Effects of Sigma-1 Receptor Ligands on Peripheral Nerve Regeneration

Peripheral nerve injuries lead to the loss of motor, sensory and autonomic functions in the territories supplied by the injured nerve. Currently, nerve injuries are managed by surgical repair procedures, and there are no effective drugs in the clinic for improving the capacity of axonal regeneration. Sigma-1 receptor (Sig-1R) is an endoplasmic reticulum chaperon protein involved in many functions, including neuroprotection and neuroplasticity. A few previous studies using Sig-1R ligands reported results that suggest this receptor as a putative target to enhance regeneration. The aim of this study was to evaluate the possible effects of Sig-1R ligands on axonal regeneration in a sciatic nerve section and repair model in mice. To this end, mice were treated either with the Sig-1R agonist PRE-084 or the antagonist BD1063, and a Sig-1R knock-out (KO) mice group was also studied. The electrophysiological and histological data showed that treatment with Sig-1R ligands, or the lack of this protein, did not markedly modify the process of axonal regeneration and target reinnervation after sciatic nerve injury. Nevertheless, the nociceptive tests provided results indicating a role of Sig-1R in sensory perception after nerve injury, and immunohistochemical labeling indicated a regulatory role in inflammatory cell infiltration in the injured nerve

Sensory Involvement in Amyotrophic Lateral Sclerosis

Although amyotrophic lateral sclerosis (ALS) is pre-eminently a motor disease, the existence of non-motor manifestations, including sensory involvement, has been described in the last few years. Although from a clinical perspective, sensory symptoms are overshadowed by their motor manifestations, this does not mean that their pathological significance is not relevant. In this review, we have made an extensive description of the involvement of sensory and autonomic systems described to date in ALS, from clinical, neurophysiological, neuroimaging, neuropathological, functional, and molecular perspectives

On the use of fast blue, fluoro-gold and diamidino yellow for retrograde tracing after peripheral nerve injury: uptake, fading, dye interactions, and toxicity

The usefulness of three retrograde fluorescent dyes for tracing injured peripheral axons was investigated. The rat sciatic was transected bilaterally and the proximal end briefly exposed to either Fast Blue (FB), Fluoro-Gold (FG) or to Diamidino Yellow (DY) on the right side, and to saline on the left side, respectively. The nerves were then resutured and allowed to regenerate. Electrophysiological tests 3 months later showed similar latencies and amplitudes of evoked muscle and nerve action potentials between tracer groups. The nerves were then cut distal to the original injury and exposed to a second (different) dye. Five days later, retrogradely labelled neurones were counted in the dorsal root ganglia (DRGs) and spinal cord ventral horn, The number of neurones labelled by the first tracer was similar for all three dyes in the DRG and ventral horn except for FG, which labelled fewer motoneurones. When used as second tracer, DY labelled fewer neurones than FG and FB in some experimental situations. The total number of neurotics labelled by the first and/or second tracer was reduced by about 30% compared with controls. The contributions of cell death as well as different optional tracer combinations for studies of nerve regeneration are discussed

Fascicular Topography of the Human Median Nerve for Neuroprosthetic Surgery

One of the most sought-after applications of neuroengineering is the communication between the arm and an artificial prosthetic device for the replacement of an amputated hand or the treatment of peripheral nerve injuries. For that, an electrode is placed around or inside the median nerve to serve as interface for recording and stimulation of nerve signals coming from the fascicles that innervate the muscles responsible for hand movements. Due to the lack of a standard procedure, the electrode implantation by the surgeon is strongly based on intuition, which may result in poor performance of the neuroprosthesis because of the suboptimal location of the neural interface. To provide morphological data that can aid the neuroprosthetic surgeon with this procedure, we investigated the fascicular topography of the human median nerve along the forearm and upper arm. We first performed a description of the fascicular content and branching patterns along the length of the arm. Next we built a 3D reconstruction of the median nerve so we could analyze the fascicle morphological features in relation to the arm level. Finally, we characterized the motor content of the median nerve fascicles in the upper arm. Collectively, these results indicate that fascicular organization occurs in a short segment distal to the epicondyles and remains unaltered until the muscular branches leave the main trunk. Based on our results, overall recommendations based on electrode type and implant location can be drawn to help and aid the neuroprosthetic procedure. Invasive interfaces would be more convenient for the upper arm and the most proximal third of the forearm. Epineural electrodes seem to be most suitable for the forearm segment after fascicles have been divided from the main trunk

Microglia Stimulation by Protein Extract of Injured Rat Spinal Cord. A Novel In vitro Model for Studying Activated Microglia

Research on microglia has established the differentiation between the so-called M1 and M2 phenotypes. However, new frameworks have been proposed attempting to discern between meaningful microglia profiles. We have set up an in vitro microglial activation model by adding an injured spinal cord (SCI) lysate to microglial cultures, obtained from postnatal rats, in order to mimic the environment of the spinal cord after injury. We found that under the presence of the SCI lysate microglial cells changed their phenotype, developing less ramified but longer processes, and proliferated. The SCI lysate also led to upregulation of pro-inflammatory cytokines, such as IL-1β, IL-6, and TNF-α, downregulation of the anti-inflammatory cytokines IL-10 and IL-4, and a biphasic profile of iNOS. In addition, a latex beads phagocytosis assay revealed the SCI lysate stimulated the phagocytic capacity of microglia. Flow cytometry analysis indicated that microglial cells showed a pro-inflammatory profile in the presence of SCI lysate. Finally, characterization of the microglial activation in the spinal cord on day 7 after contusion injury, we showed that these cells have a pro-inflammatory phenotype. Overall, these results indicate that the use of SCI lysates could be a useful tool to skew microglia towards a closer phenotype to that observed after the spinal cord contusion injury than the use of LPS or IFNγ

El trasplante de células de la glía envolvente del bulbo olfatorio tras lesión de la médula espinal: Estudio experimental en ratas.

Objetivo Evaluar el efecto a largo plazo del trasplante de células de la glía envolvente (GE) del bulbo olfatorio tras lesión de la médula espinal. Material y método Se practicó una laminectomía dorsal T8, en 16 ratas adultas Sprague-Dawley, dejando al descubierto la médula espinal subyacente, la cual se bañó con rosa de Bengala durante 10 minutos, antes de lesionarla por iluminación con una fibra óptica acoplada a una lámpara halógena, durante 2,5 minutos. A la mitad de los animales se les inyectó 180.000 células de GE, en 10 μl de medio (grupo GE), y a la otra mitad sólo 10 μl de DMEM (Dulbecco's modified Eagle's medium) (grupo DM). Los animales se sacrificaron a los 90 días de efectuar la lesión y se evaluó el área de médula espinal preservada, la recuperación locomotora y la sensibilidad nociceptiva. Resultados Los animales del grupo GE mostraron un nivel de locomoción superior y retiraron antes la pata al estímulo nociceptivo que los del grupo DM. También hubo una mayor preservación de parénquima medular y más células p75 positivas en el grupo GE que en el DM. Conclusiones El trasplante de GE favorece la preservación de parénquima medular y evita la pérdida de funciones motoras y sensoriales en la rata

On the use of Parylene C polymer as substrate for peripheral nerve electrodes

Parylene C is a highly flexible polymer used in several biomedical implants. Since previous studies have reported valuable biocompatible and manufacturing characteristics for brain and intraneural implants, we tested its suitability as a substrate for peripheral nerve electrodes. We evaluated 1-year-aged in vitro samples, where no chemical differences were observed and only a slight deviation on Young's modulus was found. The foreign body reaction (FBR) to longitudinal Parylene C devices implanted in the rat sciatic nerve for 8 months was characterized. After 2 weeks, a capsule was formed around the device, which continued increasing up to 16 and 32 weeks. Histological analyses revealed two cell types implicated in the FBR: macrophages, in contact with the device, and fibroblasts, localized in the outermost zone after 8 weeks. Molecular analysis of implanted nerves comparing Parylene C and polyimide devices revealed a peak of inflammatory cytokines after 1 day of implant, returning to low levels thereafter. Only an increase of CCL2 and CCL3 was found at chronic time-points for both materials. Although no molecular differences in the FBR to both polymers were found, the thick tissue capsule formed around Parylene C puts some concern on its use as a scaffold for intraneural electrodes

Massive Loss of Proprioceptive Ia Synapses in Rat Spinal Motoneurons after Nerve Crush Injuries in the Postnatal Period

Altres ajuts: NIH-NINDS R01 NS11196Peripheral nerve injuries (PNIs) induce the retraction from the ventral horn of the synaptic collaterals of Ia afferents injured in the nerve, effectively removing Ia synapses from α-motoneurons. The loss of Ia input impairs functional recovery and could explain, in part, better recovery after PNIs with better Ia synaptic preservation. Synaptic losses correlate with injury severity, speed, and efficiency of muscle reinnervation and requires ventral microglia activation. It is unknown whether this plasticity is age dependent. In neonates, axotomized motoneurons and sensory neurons undergo apoptosis, but after postnatal day 10 most survive. The goal of this study was to analyze vesicular glutamate transporter 1 (VGluT1)-labeled Ia synapses (which also include II afferents) after nerve crush in 10 day old rats, a PNI causing little Ia/II synapse loss in adult rats. We confirmed fast and efficient reinnervation of leg muscles; however, a massive number of VGluT1/Ia/II synapses were permanently lost. This synapse loss was similar to that after more severe nerve injuries involving full transection in adults. In adults, disappearance of ventrally directed Ia/II collaterals targeting α-motoneurons was associated with a prolonged microglia reaction and a CCR2 mechanism that included infiltration of CCR2 blood immune cells. By contrast, microgliosis after P10 injuries was fast, resolved in about a week, and there was no evidence of peripheral immune cell infiltration. We conclude that VGluT1/Ia/II synapse loss in young animals differs in mechanism, perhaps associated with higher microglia synaptic pruning activity at this age and results in larger losses after milder nerve injuries

Preferential Enhancement of Sensory and Motor Axon Regeneration by Combining Extracellular Matrix Components with Neurotrophic Factors

After peripheral nerve injury, motor and sensory axons are able to regenerate but inaccuracy of target reinnervation leads to poor functional recovery. Extracellular matrix (ECM) components and neurotrophic factors (NTFs) exert their effect on different neuronal populations creating a suitable environment to promote axonal growth. Here, we assessed in vitro and in vivo the selective effects of combining different ECM components with NTFs on motor and sensory axons regeneration and target reinnervation. Organotypic cultures with collagen, laminin and nerve growth factor (NGF)/neurotrophin-3 (NT3) or collagen, fibronectin and brain-derived neurotrophic factor (BDNF) selectively enhanced sensory neurite outgrowth of DRG neurons and motor neurite outgrowth from spinal cord slices respectively. For in vivo studies, the rat sciatic nerve was transected and repaired with a silicone tube filled with a collagen and laminin matrix with NGF/NT3 encapsulated in poly(lactic-co-glycolic acid) (PLGA) microspheres (MP) (LM + MP.NGF/NT3), or a collagen and fibronectin matrix with BDNF in PLGA MPs (FN + MP.BDNF). Retrograde labeling and functional tests showed that LM + MP.NGF/NT3 increased the number of regenerated sensory neurons and improved sensory functional recovery, whereas FN + MP.BDNF preferentially increased regenerated motoneurons and enhanced motor functional recovery. Therefore, combination of ECM molecules with NTFs may be a good approach to selectively enhance motor and sensory axons regeneration and promote appropriate target reinnervation