Implantation of cultured sensory neurons and schwann cells into lesioned neonatal rat spinal cord. I. Methods for preparing implants from dissociated cells

Authors

  • Keith R. Kuhlengel,

    1. Department of Neurological Surgery, Washington University School of Medicine, St. Louis, Missouri 63110
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  • Dr. Mary Bartlett Bunge,

    Corresponding author
    1. Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri 63110
    Current affiliation:
    1. The Miami Project, University of Miami, School of Medicine, 1600 N. W. 10th Ave., R-48, Miami, FL 33136
    • The Miami Project, University of Miami, School of Medicine, 1600 N. W. 10th Ave., R-48, Miami, FL 33136
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  • Richard P. Bunge

    1. Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri 63110
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Abstract

Our goal was to devise methods of implanting defined populations of the cellular constituents of peripheral nerve into regions of spinal cord injury. This objective derived from the knowledge that the cellular environment of peripheral nerve is known to be supportive of axon regeneration from both central and peripheral neurons. Two of the constituents of the peripheral nerve environment known to influence axonal growth are the Schwann cell and extracellular matrix (particularly basal lamina), both of which can be obtained in culture. We describe here large-scale methods of establishing purified populations of rat sensory neurons to which purified populations of Schwann cells were added. These essentially monolayer preparations were then scrolled and cut into lengths of proper shape and size to provide implants for sites of spinal cord injury in newborn rats. We also describe methods enabling the addition of leptomeningeal components to the implants; this addition contributes a proliferating population of vascular endothelial cells (identified by immunostaining) to the otherwise vasculature-free neuron/Schwann cell implant. Light and electron microscopic observations were made to characterize the implants. When the implant was ready for use, it contained Schwann cells that were differentiated, i. e., had begun to ensheathe axons and form basal lamina. The use of a medium containing human plasma to foster endothelial cell growth led to increased neurite fasciculation and Schwann cell migratory activity in the outgrowth, particularly when the neurons and Schwann cells were cultured on leptomeninges. The second paper in this series reports the deportment of these implants and their influence on corticospinal tract growth after placement into regions of dorsal column injury in neonatal rats (Kuhlengel et al., J. Comp. Neurol 293:74-91, 1990).

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