Bridges and Blues—Striking a Chord for Axon Protection

Though doctors do not have many options for treating traumatic spinal cord injuries, scientists continue to sound out new strategies for re-establishing broken neural connections and for protecting injured spinal cords from further damage. Advances on both fronts are reported in two recent papers online. In the August 2 Nature Neuroscience online, researchers led by Mark Tuszynski and Armin Blesch, both at the University of California, San Diego, report that they can coax neurons to bridge gaps between the ends of severed axons. In the July 27 early edition of PNAS, Maiken Nedergaard and colleagues at the University of Rochester Medical Center, New York, report that an analog of a common blue food coloring can protect damaged spinal cords from further deterioration and that the compound, Brilliant blue G (BBG), significantly improves functional recovery when given soon after spinal cord injury. (BBG is not to be confused with methylene blue, which is being tested as a memory enhancer; see ARF related news story and ARF update from this year’s ICAD meeting in Vienna.) While both discoveries come from rodent models, there is hope they may help researchers find ways of treating human injuries. Nedergaard’s approach may even have implications for the treatment of Alzheimer disease (AD), amyotrophic lateral sclerosis (ALS), and other neurodegenerative diseases that may be exacerbated by inflammatory responses. BBG, she shows, blocks a receptor that triggers cell death and activates potentially harmful immune reactions.

BBG is an analog of FD&C brilliant blue dye No. 1, approved by the FDA as a food coloring and reportedly one of the safest food additives on the market. BBG is commonly used as an antagonist of the P2X7 purinergic receptor (P2X7R). This receptor, which abounds in spinal cord neurons, is activated by ATP, which spills from cells after spinal cord injury. After prolonged exposure to ATP, P2X7Rs form a large pore, which can permeabilize neurons and cause toxic elevations in intracellular calcium, eventually leading to cell death. Recent evidence suggests that P2X7R pores can also form in response to amyloid-β (Aβ), leading to activation of microglia (see ARF related news story).

Nedergaard previously showed that giving rats oxidized ATP (OxATP), a P2X7 antagonist, improves recovery after spinal cord injury (SCI), but the compound has to be injected directly into the site of injury. In this paper, joint first authors Weiguo Peng, Maria Cotrina, and colleagues show that not only does BBG (10 or 50 mg/kg/day intravenously) cross the blood-brain barrier, but it accumulates at the site of damage in a rat SCI model, where it appears to reduce tissue loss after injury. The compound also accelerates motor recovery. Animals administered BBG immediately after injury and again over three consecutive days showed a statistically significant improvement in motor function at 10 days, and continued to improve compared to controls. In contrast to the latter, the BBG-treated animals had coordinated fore/hind limb movement at day 42 post-injury.

Spinal cord injuries can be exacerbated by inflammation. Activated microglia, which bump up expression of the P2X7 receptor, may be involved in the process, as might immune cells that infiltrate from the periphery. Peng and colleagues found that BBG suppressed activation of microglia following SCI, and also reduced the extent of neutrophil infiltration by about 70 percent four days after injury. BBG also significantly reduced morphological changes in astrocytes—a sign of their activation. The data suggest that BBG may help reduce tissue loss after spinal cord injury by reducing inflammatory responses.

Inflammatory responses are also a facet of other neurodegenerative diseases such as ALS and Alzheimer disease (AD). Though anti-inflammatories have proven of limited use for AD (see ARF related news story) and even for traumatic spinal cord injury (see Lee et al., 2008), Nedergaard and colleagues offer some hope that BBG might be different since P2X7 receptors are widespread, found on both neurons and glia, and are activated within seconds of injury. “BBG may thus have a unique therapeutic profile, capably suppressing the earliest steps in post-traumatic inflammatory cascades,” write the authors. Whether it may have any potential for treating AD or ALS remains to be seen.

Mind the Gap
The Nature Neuroscience paper from Tuszynski, Blesch, and colleagues addresses another major problem in treating spinal cord injury—how to get severed, withered axons to regenerate and reconnect. One approach that has shown promise is to tempt axons to traverse the injury site with morsels laden with appropriate stimuli, such as trophic factors (see ARF related news story). However, as first author Laura Taylor Alto and colleagues point out, getting the axons to bridge the lesion is one thing, but getting them to make appropriate connections on the other side, when there may be millions of potentially incorrect neuronal partners, is another.

Neurotrophic factors such as NT-3 guide axons to make appropriate connections during development. To see if NT-3 could provide similar guidance in regenerating axons, Alto and colleagues developed a complex model. They used a preconditioning lesion of the sciatic nerve (cutting or crushing peripheral axons of sensory nerves stimulates regeneration of their spinal cord axons; see Richardson et al., 1984) to help ascending sensory axons regenerate and grow through a C1 cervical lesion caused by severing ascending axonal tracts in rats. The authors labeled ascending axons with cholera toxin B subunit (CTB) and placed bone marrow stromal cell grafts at the site of the lesion to act as a cellular bridge for the axons to cross. To determine if CTB-labeled axons made appropriate connections on the other side of the bridge, they labeled neurons in the nucleus gracilis, where the ascending sensory axons normally terminate, with Flourogold.

With all this in place, the authors then injected some of the rats with lentiviral vectors carrying the NT-3 gene. They found that the combination of preconditioning lesion and the NT-3 vectors was optimal for axons to traverse the lesion site. Without the NT-3 vector, no axons crossed the lesion. The highest of two doses of viral vectors plus the conditioning lesion restored the number of axons in the nucleus gracilis to 27 percent of that in intact animals. That the NT-3 served as a guide was demonstrated in two experiments. The axons were attracted to the nearby medullary reticular formation when the lentiviruses were injected there, and they grew 900 μm beyond the nucleus gracilis into the brainstem if the viruses were injected there. “These results demonstrate that axons regenerating after adult CNS injury have classic directional and dose responsiveness to chemotropic gradients of growth factors,” write the authors.

But are the axons making the right connections? Indications are that they are. Alto and colleagues used confocal microscopy to show that CTB-labeled ascending axons had synaptic features, including synaptic boutons, and that the axons made contact with Flourogold labeled dendrites. Unfortunately, recordings from electrodes placed in the nucleus gracilis showed that those connections are not robustly active. Although 27 percent of axons were restored in the nucleus by the combination of preconditioning and NT-3 expression, only about 1.4 percent of sites tested had meaningful electrical responses, compared to 67 percent of tested sites in intact rats. One explanation for the poor electrical connectivity might be that the axons are not properly insulated. The researchers found that axons regenerating beyond the C1 lesions remained sparsely myelinated.

“The study is, to the best of our knowledge, the first to demonstrate reinnervation of a natural brainstem target by regenerating spinal cord axons,” write the authors. The next step may be to find ways to improve myelination of regenerating axons, or improve the longevity of the myelin sheaths, which are reported to be unstable in regenerating axons.—Tom Fagan

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References

News Citations

  1. Chicago: Out of the Blue—A Tau-based Treatment for AD?
  2. Vienna (and Burkina Faso): What's New With Methylene Blue?
  3. Pore It On: Purinergic Receptor Opened by Aβ, Activates Microglia
  4. NSAIDs in AD: Epi and Trial Data at Odds—Again
  5. San Diego: Getting a Grip on Glia, Part 2

Paper Citations

  1. Lee JM, Yan P, Xiao Q, Chen S, Lee KY, Hsu CY, Xu J. Methylprednisolone protects oligodendrocytes but not neurons after spinal cord injury. J Neurosci. 2008 Mar 19;28(12):3141-9. PubMed.
  2. Richardson PM, Issa VM. Peripheral injury enhances central regeneration of primary sensory neurones. Nature. 1984 Jun 28-Jul 4;309(5971):791-3. PubMed.

Further Reading

Primary Papers

  1. Alto LT, Havton LA, Conner JM, Hollis Ii ER, Blesch A, Tuszynski MH. Chemotropic guidance facilitates axonal regeneration and synapse formation after spinal cord injury. Nat Neurosci. 2009 Sep;12(9):1106-13. PubMed.
  2. Peng W, Cotrina ML, Han X, Yu H, Bekar L, Blum L, Takano T, Tian GF, Goldman SA, Nedergaard M. Systemic administration of an antagonist of the ATP-sensitive receptor P2X7 improves recovery after spinal cord injury. Proc Natl Acad Sci U S A. 2009 Jul 28;106(30):12489-93. PubMed.

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