve , PhysiologyABSTRACT
In the retina, dopaminergic cells express the receptor for brain-derived neurotrophic factor (BDNF), which is known to be retrogradely transported from higher center to the retina. This study was conducted to identify the effect of optic nerve transaction on the dopaminergic cells in the rat retina by immunocytochemistry using antityrosine hydroxylase (TH) antiserum. In the control retina, we found two types of TH-immunoreactive amacrine cells, type I and type II, in the inner nuclear layer (INL) adjacent to the inner plexiform layer (IPL). The type I amacrine cell varicosities formed ring-like structures in contact with AII amacrine cell somata in stratum 1 of the IPL. In the axotomized retinas, TH-labeled processes formed loose networks of fibers, unlike the dense networks in the control retina, and the ring-like structures were disrupted. Our data suggest that retrogradely transported neurotrophic factor affects the expression of TH immunoreactivity in the axotomized rat retina and may therefore influence the retinal dopaminergic system.
Subject(s)
Animals , Rats , Amacrine Cells , Axotomy , Brain-Derived Neurotrophic Factor , Immunohistochemistry , Optic Nerve , Retina , Retinaldehyde , Tyrosine 3-MonooxygenaseABSTRACT
This study demonstrated that xenogenic human marrow mesenchymal stem cells (hMSCs) could elicit the regeneration of the sensory nerve after axotomy in the adult rats'infraorbital nerves without immunosuppression. For this, we evaluated the behavioral testing for functional recovery of the nerve and histological findings at weeks 3 and 5 compared to controls. Xenogenic hMSCs did not evoke any significant inflammatory or immunologic reaction after systemic and local administrations. HMSCs-treated rats exhibited significant improvement on sensory recovery tested with von Frey monofilaments. At 5 postoperative weeks, in the hMSCs treated nerve, expression of myelin basic protein (MBP), neurofilament (NF) at the site of axotomy was higher than control. And mRNA expression of neurotropin receptor Trk precursor (TrkPre), nerve growth factor receptor (NGFR) and neuropeptide (NPY) in trigeminal ganglion were also higher. The number of myelinated nerve at distal stump and cells in trigeminal ganglion were higher in hMSC treated rats. So it was supposed that transplanted MSCs contributed to reducing post-traumatic degeneration and production of neurotrophic factors. Immunofluorescence labeling showed small portion of hMSCs(<10%) expressed a phenotypic marker of Schwann cell (S-100). Xenogenic or allogenic mesenchymal stem cells might have immune privileged characteristics and useful tool for cell based nerve repair.
Subject(s)
Adult , Animals , Humans , Rats , Axotomy , Bone Marrow , Fluorescent Antibody Technique , Immunosuppression Therapy , Mesenchymal Stem Cells , Myelin Basic Protein , Myelin Sheath , Nerve Growth Factor , Nerve Growth Factors , Nerve Regeneration , Neuropeptides , Regeneration , RNA, Messenger , Trigeminal GanglionABSTRACT
Changes in morphology, immunophenotypes and proliferative activity of neuroglia are key features in most forms of CNS pathology. We compared proliferative activity of neuroglial cells in response to two different types of brain injury induced by medial forebrain bundle (MFB) axotomy. In the cannula track where acute necrosis occurs due to mechanical lesion caused by cannula inserted to incise the MFB, many BrdU-immunoreactive (ir) cells appeared around the cannula track already at 1 day post-lesion (1 dpl). Their number significantly increased by 7 dpl and then decreased, but considerable number of BrdU-ir cells was still found at 14 dpl. Some of the BrdU-ir cells were double-labeled with either OX-42 or GFAP. This finding suggests that both microglia and astrocytes are activated and proliferate immediately after the mechanical damage, and the proliferative activity is maintained in a considerable number of these cells by 14 dpl. In general, the main cell type showing BrdU immunoreactivity was amoeboid microglia within the necrotic zone immediately surrounding the cannula track, and was astrocytes in the periphery of the necrotic zone more or less apart from the cannula track. Previously, we reported that MFB axotomy induces apoptosis of dopaminergic (DA) neurons in the substantia nigra (SN). In the SN where axotomized DA neurons undergo apoptosis, only a few BrdU-ir cells were found at 1 dpl. Their number increased gradually from 3 dpl and peaked at 7 dpl, then significantly reduced at 14 dpl. Most of them were double-labeled with OX -42-positive ramified microglia but not with GFAP. This data indicates that microglia but not astrocyte are the cell type that proliferate in response to apoptotic neuronal cell death, and their morphology and proliferative activity are different from those observed in the cannula track. Meanwhile, in the both cannula track and SN, some BrdU-ir cells were thought to be neither GFAP-positive nor OX-42-positive, and thus they were presumed to be infiltrated peripheral immune cells. These results demonstrate that different types of neuronal cell death are accompanied with different neurogilal proliferative activities.