THE EXTRACTION AND ASSAY OF SUBSTANCE-P IN THE RAT SCIATIC-NERVE

Published data for substance P (SP) content of sciatic nerves in contr ol rats vary greatly. This study sought possible reasons for this vari ability by examining the influence of homogenisation procedures, freez ing and selection of left/right nerve or proximal/distal segments. Sub stance P-like immunoreactivity (SP-LI) content (pg/mg nerve protein +/ - SD) was significantly greater in sciatic nerves which had been homog enised 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 +/- 7 7.9), snap-frozen and stored at - 70-degrees-C for 7 days (331.8 +/- 5 3.5), or tissue allowed to remain in the cadaver for 1 h and subsequen tly dissected and homogenised immediately (412.6 +/- 105.8). These dat a also show that storage at - 70-degrees-C imposes no further losses o n those caused by freezing and extraction of frozen tissue. Two furthe r 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 immed iately upon dissection, followed by SP extraction, for complete avoida nce of losses of SP-LI.