The extraction and assay of substance P in the rat sciatic nerve.

Published data for substance P (SP) content of sciatic nerves in control rats vary greatly. This study sought possible reasons for this variability by examining the influence of homogenisation procedures, freezing and selection of left/right nerve or proximal/distal segments. Substance P-like immunoreactivity (SP-LI) content (pg/mg nerve protein +/- SD) was significantly greater in sciatic nerves which had been homogenised using motor-powered equipment (615.4 +/- 146.3) as opposed to a hand-held pestle (445.4 +/- 111.8). Our second investigation revealed that freshly homogenised nerve tissue yielded greater SP-LI (508.8 +/- 88.7) than either tissue snap frozen in liquid nitrogen (307.6 +/- 77.9), snap-frozen and stored at -70 degrees C for 7 days (331.8 +/- 53.5), or tissue allowed to remain in the cadaver for 1 h and subsequently dissected and homogenised immediately (412.6 +/- 105.8). These data also show that storage at -70 degrees C imposes no further losses on those caused by freezing and extraction of frozen tissue. Two further studies indicated no variation between left and right sciatic nerves nor any proximal-distal gradients. Hence, this study illuminated the need for samples to be homogenised using motor-powered equipment immediately upon dissection, followed by SP extraction, for complete avoidance of losses of SP-LI.