 The top view shows a spinal cord that is cut in two pieces. The middle view shows one severed nerve fiber of the thousands within the spinal cord. In a recent study, Purdue researchers applied the polymer PEG to the fibers, fusing the segments together and restoring electrical nerve impulses through the cell. (Graphic courtesy Riyi Shi, Purdue School of Veterinary Medicine.) |
The scientists conducted their experiments on isolated spinal cords removed from adult guinea pigs. Researchers fused the cut nerve fibers using a polymer called PEG. Electrical impulses were restored in all of the guinea pig cords used in the study. The repaired spinal cords transmitted between 5 percent and 58 percent of pre-cut impulses.
Significant numbers, but not all, of the nerve fibers within the cut spinal cords were reconnected. Researchers demonstrated this by passing special dyes through the repaired nerve fibers.
"If you have even 5 percent of the nerve fibers carrying nerve impulses, you'll get significantly more than 5 percent back in terms of restored behavior," says Richard B. Borgens, professor of developmental anatomy at Purdue. He and his colleague, assistant professor Dr. Riyi Shi, both of Purdue's Center for Paralysis Research in the School of Veterinary Medicine, will report their findings Thursday (11/12) in Long Beach, Calif., at the 18th annual meeting of the Society for Physical Regulation in Biology and Medicine. Borgens also has submitted a paper on the research to the Journal of Neurotrauma.
"This technique may be a revolutionary new way of dealing with injuries to the nervous system," Borgens says. "It's too soon to know whether it would help patients with old injuries, but it is likely to be useful in treating recent injuries."
Shi and Borgens now are testing the procedure in live animals. They plan to conduct clinical trials in natural cases of paraplegia in dogs early in 1999, but human clinical trials are at least two years away.
The isolated spinal cords remain viable for about 36 hours after removal. Borgens says the results of studies in live animals will shed more light on how permanent the new technique might be.
Borgens and Shi applied polyethylene glycol, or PEG -- a nontoxic, water-soluble polymer used in medicine and cosmetics -- across the region of the guinea pig's spinal cord that had been severed but gently pressed back together. PEG was applied for two minutes, then removed. The polymer "fused" the membranes of a significant number of nerve cells together, making them continuous once again.
Using an apparatus and procedure that Shi designed, the researchers then applied a small electrical current to one end of the cord to stimulate nerve impulses to travel towards the other end. Between five and 15 minutes after the PEG application, nerve fibers were repaired, allowing some impulses to reach the other end of the severed cord in 100 percent of the cases.
"The technique is called fusion technology," Borgens explains. He compares the procedure to repairing a garden hose -- the rubber is the cell membrane and the water inside is the salty material, called cytoplasm, found inside all cells. "If we cut the hose and just hold the two ends tightly together, they're not going to reconnect or function as a hose. But if we add this special molecule, PEG, the 'rubber' melts a tiny bit on each side and literally fuses the 'hose' back together."
Borgens says the technique also can repair crushed nerve cells that develop holes in their membranes.
"In most spinal cord injuries in animals and in people, the spinal cord is not completely severed, as we have done in these first experiments," he says. "The cord is more likely to be crushed, and the nerve fibers develop holes in their membranes, which ultimately leads to separation of the nerve fiber within 24 to 72 hours."
Nerve cells consist of a cell body and trailing tail called an axon, which can extend from the spinal cord to the muscles in the leg, for example.
In mammals, if nerve fibers outside the brain and spinal cord are damaged, they can regenerate and may eventually reach their original target muscle to restore some function. Spinal cord and brain nerve axons, however, do not regenerate in mammals, and once separated from the cell body, they die. The result is paralysis.
Borgens and his colleagues at the Center for Paralysis Research have done extensive work in producing other methods to treat naturally paralyzed dogs with spinal cord injuries, and some methods have moved to tests in human spinal cord injuries.
Borgens' research is supported in part by the Department of Defense and the National Science Foundation.
Source: Richard Borgens, 765-494-7600; e-mail, cpr@vet.purdue.edu
Writer: Amanda Siegfried, 765-494-4709; e-mail, amanda_siegfried@uns.purdue.edu
Color graphic, electronic transmission, and Web and ftp download available. Graphic ID: Borgens.PEG
ABSTRACT
Polyethylene Glycol Repairs Mammalian Spinal Cord Axons After Mechanical Injury
Riyi Shi and Richard B. Borgens
Center for Paralysis Research, Department of Basic Medical Sciences
Purdue University
W. Lafayette, IN 4790
The most significant structural damage following mechanical injury to the spinal cord is caused by the disruption of large numbers of nerve fibers which interrupts sensory and motor function. At the cellular level, the loss of spinal cord function is mainly due to damage to axonal membranes which can partially or completely sever the axon. Such insults to the axon cause the dissolution of the distal segment and sometimes the atrophy of the target tissue. In order to survive and functionally recover from a mechanical injury, axons must first seal the breach in the membrane to avoid secondary deterioration leading to axotomy and cell death. Here we describe an in vitro technique using polyethylene glycol (PEG) which can functionally reconnect the two segments of transected adult guinea pig spinal axons within minutes of the injury. Strips of isolated spinal cord white matter and a double sucrose gap chamber were used for electric recording. The cord strips were cut, using a microknife, in the chamber after Compound Action Potential (CAPs) recording was stabilized. PEG, in a solution of 50% (w/w in water), was applied directly onto the lesion site for 2 minutes through a glass pipette immediately after transection. The initial recovery of CAPs was evident in 5 to 10 min. following the application of PEG. Successful fusion was documented by the restored conduction of CAPs and the diffusion of two intracellular fluorescent markers through fused axons.
In addition, PEG can also significantly improve the physiological recovery of spinal cord axons following severe compression injury. A standardized compression was carried out in the recording chamber with the use of a rod attached to a motorized micromanipulator. The compression rod was advanced at a speed of 24 llm/s. CAPs were monitored during the compression which was stopped when CAPs disappeared. A recovery of conduction (reappearance of CAPs) was evident within 60 minutes of injury. The average amplitude of recovering CAPs was significantly different between the control and PEG-treated group (4% vs. 19%,10 in each group). Examination of the relationship between stimulus and response amplitude in control and PEG-treated groups indicated that PEG equally repairs axons of different stimulus thresholds. Furthermore, 4aminopyridine, a potassium channel blocker, when used in combination with PEG, can produce an additional 70% percent increase in CAP amplitude following injury. These techniques may lead to a novel acute treatment for central nervous system trauma.
NOTE TO JOURNALISTS: A color graphic depicting the repair of spinal cord nerves is available. It is called Borgens.PEG. Richard Borgens can be reached Tuesday through Saturday (11/10-14) at the Hotel Queen Mary in Long Beach, Calif., at 562-435-3511. Journalists wishing to cover Borgens' presentation to the Society for Physical Regulation in Biology and Medicine should contact conference organizer Dr. Fred Nelson, 619-278-8300. November 12, 1998
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