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

Abstract

Abstract: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 4-28 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.

Effect of conduit repair on aberrant motor axon growth within the nerve graft in rats

“…However, the error was rather within the permissible range considering that the percentage was about 50% in a previous report with muscle injection technique. 27 The percentage in group TUBG (60.8% 6 5.1%) demonstrated no significant difference from that in group FG (57.8% 6 4.1%), whereas it significantly differed from that in group WG (45.5% 6 2.7%). This result is entirely consistent with the % CP nerve innervation of the TA muscle as measured by CMAPs (Fig.…”

“…While there was a minimal re-uptake by the CP motoneurons per section, the number was negligible as compared to the total number of primarily labeled neurons, consistent with a previous report. 27 In all groups, motoneurons labeled with either TB or FR, as well as TB-FR double labeled motoneurons were identified in the ventral horn of the spinal cord. In group CNT, most neurons were double labeled with a clear topographic prevalence, whereas in the other groups, the three types of neurons were intermingled in varying degrees (Fig.…”

“…In all rats from each group, the CP motor pool was retrogradely labeled 1 week prior to and 13 weeks following injury. One week prior to injury, the CP nerve of the anesthetized rats was isolated and 2 ll of a 2% aqueous solution of True Blue (TB, Sigma, USA), a longterm retrograde fluorescent tracer, 27 was injected into the CP nerve using a 26-gauge Hamilton syringe. Care was taken to avoid leakage of the dye into the surrounding tissue.…”

“…Care was taken to avoid leakage of the dye into the surrounding tissue. Thirteen weeks after surgery, the CP nerve of the anesthetized rats was re-exposed, and 2 ll of a 2% aqueous solution of Fluororuby (FR, Molecular Probes, USA), a short-term neural tracer, 27 was injected into the nerve distal to the repair site in the same manner. After 1 week of survival, the rats were transcardially perfused with 4% paraformaldehyde.…”

Traçadores: o uso de agentes químicos para estudos hidrológicos, ambientais, petroquímicos e biológicos

“…41 Os traçadores fluorescentes TB, FG, FE e FR são utilizados em ratos para estudar as conexões entre diferentes grupos de neurônios, bem como sua maturação e plasticidade, os neurônios que regeneram axônios, 39 e a projeção moto-neurônica do músculo. 40 A eosina (Figura 1) tem várias aplicações descritas na literatura, contudo destacam-se os exemplos recentes de seu uso como traçador: no estudo in situ dos parâmetros de um reservatório de água, tais como tamanho, porosidade, fluxo de água, transporte de contaminantes, e a influência da heterogeneidade do reservatório no fluxo de água; 42 Brouyere e colaboradores 43 estudaram, em águas subterrâneas, os mecanismos de migração de solutos e a matriz das rochas em um aquífero na Bélgica.…”