In vitro frog sural nerve test: a monitor for detecting neurotoxin solutes

In order to evaluate the in vitro neurotoxicity of MM fractions obtained by chromatographic analytical methods from plasma of uremic polyneuropathic patients and urine of healthy subjects, we performed an in vitro test on isolated frog sural nerve. The nerve is incubated in various media and stimulated by a rectangular shock (0.05ms, 1Hz, intensity supramaximal). Action potential are recorded. Results were expressed by the inhibition index Ii = 1 divided by t 1/2 x 100 where t 1/2 is the time (in min) necessary to reduce the 1/2 the spike amplitude. When the nerve is immerged in Ringer solution or plasma of healthy subjects Ii congruent to 0. Among MM fractions obtained by gel chromatography Sephadex G-15 from uremic polyneuropathic subjects' plasma and healthy subjects' urine, only fraction b gives a positive response (Ii congruent to 0.86 +/- 0.02, n = 4). From the 6 sub-fractions obtained by anion exchange chromatography DEAE Sephadex A-25 of fraction b, only sub-fraction b4-2 exhibits a reduction of the spike amplitude, Ii = 0.54 when concentration b4-2 is 6 mg/L, Ii = 1.30 when b4-2 plasma concentration is 14 mg/L. These values increase with the severity of neurologic symptoms. A positive correlation was found between Ii and various b4-2 concentrations of standard solutions and plasma ultrafiltrate (y = 0.089x + 0.046, r = 0.890, n = 13). In vitro sural nerve test demonstrated its sensitivity as a tool for monitoring analytical and preparative procedures developed for isolation of neurotoxic solutes.