Biodegradable micro/nanoparticles generated from PLGA have recently attracted attention due to their clinically proven biocompatibility, especially for immunization purposes. These polymeric particulate delivery systems are able to present antigens and activate both humoral and cellular responses. Many studies have discussed the ideal size of these particles in contributing to the generation of the different types of immune response. However, these studies do not demonstrate the effect of micro or nanoparticles, without any encapsulated bioactive, on phagocytic cells after the uptake process. In this context, the aim of this study was to analyze the in vitro inflammatory behavior of J774 murine macrophages after particles' uptake, since nano/microparticles per se can differently activate phagocytic cells, using or not appropriate receptors, inducing distinct inflammatory responses. An o/w emulsion solvent extraction-evaporation method was chosen to prepare the particles. We determined their diameters, zeta potential and morphology. Fluorescent particles' uptake by J774 murine "macrophage-like" cells was also analyzed. To evaluate the in vitro inflammatory profile of these cells after micro or nanoparticles' uptake, we conducted NF-κB translocation assay by confocal microscopy and also determined the pro-inflammatory cytokines production provoked by the particles.

The presented three-dimensional electrode provides an effective anchorage system to the nervous tissue that can improve the stability of the implant for acute and chronic studies.

No abstract

Although peripheral axons can regenerate after nerve transection and repair, functional recovery is usually poor due to inaccurate reinnervation. Neurotrophic factors promote directional guidance to regenerating axons and their selective application may help to improve functional recovery. Hence, we have characterized in organotypic cultures of spinal cord and dorsal root ganglia the effect of GDNF, FGF-2, NGF, NT-3, and BDNF at different concentrations on motor and sensory neurite outgrowth. In vitro results show that GDNF and FGF-2 enhanced both motor and sensory neurite outgrowth, NGF and NT-3 were the most selective to enhance sensory neurite outgrowth, and high doses of BDNF selectively enhanced motor neurite outgrowth. Then, NGF, NT-3, and BDNF (as the most selective factors) were delivered in a collagen matrix within a silicone tube to repair the severed sciatic nerve of rats. Quantification of Fluorogold retrolabeled neurons showed that NGF and NT-3 did not show preferential effect on sensory regeneration whereas BDNF preferentially promoted motor axons regeneration. Therefore, the selective effects of NGF and NT-3 shown in vitro are lost when they are applied in vivo, but a high dose of BDNF is able to selectively enhance motor neuron regeneration both in vitro and in vivo.

Development of new nerve guides is required for replacing autologous nerve grafts for the repair of long gap defects after nerve injury. A nerve guide comprised only of electrospun fibers able to bridge a critical (15 mm) nerve gap in a rat animal model is reported for the first time. The nerve conduits are made of poly(ethylene oxide terephthalate) and poly(butylene terephthalate) (PEOT/PBT), a biocompatible copolymer composed of alternating amorphous, hydrophilic poly(ethylene oxide terephthalate), and crystalline, hydrophobic poly(butylene terephthalate) segments. These guides show suitable mechanical properties, high porosity, and fibers aligned in the longitudinal axis of the guide. In vitro studies show that both neurites and Schwann cells exhibit growth alignment with PA fibers. In vivo studies reveal that, after rat sciatic nerve transection and repair with PEOT/PBT guides, axons grow occupying a larger area compared to silicone tubes. Moreover, after repair of limiting (10 mm) and critical (15 mm) nerve gaps, PEOT/PBT guides significantly increase the percentage of regenerated nerves, the number of regenerated myelinated axons, and improve motor, sensory, and autonomic reinnervation in both gaps. This nerve conduit design combines the properties of PEOT/PBT with electrospun structure, demonstrating that nerve regeneration through long gaps can be achieved through the design of instructive biomaterial constructs.

Neurotrophic factors (NTFs) promote nerve regeneration and neuronal survival after peripheral nerve injury. However, drawbacks related with administration and bioactivity during long periods limit their therapeutic application. In this study, PLGA microspheres (MPs) were used to locally release different NTFs and evaluate whether they accelerate axonal regeneration in comparison with free NTFs or controls. ELISA, SEM, UV/visible light microscopy, organotypic cultures of DRG explants and spinal cord slices were used to characterize MP properties and the bioactivity of the released NTFs. Results of organotypic cultures showed that encapsulated NTFs maintain longer bioactivity and enhance neurite regeneration of both sensory and motor neurons compared with free NTFs. For in vivo assays, the rat sciatic nerve was transected and repaired with a silicone tube filled with collagen gel or collagen mixed with PBS encapsulated MPs (control groups) and with free or encapsulated NGF, BDNF, GDNF or FGF-2. After 20 days, a retrotracer was applied to the regenerated nerve to quantify motor and sensory axonal regeneration. NTF encapsulation in MPs improved regeneration of both motor and sensory axons, as evidenced by increased numbers of retrolabeled neurons. Hence, our results show that slow release of NTFs with PLGA MP enhance nerve regeneration.

After peripheral nerve injuries, damaged axons can regenerate but functional recovery is limited by the specific reinnervation of targets. In this study we evaluated if motor and sensory neurites have a substrate preference for laminin and fibronectin in postnatal and adult stages. In postnatal dorsal root ganglia (DRG) explants, sensory neurons extended longer neurites on collagen matrices enriched with laminin (~50%) or fibronectin (~35%), whereas motoneurons extended longer neurites (~100%) in organotypic spinal cord slices embedded in fibronectin-enriched matrix. An increased percentage of parvalbumin-positive neurites (presumptive proprioceptive) vs. neurofilament-positive neurites was also found in DRG in fibronectin-enriched matrix. To test if the different preference of neurons for extracellular matrix components was maintained in vivo, these matrices were used to fill a chitosan guide to repair a 6-mm gap in the sciatic nerve of adult rats. However, the number of regenerating motor and sensory neurons after 1 month was similar between groups. Moreover, none of the retrotraced sensory neurons in DRG was positive for parvalbumin, suggesting that presumptive proprioceptive neurons had poor regenerative capabilities compared with other peripheral neurons. Using real-time PCR we evaluated the expression of α5β1 (receptor for fibronectin) and α7β1 integrin (receptor for laminin) in spinal cord and DRG 2 days after injury. Postnatal animals showed a higher increase of α5β1 integrin, whereas both integrins were similarly expressed in adult neurons. Therefore, we conclude that motor and sensory axons have a different substrate preference at early postnatal stages but this difference is lost in the adult.

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.