Abstract
Purpose: :
To study molecular mechanisms involved in regeneration of monkey retinal ganglion cell axons in organotypic retinal cultures in vitro.
Methods: :
Retinas were obtained from newborn to adult monkeys (Callithrix jacchus) immediately after death, freed from surrounding tissue and used to prepare stripes which were cultured in vitro. Growth of axons was monitored using microscopy and time-lapse video cinematography. Immunohistochemistry, Western blotting, qRT-PCR, proteomics and genomics were performed to characterize molecules associated with axonal growth. Then, siRNA experiments were conducted to identify the causal involvement of selected molecules in triggering axonal growth. In separate cultures the monkey RPE was examined towards responding with pro-regenerative factors.
Results: :
We have shown that RGCs exert a growth program with decreasing efficacy throughout life, which becomes fully developed with respect to growth associated molecules and axon specific markers. In particular, receptors to extracellular matrix and specifically for laminin were expressed together with neurofilaments. When the rate of growth was determined with time-lapse videography, it was similar with that reported for embryonic axons, indicating that regenerating axons recapitulate the principal molecular mechanisms of embryonic development. Proteomic profiling of the regenerating retinas in culture showed different patterns from native retinas at matching ages and from retinas without the opportunity to regenerate axons. In particular, proteins involved in Calcium homeostasis such as calmodulin and crystallin beta-b2 were regulated and stress proteins such as heat shock proteins were expressed. The molecular changes associated with axon regeneration in mature RGCs are strikingly similar to those reported during rat ganglion cell axon regeneration or peripheral nerve regeneration (e.g., GAP-43). Genomic profiling using human immobilized cDNA and hybridization with monkey mRNA showed specific regulation of genes belonging to the small nucleorprotein family.
Conclusions: :
The data show that even after maturation, the molecular mechanism for axonal growth still exists and can be reactivated to result in stump extension and growth cone formation. Understanding of the molecular mechanisms of axonal regeneration will help to develop therapeutic concepts for optic nerve injuries.
Keywords: regeneration • ganglion cells • retinal culture