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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 11, 2006 as doi:10.1096/fj.05-5121fje. |
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* Development and Regeneration of the CNS, Department of Cell Biology, IRB-PCB, University of Barcelona, Barcelona, Spain; and
Regeneration of the CNS Unit, Cajal Institute, CSIC, Madrid, Spain and National Hospital of Paraplegics, Toledo, Spain
1Correspondence: Development and Regeneration of the CNS, Department of Cell Biology, IRB-PCB, Barcelona Science Park, University of Barcelona, Josep Samitier 1–5, 08028 Barcelona, Spain. E-mail: jadelrio{at}pcb.ub.es
SPECIFIC AIMS
Damaged axons do not regenerate in the mammalian central nervous system (CNS) after injury partly because of the presence of inhibitors such as CSPG and Nogo-A. Here we examine whether the combination of two extrinsic strategies targeting CSPG and Nogo-A binding to NgR allows the regeneration of the entorhino-hippocampal pathway (EHP) in vitro. We show that both the cleavage of CSPG with ChABC and the blockade of Nogo-66/NgR binding with NEP1-40 strongly facilitate the regrowth of entorhinal axons after axotomy, although the effects of these two drugs were not additive, and only NEP1-40 was efficient for delayed treatment.
PRINCIPAL FINDINGS
1. Proliferation of glial cells after EHP axotomy in vitro
We first tested the suitability of the organotypic slice culture as a model to study axon regeneration in the EHP. Axotomy of the EHP in vitro was followed by a robust glial reaction, including glial scar-like formation by reactive astrocytes, ameboid microglia, and NG2-positive oligodendrocyte progenitors on both sides of the lesion. Astroglial phagocytosis of debris was confirmed by electron microscopy and the proliferation of reactive glial cells near the lesion and the denervated stratum lacunosum moleculare/molecular layer (slm/ml) was assessed by double GFAP- or NG2-BrdU staining. Thus, glial reaction in this model mimics that which occurs after EHP axotomy in vivo.
2. Overexpression of CSPGs, Nogo-A, and NgR after EHP axotomy
The temporal evolution of CSPG-expression in healthy and lesioned EH slices was monitored using CS-56 and 3F8 antibodies (Fig. 1
A–D). In nonlesioned co-cultures, CS-56 immunolabeling was present in all the hippocampal layers, with a lower intensity in the slm/ml (Fig. 1A, B
). From 2 to 15 days after lesion (DAL) (the last DAL analyzed), a strong increase in CS-56 immunoreactivity was observed throughout the lesioned hippocampus, including the slm/ml, but not in the entorhinal cortex (Fig. 1F
), and glial cells resembling reactive astroglia were immunostained with 3PE8 antibody in the slm/ml (Fig. 1D, E
). In parallel to CSPG overexpression, a moderated increase in NgR and Nogo-A protein expression, peaking at 2–5 DAL, was observed by Western blot (Fig. 2
A). Thus, CSPG and Nogo-A overexpression could participate in the failure of regeneration of the lesioned EHP.
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3. Effect of ChABC and NEP1-40 on axonal regeneration in vitro
To overcome CSPG- or myelin-induced inhibition of axonal regeneration, strategies based on extrinsic and intrinsic treatments have been developed. We aimed to explore whether CSPG degradation with chondroitinase ABC (ChABC) and the blockade of the Nogo-A/NgR interaction with the peptide NEP1-40 induce the regrowth of the lesioned EHP. Control experiments demonstrated that the NEP1-40 treatment did not prevent CSPG overexpression (Fig. 1G
), and conversely, that ChABC treatment did not affect Nogo-A or NgR protein levels (Fig. 2B
). Drug treatments had no apparent effect on glial scar development, microglial activation, or phagocytic activity of activated microglia after EHP axotomy.
Acute treatment of axotomized organotypic cultures for 7 days with ChABC resulted in the regrowth of numerous entorhinal axons into the denervated hippocampus (Fig. 1J-L
, Fig. 2H
). In contrast, in control cultures most of the axons stopped at the entorhino-hippocampal interface; very few entered the hippocampus (Fig. 1I
, Fig. 2H
). Degradation of CS residues of the overexpressed CSPGs in the lesioned hippocampus after ChABC treatment was corroborated by a strong decrease in CS-56 immunostaining and the appearance of the stub antigen recognized by the 2B6 antibody in Western blot analysis (Fig. 1E, H
).
Acute blockade of Nogo-66 binding to NgR with NEP1-40 led to a significant increase in the number of regenerating axons (Fig. 2C
). However, many of these axons showed lower layer specificity than that observed after ChABC treatment. Next, we examined the effect of combining these two treatments. While cultures treated with ChABC/NEP1-40 displayed more regenerating axons than those treated with NEP1-40 alone, the difference with ChABC-treated cultures was not significant (Fig. 2D-H
). Regenerating EH axons from ChABC/NEP1-40-treated co-cultures established asymmetric synaptic contacts with target hippocampal neurons (Fig. 2G
).
4. Efficiency of delayed treatment
Studies by our group have demonstrated that regenerative potential of the lesioned entorhinal neuron persisted for several days after axotomy in vitro, as when confronted with juvenile tissue several days after lesion, the EHP regenerates. Thus, we explored the effectiveness of a delayed ChABC and NEP1-40 treatment after EHP lesion. ChABC and NEP1-40 and the combined treatment induced regrowth of lesioned entorhinal axons into the hippocampus in delayed administration starting 5 DAL (Fig. 2H
). However, while the delayed application of NEP1-40 gave similar regenerative results to acute treatment, the efficiency of ChABC treatment was reduced when used either alone or in the combined treatment.
CONCLUSIONS AND SIGNIFICANCE
To achieve complete axonal regeneration, it is widely accepted that a combination of treatments will be required. Here, for the first time, we used an entorhino-hippocampal slice culture assay to compare the efficiency of two exogenous treatments, ChABC and NEP1-40, to promote axonal regeneration of a lesioned cortical connection, the EHP. We addressed the combination of these two treatments and the capacity of delayed drug delivery to enhance axonal regeneration.
Organotypic slice cultures have been widely used as a model to study diverse neuronal functions under conditions where cytoarchitecture is largely retained. Our findings demonstrate that glial reactivity after axotomy, including phagocytosis, proliferation, and overexpression of CSPG and Nogo-A, largely mimics that which occurs after EHP lesion in vivo. Therefore, our in vitro assay is a potent tool for developing and screening potential treatments to further promote regeneration across the lesion in vivo.
Our results show the relative contribution of CSPG and Nogo-66 to axonal growth inhibition in the EHP when ChABC and NEP1-40 are used alone and demonstrate that the combined use of these two drugs has no synergistic effects. Control experiments indicate that ChABC does not affect Nogo-A or NgR expression levels and, conversely, that NEP1-40 does not prevent CSPG overexpression after lesion. ChABC alone may reduce common inhibitory signaling pathways (e.g., Rho or PKC activity) to an inactivated level at which incubation with NEP1-40 has no apparent additive effect.
Finally, we determined the time window of effectiveness of ChABC and NEP1-40 treatments after lesion. Post-lesion delivery of ChABC and NEP1-40 led to axonal regeneration of the EHP and formation of mature synaptic contacts, thereby highlighting the participation of Nogo-66 and CSPG in the failure of regeneration of cortical connections. NEP1-40 delivery can be delayed for at least 5 days without loss of efficiency, in contrast to ChABC. Our results are consistent with other studies and indicate that to promote axonal regeneration, ChABC treatment must be started before or immediately after the lesion. We believe that our results provide the basis for the development of new assays and strategies to enhance axon regeneration in injured cortical connections.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5121fje;
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