Reorganization of Spinal Cord Microarchitecture by Bioluminescent Optogenetic and Rehabilitative Interventions

Spinal cord injury (SCI) induces persistent locomotor deficits that are closely associated with maladaptive structural plasticity of spinal neuronal circuits. Although motor rehabilitation improves functional outcomes, the cellular substrates underlying rehabilitation-induced recovery remain incompletely understood, particularly in relation to activity-dependent neuromodulation strategies. Here, we investigated how treadmill-based motor training (TMT) and its combination with bioluminescent optogenetic (BL-OG) stimulation of Hb9 (homebox 9)-positive motoneurons and excitatory interneurons selectively modulate microarchitectural plasticity in the injured rat spinal cord. At the level of gross locomotor assessment, Basso, Beattie and Bresnahan (BBB) scores were comparable between the BL-OG and SCI+TMT groups. Although no statistically significant differences in the total score in rung ladder were observed at 28 days post-injury, animals in the BL-OG group showed a tendency toward a higher ratio of successful hindlimb placements, indicating improved step accuracy. BL-OG stimulation was associated with a slightly greater attenuation of SCI-induced spine abnormalities compared to TMT alone, with significant differences between the experimental groups detected specifically in laminae VIII and IX. These lamina-specific alterations in dendritic integration and dendritic spine composition were accompanied by preservation of wisteria floribunda agglutinin WFA-positive perineuronal net (PNN) architecture. Against this background, reduced glypican-4 (GPC-4) expression and attenuated WFA/GPC-4 colocalization were observed in the SCI+BL-OG group relative to SCI in laminae VII-IX, consistent with activity-dependent modulation of PNN-associated synaptic organization in Hb9-positive neuronal populations. Together, these findings indicate that motor rehabilitation and bioluminescent optogenetic stimulation engage distinct but partially overlapping mechanisms of activity-dependent microarchitectural remodeling, preferentially targeting synaptic and perineuronal net-associated substrates rather than inducing large-scale circuit reorganization. Further studies are warranted to elucidate the mechanisms underlying these distinct plasticity profiles.