Our research represents the first systematic review and meta-analysis dedicated to exploring the therapeutic potential of Rapamycin in rodent models of SCI. Our findings demonstrate significant reductions in apoptosis, inflammation, and astrogliosis, along with a substantial increase in autophagic markers. These outcomes correspond with a significant improvement in long-term locomotion recovery following Rapamycin administration in the injured animals (Fig. 5).
Apoptosis, often referred to as programmed cell death, plays a pivotal role in the development of secondary injury after SCI (29). Mitochondria-associated cell death is among the critical mechanisms triggered by SCI (30). This process involves key proteins from the Bcl-2 family, including Bcl-2, Bax, and the executioner Caspase. When cells undergo apoptosis, Bax adheres to the outer mitochondrial membrane, forming pores through oligomerization. This pore formation allows the release of apoptogenic factors like Cytochrome C into the cytoplasm. Bcl-2, a(31)n anti-apoptotic protein, can inhibit apoptosis by binding to Bax, preventing the formation of these pores. In the cytoplasm, Cytochrome C contributes to the formation of the apoptosome, which activates Caspase-9. Activated Caspase-9 then transforms Pro-caspase-3 into effector Caspase-3, leading to the typical morphological changes observed in apoptotic cells (32, 33). Our study also reveals that Rapamycin treatment results in an increase in the anti-apoptotic protein Bcl-2 and a decrease in the pro-apoptotic protein Bax. While it is important to note that individual studies examining Bax levels in the Rapamycin-treated group show a significant increase, pooled data analysis did not demonstrate a significant overall change. This discrepancy may be attributed to the limited number of studies included in the meta-analysis. Furthermore, we observed an increase in the executioner caspase, Caspase-3, suggesting that Rapamycin has complex effects on the regulation of apoptosis. Specifically, it appears that Rapamycin suppresses the mitochondrial apoptosis pathway. Additionally, our assessment of Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assays to detect apoptotic cells undergoing extensive DNA degradation during the late stages of apoptosis (34), demonstrated a decrease in the groups treated with Rapamycin, further confirming the inhibitory role of Rapamycin in the apoptotic pathway.
Autophagy is the process by which cells digest their components to maintain cellular homeostasis (35). This autodigestion increases in neurons within hours after SCI, likely serving as a neuroprotective mechanism (36). The initiation of autophagy relies on the key regulatory protein Beclin-1 (37). LC3-II, a protein involved in forming autophagosomes for degradation, also reflects autophagy levels (38). Our study found that Rapamycin significantly increased Beclin-1 and LC3-II, indicating enhanced autophagic flux. Importantly, p62, which targets proteins for autophagic degradation, was lower with Rapamycin treatment in two studies (6, 20). This decline in p62, which binds to ubiquitinated proteins and targets them for degradation via autophagy, confirms that Rapamycin truly enhances autophagic digestion rather than just accumulating autophagosomes (39).
Post-trauma inflammation after SCI is characterized by increased pro-inflammatory cytokines such as TNF-α and IL-1β, attracting immune cells like macrophages and microglia, which exacerbate the inflammatory response. This inflammation contributes to secondary neuronal damage through various pathways (15, 23). Studies have shown that Rapamycin treatment can reduce inflammation by attenuating the mTOR pathway in microglia, decreasing their activation and production of pro-inflammatory cytokines (40). Our study aligns with previous research, as it demonstrates that Rapamycin significantly lowers levels of TNF-α after injury. While an IL-1β meta-analysis was not conducted due to limited studies, the two studies included also indicated significantly lower levels of IL-1β in the Rapamycin treatment group compared to the injury control group (23, 27). By dampening this inflammatory cascade, Rapamycin might help limit secondary damage after SCI.
GFAP, an intermediate filament protein expressed predominantly in astrocytes, is commonly used to identify astrocytes and to examine astrogliosis, referring to reactive astrocyte proliferation in response to injury. Activated astrocytes secrete proinflammatory cytokines and contribute to glial scar formation and inhibit axonal regeneration (14, 16). Studies suggest Rapamycin inhibits STAT3 signaling in astrocytes, which is known to promote reactive astrocytosis. By inhibiting STAT3 activation, Rapamycin reduces GFAP expression and other markers of astrocyte reactivity (41). Additionally, by inhibiting mTOR, Rapamycin shifts astrocyte metabolism from glycolysis to more efficient oxidative phosphorylation, which could be associated with reduced astrocyte reactivity (14). Consistent with these mechanisms, our study shows that Rapamycin reduces GFAP expression after SCI, potentially creating a more favorable environment for nerve regeneration.
Alpha-motor neurons, also known as lower or skeletal motor neurons, innervate muscle fibers (42). Our study indicates that there are more surviving alpha-motor neurons in the spinal cord of the Rapamycin-treated group compared to the controls. Rapamycin likely provides neuroprotection by decreasing microglial and astrocytic activation and increasing neuronal autophagy, thereby reducing secondary damage after injury (14, 15).
Rapamycin inhibits mTOR Complex 1 (mTORC1), a key regulator of cell growth and metabolism (43). mTORC1 normally phosphorylates and activates p70S6 Kinase (p70S6K), which promotes protein synthesis and cell growth (15, 44). Akt is a protein kinase that activates mTORC1 by phosphorylating and inhibiting TSC1/TSC2, negative regulators of mTORC1 (45). The p-Akt protein expression was assessed in four studies; two studies measured p-Akt/β -actin and two studies measured p-Akt/Akt. Therefore, data pooling did not apply to a meta-analysis. However, studies showed increased p-Akt levels within the first week of Rapamycin therapy compared to controls in SCI models, likely due to the negative feedback on PI3K/Akt signaling when mTORC1 is inhibited (3, 18, 27). However, one study found decreased p-Akt at 2- and 4 weeks post-injury (6). Considering Rapamycin's half-life, the early feedback-driven p-Akt upregulation may peak and normalize after prolonged treatment, leading to observable declines by 2 weeks.
Our research uncovers various potential explanations for how Rapamycin contributes to enhancing motor function recovery. Firstly, we observed that Rapamycin effectively prevents apoptosis. Secondly, it reduces astrogliosis and inflammation. Thirdly, it promotes autophagy at the site of the injury. On the flip side, treatment with the mTOR inhibitor Rapamycin hampers the Akt/mTOR/p70S6K signaling pathway and decreases the expression of proteins related to myelin formation in the damaged spinal cord (6). Additionally, mTORC1 inhibition leads to the suppression of p70S6K, responsible for promoting protein synthesis and cell growth (15). Consequently, these findings have given rise to conflicting hypotheses. In alignment with these controversies, our study demonstrated a lack of significant short-term improvement and only low to moderate long-term enhancements in motor function.
Our study has limitations. It's important to acknowledge that the literature has only reported a limited number of underlying pathways, and there is some degree of heterogeneity. This emphasizes the need for caution when interpreting these findings. Additionally, certain variables were too scarce to be combined, including the severity of the injury, the timing of treatment, the number and routes of administered doses, robust follow-up evaluations, and some outcomes were solely assessed through one method, such as TNF-α for inflammatory markers and p70S6K for Akt/mTOR/p70S6K signaling pathway. These considerations underscore the necessity for further experimental research in this field to ensure the generalizability of results. Another shortcoming arises from the low quality of the animal studies, all of which were judged to carry a high risk of bias. This limitation stems from the studies' failure to include some major elements outlined in CYRCLE's risk of bias tool. It is important to note that researchers cannot be faulted for this omission, as the ARRIVE guidelines for animal research do not mandate reporting on most of these items (46). Finally, there was evidence of publication bias in the assessment of locomotion at 2- and 3-weeks post-injury, as well as in p-P70S6K. This bias was predisposed by the limited number of studies for each outcome.
In conclusion, Rapamycin demonstrates neuroprotective, anti-inflammatory, and pro-autophagic effects in rodent SCI models, leading to modest long-term improvements in motor function recovery versus controls. The low- to moderate-level evidence from this study emphasizes the necessity for more rigorous experimental investigations to thoroughly assess the effectiveness of Rapamycin treatment in animal models with SCI.