Neuroprotective Effect of Vascular Endothelial Growth Factor on Motoneurons of the Oculomotor System

Vascular endothelial growth factor (VEGF) was initially characterized as a potent angiogenic factor based on its activity on the vascular system. However, it is now well established that VEGF also plays a crucial role as a neuroprotective factor in the nervous system. A deficit of VEGF has been related to motoneuronal degeneration, such as that occurring in amyotrophic lateral sclerosis (ALS). Strikingly, motoneurons of the oculomotor system show lesser vulnerability to neurodegeneration in ALS compared to other motoneurons. These motoneurons presented higher amounts of VEGF and its receptor Flk-1 than other brainstem pools. That higher VEGF level could be due to an enhanced retrograde input from their target muscles, but it can also be produced by the motoneurons themselves and act in an autocrine way. By contrast, VEGF’s paracrine supply from the vicinity cells, such as glial cells, seems to represent a minor source of VEGF for brainstem motoneurons. In addition, ocular motoneurons experiment an increase in VEGF and Flk-1 level in response to axotomy, not observed in facial or hypoglossal motoneurons. Therefore, in this review, we summarize the differences in VEGF availability that could contribute to the higher resistance of extraocular motoneurons to injury and neurodegenerative diseases.Ministerio de Ciencia e Innovación de España (MCI), Agencia Estatal de Investigación de España (AEI) y Fondo Europeo de Desarrollo Regional (FEDER)-BFU2015-64515-P y PGC2018-094654-B-100Junta de Andalucía-BIO-29

Vascular Endothelial Growth Factor (VEGF) Prevents the Downregulation of the Cholinergic Phenotype in Axotomized Motoneurons of the Adult Rat

Vascular endothelial growth factor (VEGF) was initially characterized by its activity on the vascular system. However, there is growing evidence indicating that VEGF also acts as a neuroprotective factor, and that its administration to neurons suffering from trauma or disease is able to rescue them from cell death. We questioned whether VEGF could also maintain damaged neurons in a neurotransmissive mode by evaluating the synthesis of their neurotransmitter, and whether its action would be direct or through its well-known angiogenic activity. Adult rat extraocular motoneurons were chosen as the experimental model. Lesion was performed by monocular enucleation and immediately a gelatine sponge soaked in VEGF was implanted intraorbitally. After 7 days, abducens, trochlear, and oculomotor nuclei were examined by immunohistochemistry against choline acetyltransferase (ChAT), the biosynthetic enzyme of the motoneuronal neurotransmitter acetylcholine. Lesioned motoneurons exhibited a noticeable ChAT downregulation which was prevented by VEGF administration. To explore whether this action was mediated via an increase in blood vessels or in their permeability, we performed immunohistochemistry against laminin, glucose transporter-1 and the plasmatic protein albumin. The quantification of the immunolabeling intensity against these three proteins showed no significant differences between VEGF-treated, axotomized and control animals. Therefore, the present data indicate that VEGF is able to sustain the cholinergic phenotype in damaged motoneurons, which is a first step for adequate neuromuscular neurotransmission, and that this action seems to be mediated directly on neurons since no sign of angiogenic activity was evident. These data reinforces the therapeutical potential of VEGF in motoneuronal diseases.España, MINECO and FEDER BFU2015-64515-PJunta de Andalucía and FEDER : P10-CVI605

Particle Swarm Optimisation Prediction Model for Surface Roughness

Acrylic sheet is a crystal clear (with transparency equal to optical glass), lightweight material having outstanding weather ability, high impact resistance, good chemical resistance, and excellent thermo-formability and machinability. This paper develops the artificial intelligent model using partial swarm optimization (PSO) to predict the optimum surface roughness when cutting acrylic sheets with laser beam cutting (LBC). Response surface method (RSM) was used to minimize the number of experiments. The effect of cutting speed, material thickness, gap of tip and power towards surface roughness were investigated. It was found that the surface roughness is significantly affected by the tip distance followed by the power requirement, cutting speed and material thickness. Surface roughness becomes larger when using low power, tip distance and material thickness. Combination of low cutting speed, high power, tip distance and material distance produce fine surface roughness. Some defects were found in microstructure such as burning, melting and wavy surface. The optimized parameters by PSO are cutting speed (2600 pulse/s), tip distance (9.70 mm), power (95%) and material thickness (9 mm) which produce roughness around 0.0129 µm

Substance P-Botulinum Mediates Long-term Silencing of Pain Pathways that can be Re-instated with a Second Injection of the Construct in Mice

Chronic pain presents an enormous personal and economic burden and there is an urgent need for effective treatments. In a mouse model of chronic neuropathic pain, selective silencing of key neurons in spinal pain signalling networks with botulinum constructs resulted in a reduction of pain behaviours associated with the peripheral nerve. However, to establish clinical relevance it was important to know how long this silencing period lasted. Now, we show that neuronal silencing and the concomitant reduction of neuropathic mechanical and thermal hypersensitivity lasts for up to 120d following a single injection of botulinum construct. Crucially, we show that silencing and analgesia can then be reinstated with a second injection of the botulinum conjugate. Here we demonstrate that single doses of botulinum-toxin conjugates are a powerful new way of providing long-term neuronal silencing and pain relief

Extraocular Motor System Exhibits a Higher Expression of Neurotrophins When Compared with Other Brainstem Motor Systems

Extraocular motoneurons resist degeneration in diseases such as amyotrophic lateral sclerosis. The main objective of the present work was to characterize the presence of neurotrophins in extraocular motoneurons and muscles of the adult rat. We also compared these results with those obtained from other cranial motor systems, such as facial and hypoglossal, which indeed suffer neurodegeneration. Immunocytochemical analysis was used to describe the expression of nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3 in oculomotor, trochlear, abducens, facial, and hypoglossal nuclei of adult rats, and Western blots were used to describe the presence of neurotrophins in extraocular, facial (buccinator), and tongue muscles, which are innervated by the above-mentioned motoneurons. In brainstem samples, brain-derived neurotrophic factor was present both in extraocular and facial motoneuron somata, and to a lesser degree, in hypoglossal motoneurons. Neurotrophin-3 was present in extraocular motor nuclei, while facial and hypoglossal motoneurons were almost devoid of this protein. Finally, nerve growth factor was not present in the soma of any group of motoneurons, although it was present in dendrites of motoneurons located in the neuropil. Neuropil optical density levels were higher in extraocular motoneuron nuclei when compared with facial and hypoglossal nuclei. Neurotrophins could be originated in target muscles, since Western blot analyses revealed the presence of the three molecules in all sampled muscles, to a larger extent in extraocular muscles when compared with facial and tongue muscles. We suggest that the different neurotrophin availability could be related to the particular resistance of extraocular motoneurons to neurodegeneration.MINECO BFU2012-33975MINECO BFU2015-64515-P

Extraocular motoneurons of the adult rat show higher levels of vascular endothelial growth factor and its receptor Flk-1 than other cranial motoneurons

Recent studies show a relationship between the deficit of vascular endothelial growth factor (VEGF) and motoneuronal degeneration, such as that occurring in amyotrophic lateral sclerosis (ALS). VEGF delivery protects motoneurons from cell death and delayed neurodegeneration in animal models of ALS. Strikingly, extraocular motoneurons show lesser vulnerability to neurodegeneration in ALS compared to other cranial or spinal motoneurons. Therefore, the present study investigates possible differences in VEGF and its main receptor VEGFR-2 or Flk-1 between extraocular and non-extraocular brainstem motoneurons. We performed immunohistochemistry and Western blot to determine the presence of VEGF and Flk-1 in rat motoneurons located in the three extraocular motor nuclei (abducens, trochlear and oculomotor) and to compare it to that observed in two other brainstem nuclei (hypoglossal and facial) that are vulnerable to degeneration. Extraocular motoneurons presented higher amounts of VEGF and its receptor Flk-1 than other brainstem motoneurons, and thus these molecules could be participating in their higher resistance to neurodegeneration. In conclusion, we hypothesize that differences in VEGF availability and signaling could be a contributing factor to the different susceptibility of extraocular motoneurons, when compared with other motoneurons, in neurodegenerative diseases

Sources and lesion-induced changes of VEGF expression in brainstem motoneurons

Motoneurons of the oculomotor system show lesser vulnerability to neurodegeneration compared to other cranial motoneurons, as seen in amyotrophic lateral sclerosis (ALS). The overexpression of vascular endothelial growth factor (VEGF) is involved in motoneuronal protection. As previously shown, motoneurons innervating extraocular muscles present a higher amount of VEGF and its receptor Flk-1 compared to facial or hypoglossal motoneurons. Therefore, we aimed to study the possible sources of VEGF to brainstem motoneurons, such as glial cells and target muscles. We also studied the regulation of VEGF in response to axotomy in ocular, facial, and hypoglossal motor nuclei. Basal VEGF expression in astrocytes and microglial cells of the cranial motor nuclei was low. Although the presence of VEGF in the different target muscles for brainstem motoneurons was similar, the presynaptic element of the ocular neuromuscular junction showed higher amounts of Flk-1, which could result in greater efficiency in the capture of the factor by oculomotor neurons. Seven days after axotomy, a clear glial reaction was observed in all the brainstem nuclei, but the levels of the neurotrophic factor remained low in glial cells. Only the injured motoneurons of the oculomotor system showed an increase in VEGF and Flk-1, but such an increase was not detected in axotomized facial or hypoglossal motoneurons. Taken together, our findings suggest that the ocular motoneurons themselves upregulate VEGF expression in response to lesion. In conclusion, the low VEGF expression observed in glial cells suggests that these cells are not the main source of VEGF for brainstem motoneurons. Therefore, the higher VEGF expression observed in motoneurons innervating extraocular muscles is likely due either to the fact that this factor is more avidly taken up from the target muscles, in basal conditions, or is produced by these motoneurons themselves, and acts in an autocrine manner after axotomy.Ministerio de Economía y Competitividad BFU2015-64515-P, PGC2018-094654-B-100Junta de Andalucía BIO-29

Factor de crecimiento del endotelio vascular (VEGF) en el sistema oculomotor: presencia y modulación tras la lesión

Estudios previos han demostrado que las motoneuronas que inervan los músculos extraoculares muestran una menor vulnerabilidad a la neurodegeneración, en enfermedades como la ELA, en comparación con otras motoneuronas craneales, como son las faciales e hipoglosas. Estos hallazgos, junto con los que otorgan al factor trófico VEGF un importante papel en la neuroprotección de las motoneuronas durante los procesos degenerativos, nos llevaron a plantear si una mayor expresión de VEGF y de su principal receptor efector Flk-1 podrían ser las claves de la resistencia selectiva que caracteriza y diferencia a estas neuronas oculomotoras de otros grupos motores más vulnerables. Realizamos inmunohistoquímica y Western blot para determinar la presencia de VEGF y Flk-1 en las motoneuronas ubicadas en los tres núcleos oculomotores (NMOE, NMOT y NMOC) de la rata adulta, y compararla con las observadas en los otros dos núcleos motores del tronco encefálico (facial e hipogloso). Las neuronas que inervan los músculos extraoculares presentaron mayores cantidades de VEGF y de su receptor Flk-1, en comparación con las otras motoneuronas troncoencefálicas, por lo que este factor trófico podría participar en su mayor resistencia a la neurodegeneración. Ante este hallazgo, se abordó el estudio de las posibles fuentes de VEGF hacia las neuronas motoras, como los astrocitos, las células microgliales, los músculos diana y los vasos sanguíneos en los cinco núcleos motores. También se estudió la regulación de VEGF y de su receptor en estas mismas motoneuronas en respuesta a la axotomía. Para llevar a cabo este objetivo, se utilizaron las técnicas de inmunohistoquímica, Western blot y qPCR. Los estudios de la vía retrógrada desvelaron que todos los músculos craneales diana resultaron inmuno-positivos para VEGF, sin detectarse diferencias entre ellos. Por el contrario, se observó una mayor expresión del receptor Flk-1 en el terminal pre-sináptico de las motoneuronas que inervaban los músculos extraoculares, en comparación con los terminales pre-sinápticos de las neuronas faciales e hipoglosas. Por otro lado, los estudios de la vía paracrina revelaron que tanto los astrocitos como la microglía expresaron un bajo nivel de VEGF en los cinco núcleos troncoencefálicos, tanto en situación control como tras inducir la axotomía, a pesar de la clara reacción glial desencadenada. Asimismo, el estudio de la densidad vascular tampoco reveló diferencias en la irrigación sanguínea entre ninguno de los núcleos en estudio, ni se detectaron cambios en la densidad vascular tras la lesión. Sin embargo, tras la axotomía se observó un incremento en la expresión de VEGF y Flk-1 en las motoneuronas oculomotoras, en comparación con la situación control y a diferencia de lo ocurrido en las motoneuronas más vulnerables, que no incrementaron su nivel del factor trófico ni de su receptor. De hecho, la técnica de qPCR reveló una bajada en la expresión de ARNm para VEGF y Flk-1 en los núcleos facial e hipogloso tras la axotomía, cambios que no se detectaron en la población oculomotora. En resumen, la baja expresión de VEGF observada en las células gliales sugiere que estas células no son las causantes de la mayor expresión de VEGF observada en las motoneuronas oculares. Así pues, esa mayor presencia del factor trófico en las neuronas oculomotoras podría deberse a un mayor aporte retrógrado procedente desde el músculo, como sugiere la mayor expresión del receptor Flk-1 en el terminal pre-sináptico de estas neuronas motoras, pero también puede ser producido por las propias motoneuronas, actuando de forma autocrina. Además, la respuesta de estas neuronas a la axotomía fue de incrementar su nivel de VEGF y de su receptor, a diferencia de lo observado en las neuronas faciales e hipoglosas, lo que puede contribuir a la distinta resistencia a la neurodegeneración descrita en las diferentes poblaciones motoneuronales. En conclusión, nuestros resultados sugieren que las diferencias en la disponibilidad de VEGF puede ser un factor clave en la desigual susceptibilidad de las motoneuronas troncoencefálicas a la neurodegeneración

Substance P-Botulinum mediates long-term silencing of pain pathways that can be re-instated with a second injection of the construct in mice

Chronic pain presents an enormous personal and economic burden and there is an urgent need for effective treatments. In a mouse model of chronic neuropathic pain, selective silencing of key neurons in spinal pain signalling networks with botulinum constructs resulted in a reduction of pain behaviours associated with the peripheral nerve. However, to establish clinical relevance it was important to know how long this silencing period lasted. Now, we show that neuronal silencing and the concomitant reduction of neuropathic mechanical and thermal hypersensitivity lasts for up to 120d following a single injection of botulinum construct. Crucially, we show that silencing and analgesia can then be reinstated with a second injection of the botulinum conjugate. Here we demonstrate that single doses of botulinum-toxin conjugates are a powerful new way of providing long-term neuronal silencing and pain relief. PERSPECTIVE: This research demonstrates that botulinum-toxin conjugates are a powerful new way of providing long-term neuronal silencing without toxicity following a single injection of the conjugate and have the potential for repeated dosing when silencing reverses