Innovative therapies for spinal cord injury : a zebrafish approach

Spinal cord injury (SCI) is a debilitating condition that affects millions of people worldwide. It leads to devastating motor and sensory dysfunctions and elicits cardiac and respiratory alterations, bladder and bowel dysfunction, as well as, sexual dysfunction and other complications. This complex neurological condition has a huge impact on the health, quality and life expectancy, thus constituting a high socioeconomic and psychological burden for patients and their families. After a traumatic SCI, current management approaches consist in the complete immobilization and constant monitorization of the patient (blood pressure, respiratory and cardiac alterations), administration of anti-inflammatory drugs, surgical decompression of the spinal cord to stabilize the spinal column and rehabilitative care. However, none of these efforts result in a cure for SCI patients. In fact, due to the complexity and multitude of SCI facets, there is still no effective treatment available on the market for this indication. The biphasic SCI pathophysiology comprises primary and secondary injuries which trigger a complex cascade of events that can lead to beneficial or detrimental responses. However, the reparative responses are limited and the fibroglial scar formation and the inhibitory microenvironment constitute the main obstacles for SCI repair in mammals. In contrast to mammalian models, zebrafish show robust spinal cord recovery without a fibroglial scar formation. Thus, in addition to having emerged as a valuable tool for drug discovery, zebrafish has also emerged as an attractive model for the study of SCI. In fact, despite having different outcomes, zebrafish and mammals share a considerable number of disease-associated targets and drug metabolic pathways. Therefore, the idea of developing a phenotypic-based in vivo screening using a larval zebrafish model of SCI to select bioactive chemical compounds with therapeutic potential to SCI, becomes particularly appealing. Thus, we designed a simple, fast and efficient drug screening platform using a zebrafish larval spinal cord transection model. This approach allows the selection of small molecules with locomotor rescue properties in this zebrafish larva model of SCI. We validated our screening platform by showing that Riluzole and Minocycline, two different pharmacologic classes of molecules that have entered in clinical trials for SCI indication, promote rescue of the motor function of the transected larvae. Further validation of the platform was demonstrated through the blind identification of D-Cycloserine, a molecule scheduled to enter phase IV clinical trials for SCI. Next, we used this larval zebrafish drug discovery platform to blindly screen 113 bioactive compounds from a FDA-approved small molecule library. As we used known FDA-approved drugs, we took advantage of previous studies and reports to further select three candidate compounds, Tranexamic acid, Pefloxacin mesylate and Eletriptan Hbr that showed significant motor rescue properties in the pro-regenerative SCI model. As we intended to validate the translational value of zebrafish larvae in the SCI context, we tested the therapeutic efficacy of the three candidate compounds in a rodent contusion (pro-fibrotic) model of SCI. We showed that Pefloxacin mesylate did not preserve its therapeutic effect on motor function in contused mice but exhibited a therapeutic effect on another important SCI sequel, the bladder dysfunction. Importantly, we showed that Tranexamic acid and Eletriptan Hbr molecules maintain their locomotor recovery properties in T9-contused mice. We showed that Tranexamic acid reduces the extension of the lesion and we proposed that its therapeutic effect on motor function could be explained by a possible role in limiting the toxic effect of the parenchymal haemorrhage, thereby controlling the extension of the lesion and improving the locomotor outcome of SCI animals. Besides proving that the therapeutic effect of Eletriptan Hbr was preserved in a mammalian SCI model, we also explore its therapeutic potential in a second rodent study. This second study aimed to determine if a longer therapeutic administration would result in greater improvement in the long term and allowed to further evaluate its therapeutic effect on the demyelination status, fibrotic scar and microglia/macrophages at a chronic phase of the injury. We suggested that perhaps the locomotor improvement properties of Eletriptan Hbr could be explain by a possible mediation by PDGFRβ+ cells. Additionally, we also demonstrated that Eletriptan Hbr led microglia to display a morphology that is more similar to that one characteristic of moderately activated microglia which suggests that maybe Eletriptan Hbr has the ability to modulate the inflammatory response. Overall, we demonstrated in this study the predictive value of zebrafish for phenotype-based drug screenings and presented a full proof-of-concept of transected spinal cord zebrafish larval model for the identification of new therapeutics for SCI. Notably, it was presented a simple, fast and automated phenotypic screening with proved efficacy in a mouse contusion model of SCI. Besides demonstrating the translational value of this zebrafish drug screening platform, we also showed the importance of testing the effectiveness of the new molecules identified on this platform, in a mammalian model before translation to clinical trials. Ultimately, this approach combined with drug repositioning allowed to identify a potential new use for two molecules (Tranexamic acid and Eletriptan Hbr) already in use in the clinic for other therapeutic indications. We believe that the strategy here presented, associated with combinatorial therapeutic approaches, promises to accelerate the discover and rapid translation of effective treatments for SCI in humans