Small-molecule trkB agonists promote axon regeneration in cut peripheral nerves.

Treatments with two-small molecule tropomyosin receptor kinase B (trkB) ligands, 7,8 dihydroxyflavone (7,8 DHF) and deoxygedunin, were evaluated for their ability to promote the regeneration of cut axons in injured peripheral nerves in mice in which sensory and motor axons are marked by YFP. Peripheral nerves were cut and repaired with grafts from strain-matched, nonfluorescent donors and secured in place with fibrin glue. Lengths of profiles of regenerating YFP(+) axons were measured 2 wk later from confocal images. Axon regeneration was enhanced when the fibrin glue contained dilutions of 500-nM solution of either small-molecule trkB agonist. In mice in which the neurotrophin receptor trkB is knocked out selectively in neurons, axon regeneration is very weak, and topical treatment with 7,8 DHF had no effect on axon regeneration. Similar treatments with deoxygedunin had only a modest effect. In conditional BDNF knockout mice, topical treatments with either 7,8 DHF or deoxygedunin resulted in a reversal of the poor regeneration found in controls and produced significant enhancement of regeneration. In WT mice treated with 2 wk of daily i.p. injections of either 7,8 DHF or deoxygedunin (5 mg/kg), regenerating axon profiles were nearly twice as long as in controls. Restoration of direct muscle responses evoked by sciatic nerve stimulation to pretransection levels over an 8-wk survival period was found only in the treated mice. Treatments with either small-molecule trkB agonist enhanced axon regeneration and muscle reinnervation after peripheral nerve injuries.

Effects of treadmill training on functional recovery following peripheral nerve injury in rats.

Exercise, in the form of moderate daily treadmill training following nerve transection and repair leads to enhanced axon regeneration, but its effect on functional recovery is less well known. Female rats were exercised by walking continuously, at a slow speed (10 m/min), for 1 h/day on a level treadmill, beginning 3 days after unilateral transection and surgical repair of the sciatic nerve, and conducted 5 days/wk for 2 wk. In Trained rats, both direct muscle responses to tibial nerve stimulation and H reflexes in soleus reappeared earlier and increased in amplitude more rapidly over time than in Untrained rats. The efficacy of the restored H reflex was greater in Trained rats than in Untrained controls. The reinnervated tibialis anterior and soleus were coactivated during treadmill locomotion in Untrained rats. In Trained animals, the pattern of activation of soleus, but not tibialis anterior, was not significantly different from that found in Intact rats. The overall length of the hindlimb during level and up- and downslope locomotion was conserved after nerve injury in both groups. This conservation was achieved by changes in limb orientation. Limb length was conserved effectively in all rats during downslope walking but only in Trained rats during level and upslope walking. Moderate daily exercise applied immediately after sciatic nerve transection is sufficient to promote axon regeneration, to restore muscle reflexes, and to improve the ability of rats to cope with different biomechanical demands of slope walking.

Chondroitinase ABC reduces time to muscle reinnervation and improves functional recovery after sciatic nerve transection in rats.

Application of chondroitinase ABC (ChABC) to injured peripheral nerves improves axon regeneration, but it is not known whether functional recovery is also improved. Recordings of EMG activity [soleus (Sol) M response and H reflexes] evoked by nerve stimulation and of Sol and tibialis anterior (TA) EMG activity and hindlimb and foot kinematics during slope walking were made to determine whether ChABC treatment of the sciatic nerve at the time of transection improves functional recovery. Recovery of evoked EMG responses began as multiple small responses with a wide range of latencies that eventually coalesced into one or two more distinctive and consistent responses (the putative M response and the putative H reflex) in both groups. Both the initial evoked responses and the time course of their maturation returned sooner in the ChABC group than in the untreated (UT) group. The reinnervated Sol and TA were coactivated during treadmill locomotion during downslope, level, and upslope walking throughout the study period in both UT and ChABC-treated rats. By 10 wk after nerve transection and repair, locomotor activity in Sol, but not TA, had returned to its pretransection pattern. There was an increased reliance on central control of Sol activation across slopes for both groups as interpreted from elevated prestance Sol EMG activity that was no longer modulated with slope. Limb length and orientation during locomotion were similar to those observed prior to nerve injury during upslope walking only in the ChABC-treated rats. Thus treatment of cut nerves with ChABC leads to improvements in functional recovery.

Effect of axon misdirection on recovery of electromyographic activity and kinematics after peripheral nerve injury.

In this study, patterns of activity in the soleus (Sol) and tibialis anterior (TA) muscles and hindlimb kinematics were evaluated during slope walking in rats after transection and surgical repair either of the entire sciatic nerve (Sci group) or of its two branches separately, the tibial and common fibular nerves (T/CF group). With the latter method, axons from the tibial and common fibular nerves could not reinnervate targets of the other nerve branch after injury, reducing the opportunity for misdirection. Activity in the TA shifted from the swing phase in intact rats to nearly the entire step cycle in both injured groups. Since these changes occur without misdirection of regenerating axons, they are interpreted as centrally generated. Sol activity was changed from reciprocal to that of TA in intact rats to coactivate with TA, but only in the Sci group rats. In the T/CF group rats, Sol activity was not altered from that observed in intact rats. Despite effects of injury that limited foot movements, hindlimb kinematics were conserved during downslope walking in both injury groups and during level walking in the T/CF group. During level walking in the Sci group and during upslope walking in both groups of injured rats, the ability to compensate for the effects of the nerve injury was less effective and resulted in longer limb lengths held at more acute angles throughout the step cycle. Changes in limb movements occur irrespective of axon misdirection and reflect compensatory changes in the outputs of the neural circuits that drive locomotion.

Effect of slope and sciatic nerve injury on ankle muscle recruitment and hindlimb kinematics during walking in the rat.

Slope-related differences in hindlimb movements and activation of the soleus and tibialis anterior muscles were studied during treadmill locomotion in intact rats and in rats 4 and 10 weeks following transection and surgical repair of the sciatic nerve. In intact rats, the tibialis anterior and soleus muscles were activated reciprocally at all slopes, and the overall intensity of activity in tibialis anterior and the mid-step activity in soleus increased with increasing slope. Based on the results of principal components analysis, the pattern of activation of soleus, but not of tibialis anterior, changed significantly with slope. Slope-related differences in hindlimb kinematics were found in intact rats, and these correlated well with the demands of walking up or down slopes. Following recovery from sciatic nerve injury, the soleus and tibialis anterior were co-activated throughout much of the step cycle and there was no difference in intensity or pattern of activation with slope for either muscle. Unlike intact rats, these animals walked with their feet flat on the treadmill belt through most of the stance phase. Even so, during downslope walking limb length and limb orientation throughout the step cycle were not significantly changed from values found in intact rats. This conservation of hindlimb kinematics was not observed during level or upslope walking. These findings are interpreted as evidence that the recovering animals adopt a novel locomotor strategy that involves stiffening of the ankle joint by antagonist co-activation and compensation at more proximal joints. Their movements are most suitable to the requirements of downslope walking but the recovering rats lack the ability to adapt to the demands of level or upslope walking.

Misdirection of regenerating axons and functional recovery following sciatic nerve injury in rats.

Poor functional recovery found after peripheral nerve injury has been attributed to the misdirection of regenerating axons to reinnervate functionally inappropriate muscles. We applied brief electrical stimulation (ES) to the common fibular (CF) but not the tibial (Tib) nerve just prior to transection and repair of the entire rat sciatic nerve, to attempt to influence the misdirection of its regenerating axons. The specificity with which regenerating axons reinnervated appropriate targets was evaluated physiologically using compound muscle action potentials (M responses) evoked from stimulation of the two nerve branches above the injury site. Functional recovery was assayed using the timing of electromyography (EMG) activity recorded from the tibialis anterior (TA) and soleus (Sol) muscles during treadmill locomotion and kinematic analysis of hindlimb locomotor movements. Selective ES of the CF nerve resulted in restored M-responses at earlier times than in unstimulated controls in both TA and Sol muscles. Stimulated CF axons reinnervated inappropriate targets to a greater extent than unstimulated Tib axons. During locomotion, functional antagonist muscles, TA and Sol, were coactivated both in stimulated rats and in unstimulated but injured rats. Hindlimb kinematics in stimulated rats were comparable to untreated rats, but significantly different from intact controls. Selective ES promotes enhanced axon regeneration but does so with decreased fidelity of muscle reinnervation. Functional recovery is neither improved nor degraded, suggesting that compensatory changes in the outputs of the spinal circuits driving locomotion may occur irrespective of the extent of misdirection of regenerating axons in the periphery.