Longitudinal effects of ketamine on cell proliferation and death in the CNS of zebrafish.

Zebrafish is known for its widespread neurogenesis and regenerative capacity, as well as several biological advantages, which turned it into a relevant animal model in several areas of research, namely in toxicological studies. Ketamine is a well-known anesthetic used both in human as well as veterinary medicine, due to its safety, short duration and unique mode of action. However, ketamine administration is associated with neurotoxic effects and neuronal death, which renders its use on pediatric medicine problematic. Thus, the evaluation of ketamine effects administration at early stages of neurogenesis is of pivotal importance. The 1-41-4 somites stage of zebrafish embryo development corresponds to the beginning of segmentation and formation of neural tube. In this species, as well as in other vertebrates, longitudinal studies are scarce, and the evaluation of ketamine long-term effects in adults is poorly understood. This study aimed to assess the effects of ketamine administration at the 1-4 somites stage, both in subanesthetic and anesthetic concentrations, in brain cellular proliferation, pluripotency and death mechanisms in place during early and adult neurogenesis. For that purpose, embryos at the 1-4 somites stage (10.5 h post fertilization - hpf) were distributed into study groups and exposed for 20 min to ketamine concentrations at 0.2/0.8 mg/mL. Animals were grown until defined check points, namely 50 hpf, 144 hpf and 7 months adults. The assessment of the expression and distribution patterns of proliferating cell nuclear antigen (PCNA), of sex-determining region Y-box 2 (Sox 2), apoptosis-inducing factor (AIF) and microtubule-associated protein 1 light chain 3 (LC3) was performed by Western-blot and immunohistochemistry. The results evidenced the main alterations in 144 hpf larvae, namely in autophagy and in cellular proliferation at the highest concentration of ketamine (0.8 mg/mL). Nonetheless, in adults no significant alterations were seen, pointing to a return to a homeostatic stage. This study allowed clarifying some of the aspects pertaining the longitudinal effects of ketamine administration regarding the CNS capacity to proliferate and activate the appropriate cell death and repair mechanisms leading to homeostasis in zebrafish. Moreover, the results indicate that ketamine administration at 1-4 somites stage in the subanesthetic and anesthetic concentrations despite some transitory detrimental effects at 144 hpf, is long-term safe for CNS, which are newly and promising results in this research field.