Stretch growth of integrated axon tracts: extremes and exploitations

Abstract

Although virtually ignored in the literature until recently, the process of 'stretch growth of integrated axon tracts' is perhaps the most remarkable axon growth mechanism of all. This process can extend axons at seemingly impossible rates without the aid of chemical cues or even growth cones. As animals grow, the organization and extremely rapid expansion of the nervous system appears to be directed purely by mechanical forces on axon tracts. This review provides the first glimpse of the astonishing features of axon tracts undergoing stretch growth and how this natural process can be exploited to facilitate repair of the damaged nervous system.

Fig. 1

(A) A 1941 diagram originally entitled, “Group of neuroplasts in three successive stages,” in the first published suggestion of a mechanical mechanism of nerve growth. Paul Weiss posited that “the nerve is drawn out by the growth and dislocations of its terminal tissues,” in a paper entitled, “Nerve patterns: the mechanics of nerve growth” in Growth, Third Growth Symposium 5:163–203. Note the initial phase of axon growth of a migrating growth cone navigating to a target. (B) Proposed second phase of axonal growth includes stretch growth of nerves and white matter tracts following synaptogenesis. As Weiss postulated, this axon growth is due to mechanical tension as the body grows. One of the most extreme examples is the growth of whale spinal axons during development as the vertebrae grow in length, reaching at least 3 cm of growth per day.

Fig. 2

(A) Schematic illustration of the experimental process of stretch growth in integrated axon tracts. Top: A short membrane (yellow) attached to a plastic block is placed on top of a long rectangular membrane (blue). A chamber is formed by the plastic block, in which neurons are plated and allowed to integrate. A neural network (red) is formed, including axon fascicles that grow across the border between the top and bottom membranes. Bottom: Movement of the plastic block via a computer controlled microstepper motor system divides the culture and progressively separates the opposing halves by sliding the top membrane across the bottom membrane. This technique results in the creation of long fascicular tracts of axons spanning the two membranes. (B) Components of elongation device: Left, cell culture bioreactor chamber capable of producing stretch grown axon fascicles to 15 cm. Right, two types of fully assembled elongator apparati, including the bioreactors, linear tables and microstepper motor. Computer driving system not shown.

Fig. 3

(A) Stretch-growth-induced organization of axon tracts. Phase photomicrographs of a live cortical neuron cultures demonstrating a region of stretch-grown axons at the border of the top membrane at successive days (2, 4, and 7) of elongation. Note the gradual coalescing of neighboring axon bundles and thickening of the bundles at the edge of top membrane. Bar = 50 μm. (B) Recapitulation of a natural stretch growth process of axon tracts. Fluorescence confocal photomicrograph of axon fascicles from cortical neurons at 7 days of stretch-induced growth, elucidated by immunostained microtubule protein in fixed cultures. Left: Note the harpstring appearance of multiple long fascicular axon tracts arranged in parallel (Bar = 50 μm). Right: Two large fascicular tracts composed of thousands of axons that were produced by stretch-induced growth in vitro (each approximately 50 μm wide).

Fig. 4

(A) Stretch growth boundaries of integrated axon tracts. Left: Graphic representation of stretching conditions that define the boundaries of axon growth or disconnection for DRG neurons. Each line represents individual paradigms of accelerating displacement (elongation) of integrated axon tracts in culture. X’s in shaded area denote disconnection of axon tracts during stretching. Lines without X’s represent successful growth of axon tracts in response to escalating stretch rates. Right: Photomicrograph of immuno-labeled disconnected axon. Disruption resulted from a too rapidly accelerated elongation program. (B) Not jellyfish; stretch grown axon tracts. Simple light photograph of axon tracts from DRG neurons stretch grown to 5-cm long (specimen and background colors are modified to highlight axon tracts). Axon tracts (middle) bridge two populations of neurons (top and bottom). Prior to the initiation of stretch growth, the two populations of neurons were adjacent and the bridging axons were only approximately 100-μm long. Progressively separating the neuron populations induced mechanical tension on the axon tracts resulting in enormous and rapid growth. (C) Normal ultrastructure of axons despite extreme stretch growth reaching 5-cm lengths in 14 days. Left: Scanning electron micrographs (boxed area on top expanded below) illustrating a small fascicle of stretch grown DRG axons. Right: Transmission electron micrograph of cross-sections near the center length of stretch grown axon fascicles shows a normal complement of cytoskeletal structures. Notably, however, the diameter of stretch grown axons is actually greater than that of short, non-stretch grown axons. Scale bar = 500 nm. (D) Schematic illustration at top showing communication across stretch grown axons. Application of KCl on neuron cell bodies at one end results in action potential transmission that can be recorded from neurons at the other side with recording trace shown at bottom.

Fig. 5

Left: Survival and integration of transplanted living nervous tissue constructs for peripheral nerve repair. Top, grossly normal appearing 1.5-cm long region of nerve bridged by a nervous tissue construct 6 weeks post-transplantation; Middle, Surviving transplanted neuron cell bodies (red) and axons (green) at one end of the transplanted region; Bottom, confocal projection in the same region depicts host axons (red) and graft axons (red/yellow) assembled in a network extending through the transplanted region from the neuron clusters. Right: Top two panels show the center of the transplanted region, where the axons from host (red) have penetrated and are remarkably intertwined with transplanted axons (green). A cross-section of this region shows myelination (red) circumscribing axons (green).

Fig. 6

A new approach for a brain machine interface. A microelectrode array (gray, right) serves as the plating membrane under one population of neuron cell bodies. An adjacent population is pulled away inducing stretch growth of the connecting axon tracts (bottom, right), creating a transplantable neuro-electric construct. This process creates a conceptual interface with the nervous system. For example, transplanting the non-electrode end onto a peripheral nerve may allow for bi-directional communication. Stimuli from a device (e.g., upper limb prosthetic) passed to an electrode (bottom right) could be transferred through the transplanted construct into the integrated nerve and onto the brain for sensory detection. Likewise, motor signals from the brain through the nerve to the electrodes could be detected to drive functions of the device.

Fig. 7

Tissue-engineering adult human neurons. Top: Fluorescent micrographs using multiple stains to identify harvested individual DRG neurons that survive for months in culture. Bottom: The axons from these neurons can be stretch grown to create long living nervous tissue constructs—a type of mini human nervous system that can be transplanted.