Consequences of Neurite Transection In Vitro

Abstract

In order to quantify degenerative and regenerative changes and analyze the contribution of multiple factors to the outcome after neurite transection, we cultured adult mouse dorsal root ganglion neurons, and with a precise laser beam, we transected the nerve fibers they extended. Cell preparations were continuously visualized for 24 h with time-lapse microscopy. More distal cuts caused a more elongated field of degeneration, while thicker neurites degenerated faster than thinner ones. Transected neurites degenerated more if the uncut neurites of the same neuron simultaneously degenerated. If any of these uncut processes regenerated, the transected neurites underwent less degeneration. Regeneration of neurites was limited to distal cuts. Unipolar neurons had shorter regeneration than multipolar ones. Branching slowed the regenerative process, while simultaneous degeneration of uncut neurites increased it. Proximal lesions, small neuronal size, and extensive and rapid neurite degeneration were predictive of death of an injured neuron, which typically displayed necrotic rather than apoptotic form. In conclusion, this in vitro model proved useful in unmasking many new aspects and correlates of mechanically-induced neurite injury.

The microtubule-stabilizing drug Epothilone D increases axonal sprouting following transection injury in vitro

“…Our data suggests that in an isolated system of cortical neurons, rate of outgrowth may not be reliant upon microtubule dynamics. However, it has been reported that the regenerative response of cut axons is correlated with the length of the remaining stump (Gomis-Ruth et al, 2008;Cengiz et al, 2012). Thus it is plausible that by using the scratch injury model where numerous axons are severed at once could have important cofounders possibly masking drug effects on post-injury sprout length and outgrowth speed.…”

Two distinct types of dying back axonal degeneration in vitro

“…We also found that axoplasmic compaction and subsequent axial collapse is more effective in thinner axons suggesting a potentially more effective cut-end sealing. Indeed we previously demonstrated that axons with smaller diameters tend to degenerate less [38]. The collapse may be passive due to lose of internal scaffold in most part, but may also depend, at least partially, on retained actomyosin contractility [18,39].…”

Determining of Apoptosis at in vitro Conditioned DRG Neurons with TUNEL Method

“…For this, the cell suspension was carefully pipetted on top of a three-layer percol (Sigma) gradient (60, 35 and 10% from bottom to top) prepared with NBA-B27 and spun at 3000g for 20 mins in a centrifuge cooled down to 4 0 C. The neurons that were collected from 35 % layer were washed with NBA-B27 and spun once more at 120 g for 3 mins then supernatant was discharted and pellet was re-suspended in 1 ml NBA-B27. (Cengiz et al 2012). The cell suspension were seeded on silicon (WPI-Dow-corning) coated glass dish and conditioned in incubator for 3 days (37 C 0 and 5% CO2).…”