A novel molluscan CNS organ culture for the examination of adult nerve regeneration.

BACKGROUND

Central nervous system (CNS) regeneration is crucial for re-establishing neural connections following injury. Though some adult vertebrates have regeneration-competent CNS, invertebrate CNS also provide useful model systems for examining cellular and molecular mechanisms promoting regeneration.

NEW METHOD: Using the mollusc Lymnaea stagnalis, we have developed a novel ex vivo CNS organ culture to examine regenerative neurite outgrowth from cut nerves floating at the air-liquid interface.

RESULTS

We determined that single CNS organ cultures produced more outgrowth than co-cultures of 3 CNS, and also determined that CNS from 3 month old animals exhibited more outgrowth than older CNS. We characterized the extent of outgrowth from 11 individual cut nerves emanating from the central ring ganglia (CNS), and report 4 nerves with higher regenerative capacity. We also examined how nerve outgrowth was affected by retinoic acid, the active metabolite of vitamin A, and a known inducer of neurite outgrowth in vertebrates and Lymnaea. We found that the growth-promoting effects of RA were both age and concentration-dependent, with lower levels of RA promoting outgrowth mainly from CNS of younger animals. Higher levels of RA, however, inhibited neurite outgrowth from both young and aged CNS.

COMPARISON WITH EXISTING

METHODS

Previous studies have examined adult neuronal regeneration in molluscs using either cultured neurons, or using dye injections after nerve crush injury in the CNS.

CONCLUSION

We have established a versatile new organ culture allowing for easy visualization and quantification of neurite outgrowth for future examination of mechanisms that affect nerve regeneration.