Research on Neuroregeneration Thriving

They can't reproduce, but mature neurons can grow. Indeed, for more than a century, neurologists have been able to restore at least partial function to severed peripheral nerve networks through neuronal transplantation. But the CNS has proved more recalcitrant: there, scar tissue and a complex molecular brew stymie the efforts of damaged axons to sprout new fibers and restore old connections.

Investigators around the world are slowly elucidating the mechanisms the CNS uses to prevent injured neurons from regenerating. The field is young, and many questions still remain, but the challenges of neuroregeneration are eliciting some of the most creative thinking in neurology.

BARRIERS TO REGENERATION

The apparent obstacles to CNS regeneration have been a source of frustration to researchers, not to mention the patients who must live with the effects of wounded neurons. "It's a cruel paradox that when axons stop sprouting at an early age, the CNS secretes proteins that inhibit them from further growth," said Douglas Smith, MD, professor of neurosurgery and director of the Center for Brain Injury and Repair at the University of Pennsylvania in Philadelphia.

Currently, spinal cord injuries (SCIs) are incurable because they disrupt the neural circuitry on which the nervous system depends. Taking a cue from observations of the peripheral nervous system, in which transplanted neurons can grow and form new functional connections, researchers have tried transplanting peripheral nerve segments and cultured Schwann cells into the spinal cord. However, these efforts proved disappointing because the regenerating nerve cells could not re-enter the host CNS.¹ Severed spinal nerves form scars that contain chondroitin sulfate proteoglycans that act as a roadblock, preventing regenerating axons from penetrating the scar tissue and connecting with fibers on the other side of the scar, which might help restore function. Oligodendrocytes and myelin also appear to dampen axonal growth. The result has been likened to a roadblock that stops neural traffic from moving forward.²

Some investigators are trying to dismantle the roadblock by neutralizing the compounds that inhibit axonal regeneration. One group used the enzyme chondroitinase-ABC (ChABC) to break down the proteoglycans that accumulate in spinal scars, in the hopes that transplanted peripheral nerves would penetrate the scar and establish new, functional connections.

The researchers gave rats hemisection lesions at C3 and then transplanted a 1.5-cm piece of the tibial branch of the sciatic nerve into the resulting cavity. After approximately 2½ weeks, when the transplanted nerve sprouted new fibers, the researchers implanted a pump that delivered a steady dose of ChABC to C5 to get the regenerated axons to extend to that level.

The animals that received ChABC demonstrated some restoration of locomotion. Control animals that received a saline solution exhibited no functional change. The team, led by Jerry Silver, MD, professor of neurosciences at Case Western Reserve University School of Medicine in Cleveland, concluded that the axons in the ChABC-treated rats extended for much longer distances in the spinal cord than those in the control rats.²

GROWTH GUIDES

That the CNS would try to prevent new connections from forming seems counterintuitive.³ After all, humans are capable of learning and making new neural connections virtually throughout their lives.

Investigators in Smith's laboratory have demonstrated that test rats respond to traumatic brain injuries with a persistent proliferation of neurons, as well as glial cells, that is not seen in intact animals.⁴ Research in other laboratories has shown that neurotrophins figure in CNS plasticity and may contribute to the functional recovery associated with neural stem cell transplantation.⁵ Thus, a more likely explanation is that so-called inhibitory molecules actually are guides, the purpose of which is not to stop neuronal growth but to direct it. While the organism is growing, these factors help the developing neurons make the right connections and form the proper pathways. Nerves that are severed after they reach maturity attempt to grow, but they now encounter an environment very different from that of an organism's developmental stage. That mature environment includes longer distances, other fibers in well-established pathways, and a more complex brain and nervous system.³

Following this line of reasoning, Geoffrey Raisman, MD, chair of Neurological Regeneration at the Institute of Neurology, University College, London, and associates have been trying to exploit these molecules' natural guiding properties by transplanting ensheathing cells that surround adult olfactory nerves at the site of SCIs in rat models. Within 3 to 4 weeks, the cells encase the newly sprouting fibers in myelin and shepherd them through the spinal cord toward their normal target areas. Once there, the fledgling fibers can form new connections and restore function. According to Raisman, these olfactory ensheathing cells (OECs), which are in a state of continuous growth and replacement, restore the integrity of the original neural pathway by acting as a patch over the injured site.^1,3

Raisman and colleagues have been experimenting with OECs in animals since 1985. They still haven't identified the mechanisms by which the cells exert their salutary effects, he tells Applied Neurology, nor is it known whether neural cells from other sites have similar properties. They began human testing this year, but Carlos Lima, MD, of Lisbon's Hospital de Egas Moniz, and associates, have used this technique in more than 120 patients since 2001,⁶ and Hongyun Huang, MD, of Beijing, has already tried it in more than 450 patients.⁷ Both researchers report promising findings. Clinical trials in Australia and elsewhere in Beijing also are under way.

STRETCHING THE POSSIBILITIES

Smith and associates are taking a different approach to neuronal growth. They have developed a tissue-engineering technique that induces long axon tracts to form bridges over spinal cord lesions and connect to the fibers on either side. As with OECs, the result is restoration of the severed neural circuit.

They start by culturing neurons from dorsal root ganglia on 2 nutrient-rich plates placed beside each other. As the axons on each plate grow, they reach out and form connections with the axons on the other plate. One of the plates is attached to a block equipped with a computer-controlled motor that slowly moves the plate away from its counterpart, stretching the axons. The cells thrive in this environment and grow to 10 mm (about 4 in) within 7 days. The architecture of the complex resembles the longitudinal arrangement of an intact spinal cord.

In a proof-of-principle study, these axons were encased in a collagen gel enriched with growth factors and folded into a form resembling a jelly roll. Smith described this structure as a nervous system "construct." The investigators modified hemisection SCIs in rats by creating 1-cm long cavities in the spinal cord. Ten days after the injury, they implanted the constructs into the lesions and sacrificed the animals 4 weeks later. The cells in the constructs not only survived, they sprouted axons from the construct into the host spinal cord tissue where they have the potential to form new functional connections.^8,9

Placed in spinal cord lesions, the constructs--or, as Smith likes to call them, "mini nervous systems"--function like jumper cables by forming bridges across the severed fibers and providing a jolt that gets the host's neurons to sprout new axons.

The ability of axons to lengthen through stretch growth has only recently been recognized, but it isn't surprising, Smith said. After all, "a blue whale can do it." Baby blue whales grow 4 cm per day and ultimately reach lengths of as much as 30 m, or 100 ft. That means they must have neurons with enormous axons that span the distance from head to tail. Giraffes have similarly long neurons. Growth of this magnitude "defies anything in our textbooks," he remarked. Yet it can't occur through growth cones because by the time the animal is born, most of these neurons no longer have an exposed axon tip. Lengthening must occur from somewhere in the middle of the axon, fueled by the tension forces that drive growth overall.

The next step will be to determine whether the constructs really can form functional neural networks, he continued. Smith and his colleagues also are evaluating other animal models. Conducting spinal cord studies only on rats can give misleading results because rats recover from SCIs better than humans regardless of the treatment involved.

THE DOPAMINE STRATEGY

Neurotransmitters play an integral role in nervous system function and malfunction, so many investigators have focused on these compounds as possible agents in neural regeneration. In one novel example of this research, a team of researchers led by Yadong Wang, PhD, associate professor of biomedical engineering at Georgia Institute of Technology in Atlanta, designed a polymer that derives its activity from dopamine. Explanted neurons from the dorsal root ganglion cultured on the polymer grew very well, as did rat pheochromocytoma cells primed with nerve growth factor.¹⁰ "What I envision is that this will be a material that you can spin into fibers of about 100 nanometers and form into a cord or a hollow tube," Wang explained. The resulting tube can be put in a lesion to act as bridge; the dopamine component would accelerate neuronal growth across it. He and his colleagues are experimenting with other neurotransmitters, including acetylcholine and g-aminobutyric acid.

APPLIED TO PDB

Regeneration of dopaminergic neurons specifically is garnering research interest because of its potential in treating--and perhaps even curing--Parkinson disease (PD). Transplantation of dopaminergic cells taken from fetuses or derived from embryonic stem cells has been associated with improved motor function both in animal models of PD and patients with PD. What's more, the transplanted cells remain viable over time, even as nigrostriatal deterioration progresses.¹¹

Although both types of cells are effective, fetal tissue is associated with problems including low efficiency, inconsistent clinical outcomes, and concerns over the ethics of using fetuses as cell donors. Stem cells may be a better choice because they can be cultivated in unlimited quantities and provide a more consistent source of dopaminergic cells. However, they, too, have their problems. It is difficult to cultivate, in sufficient quantity, embryonic stem cells that develop into the right type of dopaminergic neuron. And, of course, use of human embryonic stem cells is mired in ethical questions and controversy.^12,13

PD is an attractive target of study "because it is the least complicated of the neurologic diseases," explained Ole Isacson, MD, PhD, head of the Neuroregeneration Laboratories at Harvard Medical School in Boston. "It is a systemic disease, but a lot of its pathology is understood. Also, it is more tractable than Alzheimer disease, which produces diffuse damage. [PD] leaves most of the brain relatively intact. The brunt of the disorder affects neurons that respond to levodopa."

Isacson believes the most important work he and his associates are engaged in right now lies in elucidating why PD seems to attack only one type of dopaminergic cell. They recently analyzed gene expression profiles of A9 and A10 neurons taken from mice and found that each cell type expressed different genes at different levels, with corresponding differences in the expression of certain molecules, including peptides and growth factors, that resulted either in resistance or vulnerability to attack by PD.¹³

Studies like these are part of the rapidly developing discipline known as molecular interference, "using molecular or genetic means to modify cellular function from the inside," Isacson said. "In this way, we can harness the cell's regenerative capacity by introducing new genes or blocking old ones." Other groups have used molecular interference in the treatment of mouse models of Huntington disease.¹⁴

REMAINING QUESTIONS

With so much research on so many fronts, it's easy to forget that this field is still in its early stages. Investigators have yet to identify the mechanisms behind many of their interventions. Bench and animal studies have yielded promising findings, but the question of their clinical viability is still largely unanswered. "Our goal is to ensure that [regenerated] cells will do what you want them to once they're in the brain," Isacson said. "The challenge is to get the right cells to survive and make the right connections."

REFERENCES1. Raisman G. Olfactory ensheathing cells and repair of brain and spinal cord injuries. Cloning Stem Cells. 2004;6:364-368.
2. Houle JD, Tom VJ, Mayes D, et al. Combining an autologous peripheral nervous system "bridge" and matrix modification by chondroitinase allows robust, functional regeneration beyond a hemisection lesion of the adult rat spinal cord. J Neurosci. 2006;26:7405-7415.
3. Raisman G. A promising therapeutic approach to spinal cord repair. J Royal Soc Med. 2003;96:259-261.
4. Chen XH, Iwata A, Nonaka M, et al. Neurogenesis and glial proliferation persist for at least one year in the subventricular zone following brain trauma in rats. J Neurotrauma. 2003;20:623-631.
5. Sun CR, Wang CC, Tsang KS, et al. Modulation and impact of class I major histocompatibility complex by neural stem cell-derived neurotrophins on neuroregeneration. Med Hypotheses. 2007;68:176-179.
6. Lima C, Pratas-Vital J, Escada P, et al. Olfactory mucosa autografts in human spinal cord injury: a pilot clinical study. J Spinal Cord Med. 2006;29:191-203.
7. Mooney P. Fetal cells used to treat ALS. The Scientist. 2004;5:20040730-02.
8. Iwata A, Browne KD, Pfister BJ, et al. Long-term survival and outgrowth of mechanically engineered nervous tissue constructs implanted into spinal cord lesions. Tissue Eng. 2006;12:101-110.
9. Pfister BJ, Iwata A, Taylor AG, et al. Development of transplantable nervous system constructs comprised of stretch-grown axons. J Neurosci Methods. 2006;153:95-103.
10. Gao J, Kim YM, Coe H, et al. A neuroinductive biomaterial based on dopamine. Proc Natl Acad Sci U S A. 2006;103:16681-16686.
11. Isacson O, Bjorklund LM, Schumacher JM. Toward full restoration of synaptic and terminal function of the dopaminergic system in Parkinson's disease by stem cells. Ann Neurol. 2003;53(suppl 3):5135-5146.
12. Sonntag KC, Simantov R, Isacson O, et al. Stem cells may reshape the prospect of Parkinson's disease therapy. Brain Res Mol Brain Res. 2005;134:34-51.
13. Chung CY, Seo H, Sonntag KC, et al. Cell type-specific gene expression of midbrain dopaminergic neurons reveals molecules involved in the vulnerability and protection. Hum Mol Genet. 2005;14:1709-1725.
14. Harper SQ, Staber PD, He X, et al. RNA interference improves motor and neuropathological abnormalities in a Huntington's disease mouse model. Proc Natl Acad Sci U S A. 2005;102:5820-5825.