Controlled Nerve Growth Factor Aids Brain Cell Transplant

The system, first tested with rat fetal brain cells and nerve growth factor (NGF) implanted in the brains of adult rats, has yet to be demonstrated in humans. But the technique to create microenvironments for tissue growth is said to be adaptable to a variety of other transplantation needs, including the treatment of neurodegenerative disease and spinal cord injuries.

The achievement is reported in the latest issue (October 2001) of the journal Nature Biotechnology by Melissa J. Mahoney and W. Mark Saltzman, the BP Amoco/W.F. Fuller Professor of Chemical Engineering at Cornell. Mahoney, a graduate student at Cornell's Department of Neurobiology and Behavior at the time of the work, is a postdoctoral fellow in neurobiology at Duke University.

"We have been trying to overcome some of the barriers to successful nerve cell transplantation and regeneration," Saltzman explains. "After the fetal stage of life, we cannot -- normally -- produce new cells in the central nervous system, including the brain, to replace nerve cells that are lost to injury, disease or aging processes."

The body has a variety of natural ways to resist nerve cell regeneration or transplantation. For example, the adult brain produces molecules that inhibit cell migration and the growth of axons (the part of the nerve cell that carries the nerve impulse) that could connect nerve cells, while scars that form on the glial (or connecting) cells after brain injuries also inhibit the elongation of axons.

Mahoney says: "Neurotrophins, such as nerve growth factor, can overcome some of these inhibitions to nerve cell differentiation and circuitization. But we can't just flood the body with neurotrophins because of the adverse side effects. These are potent molecules that influence other cell functions, besides survival and axonal growth, and they can promote inappropriate growth among other tissue. We needed to create a local delivery system to ensure activity within the microenvironment of the brain, where the transplanted tissue should thrive, without side effects."

Mahoney and Saltzman expanded on a technique they previously developed to deliver NGF to the brains of patients with neurodegenerative diseases, such as Alzheimer's. They filled the spaces of porous co-polymer microspheres, ranging in size from 0.5 to 7 microns (1 micron is 1 millionth of a meter) with NGF. In previous work, Saltzman and his students had shown that versatile, long-acting NFG delivery systems could be produced with biocompatible polymers. Working with brain cells, their plan was for some NGF to be released almost immediately, when the cells were implanted, while remaining amounts were to be released over a two-week period.

The bioengineers then assembled the NGF-filled microspheres, including some with fluorescent markers, together with rat fetal brain cells, into spherical clusters about 170 microns in diameter. The spheres of neo-tissue contained both neurons as well as glia, the connecting cells the engineers hoped would grow and attach to others cells in the rat brains. They also created some neo-tissue clusters without the NGF microspheres, as controls to gauge the activity of transplanted cells without the presumed benefits of growth-enhancing microenvironments.

The neo-tissue clusters with NGF microenvironment spheres were injected into the brains of adult rats, while other rats received transplanted neo-tissue without NGF. Over the next four weeks, the researchers periodically tested the rat brains, watching for evidence that NGF would remain in the transplanted neo-tissues and would not spread elsewhere in the brain. They also tested for the production by transplanted cells of cholinacetyl transferase, or ChAT, an enzyme important in the synthesis of the neurotransmitter acetylcholine and a sign that transplanted cells are beginning to grow, connect and function.

Their tests showed that most NGF from the microspheres remained in the regions of transplanted neo-tissue, although low levels were in the brain tissue immediately adjacent to the implant. Neo-tissue that was transplanted with NGF microenvironment spheres produced abundant ChAT, but ChAT production from neo-tissue without the growth-enhancing microenvironment was negligible. This was a clear demonstration, the researchers say, that transplanted brain cells can survive and differentiate when pre-assembled with NGF-released particles.

The Cornell researchers said they can now envision further uses of the microenvironment delivery system with other substances to encourage nerve cell regeneration, including axon-guidance factors and stimulants to axon extension, antibodies against the formation of myelin scars on injured nerves, and molecules to modify the local immune response to transplanted cells. These are all problems that must be overcome in the treatment of neurodegenerative diseases and spinal cord injuries.

Mahoney's and Saltzman's Nature Biotechnology article is titled, "Transplantation of brain cells assembled around a programmable synthetic microenvironment." The work was supported by NASA, including a grant from the agency's Graduate Student Research Program.

Related Web sites:

o Cornell Bioengineering Program: http://biomedeng.cheme.cornell.edu/

o Saltzman laboratory: http://www.cheme.cornell.edu/~saltzman/