Studying Axonal Outgrowth and Regeneration of the Corticospinal Tract in Organotypic Slice Cultures

Abstract

Studies of axonal outgrowth and regeneration after spinal cord injury are hampered by the complexity of the events involved. Here, we present a simple and improved in vitro approach to investigate outgrowth, regeneration of the corticospinal tract, and intrinsic parenchymal responses. We prepared organotypic co-cultures using explants from the motor cortex of postnatal donor mice ubiquitously expressing green fluorescent protein and cervical spinal cord from wild type pups of the same age. Our data show that: a) motor-cortical outgrowth is already detectable after 1 d in culture and is source specific; b) treatment with neurotrophin-3 and C3 transferase from Clostridium botulinum significantly enhances axonal outgrowth during the course of cultivation; c) outgrowing axons form synaptic connections, as demonstrated by immunohistochemistry and calcium imaging; and d) migrating cells of motor-cortical origin can be reliably identified without previous tracing and are mostly neural precursors that survive and mature in the spinal cord parenchyma. Thus, our model is suitable for screening for candidate substances that enhance outgrowth and regeneration of the corticospinal tract and for studying the role of endogenous neural precursors after lesion induction.

Cell viability in three ex vivo rat models of spinal cord injury

“…; Pohland et al. ). The model serves to control the extracellular environment and has the promise of lower cost and faster discovery (Morrison et al.…”

“…Cell death is one of the consequences of traumatic SCI, affecting both neurons and supporting glial cells (Beattie et al 2002). Numerous studies have reported spinal cord tissue samples isolated from embryonic or newborn rats (Krassioukov et al 2002;Cho et al 2009;Kim et al 2010;Ravikumar et al 2012;Gerardo-Nava et al 2013Pinkernelle et al 2013;Hashemian et al 2014;Weightman et al 2014;Pohland et al 2015;Sypecka et al 2015). The organotypic slice culture (OSC) model appears to reproduce much of the post-traumatic events following injury, including acute and secondary injury mechanisms.…”

“…It has been reported that there is a significant difference in function, circuitry connections and regenerative capacities between the immature spinal cord and the adult cord (Krassioukov et al 2002). The heterogeneous populations of cells found in vivo are maintained within OSCs with three-dimensional connections between neurons and supporting cells (Cho et al 2009;Kim et al 2010;Ravikumar et al 2012;Gerardo-Nava et al 2013Pinkernelle et al 2013;Hashemian et al 2014;Weightman et al 2014;Pellowska et al 2015;Pohland et al 2015) to avoid hypoxia and necrosis of the central tissue (Krassioukov et al 2002).…”

In vitro models of axon regeneration

“…Recently, spinal cord slice cultures were used to study axon growth and remyelination, as well as astrocyte migratory and growth-supportive behavior 194–196 . Several groups have reported co-culturing cortex and spinal cord tissue to investigate the regrowth of cortex derived axons into spinal cord tracts 197,198 .…”

Biocompatibility of very small superparamagnetic iron oxide nanoparticles in murine organotypic hippocampal slice cultures and the role of microglia

“…41 Briefly, nonspecific binding caused by hydrophobic interactions of the secondary goat anti-mouse antibodies with endogenous mouse IgG or other cells was blocked for 30 min using 1% Triton X-100, 1% bovine serum albumin (both from Carl Roth, Karlsruhe, Germany) and 5% normal goat serum (Vector Laboratories, Burlingame, CA, USA) diluted in 0.1 M phosphate-buffered saline (PBS). Afterward OHSC were washed three times for 20 min each in 0.1 M PBS.…”