Regeneration of lesioned entorhino‐hippocampal axons in vitro by combined degradation of inhibitory proteoglycans and blockade of Nogo‐66/NgR signaling

Abstract

Damaged axons do not regenerate after axotomy in the adult mammalian central nervous system (CNS). This may be due to local inhibitory factors at the site of injury, such as overexpression of chondroitin sulfate (CS) proteoglycans (CSPG), and the presence of myelin-associated inhibitors (MAI). To overcome CSPG- or myelin-induced inhibition, strategies based on extrinsic and intrinsic treatments have been developed. For example, NEP1-40 is a synthetic peptide that promotes axonal regeneration by blocking Nogo-66/NgR interaction and chondroitinase ABC (ChABC), which degrades CS, thereby also promoting axon regrowth. Here, we examined whether the combination of these complementary strategies facilitates regeneration of the lesioned entorhino-hippocampal pathway (EHP) in slice cultures. In this model, overexpressed CSPG and MAI impaired axon regrowth, which mimics regeneration failure in vivo. Both CS cleavage with ChABC and NEP1-40 strongly facilitated the regrowth of entorhinal axons after axotomy, permitting the re-establishment of synaptic contacts with target cells. However, the combined treatment did not improve the regeneration induced by ChABC alone, and the delayed treatment of ChABC, but not NEP1-40, had a less pronounced effect on axonal regrowth compared with acute treatment. These results provide insight into the development of new assays and strategies to enhance axon regeneration in injured cortical connections.

Chondroitinase ABC Enhances Pericontusion Axonal Sprouting But Does Not Confer Robust Improvements in Behavioral Recovery

“…It is therefore not surprising that axonal sprouting was not prolonged. Furthermore, it appears unlikely that additional, more chronic interventions to maintain reduced intact glycosaminoglycan-containing CSPG levels would be beneficial, since acute rather than delayed intervention with chondroitinase is more optimal for axonal sprouting (Mingorance et al, 2006), and for some but not all tests of behavioral outcome (García-Alías et al, 2009). Combinatorial therapies with neurotrophins are required to induce significant increases in synapse formation of newly-sprouting axons after denervation (Tropea et al, 2003).…”

Chondroitinase Enhances Cortical Map Plasticity and Increases Functionally Active Sprouting Axons after Brain Injury

“…Interventions after central nervous system (CNS) injury aimed at readjusting the balance toward growth promotion by reduction of inhibitory proteins using digestive enzymes and/or in combination with other therapies report enhanced axon sprouting, which mostly leads to improved outcome. [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] However, at least after TBI, the degree of functional-behavioral improvements do not always follow the magnitude of the anatomical response. 1 It is difficult to directly determine whether newly sprouting axons support function and do, in fact, connect and contribute to the circuitry within which they are growing.…”

Regenerating cortical connections in a dish: the entorhino-hippocampal organotypic slice co-culture as tool for pharmacological screening of molecules promoting axon regeneration

“…Though relevant, these methods do not bring the researcher close to the in vivo situation, since most factors in these preparations are lacking. The loss of regeneration of the EHC in vitro closely followed those seen in vivo 26,30,40 and most of the factors involved in the absence of regeneration are present in these culture platforms 29 . In technical terms, in our experience, TaueGFP cultures are more appropriate than Biocytin-labeled cultures for a fast evaluation of a molecular screening, since all possible regenerating axons will be fluorescent.…”