Regeneration of entorhinal fibers in mouse slice cultures is age dependentand 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 ce ntral nervous system, but occurs in the embryonic and neonatal nervous syst em. Slice cultures offer a convenient experimental system to study the decl ine of axonal regeneration with increasing maturation of central nervous sy stem tissue. We have used mouse entorhinohippocampal slice cultures to asse ss regeneration of entorhinal fibers after mechanical lesions in vitro. We found that entorhinal axons regenerate well in cultures derived from postna tal days 5-7 mouse pups when the lesion is made at the second and fourth da ys in vitro (DIV 2 and DIV 4), Only little regenerative outgrowth is seen a fter 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 neur otrophic factors and pharmacological compounds that may promote axonal rege neration. 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 regenerati on in cultures where normally little regeneration is found. A similar impro vement of regeneration was found after treatment with pertussis toxin, an i nhibitor of G(i)-proteins, and with GF109203X, an inhibitor of protein kina se C. These substances may promote regeneration by interfering with intrace llular signaling pathways activated by outgrowth inhibitors. Our findings i ndicate that the application of neurotrophic factors and the modulation of intracellular signal transduction pathways could be useful strategies to en hance axonal regeneration in a complex microenvironment. (C) 2001 Academic Press.