Given our previous findings on CEFFE’s positive effects on wound healing, [8–11, 15] we examined its effects on injured CNS repair. In the present study, we demonstrated that CEFFE has neuroprotective and axon regenerating effects on injured RGCs, which suggest its potential clinical applications.
As early as in 1988, R M Lindsay found BDNF could enhance axonal regeneration . In the decades followed, more and more neurotrophic factors such as CNTF, GDNF, and neurotrophin-4/5 etc. were founded to promote axon regeneration [17–20]. However, their use is limited due to the instability of neurotrophic factors in adverse environment associated with CNS injury. In the present study, a single injection of CEFFE did not effectively promote axon regeneration (Fig. 1); multiple injections were required. RGCs death is a gradual process, associated with different characteristics at different stages. As reported previously, RGCs underwent a series of changes within 6 h after injury; however, the number of RGCs did not decrease. On days 3–7 post-injury, the rate of RGCs increased gradually, peaking on day 7; accordingly, we injected CEFFE at the corresponding time points; multiple injections of CEFFE promoted RGCs axon regeneration more robustly than did a single injection (Fig. 1).
The use of a single neurotrophic factor may promote RGCs survival or axon regeneration to some extent, as previously reported; however, this method has shown unsatisfactory results in promoting sustained and abundant axon regeneration[22–24]; overcoming these limitations requires further research. Notably, researchers found use of stem cells or signaling inhibitors combined with neurotrophic factors may have a better effect on RGCs survival or axon regeneration than does the use of neurotrophic factors alone.[25–27]. Synergy among neurotrophic factors used is required for effective axon regeneration. The CEFFE is a mixture of diverse proteins, including trophic factors, inflammation factors, and chemokines. Previous studies have shown the role of trophic and inflammation factors in axon regeneration. To further evaluate the effects of the CEFFE, we chose BDNF, one of the most typical trophic factors involved in axon regeneration, as a control. Both CEFFE and BDNF were intravitreally injected on days 0, 3, and 7 post-crush (Fig. 2). The concentration of the CEFFE was approximately 5 ug/ul, which contained 2 pg/ul of BDNF. As we have shown, CEFFE contains diverse neurotrophic factors[8, 15] and may promote axon regeneration that is more robust than that promoted by the use of a single neurotrophic factor; this finding is consistent with those of previous studies.
In addition, the concentration of any single trophic factor in the CEFFE was not very high. Applying the BDNF at the concentration found in CEFFE alone did not promote axon regeneration; meanwhile, the use of CEFFE promoted axon regeneration effectively. This phenomenon may be accounted for by the synergistic action of neurotrophic factors. Many trophic factors share a common receptor; for example, the NGF binds to the P75 receptor, as does the BDNF. In addition, the BDNF and NT-4/5 share a common receptor, namely, trkB[26, 28] [29, 30]. Various factors may share receptors, activating downstream cascade signals. Concurrently, different factors may act through different signaling pathways, which together may promote axon regeneration. Rho-ROCK and PTEN-mTOR pathways, two signaling pathways that control axon regeneration, were activated by CEFFE [5, 31–33]. As shown in Fig. 4, ROCK2 and PTEN, well-known inhibitors of axon regeneration in the pathways mentioned above, reduced in CEFFE treated group.
The stem cell secretome is defined as a mixture of soluble factors and membrane vesicles, which comprises neurotrophins, microRNAs, and hormones, among others[34, 35]. The secretome from the ADSCs has been used in pre-clinical studies of traumatic brain injury[36–38]. Moreover, in some studies, the secretome from the ADSCs emerged as superior to that from the BMSCs in the treatment of neurodegenerative diseases[39, 40]. Unlike the secretome of the ADSCs, CEFFE includes bioactive extracellular and intracellular components, as shown by our proteomics findings. Some proteins, such as Signal transducer and activator of transcription 3 (STAT3), Ras-related C3 botulinum toxin substrate 1 (RAC1), and cell division control protein 42 homolog (CDC42), have been shown to regulate axon regeneration[41–43]. The concentration of some factors in CEFFE is much higher than that in the secretome from stem cells[44–46]. For example, in the present study, the concentration of the BDNF in CEFFE was approximately 1860.99 pg/ml, while that reported in other studies was approximately 13.22 or 37.03 pg/ml[8, 44, 45]. Inflammatory factors play an important role in promoting axon regeneration[14, 47]. In CEFFE, the concentration of inflammatory factors was higher than that observed elsewhere.
Gene editing technology allows to transform the injured neuron into a regenerative state by manipulating a certain gene[5, 13, 48]. Promoting neuronal survival and axon regeneration is among basic strategies to repair injuries in mature CNS. However, although changing intrinsic regulators can affect axon regeneration or neuronal survival, a single gene manipulation may also cause dissonance in axon regeneration or neuron survival, as is the case of Armcx and Sox11, among others[49, 50]. Our study has shown that the use of the CEFFE may satisfy both conditions, as it reduces the expression of cleaved-calpain that suppresses RGCs apoptosis , and the expression of p-mTOR indicated inhibition of autophagy allowing injured RGCs to survive. CRMP2 can stabilize cytoskeletal polymerization, leading to axonal growth.
This study has some limitations. From the results we obtained, we prefer to consider CEFFE work in the manner of a whole unit. However, given the large and complex network of factors involved, we could not determine the exact combination of CEFFE factors that affects neuron regeneration. The effects of CEFFE may involve several mechanisms, including activating signaling and pro-inflammatory pathways, and promoting vascularization; we could not determine which of these pathways was the main mechanism of action. Future studies are required to elucidate these mechanisms. Our future research aims to examine the duration of CEFFE action in vivo and to optimize its active components for potential clinical applications.