Extreme stretch growth of integrated axons

Abstract

Large animals can undergo enormous growth during development, suggesting that axons in nerves and white matter tracts rapidly expand as well. Because integrated axons have no growth cones to extend from, it has been postulated that mechanical forces may stimulate axon elongation matching the growth of the animal. However, this distinct form of rapid and sustained growth of integrated axons has never been demonstrated. Here, we used a microstepper motor system to evaluate the effects of escalating rates of stretch on integrated axon tracts over days to weeks in culture. We found that axon tracts could be stretch grown at rates of 8 mm/d and reach lengths of 10 cm without disconnection. Despite dynamic and long-term elongation, stretched axons increased in caliber by 35%, while the morphology and density of cytoskeletal constituents and organelles were maintained. These data provide the first evidence that mechanical stimuli can induce extreme "stretch growth" of integrated axon tracts, far exceeding any previously observed limits of axon growth.

Figure 1.

Graphic representation of stretching conditions that define the boundaries of axon growth or disconnection. Each line represents an individual paradigm of accelerating displacement (elongation) of integrated axon tracts in culture. Lines with an X in shaded area denote disconnection of axon tracts during stretching. Lines without an X represent successful growth of axon tracts in response to escalating stretch rates up to 8 mm/d. Inset figure shows a disconnected axon labeled with an antibody against phosphorylated neurofilament protein.

Figure 2.

Flowchart detailing two individual axon stretch-growth paradigms. Top, Scheme to reach an axon growth rate of 8 mm/d. Bottom, Scheme to produce 5-cm-long axon tracts.

Figure 3.

Axon tracts stretch grown to 5 cm long. Axon tracts (middle) bridge two populations of neurons (top and bottom). Before the initiation of stretch growth, the two populations of neurons were adjacent and the bridging axons were only ∼100 μm long. Progressively separating the neuron populations induced mechanical tension on the axon tracts, resulting in enormous and rapid growth (colors are inverted to highlight axon tracts).

Figure 4.

Electron microscopy of stretch-grown axons. Scanning electron micrographs illustrating a small fascicle composed of axons ∼100-250 nm in diameter (A, B). Fasciculation of axons occurs during the elongation process as smaller bundles and individual axons coalesce and adhere to one another, forming larger bundles similar to the one depicted here. Transmission electron micrograph of cross sections near the center of axon fascicles in nonstretch conditions (C) and axons stretched to a length of 5 cm in 14 d (D), showing no change in axon cytoskeletal structures. Scale bars: A, 10 μm; B, 1 μm; C, D, 500 nm.