Femke Mathot

Chapter 1 20 Evaluation methods Many different evaluation techniques have been described to quantify the level of nerve regeneration. Histology enables us to analyze the inside of the nerve. Stained ultrathin sections of nerves reveal information about the number of axons, the axon area, the myelinated fiber area and the total nerve area. The N-ratio, which is composed of the total myelinated fiber area divided by the total nerve area, is an often used measure to express the amount of sprouting axons. 71 Nerve conduction capacities can be quantified by electrophysiology, compound muscle action potentials (CMAP). An artificial action potential is evoked proximal to the nerve graft. The amplitude of that signal is measured in the muscle distal to the nerve graft. The bigger the amplitude of the distally measured signal, the better the nerve regeneration process has occurred. 65, 72 Although one could argue if histology and electrophysiology measurements are direct or indirect measures of nerve regeneration, muscle function represents the most relevant outcome factor for motion. Isometric tetanic force has been described to most reliably represent muscle function and has been validated in rodent models. A stimulus is introduced to the nerve graft, and the resulting muscle contraction force is quantified. 73 As denervation of a muscle will result in muscle atrophy and shrinking of the muscle, the size and mass of a muscle after nerve reconstruction also provide information on successful reinnervation. Although the muscle mass is easy to obtain, it does require the sacrifice of the animal. Ultrasound measurements of the cross-sectional area of (re)innervated muscles can be obtained non-invasively and can therefore be used to study nerve regeneration over time. 74, 75 GENERAL AIMS AND OUTLINE OF THIS THESIS The ideal future perspective is an off-the-shelf nerve substitute that can be used in peripheral nerve injuries, resulting in functional outcomes equal to those of autograft nerves. The specific aim of this thesis was to examine whether the in vivo outcomes of nerve allografts and conduits can be improved by the addition of adipose derived Mesenchymal Stem Cells, either undifferentiated or differentiated into Schwann cell-like cells. If so, MSC seeding might have clinical potential in peripheral nerve injury cases with shortage or inadequate autologous nerve graft material. As part of this objective we aimed to get more insight through which mechanisms these cells potentially influence nerve regeneration. We first intended to examine in vitro if and how differentiated MSCs could be dynamically seeded on nerve grafts and then we aimed to study the gene expression profiles of the seeded MSCs. Subsequently, we set the goal to analyze the in vivo effects of MSCs regarding vascularity and functionality. This hopefully would give us the opportunity to be able to explain if and how MSCs affect nerve regeneration. In order to evaluate if the described techniques can be applicable to a clinical setting, we eventually aimed to dynamically