A sequential double labeling technique for studying changes in motoneuronal projections to muscle following nerve injury and reinnervation

The purpose of this study was to develop an anatomical technique that could directly demonstrate the motoneuron projections to the muscle both before injury and again following reinnervation. Investigation focused on the identification of a long-term retrograde fluorescent tracer that would label original motoneurons and persist long enough for reinnervating motoneurons to become labeled by a second fluorescent tracer. True Blue (TB) was evaluated as a potential long-term tracer, Fluoro-ruby (FR) and Fluoro-emerald (FE) were tested as potential short-term tracers in 45 adult Sprague-Dawley rats. In the initial phase of the study, TB was injected into the tibialis anterior (TA) muscle in 16 rats and sacrificed 1 week to 6 months later, to study its persistence. During the second stage, a short-term tracer was injected into the TA muscles bilaterally in 15 rats with survival time ranging from 4 to 28 days. Sequential double labeling was subsequently performed using the combination of TB and FR in 14 rats. The number and brightness of TB cells did not change over 6 months time, a period sufficient for complete reinnervation. FR and FE showed maximum labeling of motoneurons at 1 week after tracer application. In the double labeling study, we could easily distinguish double-labeled cells from those labeled only by TB or FR. These results suggest that sequential double labeling of TB and FR is a valuable method for long-term muscle reinnervation studies.