Promoting Recovery In The Central and Peripheral Nervous System Following Axonal Injury

Background

Injured nerve fibers in the adult mammalian central nervous system (CNS) usually do not regenerate over long distances which often results in permanent disability. A graphic example is the patient suffering a severe injury to the spinal cord who remains wheelchair dependent. In striking contrast, injured nerve fibers in the adult mammalian peripheral nervous system (PNS) can and often do regenerate, thereby restoring at least some lost function. This dichotomous behavior on the part of damaged axons is beautifully illustrated by crushed dorsal root sensory fibers which represent a single axonal pathway that traverses both the PNS and CNS.

Figure 1- Black-labeled axons (arrowhead) in a rat dorsal root crushed 6 weeks previously regenerate up to but not into the spinal cord.

As shown in Figure 1, such fibers will readily regenerate past a crush lesion in the PNS but normally will not enter the CNS. Such axonal behavior highlights the importance of local environmental factors in determining whether or not an axon will regenerate.

Research Question:

What biological responses can be modified to promote axonal regeneration in the CNS and PNS following injury?

We have been investigating cellular and molecular factors modulating axonal regeneration as part of an effort to devise new strategies to promote recovery following axonal injury in both the CNS and PNS. We initially found that implants of embryonic glia (astrocytes) seeded on a Millipore filter scaffolding could promote to a limited degree the regeneration of crushed dorsal root sensory fibers into the spinal cord(Figure 2). This limited regeneration was found in association with a local inflammatory response characterized by the presence of macrophages and activated microglia. We then became interested in the response of macrophages and microglia following axonal injury in the PNS and CNS, as well as their role in influencing axonal regeneration. Interestingly, we found that following a dorsal root injury, macrophages would rapidly infiltrate the PNS portion of this pathway but not the CNS portion within the spinal cord (Figure 3).

Figure 2- A millipore implant coated with embryonic astrocytes (arrow) promotes the regeneration of a few crushed dorsal root axons (arrowhead) into the spinal cord.   Figure 3- A cut dorsal root showing macrophages infiltrating into its PNS portion (arrowhead) but not extending into the spinal cord (arrow). Figure 4- A cut dorsal root combined with LPS treatment showing macrophages and activated microglia within the PNS (arrowhead) that also extend across the PNS/CNS interface (black arow) rostrally up the spinal cord (open arrow).

We began to study factors influencing the macrophage response following peripheral nerve injury such as various cytokines, cell adhesion molecules, and serum complement. We showed that depleting serum complement reduced the macrophage response following peripheral nerve injury and also delayed axonal regeneration. This finding supported our hypothesis that the macrophage response during axonal degeneration was an important determinant of subsequent axonal regeneration. We then succeeded in using an immunomodulator, lipopolysaccaride (LPS), to induce a macrophage/microglia response in the CNS similar to that found in the PNS following a dorsal root lesion (see Figure 4). We are interested in determining whether such a macrophage/microlgia response will allow crushed dorsal root fibers to regenerate into the spinal cord. We are also studying the role of specific cell adhesion molecules, present on circulating monocytes and endothelial cells, in mediating macrophage infiltration following peripheral nerve injury.

Peripheral nerve injury is common and often results in significant disability to patients and costs to society. Although damaged peripheral nerves fibers can and often do regenerate in the PNS, recovery of function is often incomplete due to a variety of biological factors. First, neurons must survive the injury. Second, neurons must regenerate their axons. Third, axons must navigate their way back to and reconnect with their appropriate target structures. Peripheral nerves regenerate at a rate of about one inch per month. Many months may elapse before a peripheral nerve regenerates back to its original target muscle or sensory structure. It would therefore be of great clinical benefit to develop methods of accelerating and improving the recovery of damaged peripheral nerves. Working in collaboration with ultrasound experts in the Applied Physics Laboratory at the University of Washington, we have succeeded in demonstrating that local application of ultrasound to the crushed sciatic nerve of a rat accelerates the recovery of function (Figure 5a and b). We are trying to understand the biological mechanisms by which ultrasound exerts its therapeutic effect following peripheral nerve injury. We hope to eventually develop an ultrasound treatment which can be used on patients with peripheral nerve injury that will accelerate and improve their recovery.

Figure 5- Graph in panel b shows how ultrasound accelerates functional recovery, assessed by a toe spread index (panel a), following a complete sciatic nerve crush injury in the rat.

This schematic illustrates different components of the central and peripheral nervous system. We are studying and attempting to modulate certain cellular and molecular responses of neurons, their axonal pathways, and their target structures following injury in order to improve outcome.

 

Research Highlights:

1. The macrophage/microglial response in the PNS and CNS is very different following axonal injury.

In the adult mammalian PNS, circulating monocytes rapidly infiltrate within days a peripheral nerve undergoing Wallerian degeneration of axons. This reponse is mediated by a combination of cell adhesion molecules, cytokines, and other factors. In the adult mammalian CNS, circulating monocytes do not rapidly infiltrate degenerating axons and microglia do not become activated for several weeks following the onset of axonal degeneration. Axons normally more readily regenerate in the PNS than in the CNS following injury.

2. Axonal regeneration in the PNS is reduced by decreasing the macrophage response that occurs during Wallerian degeneration.

Depletion of serum complement through intravenous administration of cobra venom toxin significantly reduces macrophage infiltration in degenerating peripheral nerve. The activation state of infiltrating macrophages was also dramatically diminished. Complement depeltion resulted in slowing the rate of axonal regeneration as well.

3. An immunomodulator like LPS can enahnce the macrophage/microglial response to degenerating axons in the CNS.

Lipopolysaccharide (LPS) when administered systematically can “prime” macrophages and microglia. In LPS primed animals, macrophages/microglia associated with degenerating axons in the CNS now exhibit an enhanced response more closely resembling that occurring normally in the PNS.

4. Ultrasound can accelerate functional recovery following peripheral nerve injury.

We have shown that ultrasound administered transcutaneously to rodents in the region of their sciatic nerve crush can reduce the amount of time it takes to recover their normal foot function as determined by analysis of their gait. New Areas Of Research Under Investigation:
During development stem cells generate all the different cell and tissue types. Stem and progenitor cells have also been identified in many adult tissues and organs including the nervous system. Evidence is mounting that these multipotential cells respond to injury and may have important ongoing roles throughout life. We are currently investigating ways of modulating the response of endogenous stem cells in adult mammalian animals in the setting of trauma and disease.

Research Methods:

• Immunohisotchemical staining to identify specific cell types and molecules in vivo.
  
• Techniques to label dividing cells in vivo.
  
• Behavioral and electrophysiological techniques are used to assess functional recovery following peripheral nerve injury.
  
• Special MRI techniques have been developed to non-invasively visualize degenerating and regenerating axons in both animals and human patients.
  
• Use of transgenic animals with specific diseases.
  
• Animal models of traumatic injury.
  
• Delivery of specific factors in vivo by means of implanted osmotic mini-pumps.
  
  
  

Bibliography:

SELECTED PUBLISHED AND ACCEPTED ARTICLES IN REFEREED JOURNALS:

1. M. Kliot and C.E. Poletti. Hippocampal afterdischarges: Differential spread of activity shown by the 14C Deoxyglucose technique. Science 204: 641-643, 1979.

2. C.J. Shatz and M. Kliot. Prenatal misrouting of the retino-geniculate pathway in Siamese cats. Nature 300: 525-529, 1982.

3. M. Kliot and C.J. Shatz. Abnormal development of the retino-geniculate projection in Siamese cats. J. Neuroscience 5: 2641-2653. 1985.

4. M. Kliot, G.M. Smith, J. Siegal, S. Tyrrell, and J. Silver. Astrocyte-polymer implants promote regeneration of dorsal root fibers into the adult mammalian spinal cord. Exp. Neurol. 109: 57-69. 1990.

5. J.D. Siegal, M. Kliot, G.M. Smith, and J. Silver. A comparison of the regeneration potential of dorsal root fibers into the adult mammalian spinal cord. Exp. Neurol. 109: 90-97, 1990.

6. A.M. Avellino, D. Hart, A.T. Dailey, M. MacKinnon, D.B. Ellegala, and M. Kliot. Differential macrophage responses in the peripheral and central nervous system during Wallerian degeneration of axons. Experimental Neurology 136: 183-198. 1995.

7. A.T. Dailey, J.S. Tsuruda, A. Filler, K. Maravilla, R. Goodkin, and M. Kliot. Magnetic resonance neurography of peripheral nerve degeneration and regeneration: A clinical case presentation. Lancet 350: 1221-1222. 1997.

8. A.T. Dailey, A.M. Avellino, L. Benthem, J. Silver, and M. Kliot. Complement depletion reduces macrophage infiltration and activation during Wallerian degeneration and axonal regeneration. J. Neuroscience 18: 6713-6722. 1998.

9. D.A. Lazar, D.B. Ellegala, A.M. Avellino, A.T. Dailey, K. Andrus, and M. Kliot. Modulation of macrophage and microlgial responses to axonal injury in the peripheral and central nervous system. Neurosurgery 45: 593-600. 1999.

10. P.D. Mourad, D. Lazar, F.P. Curra, A. Avellino, L. McNutt, K. Andrus, L. Crum, and M. Kliot. Ultrasound accelerates functional recovery after peripheral nerve damage. Neurosurgery 48: 1136-1141. 2001.
G. Grant, R. Goodkin, M. Kliot. MRI in evaluating and treating peripheral nerve problems. Nerve and Muscle 25: 314-331. 2002.