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.

FIG. 1.

Transection of a neurite with a laser beam and passive recoil of the cut ends. Cultured adult mouse primary sensory neurons grow neurites that are long enough by day 2 to be cut with a laser beam focused and activated for about 1 sec (A). The cut ends suddenly retract and are separated by a few (B) to tens of micrometers (C), depending on various factors (arrowhead indicates site of transection; p, proximal part of the neurite; see Supplementary Video S1; see online supplementary material at http://www.liebertonline.com).

FIG. 2.

Regeneration of a transected neurite (arrowhead indicates site of laser transection). A growth cone (bold arrow) is formed from a retraction bulb (arrow). Note that the first image shows the intact neurite before transection, and that the time intervals at which the images were taken are shown between frames (see Supplementary Video S2; see online supplementary material at http://www.liebertonline.com).

FIG. 3.

Size distribution of primary sensory neurons that survived, died, or regenerated within 24 h after neurite transection at three different distances from the cell body.

FIG. 4.

The effect of the distance of neurite transection from the soma on the survival of the neurons. A close neurite transection caused the upper neuron to die, while the lower neuron, with more distant transection, was still alive and regenerated the cut neurite (arrow). Note that the first image demonstrates the intact neurite before transection, and that the time intervals at which the images were taken are shown between frames. Arrowheads indicate the sites of laser transection. (See Supplementary Video S3; see online supplementary material at http://www.liebertonline.com.)

FIG. 5.

The effect of cell size on the survival of neurons after neurite transection. The small neuron dies while the larger one was still alive. Note that the first image demonstrates the intact neurite before transection, and that the time intervals at which the images were taken are shown between frames. Arrowheads indicate sites of laser transection. (See Supplementary Video S4; see online supplementary material at http://www.liebertonline.com.)

FIG. 6.

Death by apoptosis and necrosis after neurite transection. Necrotic death occurs earlier than apoptotic death. The bold arrow indicates a growing bleb that has burst in the next frame. Thin arrows indicate apoptotic bodies being formed. Note that the first images in both rows demonstrate intact neurites before transection, and that the time intervals at which the images were taken are shown between frames. Arrowheads indicate sites of laser transection. (See Supplementary Videos S5a and S5b; see online supplementary material at http://www.liebertonline.com.)