Analysis of axonal growth in organotypic neural cultures

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.

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“…DRG explants were cultured for 2 days and SC slices for 4 days. A detailed description of this protocol has been previously reported [16]. …”

Section: Methodsmentioning

confidence: 99%

Dose-Dependent Differential Effect of Neurotrophic Factors on In Vitro and In Vivo Regeneration of Motor and Sensory Neurons

et al. 2016

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Epi/perineural and Schwann Cells as Well as Perineural Sheath Integrity are Affected Following 2,4-D Exposure

et al. 2017

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2,4-dicholorophenoxy acetic acid (2,4-D) is a worldwide-known hormone herbicide. However, there are increasing concerns about its exposure and risks of developing pathological conditions for the peripheral nervous system. The aim of this study was to investigate the mechanism(s) involved in the toxicity of 2,4-D on peripheral nerve's cellular components. The epi/perineural and Schwann cells and a total of three cell lines were treated with 2,4-D. The viability of cells at different doses of 2,4-D was measured by MTT assay. The cell cycle analyses, cumulative cell counting, fluorescent staining, antioxidant and caspase enzymes activity were examined on epi/perineural and Schwann cells. The epi/perineural cells were assessed as having biological macromolecular changes. Some tight junction-related genes and proteins were also tested on explants of 2,4-D treated epi/perineural tissue. The viability of 2,4-D treated cells was reduced in a dose-dependent manner. Reduced growth rate and G1 cell cycle arrest were verified in 2,4-D treated epi/perineural and Schwann cells. The use of staining methods (acridine orange/ethidium bromide and DAPI) and caspase 3/7 activity assay along with malondialdehyde, glutathione peroxidase, and superoxide dismutase activity assays indicated the apoptotic and oxidant effects of 2,4-D on epi/perineural and Schwann cells. Data obtained from FTIR revealed changes in epi/perineural proteins and cell membrane lipids. Additionally, claudin-1, occludin, and ZO-1 gene/protein expression profiles were significantly reduced in 2,4-D-treated epi/perineural pieces. Our data indicated that oxidative stress, apoptosis of epi/perineural and Schwann cell and impaired blood-nerve barrier may have contributed to nerve damage following 2,4-D exposure.

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

Santos

González-Pérez

Giudetti

et al. 2016

IJMS

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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.

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Analysis of axonal growth in organotypic neural cultures

Cited by 3 publications

References 22 publications

Dose-Dependent Differential Effect of Neurotrophic Factors on In Vitro and In Vivo Regeneration of Motor and Sensory Neurons

Dose-Dependent Differential Effect of Neurotrophic Factors on In Vitro and In Vivo Regeneration of Motor and Sensory Neurons

Epi/perineural and Schwann Cells as Well as Perineural Sheath Integrity are Affected Following 2,4-D Exposure

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

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