Timm Danker

Expansion and differentiation of neural progenitors derived from the human adult enteric nervous system.

BACKGROUND & AIMS Neural stem and progenitor cells from the enteric nervous system have been proposed for use in cell-based therapies against specific neurogastrointestinal disorders. Recently, enteric neural progenitors were generated from human neonatal and early postnatal (until 5 years after birth) gastrointestinal tract tissues. We investigated the proliferation and differentiation of enteric nervous system progenitors isolated from human adult gastrointestinal tract. METHODS Human enteric spheroids were generated from adult small and large intestine tissues and then expanded and differentiated, depending on the applied cell culture conditions. For implantation studies, spheres were grafted into fetal slice cultures and embryonic aganglionic hindgut explants from mice. Differentiating enteric neural progenitors were characterized by 5-bromo-2-deoxyuridine labeling, in situ hybridization, immunocytochemistry, quantitative real-time polymerase chain reaction, and electrophysiological studies. RESULTS The yield of human neurosphere-like bodies was increased by culture in conditional medium derived from fetal mouse enteric progenitors. We were able to generate proliferating enterospheres from adult human small or large intestine tissues; these enterospheres could be subcultured and maintained for several weeks in vitro. Spheroid-derived cells could be differentiated into a variety of neuronal subtypes and glial cells with characteristics of the enteric nervous system. Experiments involving implantation into organotypic intestinal cultures showed the differentiation capacity of neural progenitors in a 3-dimensional environment. CONCLUSIONS It is feasible to isolate and expand enteric progenitor cells from human adult tissue. These findings offer new strategies for enteric stem cell research and future cell-based therapies.

Dominant-Negative Effects of KCNQ2 Mutations Are Associated with Epileptic Encephalopathy

Mutations in KCNQ2 and KCNQ3, encoding the voltage‐gated potassium channels KV7.2 and KV7.3, are known to cause benign familial neonatal seizures mainly by haploinsufficiency. Here, we set out to determine the disease mechanism of 7 de novo missense KCNQ2 mutations that were recently described in patients with a severe epileptic encephalopathy including pharmacoresistant seizures and pronounced intellectual disability.

An improved electrophysiological method to study peripheral nerve regeneration in rats.

After restitution of motor function the grasping test alone is insufficient to figure out any further differences of axonal nerve regeneration of the median nerve in rats. To avoid this problem we developed a standardized electrophysiologic method for testing median nerve regeneration. Threshold, latency, compound muscle action potentials (CMAP) and velocity of neuromuscular transduction were recorded in 54 rats 20 weeks post-operatively. Animals of group 1 served as control group, no transection of the median nerve was carried out. Animals of groups 2 and 3 underwent either primary nerve coaptation or autologous nerve grafting after transection of the median nerve. To ensure validity of the method additional correlation between all parameters was investigated. Reliable electrophysiological results were observed in all animals. As expected, group 1 animals showed lowest threshold and latency and highest CMAP levels. Transection of the median nerve and additional nerve repair leads to significant increase of threshold and latency as well as reduction of CMAP. Furthermore, animals of group 3 showed higher levels for threshold and latency and reduced CMAP levels compared with animals of group 2. The grasping test alone could not demonstrate these slight differences 20 weeks post-operatively. Additionally, we observed strong correlations between threshold, latency and CMAP using the Spearman correlation ranking. We describe the usage of motor neurography as a reproducible and valid tool which should be mandatory for detailed analysis of regeneration in the rat median nerve model.

Local administration of DFO-loaded lipid particles improves recovery after end-to-end reconstruction of rat median nerve.

PURPOSE The improvement of regeneration and functional recovery after peripheral nerve injury is a major challenge in neurosurgery. Although microsurgical techniques for nerve reconstruction have seen great advancements over the last years, the clinical outcome with patients is often unsatisfactory. The aim of the present study was to investigate if administration of the iron chelator Deferroxamine (DFO), can improve postoperative outcome in the rat median nerve reconstruction model. METHODS After complete transection, the right median nerve was repaired by end-to-end neurorrhaphy. The suture site was wrapped by a 1-cm-long external jugular vein segment, either empty or filled with DFO-loaded lipid particles (Perineurin or with a vehicle (unloaded lipid particles) alone. Functional testing was carried out weekly by means of the grasping test. At the time of withdrawal, 12 weeks post-operatively, muscle tropism recovery was assessed by weighing flexor digitorum sublimis muscle that is innervated by the median nerve only. Before harvesting of the nerve specimens electrophysiological analyses were performed with measuring the latency, the threshold and the conduction velocity. Finally, the repaired nerves were withdrawn for immunocytochemistry with a neurofilament antibody and axon quantitative morphology. RESULTS The comparison between the groups showed that intraoperative application of the DFO-loaded lipid particles at the neurorrhaphy site led to a significant increase in the density of regenerating axons as well as to an accelerated recovery of both muscle tropism and motor function. The electrophysiological results demonstrated a decrease of the threshold, a lower latency, and a higher conduction velocity in the Perineurin-treated animals. CONCLUSIONS The results of the present study suggest that local administration of Perineurin might have a therapeutic potential for improving the postoperative outcome after microsurgical nerve reconstruction in patients.