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Previous studies have reported that the Rho-associated coiled-coil containing protein kinase 2 (ROCKII) and glycogen synthase kinase‑3β (GSK)‑3β signaling pathways are involved in axonal regeneration. The present study investigated the effects of the combined application of Y27632 (a ROCKII inhibitor) and 4-benzyl-2‑methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8; a GSK‑3β inhibitor) on neurite outgrowth and functional recovery in rats with spinal cord injury (SCI). A total of 90 female Sprague‑Dawley rats were randomly allocated into six groups, and the SCI rats received daily administration of 1.6 mg/kg Y27632 for 2 weeks and/or 1 mg/kg TDZD‑8 for 3 weeks via a catheter. Cellular apoptosis in the injured spinal cords was measured at each time point using a terminal deoxynucleotidyl transferase‑mediated dUTP nick end labeling assay. The expression levels of growth‑associated protein‑43 (GAP‑43) were determined by immunohistochemical staining. In addition, an anterograde tracer was used to analyze axonal regeneration, the Basso Beattie Bresnahan locomotor rating scale (BBB) was analyzed, and the somatosensory evoked potential (SEP) test was conducted. The results demonstrated that SCI upregulated the number of apoptotic cells, increased GAP‑43 expression and enhanced the latent periods of SEP, as compared with in mice that underwent a sham operation. Furthermore, SCI decreased the BBB scores and the SEP amplitudes. These injuries in the spinal cord were reduced following treatment with Y27632, TDZD‑8, or their combined application, as detected by decreased apoptosis, the induction of axonal regeneration, and the promotion of functional recovery of the lower limbs. Although the BBB scores, and SEP amplitudes and latent periods were not significantly different among the three drug treatment groups, the combined application of Y27632 and TDZD‑8 resulted in stronger axonal regenerative potency and a greater protective effect on secondary SCI. These results indicated that the combined application of Y27632 and TDZD‑8 may more effectively protect against secondary SCI by inhibiting cellular apoptosis, enhancing GAP-43 expression and promoting neurite outgrowth in SCI rats, compared with Y27632 or TDZD-8 alone.

Combined application of Rho-ROCKII and GSK-3β inhibitors exerts an improved protective effect on axonal regeneration in rats with spinal cord injury