"Axon Regeneration Induced by cAMP"

Student Project: Eunice Chan, swtnsmiling@aol.com

This is my summary of recent research in the field of axon regeneration in injured spinal cords, based on the research article "Regeneration of Sensory Axons within the Injured Spinal Cord Induced by Intraganglionic cAMP Elevation", Simona Neumann, Frank Bradke, Marc Tessier-Lavigne, and Allan I. Basbaum, Neuron (2002), 34: 885-893.  

This project is for the course BIOL 475, Neurobiology, Dr. Grant Mastick, Biology Department, University of Nevada, Reno.  link to course.

INTRODUCTION:

• Each year there are about 10,000 spinal cord injuries.  A car accident, gunshot, diseases, or a rough football tackle can cause injury to the spinal cord.

• The spinal cord is injured even if it's not severed.  Most people with spinal cord injuries still have an intact spinal cord.

• Damaged spinal cord can cause the loss of motility or feeling to other parts of the body.

• CNS (central nervous system) is incapable of regenerating axons after an injury unlike the PNS (peripheral nervous system).

• db-cAMP, dibutyrate cAMP, an analog of cAMP.  It is a secondary messenger that carries signals from the surface of the cell to proteins within the cell.

• Forskolin stimulates cAMP production.

Hypothesis of this study:  Does the exposure of sensory neurons in the dorsal root ganglia to db-cAMP enhance the regeneration of the central branch of primary sensory neurons.  

EXPERIMENTAL SYSTEM:

In the vivo experiments, the researchers used the dorsal columns of rats to study the regeneration of CNS axons.  They studied specifically on medium and large sensory neurons with the cells bodies located in the L4, L5, and L6 (lumbar spinal cord) dorsal root ganglion.  The researchers then injected a tracer into the sciatic nerve to help visualize the axons because the medium and large myelinated axons will take up the tracer.  Researchers then used microscissors and cut the dorsal columns bilaterally at the T6-T7 regions.  The dorsal column was cut down to the central canal. 

Control experiment:

• In 6 rats, nothing was injected into the dorsal root ganglion prior to the spinal cord lesion.

• In another set of 6 rats, saline was injected into the dorsal root ganglion 48 hours prior to the spinal cord lesion.  

• In sensory neurons that were dissociated from L4 and L5 dorsal root ganglion were plated on dishes and were not treated with forskolin.  

To test the gain of function:

• db-cAMP was injected into the dorsal root ganglion 48 hours prior to the spinal cord lesion.  

•  Sensory neurons that were dissociated from L4 and L5 dorsal root ganglion were plated on dishes and treated with forskolin.

RESULTS: 

6 to 8 weeks after the dorsal column lesions, the researchers began looking at the results:

• The rats that were untreated had no regeneration of the injured axons in the dorsal columns.  The fibers stopped at the lesion site and most of the axons just swelled up.  

(a)                       (b)

Figure 1.  (a) a diagram of a spinal cord fibers in an unoperated animal.  The fibers are in a tight bundle.  In figure (b), it shows a section of a spinal cord with a lesion.  There are no fiber growing through the lesion site.

• The rats that were treated with saline had little to no regeneration on the injured axons in the dorsal columns.  Only a few fibers are near the lesion region, but most of them stopped at the lesion site.  

Figure 2.  This is a diagram of a spinal cord lesion, and there are a few small fibers growing into the lesion site. The arrow shows the fibers growing into the lesion site.

• The rats that were injected with db-cAMP 48 hours prior to the lesion showed a massive amount of fibers growing into the lesion.  

Figure 3.  This is a diagram of spinal cord lesion , showing fibers growing into the lesion site.  The fibers are much longer than the fibers in figure 2.  

• The sensory neurons that were not treated with forskolin, had little growths of the neurites of the neurons.

Figure 4.  This is a diagram of the sensory neurons that were not treated with forskolin.  The lengths of the neurites were not very long.

• The sensory neurons that were treated with forskolin, the neurites of the neurons grew much longer in length.  

Figure 5.  This is a diagram of a sensory neuron that was treated with forskolin.  Since forskolin induces cAMP production, it shows that there was much more growth in the lengths of the neurites.  

CONCLUSION:

• The dorsal root ganglia that was not injected had no regeneration of the injured axons.

• The dorsal root ganglia that was injected with saline, had very little growth of the injured axons into the regions of the lesions.  

• The dorsal root ganglia that was injected with db-cAMP, had growth of the injured axons.

• The neurons that were treated with the forskolin, the neurites grew longer in length that the non-treated neurons.

• From the above data, it shows that the induction of cAMP can induce the growth of injured axons and neurites.

SIGNIFICANCE:

• The results showed in the experiment, the manipulation of the cAMP signaling pathways in neurons promoted axonal regeneration in the central nervous system. 

• Axonal regeneration in CNS can help patients with spinal cord injuries.   

FUTURE DIRECTION:

• Test different doses of db-cAMP to determine if different amounts would increase or decrease the growth on the axons in the CNS.

• Since db-cAMP was injected 48 hours prior to a lesion, researchers should inject db-cAMP at different times before and after a spinal cord lesion to determine if the db-cAMP will induce axon regeneration.

• There are more studies that still need to be performed on signaling pathway to determine if it can help the regeneration of axons further.  

REFERENCES:

Primary research article: Neumann, S., Branke, F., Tessier-Lavigne, M., and Basbaum, A.  "Regeneration of Sensory Axons within the Injured Spinal Cord Induced by Intraganglionic cAMP Elevation."  Neuron, 2002.  34: 885-893.  link to summary.

Review Articles: Snider, W. D., Zhou, F. Q., Zhong, J., and Markus, A.  "Signaling the Pathway to Regeneration."  Neuron, 2002.  35:13-6.  link to summary.

Related Articles: Qiu, J., Cai, D., Dai, H., McAtee, M., Hoffman, P. N., Bregman, B. S., and Filbin, M. T.  "Spinal Axon Regeneration Induced by Elevation of Cyclic AMP."  Neuron, 2002.  34: 895-903.  link to summary.

This page was created by: Eunice Chan.  If you have any questions or comments please feel free to e-mail me click here.  All diagrams were hand-drawn and not copied from the original published paper.