Regenerating cortical connections in a dish: the entorhino-hippocampal organotypic slice co-culture as tool for pharmacological screening of molecules promoting axon regeneration

We present a method for using long-term organotypic slice co-cultures of the entorhino-hippocampal formation to analyze the axon-regenerative properties of a determined compound. The culture method is based on the membrane interphase method, which is easy to perform and is generally reproducible. The degree of axonal regeneration after treatment in lesioned cultures can be seen directly using green fluorescent protein (GFP) transgenic mice or by axon tracing and histological methods. Possible changes in cell morphology after pharmacological treatment can be determined easily by focal in vitro electroporation. The well-preserved cytoarchitectonics in the co-culture facilitate the analysis of identified cells or regenerating axons. The protocol takes up to a month

Nogo, myelin and axonal regeneration

El sistema nerviós dels mamífers té una baixa capacitat de reparació axonal després d'una lesió. En els últims anys, diversos estudis han demostrat que els axons lesionats no poden recréixer a causa de la presència d'un gran nombre de molècules inhibitòries. Les molècules associades a la mielina limiten el creixement axonal i el seu bloqueig afavoreix la regeneració de diverses connexions. Tres d'aquestes proteïnes, Nogo, MAG i OMgp, comparteixen un mateix receptor: NgR. El clonatge recent de Nogo ha obert noves vies per estudiar la regeneració axonal. No obstant això, molts dels elements involucrats en la via inhibitòria de la mielina són desconeguts, i els primers estudis amb animals knockout són, a més, contradictoris. Per aquesta raó, Nogo i el seu receptor han de caracteritzar-se abans de desenvolupar noves tècniques per promoure regeneració axonal.Adult mammalian central nervous system (CNS) axons have very limited capacity of regrowth after injury. In recent years, advances in the field of axonal regeneration have proved that neurons do not regenerate, mainly because of the presence of inhibitory molecules. Myelin-associated proteins limit axonal outgrowth and their blockage improves the regeneration of damaged fiber tracts. Three of these proteins, Nogo, MAG and OMgp, share a common neuronal receptor (NgR), and together represent one of the main hindrances to neuronal regeneration. The recent molecular cloning of Nogo and its receptors opened a new door to the study of axon regeneration. However, many of the elements involved in the myelin inhibitory pathway are still unknown, and the preliminary experiments with knockout mice are rather contradictory. Because of this complexity, Nogo and NgR need to be characterized before precise strategies to promote axon regeneration in the CNS can be designed