by Janis Kelly July 30, 2009

Brilliant Blue G (BBG), a dye used to visualize transparent tissues in eye surgery and a cousin to the food additive that makes Gatorade blue, is also a candidate for preventing paralysis after spinal-cord injury (SCI).

Intravenous (IV) injections of BBG dramatically reduced secondary damage in rats with SCI and had the rats walking within days, according to data reported in the July 28 issue of the Proceedings of the National Academy of Sciences.

BBG was given by systemic injection, unlike most other neuroprotectants, which require injection into the damaged spinal cord.

Blue Dye Blocks the Spinal-Cord "Death Receptor"
Senior author Maiken Nedergaard, MD, DMSci, professor of neurosurgery and director of the Center for Translational Neuromedicine at the University of Rochester Medical Center, in New York, told Medscape Neurology that her research team decided to test BBG as part of ongoing work on the role of excess ATP in SCI. They wanted a compound that would block the ATP-sensitive P2X7 "death receptor" and could be given systemically. A search of chemical structures quickly turned up BBG, which is already approved for use in eye surgery.

Dr. Nedergaard's team had earlier discovered that SCI is quickly followed by a flood of ATP to the damaged area, at levels hundreds of times higher than normal. The ATP excess overstimulates motor neurons and causes them to die from metabolic stress, a process mediated by the P2X7 "death receptor."

Blocking P2X7 might stop the motor-neuron death spiral and prevent the spread of spinal-cord damage that occurs in the hours after injury and often results in paralysis in patients who had been able to move immediately after injury. The researchers achieved proof-of-principle for this approach by injecting oxidized ATP into the spinal cord, but this would not be a clinically useful technique.

"Because BBG is so similar to this commonly used blue food dye, we felt that if it had the same potency in stopping the secondary injury as oxidized ATP, but with none of its side effects, then it might be great potential treatment for cord injury," Dr. Nedergaard said.

BBG Rats Up and Moving After SCI
The researchers tested this idea by giving rats intravenous injections of BBG 15 minutes after weight-drop thoracic spinal-cord injuries, 2 doses (10 or 50 mg/kg/day) per day immediately after injury and for 3 consecutive days. Control rats were injected with vehicle. Dr. Nedergaard told Medscape Neurology that although the rats were given BBG within 15 minutes after injury in this study, it appears that treatment can be delayed up to 6 hours and still have some effect.
BBG significantly reduced secondary injury in spinal-cord–injured rats, which improved to the point of being able to walk, although with a limp. Rats that had not received the BBG solution never regained the ability to walk.

BBG also significantly reduced microglial activation and reactive gliosis in the injured spinal cord, demonstrated by a reduction in the number of CD68+ cells in the peritraumatic area. "These data indicate that BBG clearly reduced both astrocytosis and microglial invasion of the peritraumatic zone," the authors wrote. It also reduced neutrophil infiltration into the damaged area.

Turning Their Pink Eyes Blue
"Many of the P2-receptor antagonists are blue," Dr. Nedergaard said. The result is that BBG not only stops the cascade of molecular events that cause secondary damage to the spinal cord in the hours following a spinal-cord injury but also turns the albino lab rats' normally pink eyes, paws, ears, and noses blue.

The color change was, however, the only side effect seen with the experimental treatment. BBG, a known P2X7R antagonist, is both structurally and functionally equivalent to the commonly used FD&C blue dye #1. More than 1 million pounds of this dye, approved by the Food and Drug Administration as a food additive in 1982, are consumed yearly in the US; each day, the average American ingests 16 mg of FD&C blue dye #1.

"While we achieved great results when oxidized ATP was injected directly into the spinal cord, this method would not be practical for use with spinal-cord–injured patients," said Dr. Nedergaard. "First, no one wants to put a needle into a spinal cord that has just been severely injured, so we knew we needed to find another way to quickly deliver an agent that would stop ATP from killing healthy motor neurons. Second, the compound we initially used, oxidized ATP, cannot be injected into the bloodstream because of its dangerous side effects."

One Small Step for SCI Repair?
Edward D. Hall, PhD, director of the Spinal Cord and Brain Injury Research Center at the University of Kentucky Medical Center, in Lexington, who was asked by Medscape Neurology to comment on the study, said, "The work in this manuscript, which comes from an excellent laboratory, appears to be very well done. However, they leave a lot that remains to be done before the Brilliant Blue dye would be ready for clinical trials."

Specifically, Dr. Hall said that researchers need a more complete dose-response analysis to establish the optimal dose for acute administration, the optimal treatment regimen, and the therapeutic window in animal studies before heading to clinical trials.

"We are going to have to ultimately look at combination drug therapies that target different secondary-injury mechanisms. This P2X7 approach could theoretically be added on to high-dose methylprednisolone therapy, which my laboratory pioneered and which remains the controversial unofficial standard for acute SCI. However, research is full of surprises, and the combination therapy would need to be carefully vetted in animal studies prior to any human trial," said Dr. Hall.

The study was supported by the NY State Spinal Cord Injury Program, the Miriam and Sheldon Adelson Medical Research Foundation, and the National Institutes of Health. The authors report no conflict of interest.