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

Zhang,Ge; Lei,Fei; Zhou,Qingzhong; Feng,Daxiong; Bai,Yongheng

doi:10.3892/mmr.2016.5918

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

Authors:
- Ge Zhang
- Fei Lei
- Qingzhong Zhou
- Daxiong Feng
- Yongheng Bai
View Affiliations

Affiliations: Department of Orthopedics, The Luzhou People's Hospital, Luzhou, Sichuan 646000, P.R. China, Department of Spinal Surgery, The First Affiliated Hospital of Luzhou Medical College, Luzhou, Sichuan 646000, P.R. China, Key Laboratory of Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325000, P.R. China
Published online on: November 1, 2016 https://doi.org/10.3892/mmr.2016.5918
Pages: 5180-5188
Copyright: © Zhang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

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.

View Figures

View References

1	Horner PJ and Gage FH: Regenerating the damaged central nervous system. Nature. 407:963–970. 2000. View Article : Google Scholar : PubMed/NCBI
2	Chen MS, Huber AB, van der Haar ME, Frank M, Schnell L, Spillmann AA, Christ F and Schwab ME: Nogo-A is a myelin-associated neurite outgrowth inhibitor and an antigen for monoclonal antibody IN-1. Nature. 403:434–439. 2000. View Article : Google Scholar : PubMed/NCBI
3	Domeniconi M, Cao Z, Spencer T, Sivasankaran R, Wang K, Nikulina E, Kimura N, Cai H, Deng K, Gao Y, et al: Myelin-associated glycoprotein interacts with the Nogo66 receptor to inhibit neurite outgrowth. Neuron. 35:283–290. 2002. View Article : Google Scholar : PubMed/NCBI
4	Wang KC, Koprivica V, Kim JA, Sivasankaran R, Guo Y, Neve RL and He Z: Oligodendrocyte-myelin glycoprotein is a nogo receptor ligand that inhibits neurite outgrowth. Nature. 417:941–944. 2002. View Article : Google Scholar : PubMed/NCBI
5	Madura T, Yamashita T, Kubo T, Fujitani M, Hosokawa K and Tohyama M: Activation of Rho in the injured axons following spinal cord injury. EMBO Rep. 5:412–417. 2004. View Article : Google Scholar : PubMed/NCBI
6	Alabed YZ, Pool M, Ong Tone S, Sutherland C and Fournier AE: GSK3 beta regulates myelin-dependent axon outgrowth inhibition through CRMP4. J Neurosci. 30:5635–5643. 2010. View Article : Google Scholar : PubMed/NCBI
7	Schmandke A and Strittmatter SM and Strittmatter SM: ROCK and Rho: Biochemistry and neuronal functions of Rho-associated protein kinases. Neuroscientist. 13:454–469. 2007. View Article : Google Scholar : PubMed/NCBI
8	Fournier AE, Takizawa BT and Strittmatter SM: Rho kinase inhibition enhances axonal regeneration in the injured CNS. J Neurosci. 23:1416–1423. 2003.PubMed/NCBI
9	Shao Z, Browning JL, Lee X, Scott ML, Shulga-Morskaya S, Allaire N, Thill G, Levesque M, Sah D, McCoy JM, et al: TAJ/TROY, an orphan TNF receptor family member, binds Nogo-66 receptor 1 and regulates axonal regeneration. Neuron. 45:353–359. 2005. View Article : Google Scholar : PubMed/NCBI
10	Arimura N, Inagaki N, Chihara K, Ménager C, Nakamura N, Amano M, Iwamatsu A, Goshima Y and Kaibuchi K: Phosphorylation of collapsin response mediator protein-2 by Rho-kinase. evidence for two separate signaling pathways for growth cone collapse. J Biol Chem. 275:23973–23980. 2000. View Article : Google Scholar : PubMed/NCBI
11	Yoshimura T, Kawano Y, Arimura N, Kawabata S, Kikuchi A and Kaibuchi K: GSK-3beta regulates phosphorylation of CRMP-2 and neuronal polarity. Cell. 120:137–149. 2005. View Article : Google Scholar : PubMed/NCBI
12	Zhou FQ and Snider WD: Cell biology. GSK-3beta and microtubule assembly in axons. Science. 308:211–214. 2005. View Article : Google Scholar : PubMed/NCBI
13	Mueller BK, Mack H and Teusch N: Rho kinase, a promising drug target for neurological disorders. Nat Rev Drug Discov. 4:387–398. 2005. View Article : Google Scholar : PubMed/NCBI
14	Kim WY, Zhou FQ, Zhou J, Yokota Y, Wang YM, Yoshimura T, Kaibuchi K, Woodgett JR, Anton ES and Snider WD: Essential roles for GSK-3s and GSK-3-primed substrates in neurotrophin-induced and hippocampal axon growth. Neuron. 52:981–996. 2006. View Article : Google Scholar : PubMed/NCBI
15	Lingor P, Teusch N, Schwarz K, Mueller R, Mack H, Bähr M and Mueller BK: Inhibition of Rho kinase (ROCK) increases neurite outgrowth on chondroitin sulphate proteoglycan in vitro and axonal regeneration in the adult optic nerve in vivo. J Neurochem. 103:181–189. 2007.PubMed/NCBI
16	Chan CC, Khodarahmi K, Liu J, Sutherland D, Oschipok LW, Steeves JD and Tetzlaff W: Dose-dependent beneficial and detrimental effects of ROCK inhibitor Y27632 on axonal sprouting and functional recovery after rat spinal cord injury. Exp Neurol. 196:352–364. 2005. View Article : Google Scholar : PubMed/NCBI
17	Cuzzocrea S, Genovese T, Mazzon E, Crisafulli C, Di Paola R, Muià C, Collin M, Esposito E, Bramanti P and Thiemermann C: Glycogen synthase kinase-3 beta inhibition reduces secondary damage in experimental spinal cord trauma. J Pharmacol Exp Ther. 318:79–89. 2006. View Article : Google Scholar : PubMed/NCBI
18	Kawasaki T, Nishio T, Kawaguchi S and Kurosawa H: Spatiotemporal distribution of GAP-43 in the developing rat spinal cord: A histological and quantitative immunofluorescence study. Neurosci Res. 39:347–358. 2001. View Article : Google Scholar : PubMed/NCBI
19	Park DY, Mayle RE, Smith RL, Corcoran-Schwartz I, Kharazi AI and Cheng I: Combined transplantation of human neuronal and mesenchymal stem cells following spinal cord injury. Global Spine J. 3:1–6. 2014. View Article : Google Scholar
20	Basso DM, Beattie MS and Bresnahan JC: A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma. 12:1–21. 1995. View Article : Google Scholar : PubMed/NCBI
21	Caizhong X, Chunlei S, Beibei L, Zhiqing D, Qinneng D and Tong W: The application of somatosensory evoked potentials in spinal cord injury rehabilitation. NeuroRehabilitation. 35:835–840. 2014.PubMed/NCBI
22	Kim DH and Jahng TA: Continuous brain-derived neurotrophic factor (BDNF) infusion after methylprednisolone treatment in severe spinal cord injury. J Korean Med Sci. 19:113–122. 2004. View Article : Google Scholar : PubMed/NCBI
23	Kubo T, Hata K, Yamaguchi A and Yamashita T: Rho-ROCK inhibitors as emerging strategies to promote nerve regeneration. Curr Pharm Des. 13:2493–2499. 2007. View Article : Google Scholar : PubMed/NCBI
24	Jeon BT, Jeong EA, Park SY, Son H, Shin HJ, Lee DH, Kim HJ, Kang SS, Cho GJ, Choi WS and Roh GS: The Rho-kinase (ROCK) inhibitor Y-27632 protects against excitotoxicity-induced neuronal death in vivo and in vitro. Neurotox Res. 23:238–248. 2012. View Article : Google Scholar : PubMed/NCBI
25	Fournier AE and Strittmatter SM: Repulsive factors and axon regeneration in the CNS. Curr Opin Neurobiol. 11:89–94. 2001. View Article : Google Scholar : PubMed/NCBI
26	Liu BP, Fournier A, GrandPre T and Strittmatter SM: Myelin-associated glycoprotein as a functional ligand for the Nogo-66 receptor. Science. 297:1190–1193. 2002. View Article : Google Scholar : PubMed/NCBI
27	Buchsbaum RJ: Rho activation at a glance. J Cell Sci. 120:1149–1152. 2007. View Article : Google Scholar : PubMed/NCBI
28	Joshi AR, Bobylev I, Zhang G, Sheikh KA and Lehmann HC: Inhibition of Rho-kinase differentially affects axon regeneration of peripheral motor and sensory nerves. Exp Neurol. 263:28–38. 2015. View Article : Google Scholar : PubMed/NCBI
29	Dill J, Wang H, Zhou F and Li S: Inactivation of glycogen synthase kinase 3 promotes axonal growth and recovery in the CNS. J Neurosci. 28:8914–8928. 2008. View Article : Google Scholar : PubMed/NCBI
30	Gopalakrishnan SM, Teusch N, Imhof C, Bakker MH, Schurdak M, Burns DJ and Warrior U: Role of Rho kinase pathway in chondroitin sulfate proteoglycan-mediated inhibition of neurite outgrowth in PC12 cells. J Neurosci Res. 86:2214–2226. 2008. View Article : Google Scholar : PubMed/NCBI
31	Dubreuil CI, Winton MJ and McKerracher L: Rho activation patterns after spinal cord injury and the role of activated Rho in apoptosis in the central nervous system. J Cell Biol. 162:233–243. 2003. View Article : Google Scholar : PubMed/NCBI
32	Collino M, Thiemermann C, Mastrocola R, Gallicchio M, Benetti E, Miglio G, Castiglia S, Danni O, Murch O, et al: Treatment with the glycogen synthase kinase-3beta inhibitor, TDZD-8, affects transient cerebral ischemia/reperfusion injury in the rat hippocampus. Shock. 30:299–307. 2008. View Article : Google Scholar : PubMed/NCBI