Hyaluronan tetrasaccharide promotes regeneration of peripheral nerve: In vivo analysis by film model method

Hyaluronan (HA) is known to inhibit neurons from regenerating in the central nervous system. However, hyaluronan tetrasaccharide (HA4) was found in in vitro experiments to promote outgrowth of neurons. To investigate the promotion by HA4 of nerve regeneration in vivo, we analyzed outgrowth of regenerating axons treated with HA4, using a film model method. After the common peroneal nerve in mice was transected, the proximal end of cut nerve was placed on a sheet of thin plastic film, immersed in several drops of HA4 solution, covered with another sheet of film, and then kept in vivo. Six hours after the procedure, terminal sprouts had grown out from ending bulbs formed at the cut end of parent nerve administered with HA4 solution 100 or 1000 μg/mL, while no sprouts were observed in groups treated with 10 μg/mL of HA4 or in controls. On the 2nd day after axotomy (day 2), many regenerating axons in the group treated with 100 μg/mL of HA4 extended onto the flat film for a longer distance than those treated with 1000 μg/mL of HA4 and controls. With the optimal dose of HA4 (100 μg/mL), axonal outgrowth was significantly (p < 0.01) greater than that in controls at each time point. Schwann cells appeared migrating from parent nerve onto the film from day 3 as well as in controls. Thus, enhanced outgrowth of regenerating axons and normal behavior of migratory Schwann cells suggested that HA4 promoted regeneration of neurons without the mediation of Schwann cells.