Seeing is Believing: Imaging Injured Spinal Axons with Two-Photon Microscopy

Binhai Zheng, Ph.D.

University of California, San Diego, La Jolla, CA

Grant Program:

David Mahoney Neuroimaging Program

Funded in:

December 2006, for 3 years

Funding Amount:


Lay Summary

Can a New Imaging Technique Show Axonal Regeneration in Spinal Cord Injury?

These researchers will see, in an animal model of spinal cord injury, whether repeated two-photon cellular imaging can accurately identify injured axons that are regenerating.

Lack of scientific tools has hampered efforts to understand the molecular events that occur after spinal cord injury and during efforts to stimulate regeneration of injured axons, the nerve cells’ communication cables. Currently used spinal cord tracing and chemical analyses fail to distinguish spared axons from regenerated ones, and axonal sprouts from uninjured axons are often mistaken as evidence of regeneration.  In contrast, repeated visualization of the same axons over time, using time-lapse two-photon cellular imaging, may profoundly improve scientists’ capacity to study the processes that impede axonal regeneration and to assess the effects of experimental methods for stimulating axonal repair.

The UCSD investigators will work to validate this approach as a reliable and minimally invasive method for imaging sensory axons in mice.  As part of this validation, they will study axonal responses to known growth-promoting and growth-inhibiting molecules in the mouse model.  Additionally, they will image the interaction between the growth-promoting chemical (cyclic AMP) and the inhibitory molecule (“Nogo,” which is made by myelin that insulates axons), to reveal the dynamics between these opposing forces that occur following axonal injury in the mouse model.

Significance: If the investigators can unequivocally identify spared from re-growing axons using time-lapse two-photon imaging, they will have proof of this new technique’s utility in studying spinal cord injury repair, and effects of regenerative therapies in the animal model.


Seeing is Believing: Imaging Injured Spinal Axons with Two-Photon Microscopy

The goals of this proposal are to establish a method to repetitively image injured spinal axons in live mice with two-photon microscopy over an extended period of time and to start to explore this methodology to study spinal axon regeneration following various manipulations, including growth stimulation and disrupting axon growth inhibitors.

The capacity to regenerate axons after traumatic injury in the adult mammalian central nervous system (CNS) is rather limited. Injured axons attempt to regenerate, but this attempt generally fails in the CNS. The failure of CNS axon regeneration underlies the permanent functional deficits and paralysis in spinal cord injury patients. Understanding the molecular mechanisms of CNS axon regeneration failure will significantly enhance our ability to design rational therapeutic intervention for spinal cord injury, white matter stroke, and certain neurodegenerative disorders. Several hypotheses have been intensively investigated to explain the failure of CNS axon regeneration, including a lack of intrinsic growth potential in the CNS neurons, a lack of growth promoting factors or tissue bridges, and the presence of axon growth inhibitors that actively block axon regeneration.

Despite rapid progress in this field in the past decade, it remains difficult to convincingly demonstrate axon regeneration after various cellular and molecular interventions in animal models of spinal cord injury. Conventional methods entail tract tracing and immunohistochemistry in end-point analyses to assess axonal response to injury. A major problem in such analyses is the difficulty in distinguishing regenerated axons from axons that have been spared by incomplete lesion. The ability to follow axonal response in real time and at various time intervals after injury would be highly desirable, as it would allow for unequivocal identification of regenerating axons and for temporal dynamics of axonal response to injury to be revealed.

We have experimented with in vivo imaging of genetically labeled sensory axons in live mice with two-photon microscopy. Here we present preliminary data to prove feasibility of this approach and propose to develop this technology to monitor axonal response to injury for up to 8 weeks after injury. We then propose to validate this technology as a novel method to analyze axon regeneration by examining the effect of two manipulations that are known to promote sensory axon regeneration: cAMP elevation and a conditioning peripheral nerve lesion. Finally, we propose to apply this technology to examine the role of Nogo and NgR, which have been hypothesized to be a major myelin-derived axon growth inhibitor and receptor respectively, and to determine whether disrupting such inhibitory molecules have a synergistic effect with growth stimulation.

Together, such analyses will reveal axonal dynamics after injury that have not been possible with conventional methods following various genetic, pharmacological, and surgical manipulations, and will provide a proof of principle for future studies of spinal axon regeneration. Furthermore, our in vivo imaging experiments involve laser ablation of single axons that will minimize scar formation, which would allow us to separate the contribution of myelin inhibitors from that of glial scar to CNS axon regeneration failure.

Investigator Biographies

Binhai Zheng, Ph.D.

Assistant Professor, University of California, San Diego



The progress in axon regeneration and spinal cord repair research will significantly benefit from a powerful tool to unequivocally identify regenerating axons from spared axons. We hypothesize that optical imaging of injured spinal axons with two-photon microscopy has the potential to unequivocally identify regenerating axons and to reveal detailed dynamics of axonal response to injury that are otherwise undetectable with conventional methods, and will be a powerful method to study the molecular mechanisms of CNS axon regeneration failure with mouse models of spinal cord injury.

1. To develop the methodology to repetitively image injured dorsal column sensory axons that are genetically labeled with a fluorescent protein over an extended period of several weeks.
2. To examine the injury response of fluorescently labeled dorsal column sensory axons by applying two growth stimulating manipulations that are known to promote sensory axon regeneration: cAMP elevation and a conditioning peripheral nerve lesion.
3. To determine the contribution of Nogo and NgR in sensory axon regeneration failure by examining axonal response to injury in mice deficient in Nogo/NgR, in the presence and absence of growth stimulating manipulations.

We will use two-photon microscopy to image fluorescently labeled ascending sensory axons in live mice. We will laser-ablate single axons and follow the axonal behavior for minutes to hours at a time, and repetitively image the same axons days and weeks after axotomy. We will explore the potential of this technology to analyze the effect of growth stimulation and removing myelin inhibitory factors in promoting spinal axon regeneration after axotomy.

Selected Publications

Zheng B., Ho C., Li S., Keirstead H., Steward O., and Tessier-Lavigne M. Lack of enhanced spinal regeneration in Nogo-deficient mice. Neuron. 2003 Apr 24;38(2):213-24.

Zheng B., Atwal J., Ho C., Case L., He X. L., Garcia K. C., Steward O., and Tessier-Lavigne M. Genetic deletion of Nogo receptor does not reduce neurite inhibition in vitro or promote corticospinal tract regeneration in vivoProc Natl Acad Sci U S A. 2005 Jan 25;102(4):1205-10.

Zheng B., Lee J. K., and Xie F. Genetic mouse models for studying inhibitors of spinal axon regeneration. Trends Neurosci. 2006 Nov;29(11):640-6.