Oct. 27, 2004 Novel methods for transplanting cells into areas damaged by spinal cord injury and experimental drug treatments show promise for aiding those suffering from injury to their spinal cord.
“New animal research brings increasing hope for sufferers of spinal cord injury,” says Oswald Steward, PhD, of the Reeve-Irvine Research Center at the University of California, Irvine, College of Medicine. “Studies are beginning to invalidate one of the longest held 'truths' in medicine—that nerve cells of the spinal cord are not able to regrow once damaged.”
New research shows that a special type of cell transplanted into injured rat spinal cord forms myelin-the insulating material around nerves that speeds conduction of nerve impulses-and improves rats' functioning, according to Masanori Sasaki, MD, Jeffery Kocsis, PhD, Karen Lankford, PhD, and Micheas Zemedkun, BS, in the department of neurology at Yale University School of Medicine.
Olfactory ensheathing cells (OECs) are specialized glial cells found in nerves and brain tissue associated with the sense of smell. Nerve cells within the olfactory tissue in the nose divide throughout life and send new axons-or nerve fibers-to transmit smell sensations to the brain. Scientists have long thought that OECs assist the normal regeneration of these axons and guide them into the brain where they make new functional connections. Because axons lose myelin after a trauma such as spinal cord injury, scientists have explored using OECs as a possible treatment. Several clinical trials to study transplantation of OECs into spinal cord injury patients are either ongoing or in the planning stages.
The Yale researchers obtained OECs from the olfactory bulbs of adult transgenic—genetically altered—rats expressing green fluorescent protein and transplanted them into other rats' spinal cords that had been completely cut at the dorsal funicular location. The green fluorescent protein allowed the cells to be easily seen in the spinal cord.
The researchers observed groups of regenerating nerve fibers crossing the spinal cord injury site and the alignment of green cells forming myelin. Electron microscopic examination of the tissue showed that myelin was indeed produced around the axons by the transplanted cells.
“These results indicate that a number of factors including remyelination of axons may contribute to improvement in function following transplantation of OECs into the injured spinal cord,” Sasaki says.
In other work, “tiny beads” (nanospheres) were found to release the enzyme that breaks down a component of the scar that forms after spinal cord injury. Dennis Stelzner, PhD, and his colleagues Donna Osterhout, PhD, and Julie Hasenwinkel, PhD, at SUNY Upstate Medical University and Syracuse University found that axonal growth, normally blocked by a component of the scar, is seen when the biodegradable nanospheres are injected.
Failure of axons to regenerate after spinal cord damage is attributed to a number of molecules present after injury that inhibit regrowth, including myelin (nerve covering) components and the scar tissue that forms after spinal cord injury, including the molecule chondroitin sulfate.
“We hypothesized that it would be possible to remove the inhibiting elements of chondroitin sulfate with the enzyme chondroitinase ABC (cABC) by delivering this enzyme in biodegradable nanospheres to the spinal injury, and thus enhance axonal regrowth and recovery of function,” Stelzner says.
Stelzner and his colleagues created and injected nanospheres (3 m l of 10 mg/ml of cABC) directly into eight different tissue cultures containing chondroitin sulfate that blocked axonal growth, and axonal growth was seen within two days. The cABC continued to be released for at least two weeks, assessed by analyzing its carrier molecule, as the nanospheres slowly degraded. Other experiments confirmed that the design of the nanospheres is effective in adhering the nanospheres to the chondroitin sulfate and that it has no toxic effects on cultured neurons.
The researchers are currently using these cABC nanospheres to treat spinal cord injury in rats. They have injected 3 or 6 m l of nanospheres immediately or one week following spinal contusion injury, or into uninjured spinal cord. Results so far show that the nanospheres remain near the injection site, and do not cause any major inflammation of spinal tissue, Stelzner says.
“A unique feature of the nanosphere delivery system,” Stelzner says, “is its ability to encapsulate other agents, in addition to cABC, that can be administered together, but released at various times, to counteract the inhibitory substances, and to target and promote regrowth in the spinal cord.”
Aside from blocking the growth-inhibiting molecules present after injury to aid in spinal cord regeneration, nerve cells also can be altered internally so they no longer recognize the inhibitory molecules as hindering growth, and instead grow right through them. Marie Filbin, PhD, in the department of biological sciences at Hunter College and her team were able to use this approach to regrow spinal cord nerve cells using a drug called Rolipram.
First, researchers conducted in vitro studies in which nerve cells were removed from Rolipram-treated rats and exposed them to molecules that inhibited nerve regrowth. They found that if cyclic AMP (cAMP)—a molecule found in every cell in our bodies—was elevated in nerve cells, the blockers of growth did not inhibit regeneration. The drug Rolipram blocks an enzyme—phosphodiesterase (PDE)—that breaks down cAMP, thus inhibiting PDE , which leads to an accumulation of cAMP and nerve regrowth.
To treat seven female rats injured on one side of their spinal cords at the C3/4 location, researchers implanted embryonic tissue into the injury site to effectively “fill the hole,” and then administered 0.4 or 0.8 µmol/kg/hr of Rolipram via a mini-pump inserted under the animals' skin. The drug was delivered continuously for 10 days, beginning two weeks after the injury. Six weeks after drug administration, scientists found improvement in front paw control and movement and in nerve regeneration. Less of the scar formed in the Rolipram-treated animals as well.
In a control group of five rats, researchers placed only embryonic cells in the spinal cord injury site, without Rolipram, but no nerve cell regrowth or functional recovery occurred. “These results suggest that drugs that elevate cAMP (i.e., Rolipram), are likely to be effective in treating spinal cord injuries,” says Filbin.
As a result of Rolipram's success in regenerating spinal cord nerve cells, which was an off-label use at the time of Filbin's study, the company Renovis now has licensed patents on the drug Rolipram.
Another animal study demonstrated nerve regrowth and restoration of function of sensory nerves leading directly into the spinal cord, when two agents—Zymosan and cABC—were used in combination.
Zymosan is an agent that is theorized to aid in the regeneration of neurons by escalating mechanisms responsible for neuronal regrowth. “Just as the inhibition of neuronal regrowth in spinal cord injury is caused by many factors, such as growth-inhibiting molecules and scarring, repair may be best achieved with several agents,” says Jerry Silver, PhD, at Case Western Reserve University . “Combinations of agents to treat spinal cord injury may result in robust regrowth of spinal cord neurons.”
Silver and his colleagues microinjected 31 µg/µl of Zymosan into one of the nerves (C8) of the upper limb of adult, female rats at the C8 dorsal root entry zone (DREZ) . Seven days later, the C8 dorsal root was crushed three times with #3 jeweler's forceps. Using a subset of 12 animals, the investigators microinjected 2 µl (20 U/µl) of cABC, in addition to the previous injection of Zymosan, directly where the nerve enters the spinal cord. Twenty rats received no injection, 10 rats received cABC injection alone, and eight rats received Zymosan injection alone.
Two weeks after nerve injury, the animals were euthanized and the neurons were labeled and analyzed for any regrowth from the injured nerve back into the spinal cord. The researchers also tested to see if the new nerves actually worked by performing motor evoked potentials (H-reflex) via stimulating the C8 sensory root and recording action potentials in the triceps muscles.
Eight of the 12 rats that received a combination of Zymosan and cABC had significant nerve regrowth, while two of the 10 rats that received the cABC alone, and five of the eight that received the Zymosan alone showed minimal regeneration. Untreated animals did not show evidence of regeneration through the dorsal root entry zone.
Furthermore, one-third of the group that received the combination Zymosan and cABC positively responded to electrical stimulation, confirming that the nerves that regrew actually worked. Re-severing the nerve resulted in a loss of the electrical response, which indicated that the response was truly due to the regrowth of injured nerves into the spinal cord.
“The finding that nerves can regrow and actually work after injury,” says Silver, “strongly suggests that the combination of Zymosan and cABC results in robust and functional regeneration of sensory nerves through the dorsal root entry zone following injury.”
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