Detecting necrotic neurons with fluoro-jade stain

Fluoro-jade, a novel stain for detection of neuropathic lesions by fluoresc ence microscopy, was validated on the models of toxic neuropathy induced wi th 3-acetylpyridine (3-AP) or with acrylamide (ACR). Groups of male and fem ale albino rats of Wistar strain were either exposed to a single administra tion of 80 mg/kg i.p. 3-AP followed 5 hours later by 300 mg/kg of nicotinam ide i.p. and examined at days 3 and 15, or to 15 daily doses of 30 mg/kg p. o. ACR and examined at day 15. Following in-life behavioral observations an d measurements, the rats were fixed by perfusion with formalin. Additional animals treated with same dose of 3-AP and nicotinamide were submitted to p urposeful autolysis for 4 or 16 hours before immersion fixation with formal in on test day 3. In-life observations showed in 3-AP-treated animals signs of severe general toxicity, sensorimotor dysfunction and decreased motor a ctivity starting shortly after the treatment and persisting throughout the observation period. ACR-treated rats started to develop abnormal gait on te st day 8 and by day 15 developed reduced grip strength, increased landing f oot-splay and decreased motor activity. Fluoro-jade, applied to paraffin se ctions of the nervous system, detected selectively and sensitively the necr otic neurons in the brain, especially those in the inferior olivary nucleus of animals treated with 3-AP, at test day 3, as well as the necrotic Purki nje cells in the cerebellum of ACR-treated animals at test day 15. Chromato lytic neurons in the dorsal root ganglia of ACR-treated animals did not sta in positively, indicating that this kind of reversible neuronal remodeling is not detectable using fluoro-jade. Necrotic neurons were still stained by fluoro-jade after 4 hour autolysis, but following 16 hour autolysis the re sults became false negative. There was no false positive fluorescence in fr esh or autolytic tissues, except that emitted by red blood cells in unperfu sed specimens. The study confirmed the validity of fluoro-jade as a stain s uitable for detecting necrotic neurons in toxicological safety studies.