Supravital diffusion of fluorescent Evans blue in brain and spinal cord tissue

The influence of the fixation method on the distribution of fluorescent Evans blue (EB) was studied in the cervical grey matter of rats treated with 6-aminonicotinamide (6-AN). 6-AN leads to selective injury to the neuroglial cells and blood brain barrier without primary neuronal damage. In this model hyperchromasia and selective fluorescence of neurons were closely related but only found in tissue fixed by immersion, indicating a supravital shift of EB to shrinking neurons. Adequate perfusion fixation with Bouin solution prevents this artifactitious neuronal staining and demonstrates that EB is primarily bound by the vessel wall and the perivascular neuropil. The supravital diffusion of EB in the CNS and its dependence from the fixation method represents relevant methodological factors in morphological studies on the blood brain barrier.