27 Mar 2009

Transplanted nerve cells with artificially stretched axons may act as “jumper cables” to guide and accelerate axon regeneration across nerve lesions, potentially restoring limb function to people with peripheral nerve injury. So suggests a paper published in this month’s Tissue Engineering: Part A. Scientists from the University of Rochester, New York, and the University of Pennsylvania in Philadelphia report that lab-stretched, transplanted nerves survive for up to four months in host rats, where the new neurons entwine with native axons to bridge an excised section of sciatic nerve. The work first appeared online last month. Although the authors are the first to admit that any clinical applications are years, possibly a decade, away, they suggest that the technique could potentially save motor function in people with large or multiple peripheral nerve injuries.

When a peripheral nerve is severed, the cells will regenerate new axons. However, they require some guidance to grow in the correct direction. For small gaps, less than a centimeter, a simple tube will often suffice. To bridge a larger gap, neurosurgeons frequently use an autograft, replacing the missing axons with tissue excised from another nerve, such as the sural nerve in the leg. This technique, then, sacrifices some sensory input in favor of increased motor function. Autografts can induce growth at a rate of one millimeter per day, but over weeks the support cells at the far end of the injury degenerate, so the replacement axons might not make it all the way across before the pathway on the other side is gone. That means, for example, that a person with a shoulder injury and autograft might achieve nerve function as far as the elbow, but not all the way out to the hand, said D. Kacy Cullen of the University of Pennsylvania, joint first author along with Jason Huang at the University of Rochester. Autografts are also limited by the amount of tissue available—for injuries beyond several centimeters, or multiple injuries, the surgeon may simply run out of replacement nerve to graft.

Cullen and Huang believe their technique may someday fill in the gaps that autografts can’t cross. Accidents can cause these large gaps, and tumor removal may also necessitate excising several centimeters of nerves. Multiple peripheral nerve injuries are common in the military, Cullen said. “If you’re hit with any projectile, shrapnel, anything ballistic…it’s easy to see how you will overwhelm what’s available from autografts in a patient.” Instead, the authors envision off-the-shelf transplant tissue, grown in the lab from cells harvested from organ donors. This would be a boon in the battlefield, said Huang, who recently spent four months working with soldiers in Iraq.

Principal investigator Douglas Smith, also at the University of Pennsylvania, first imagined such a treatment 10 years ago. In nature, nerve cells that formed their connections early in development must stretch as an animal grows—and they may stretch quite a way, for example, in the spine of a blue whale or the neck of a giraffe. Smith developed methods to recapitulate this stretching in culture.

In this study, the authors isolated neurons from the dorsal root ganglia of embryonic rats. They arrayed the cells in parallel lines 100 microns apart on a custom-built, movable platform. After a few days the axons had bridged the divide, and the scientists used a computer-controlled motor to gently pull them farther and farther apart, a millimeter at a time. For this study they aimed for axons 1-1.5 centimeters long; the lab’s record is 10 centimeters (Pfister et al., 2004).

The researchers then packaged the cultured neurons into a collagen matrix and wrapped the bundle in a surgical tube. They excised a centimeter of sciatic nerve from adult rats and sewed in the engineered replacement. By using transgenic, GFP-expressing rats as donors, the authors were able to follow growth and survival of the new tissue. Similarly, a strain of rats globally expressing human placental alkaline phosphatase (AP) allowed the scientists to tag host cells with AP antibodies. After four weeks, not only did the GFP-labeled cells survive within the tube, they also projected at least five millimeters beyond it, into the host tissue. GFP-negative, AP-positive axons from the host grew into the tube as well, and were often found tightly wound with the transplanted axons. By 16 weeks post-transplant, the neural bridge looked fairly normal, the authors wrote.

The scientists also looked for nerve function in the transplant recipients, although those data are not reported in the current paper. Rats that received the new axonal tissue were quick to withdraw their feet from a hot plate, indicating pain sensation, and were able to hang on to an incline longer than untreated animals, suggesting they recovered some motor function, Cullen and Huang told ARF.

Any transplant procedure carries a risk that the host will reject the new tissue, and the authors were surprised when the rats showed no evidence of rejection—no macrophages or neutrophils infiltrating the surgical site. Pure nerve cells or embryonic tissue might be less immunogenic than other kinds of cells, the authors suggested. The explanation could also be that the transplants were from and grafted to Sprague-Dawley rats. “They’re so inbred, I wasn’t that surprised,” said Jerry Silver of Case Western Reserve University in Cleveland, Ohio, who was not involved in the study.

Axons, Silver noted, do not a functional nerve make. “It’s the support cells that are critical…without glia you have no myelin, without myelin you have no action potential.” The authors did note that many new axons were surrounded by cells expressing myelin basic protein, suggesting myelination, and Cullen told ARF in an e-mail that in unpublished experiments, they have demonstrated action potentials across the regenerated nerve.

It is not quite clear how the transplants mediate regeneration, but the authors suggested they may provide both biochemical signals that promote growth, as well as a physical scaffold for new axons to follow. The cultured nerves act like “jumper cables,” Huang said, connecting the host nerves together until they can function on their own again.

Huang and Cullen imagine that someday these nerve replacements might be available as off-the-shelf components. Smith’s group have previously shown they can culture and stretch dorsal ganglia neurons from human cadavers and from people who had a ganglionectomy to cure headaches (Huang et al., 2008). Organ donors, then, are a potential supply of starting material, Huang said, although they would still need a way to mass produce and store the tissue constructs. Humans, being less alike than lab rats, might have a bigger issue with rejection. However, it’s possible that the grafts would need to last for only a limited time, Cullen said. The grafts could “babysit” the gap during regeneration, but it might be acceptable to let the immune system destroy them after healing. In that case, graft recipients would not be sentenced to a lifelong immunosuppressive regime. Even if immunosuppressants were required, it would probably be worthwhile for a person otherwise facing amputation, Huang wrote in an e-mail to ARF.

In order to show that their methods have clinical merit, the authors must show that they are better than the autograft “gold standard,” Silver said. “The fact that they can stretch nerves that far, that’s really cool, but the clinical utility is still a ways away.” The authors agree that they have plenty left to prove, but say that for large peripheral nerve injuries, no other technique does the job. They have also considered using the technique to rebuild injured spinal cord (Iwata et al., 2006) or even nerves damaged by degenerative disease, although such applications are further down the road, Huang said.—Amber Dance

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References

Paper Citations

1. Pfister BJ, Iwata A, Meaney DF, Smith DH. Extreme stretch growth of integrated axons. J Neurosci. 2004 Sep 8;24(36):7978-83. PubMed.
2. Huang JH, Zager EL, Zhang J, Groff RF, Pfister BJ, Cohen AS, Grady MS, Maloney-Wilensky E, Smith DH. Harvested human neurons engineered as live nervous tissue constructs: implications for transplantation. Laboratory investigation. J Neurosurg. 2008 Feb;108(2):343-7. PubMed.
3. Iwata A, Browne KD, Pfister BJ, Gruner JA, Smith DH. Long-term survival and outgrowth of mechanically engineered nervous tissue constructs implanted into spinal cord lesions. Tissue Eng. 2006 Jan;12(1):101-10. PubMed.

Further Reading

Papers

1. Hilliard MA. Axonal degeneration and regeneration: a mechanistic tug-of-war. J Neurochem. 2009 Jan;108(1):23-32. PubMed.
2. Sweet ES, Firestein BL. Neuronal polarization: old cells can learn new tricks. Curr Biol. 2008 Aug 5;18(15):R661-R663. PubMed.
3. Maden M. Retinoic acid in the development, regeneration and maintenance of the nervous system. Nat Rev Neurosci. 2007 Oct;8(10):755-65. PubMed.
4. Kerschensteiner M, Schwab ME, Lichtman JW, Misgeld T. In vivo imaging of axonal degeneration and regeneration in the injured spinal cord. Nat Med. 2005 May;11(5):572-7. PubMed.
5. Brittis PA, Flanagan JG. Nogo domains and a Nogo receptor: implications for axon regeneration. Neuron. 2001 Apr;30(1):11-4. PubMed.
6. Menet V, Prieto M, Privat A, Giménez y Ribotta M. Axonal plasticity and functional recovery after spinal cord injury in mice deficient in both glial fibrillary acidic protein and vimentin genes. Proc Natl Acad Sci U S A. 2003 Jul 22;100(15):8999-9004. PubMed.

News

1. New Direction for Axonal Regeneration? Navigation Is Key 13 Apr 2005
2. Two Other Faces of Inflammation—Stem Cell Guidance and Axon Repair 16 Dec 2004
3. Shedding Light on the Dark Side of Astrocytes Implicates Vimentin, GFAP 11 Jul 2003
4. Can Adult Mice Grow New, Functional Cortical Neurons? 22 Nov 2002