Escola Universitária Vasco da Gama (EUVG), Centro de Investigação Vasco da Gama (CIVG), EUVG, Coimbra, Portugal; Faculdade de Medicina da Universidade de Coimbra (FMUC), Coimbra, Portugal; Centro de Ciência Animal e Veterinária (CECAV), Universidade de Trás-os-Montes e Alto Douro (UTAD), Vila Real, Portugal.
Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Porto, Portugal; Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Porto, Portugal.
Neurotoxicology. 2023 Jul;97:78-88. doi: 10.1016/j.neuro.2023.05.008. Epub 2023 May 15.
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.
斑马鱼因其广泛的神经发生和再生能力以及一些生物学优势而闻名，这些优势使其成为多个研究领域的相关动物模型，特别是在毒理学研究中。氯胺酮是一种众所周知的麻醉剂，在人类和兽医医学中都有使用，因为它具有安全性、作用时间短和独特的作用模式。然而，氯胺酮的给药与神经毒性和神经元死亡有关，这使得它在儿科医学中的应用存在问题。因此，评估神经发生早期阶段氯胺酮的给药效果至关重要。斑马鱼胚胎发育的 1-41-4 体节阶段对应于分段和神经管形成的开始。在这种物种以及其他脊椎动物中，纵向研究很少，对成年动物中氯胺酮的长期影响也知之甚少。本研究旨在评估在早期和成年神经发生过程中，在亚麻醉和麻醉浓度下，于 1-4 体节阶段（受精后 10.5 小时 - hpf）给予氯胺酮对脑细胞增殖、多能性和死亡机制的影响。为此，将处于 1-4 体节阶段（10.5 hpf）的胚胎分为研究组，并暴露于 0.2/0.8 mg/mL 的氯胺酮浓度下 20 分钟。动物生长至特定的检查点，即 50 hpf、144 hpf 和 7 个月龄成年。通过 Western-blot 和免疫组织化学评估增殖细胞核抗原（PCNA）、性别决定区 Y 盒 2（Sox 2）、凋亡诱导因子（AIF）和微管相关蛋白 1 轻链 3（LC3）的表达和分布模式。结果表明，在最高浓度的氯胺酮（0.8 mg/mL）下，144 hpf 幼虫中出现了主要的变化，即自噬和细胞增殖。然而，在成年动物中没有发现显著的变化，这表明其恢复到了一个稳定状态。本研究澄清了关于氯胺酮给药的纵向作用的一些方面，这些作用与中枢神经系统增殖和激活适当的细胞死亡和修复机制的能力有关，从而导致斑马鱼的体内平衡。此外，结果表明，在亚麻醉和麻醉浓度下于 1-4 体节阶段给予氯胺酮，尽管在 144 hpf 时会产生一些暂时的有害影响，但对中枢神经系统是长期安全的，这是该研究领域的新的有前景的结果。
