Mammalian endoneurial fluid: Collection and protein analysis from normal and crushed nerves

Abstract

An elution procedure was developed for the extraction of endoneurial fluid from desheathed, rat sciatic nerves. The endoneurial fluid elutions (EFE) from normal and crushed nerves were evaluated for extracellular proteins by sodium dodecyl sulphate-pore gradient electrophoresis (SDS-PGE) after silver stain and after immune overlay following electrophoretic transfer to nitrocellulose using antisera to albumin (ALB), neurons specific enolase (NSE), and the major myelin glycoprotein (P 0). Afer removal of the epineurium/ perineurium, the EFE protein accounted for 3.6% (34–37 μg) of the total endoneurial protein which did not change with intra-arterial perfusion. The endoneurium was further fractionated to obtain an aqueous supernatant (S-I), and SDS-solubilized supernatant (S-II), and an SDS-insoluble fraction. SDS-PGE analysis revealed that the EFE has a distinctive protein composition relative to the other endoneurial fractions. A predominant band with M r of 64,000; 4 major bands with M r of 86,200, 61,000, 54,500 and 46,900; and several other minor bands were observed. The predominant band at 64,600 co-migrates with ALB and was demonstrated by immune overlay to be ALB, which was also the major protein in the S-I fraction. The uniqueness of the EFE was established by the absence of NSE, an enzyme marker for the cytoplasmic fraction of axons which was found to be present only in S-I (subunit M r= 49,600), the absence of P 0, and the distinctive protein profiles as determined by silver stain. Crush injury resulted in a progressive increase in the amount of protein found in the EFE as well as the S-I with a corresponding decrease in S-II protein as a function of time after crush (1, 2, 5, 10, 24, 48, 96, 168 h). Dramatic alterations in the protein profile were demonstrated in the EFE from crushed nerves after SDS-PGE indicating substantial changes in the endoneurium as a result of the axonal degeneration, demyelination, and breakdown of the blood-nerve barrier. Alterations in EFE proteins after crush were also observed by lectin overlay experiments after SDS-PGE. Analysis of EFE collected after crush for NSE and P 0 were negative. It is concluded that the distinctive pattern of EFE proteins identified from normal nerve will permit their further characterization as a separate endoneurial protein compartment. Such an elution procedure for collection of endoneurial fluid can readily be adapted to human sural nerve biopsies from patients with peripheral neuropathy for characterization of the appearance of new proteins which may be used as markers of disease activity.