Overexpression of ATF3 or the combination of ATF3, c-Jun, STAT3 and Smad1 promotes regeneration of the central axon branch of sensory neurons but without synergistic effects.

Peripheral nerve injury results in the activation of a number of transcription factors (TFs) in injured neurons, some of which may be key regulators of the regeneration-associated gene (RAG) programme. Among known RAG TFs, ATF3, Smad1, STAT3 and c-Jun have all been linked to successful axonal regeneration and have known functional and physical interactions. We hypothesised that TF expression would promote regeneration of the central axon branch of DRG neurons in the absence of a peripheral nerve lesion and that simultaneous overexpression of multiple RAG TFs would lead to greater effects than delivery of a single TF. Using adeno-associated viral vectors, we overexpressed either the combination of ATF3, Smad1, STAT3 and c-Jun with farnesylated GFP (fGFP), ATF3 only with fGFP, or fGFP only, in DRG neurons and assessed axonal regeneration after dorsal root transection or dorsal column injury and functional improvement after dorsal root injury. ATF3 alone and the combination of TFs promoted faster regeneration in the injured dorsal root. Surprisingly, however, the combination did not perform better than ATF3 alone. Neither treatment was able to induce functional improvement on sensory tests after dorsal root injury or promote regeneration in a dorsal column injury model. The lack of synergistic effects among these factors indicates that while they do increase the speed of axon growth, there may be functional redundancy between these TFs. Because axon growth is considerably less than that seen after a conditioning lesion, it appears these TFs do not induce the full regeneration programme.

Evaluation of Five Tests for Sensitivity to Functional Deficits following Cervical or Thoracic Dorsal Column Transection in the Rat.

The dorsal column lesion model of spinal cord injury targets sensory fibres which originate from the dorsal root ganglia and ascend in the dorsal funiculus. It has the advantages that fibres can be specifically traced from the sciatic nerve, verifiably complete lesions can be performed of the labelled fibres, and it can be used to study sprouting in the central nervous system from the conditioning lesion effect. However, functional deficits from this type of lesion are mild, making assessment of experimental treatment-induced functional recovery difficult. Here, five functional tests were compared for their sensitivity to functional deficits, and hence their suitability to reliably measure recovery of function after dorsal column injury. We assessed the tape removal test, the rope crossing test, CatWalk gait analysis, and the horizontal ladder, and introduce a new test, the inclined rolling ladder. Animals with dorsal column injuries at C4 or T7 level were compared to sham-operated animals for a duration of eight weeks. As well as comparing groups at individual timepoints we also compared the longitudinal data over the whole time course with linear mixed models (LMMs), and for tests where steps are scored as success/error, using generalized LMMs for binomial data. Although, generally, function recovered to sham levels within 2-6 weeks, in most tests we were able to detect significant deficits with whole time-course comparisons. On the horizontal ladder deficits were detected until 5-6 weeks. With the new inclined rolling ladder functional deficits were somewhat more consistent over the testing period and appeared to last for 6-7 weeks. Of the CatWalk parameters base of support was sensitive to cervical and thoracic lesions while hind-paw print-width was affected by cervical lesion only. The inclined rolling ladder test in combination with the horizontal ladder and the CatWalk may prove useful to monitor functional recovery after experimental treatment in this lesion model.