13 April 2005. Navigational failure is the rock on which many a traveler has perished. It may also be the reason why many spinal cord neurons fail to recover from injury. So conclude Thomas Misgeld and colleagues at Harvard University and the University of Zurich, Switzerland, in a report in the April 10 Nature Medicine online. The authors have pulled off a technical tour de force in using newly developed in vivo microscopy to follow regeneration of injured spinal cord axons in real time. Their videos suggest that when broken axons fail to reconnect, it is not from lack of trying, but rather from lack of direction. The new technique, in addition to facilitating the study of axonal regeneration, may also prove useful in studying neurodegeneration in models of motor neuron diseases such as ALS.
The severing of a spinal cord axon usually spells doom for a neuron. The axon on the proximal side of injury retreats back toward the cell body, while that on the distal side undergoes Wallerian degeneration (see ARF related news story), a slow and inexorable demise. It is rare that the two ends shall ever meet again. But why is that, exactly?
Neuroscientists have faced one major problem in trying to answer that question, namely, their inability to watch, in real time, what happens to the two ends of the axon. First author Martin Kerschensteiner and colleagues got over that hurdle by using a “dipping-cone” water immersion objective lens that could be placed right on a mouse spinal cord. They coupled that with a computer-assisted wide field microscope, which allowed them to visualize axons up to several vertebrae away from the objective. The use of GFP-S mice, which express green fluorescent protein in neurons of the dorsal root ganglia, provided sufficient sensitivity to visualize the neurons.
The first thing the authors noticed when they got a look at damaged neurons is that the axon behaves more like a reluctant bungee cord than a piece of string. About two minutes after injury, both distal and proximal ends of the axon begin to retract, eventually ending up several hundred micrometers from the lesion site in a process the authors call “acute axonal degeneration” or AAD (see video below). Though this happens on both sides of the lesion, it appears to be related to Wallerian degeneration because AAD was largely absent in Wlds mice, which exhibit slow degeneration, and in the presence of calpain inhibitors, which block the Wallerian process.
Kerschensteiner and colleagues show that after this initial AAD, the axons on the proximal end stabilize about 300 micrometers from the site of lesion for at least seven days (as long as they were observed). But the axons are not just resting; they are actively trying to grow back. Thirty percent of them started sprouting within two days, while the really gung ho axons began to sprout after 6 hours. The authors followed these growth spurts in individual axons for several days. Growth originated in two places: at the terminal end of the axon and at the proximal node of Ranvier, the joint formed by the Schwann cells that wrap the axons. In some axons, growth occurred in both places; in others it was rapid and extended mostly from the tip of the axon.
But what all these growth spurts had in common was lack of directionality. Instead of growing straight toward the severed distal axon, trajectories were all over the map. Despite the fact that they grew, on average, 200 micrometers during two days of observation—almost enough to bridge the gap to the remains of the distal end of the neuron—the axons grew laterally or did a complete U-turn. “Their erratic growth pattern suggests that this is at least partly the result of a lack of directional information needed to reiterate the original growth trajectory,” write the authors.
Overall, the data indicate that axonal degeneration, on both sides of the lesion, may be a form of self-destruction, while attempts to reconnect the two are hampered by poor or nonexistent navigation. In addition, “AAD is potentially of considerable clinical relevance as it augments denervation by removing spared side branches of axons proximal to the lesion,” suggest the authors.—Tom Fagan
Kerschensteiner M, Schwab ME, Lichtman JW, Misgeld T. In vivo imaging of axonal degeneration and regeneration in the injured spinal cord. Nat Med. 2005 Apr 10; [Epub ahead of print] Abstract