Determination of the essential number of motoneurons required to produce functionally useful hind limb locomotion

Avulsion injury of one or more spinal ventral roots induces a critical loss of motoneurons, followed by irreversible locomotor function impairment ranging from inadequate limb movement to complete paralysis of the limb. Recent surgical techniques facilitate improvement of limb function, but it remains to be determined exactly how many motoneurons are needed to survive and grow new axons to achieve sufficient muscle reinnervation. The aim of this study was to determine the minimum motoneuron quantity required to reinnervate the denervated skeletal muscles of the limb and produce a functionally satisfactory locomotor pattern. Since none of the commercially available methods and equipment were able to provide a quantifiable and in-depth analysis of the motor pattern of the entire hind limb, we have developed and applied a sensitive movement recording and analyzing system in order to determine the threshold of satisfactory functional reinnervation; we combined video-based footprint analysis and hind limb motion analysis to achieve a new and reliable assessment. Sprague-Dawley rats underwent a lumbar 4-5 ventral root avulsion, and their L4 ventral roots were subsequently reimplanted. The animals received different doses of riluzole treatment in order to rescue incremental numbers of the damaged motoneuron pool. We were able to assess one rear-view and six lateral parameters of the hind limb movement pattern by measuring specific joint angles, footprint, and gait parameters in single video frames. Four months after the operation, we performed Fast Blue retrograde tracing to label and count the reinnervating motoneurons. We then compared the numbers of reinnervating motoneurons and the functional improvement. Our results confirmed a strong relationship between functional restoration of the original movement pattern and morphological reinnervation; approximately 30% of the original motor pool was able to produce a useful locomotor pattern. We believe that our knowledge of the minimal motoneuron numbers required to reinnervate target muscles may help plan the segmental redistribution of the motoneuron pools for reinnervation surgeries.