Regeneration of entorhinal fibers in mouse slice cultures is age dependent and can be stimulated by NT-4, GDNF, and modulators of G-proteins and protein kinase C.

Axonal regeneration after lesions is normally not possible in the mature central nervous system, but occurs in the embryonic and neonatal nervous system. Slice cultures offer a convenient experimental system to study the decline of axonal regeneration with increasing maturation of central nervous system tissue. We have used mouse entorhinohippocampal slice cultures to assess regeneration of entorhinal fibers after mechanical lesions in vitro. We found that entorhinal axons regenerate well in cultures derived from postnatal days 5-7 mouse pups when the lesion is made at the second and fourth days in vitro (DIV 2 and DIV 4). Only little regenerative outgrowth is seen after lesions made at DIV 6 and DIV 10. This indicates that a maturation of the cultures occurs within a short time period in vitro resulting in a loss of the regenerative potential. We have used this system to screen for neurotrophic factors and pharmacological compounds that may promote axonal regeneration. Treatments were added to the cultures 1 day before the lesion was made. We found that most added factors did not promote regeneration. Only treatment with the neurotrophic factors NT-4 and GDNF stimulated regeneration in cultures where normally little regeneration is found. A similar improvement of regeneration was found after treatment with pertussis toxin, an inhibitor of G(i)-proteins, and with GF109203X, an inhibitor of protein kinase C. These substances may promote regeneration by interfering with intracellular signaling pathways activated by outgrowth inhibitors. Our findings indicate that the application of neurotrophic factors and the modulation of intracellular signal transduction pathways could be useful strategies to enhance axonal regeneration in a complex microenvironment. Copyright 2001 Academic Press.