Cellular changes in chronic nerve injury wins Young Investigator Award

By Elaine Fiedler

The 2006 Kappa Delta Young Investigator Award went to Ranjan Gupta, MD, for his paper on “Schwann Cell Regulation of Chronic Nerve Injury.”

During the last six years, Dr. Gupta and his team—Laura S. Rummler, MS; Tahseen Mozaffar, MD; and Oswald Steward, PhD—have investigated the cellular and molecular changes that occur with chronic nerve compression (CNC) injuries. Using both in-vivo and in-vitro models, his research is helping to define the cellular pathology of these injuries.

“As we continue to gain a better understanding of the altered signaling pathways after CNC injury,” said Dr. Gupta, “we move closer to the eventual direction of this research program — to develop alternative therapeutic regimens, including targeted gene therapy.”

Noting that traditional therapies are often unpredictable and that surgical management alone has limited success , Dr. Gupta hoped a better understanding of the origin and development of CNC injuries would result in better treatments.

Rajan Gupta, MD.

Schwann cell regulation studies

Schwann cells support the peripheral nervous system. They help form the myelin sheath of the neuron’s axon, which insulates it and aids in the conduction of the action potential propogation (nerve conduction velocity). Schwann cells also help clean up debris. Dr. Gupta’s study of Schwanncell regulation reveals that chronic nerve injury stimulates a massive increase in their numbers.

“The initial response in chronic nerve compression injuries is primarily Schwann cell-mediated, which in turn secondarily affects neuronal function,” he said. “Schwann cells undergo significant cellular changes early in the disease process before overt changes in nerve conduction velocity occur.”

When Schwann cells receive signals from the axon, changes occur that enable them to myelinate. For myelination to proceed, Schwann cells must produce large amounts of membrane, and increase myelin-associated proteins. Schwann cells’ proliferation at the site of injury is critical for regeneration.

Dr. Gupta’s initial study and analysis of light microscopy images “revealed that nerve compression injury stimulates a massive increase in Schwann cell numbers,” contradicting previous reports that they decreased. Based on this study, Dr. Gupta hypothesized that chronic compression might trigger Schwann cell proliferation.

After CNC injury, the Schwann cell numbers gradually decrease. To examine apoptosis, Dr. Gupta’s team used four techniques: cell morphology analysis; DAPI staining (nuclear condensation); TUNEL assays (late-stage apoptosis); and immuno-localization of caspase activity (early-stage apoptosis).

“Surprisingly, all of these studies showed that at the same time the dramatic increase in Schwann cell number was occurring, Schwann cells were simultaneously undergoing apoptosis,” he wrote.

Dr. Gupta explored possible mechanisms for Schwann cell proliferation, including Wallerian degeneration, axonal sprouting, and mechanical stimulation of Schwann cells. When his findings showed that Schwann cells undergo proliferation and concurrent apoptosis in CNC injury, he hypothesized that demyelination and remyelination may occur in early stages of CNC injury.

Immunolabeling of teased nerve fibers confirmed this and also showed that the proliferating Schwann cells produce new, thinner myelin. Neuronal or axonal degeneration—such as Wallerian degeneration—had been thought to be the primary mediator of altered Schwann cell function. Yet, photomicrographs at the one-month point of CNC injury failed to reveal evidence of axonal degeneration or muscular atrophy.

Dr. Gupta’s analysis of electron micrographs showed a significant increase in unmyelinated axons at the one-month point, leading him to hypothesize their increase may be due to axonal sprouting. The data indicated that there are axonal sprouts within the injury zone without morphometric evidence of axonal degeneration.

Altered gene expression

Dr. Gupta hoped to define the mechanisms responsible for the changes in neural form and function, including the alteration of gene expression of myelin-associated glycoprotein (MAG), myelin basic protein (MBP) , inducible nitric oxide synthase (iNOS), and vascular endothelial growth factor (VEGF).

His research suggested that Schwann cells function in regulating the maturation, maintenance and viability of axons. They may mediate the pathogenesis of CNC injuries. To understand this process, Dr. Gupta examined how Schwann cells responded to mechanical stimuli.

To test the ability of shear stress to change the phenotype of Schwann cells in vitro, Dr. Gupta used a laminar flow chamber model. After mechanical stimulation of Schwann cells with shear stress, expression of MBP and MAG was positive in 99 percent of experimental and control groups, which indicated that the cells maintained their pro-myelinating phenotype.

MAG is important in the early stages of myelination in the peripheral nervous system. Western blot analysis and RT-PCR data demonstrated a significant down-regulation of MAG after exposure to shear stress. Data from in-vivo models demonstrated that a decrease in MAG production led to a decrease in the diameter of myelinated axons and a localized axonal sprouting.

MBP is essential to the myelin structure, helping develop its thickness and degree of compaction. Western blot analysis and RT-PCR data also demonstrated a down-regulation of MBP proteins after exposure to shear stress and may be responsible for the altered myelin observed with CNC injury.

To explore the role ischemia plays in chronic nerve injuries, Dr. Gupta examined the expression of VEGF, which increases vascular permeabilization. Nerves determine blood vessel organization and Schwann cells are required for VEGF expression in the peripheral nervous system.

Using in situ hybridization and immunohistochemistry, Dr. Gupta’s team showed that Schwann cells produce significant levels of VEGF in the compressed nerve as early as two weeks post-compression. He theorized that the increased numbers of Schwann cells produce increasing amounts of VEGF, which leads to neovascularization and the recruitment of macrophages to the injury site.

His data provides evidence that macrophage response is gradual and progressive in the early stages of chronic nerve injury, unlike the rapid macrophage response of an acute nerve injury such as an axonotomy or crush injury.

The gradual recruitment of macrophages leads to the up-regulation of iNOS, generating increases in nitric oxide. In-situ hybridization and immunohistochemistry data on iNOS expression found an increase at three months after CNC injury.

Dr. Gupta’s studies suggest that “Schwann cells recruit macrophages and participate in the early inflammatory response after injury rather than simply acting as secondary response cells.”

The team is currently developing more modeling systems with neuronal-glial cell co-cultures to study the effects of mechanical stimuli and altered signaling pathways after CNC injury.

Dr. Gupta is an associate professor in the departments of orthopaedic surgery, anatomy & neurobiology and department of biomedical engineering at the University of California, Irvine.

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