Inhibitor Shows Potential To Protect Infant Brain

"To our knowledge, this is the first demonstration that delayed treatment with a caspase inhibitor, even when given systemically, can be neuroprotective in an injury model," says David M. Holtzman, M.D., chief author and assistant professor of neurology and molecular biology & pharmacology. "If these compounds prove not to be toxic and are able to come into clinical use, it appears there may be a window of at least several hours when one could administer them."

The findings are published in the May 1 issue of Journal of Clinical Investigation. Former postdoctoral fellow Yu Cheng, M.D., Ph.D., is first author.

An interruption in the supply of blood and oxygen to the brain in the womb or during or after birth is the largest contributor to brain degeneration in children. Life-time consequences include mental retardation, seizures and cerebral palsy, which affects 500,000 Americans.

Holtzman mimics the condition by interrupting the blood supply to one side of a newborn rat's brain and briefly lowering the oxygen content of the air. This procedure kills neurons, which results in loss of brain tissue. Holtzman and colleagues showed, however, that while some neurons swell up and burst, as happens rapidly in adults with head injury or stroke, many neurons slowly shrink through a process resembling cell suicide.

A series of genetically programmed steps lead to cell suicide or apoptosis. The rat brain cells appeared to die this way because their demise was delayed, beginning six hours after the procedure and peaking between 18 and 24 hours. Just before they died, the cells activated their caspases and chopped up their DNA, two events that typify apoptosis. Protein-cutting caspases are the executioners that finalize a cell's decision to self-destruct. If they're prevented from doing their job, cells can't commit suicide.

"A major question in the field is whether it would be clinically useful to use caspase inhibitors to prevent apoptosis," Holtzman says. "So we tested the potential of a compound called BAF."

BAF - boc-aspartyl(OMe)-fluoromethylketone - is a modified amino acid produced by Enzyme Systems Products in Livermore, Calif. Eugene M. Johnson Jr., Ph.D., the Norman J. Stupp Professor of Neurology and professor of molecular biology & pharmacology, previously had discovered that it saves the lives of cultured sympathetic neurons under apoptotic conditions.

Holtzman's team injected BAF into the brains of some rats right before the damaging procedure and into others three hours later. After a week, the researchers looked at brain slices.

The rats that received no BAF had lost extensive amounts of tissue in the affected hemisphere. The cortex, hippocampus and striatum had shrunk, and the fluid-filled ventricles had expanded. But the researchers saw quite a different picture when they analyzed the BAF-treated rats. "On average, the control animals lost about 50 percent of the tissue in these regions, whereas the BAF-treated animals lost only about 20 percent," Holtzman says.

Administering BAF three hours after the procedure was just as effective as giving the drug ahead of time. BAF also was protective when injected systemically instead of into the brain, the researchers discovered. Systemically treated rats lost 66 percent less tissue from the hippocampus, a site of learning and memory, than control rats. And they lost 53 percent less tissue from the striatum and 33 percent less from the cortex, the brain's wrinkled top layer. The number of neurons that vanished from these regions showed a corresponding decline, suggesting that BAF really had protected neuronal tissue. Moreover, the activity of an enzyme called caspase-3 was lower than normal in the BAF-treated rats, whereas it was three-to-five times higher in the control animals.

"These results suggest that BAF protects brain tissue by inhibiting caspases and thus preventing apoptosis," Holtzman says. "The difference between the BAF-treated animals and the control animals was so great that it's hard to imagine that treatment with BAF or similar compounds wouldn't be worth exploring as potential treatments in humans. That issue obviously needs to be investigated further, however."

Cheng Y, Deshmukh M, D'Costa A, Demaro JA, Gidday JM, Shah A, Sun Y, Jacquin MF, Johnson EM, Holtzman DM. Caspase inhibitor affords neuroprotection with delayed administration in a rat model of neonatal hypoxic-ischemic brain injury. Journal of Clinical Investigation, 101(9), May 1998.

The study was funded by the National Institute of Nervous Disorders and Stroke, the National Institute on Aging, the Spinal Cord Research Foundation and a Paul Beeson Physician Faculty Scholar Award from the American Federation for Aging Research.