Fluorescence measurement of lesion-induced fiber growth. I. A microfluorometric technique to measure density and intensity of varicosities and fibers.

A microfluorometric (MF) technique was developed and validated as a first step in quantifying the growth of sympathohippocampal fibers. This MF technique measures independent components of areal density and fiber intensity. Areal density refers to a basic stereological measure of percentage surface or volume of fluorescent fibers. Fiber intensity refers to the amount of fluorescence (normalized for fading) emitted by varicosities or fibers at a specific wavelength. The first phase of this research was undertaken to identify the criteria yielding the best correlations with the results of classical stereological methods. The second phase then demonstrated that the MF procedure was insensitive to shifts in the use of different criteria. Five areas of the hippocampus, and the pineal and habenula, were measured in 55 animals whose survival times after septal lesions varied widely. With 309 areas finally measured, the strongest intercorrelations separated intensity from density variables. Error variance contributed little to the final solution. We concluded that this MF method was a simple and specific way to measure density and intensity of fluorescent fibers and varicosities. Slight variations in equipment characteristics or in the use of criteria across several laboratories should not affect the results obtained with this method.