A method for in vitro enzymatic dissociation of nerve roots and peripheral nerves from adult mammals.
Preparations yielding a high percentage of undamaged axons from fresh peripheral nerve or nerve root were made using an enzymatic dissociation regimen. The nerve was placed in a temperature-controlled chamber mounted over an inverted phase-contrast microscope. An oxygenated solution (Brimijoins) or modified Hank's solution was pumped through the chamber, first in a calcium-free form and then containing enzymes. The enzymes for dissociation were collagenase and trypsin, alternated. Enzymatic dissociation of the epineurium, perineurium and extracellular matrix was achieved. We supplemented the gentle agitation of a 10-roller peristaltic pump by periodically raising and lowering the fluid level in the chamber to provide a controlled mechanical agitation that promoted dissociation. A large percentage of the axons can be dissociated from the nerve, varying from approximately one-quarter to occasional complete dissociation. Action potentials were still conducted through dissociated axons, and axon transport was also still present, as documented by direct visualization using an AVEC-DIC type of microscope system. The axons had a better morphological appearance and displayed better transport than comparison preparations prepared by the usual mechanical teasing method, in our hands. The enzymatic method allows study of axons in an adult or developing mammal with regard to their electrical conduction and axon transport mechanisms. It should help to avoid a selection process for more hardy axons which may be imposed by traditional mechanical teasing methods. Mechanical stress was observed to cause widened Schmidt-Lanterman clefts, widened nodes, myelin bubbles, and other abnormal morphology as evidence of damage.
Levy
W J
WJ
Spagnolia
T
T
Rumpf
R
R
York
D H
DH
D000818:Animals; D001370:Axonal Transport; D002469:Cell Separation / Q000379:methods*; D066298:In Vitro Techniques; D003012:Microbial Collagenase; D009431:Neural Conduction; D010525:Peripheral Nerves / Q000166:cytology* / Q000502:physiology; D051381:Rats; D011919:Rats, Inbred Strains; D013126:Spinal Nerve Roots / Q000166:cytology; D014357:Trypsin
D014357:Trypsin; D003012:Microbial Collagenase