Significance: Ferroptosis is featured by the accumulation of polyunsaturated-lipid peroxidation on cellular membranes in an iron-dependent manner. Ferroptosis has been implicated in various pathophysiological processes, including cancer, neurodegeneration, and ischemia-reperfusion tissue injury. However, our understanding about the dynamic and context-specific regulation of ferroptosis remains incomplete. Recent Advances: As the major substrate for peroxidation, the cellular lipidome regulates ferroptosis sensitivity and execution by controlling the abundance and availability of polyunsaturated-lipids for peroxidative modifications. In turn, the cellular lipidome is regulated by a complex network of enzymes and transporters, as well as upstream layers of receptors, kinases, and transcription factors. A number of research has shed light on the link between lipid metabolism and ferroptosis. Here, we summarize our current knowledge on the role of the lipidome and associated protein regulators in various stages of ferroptosis, ranging from initiation, execution to cell death evasion by cells experiencing ferroptotic stress. Critical Issues: This review provides an overview of the mechanisms underlying lipid peroxidation and ferroptosis by discussing the lipid species that directly contribute to lipid peroxidation and ferroptosis, how cells regulate the abundances of these pro-ferroptosis lipids, how lipid peroxidation causes cell death, and how cells prevent and repair membrane lipid damage under ferroptotic conditions. Future Directions: Cell fate regulation in vivo could be different from in vitro culture settings. We envision that a comprehensive and detailed understanding about these important questions in the dynamic regulation of ferroptosis in vivo will accelerate our development of ferroptosis-targeted therapies to improve human health.

Cerebrospinal fluid (CSF) is an important sample source for diagnosing diseases in the central nervous system (CNS), but collecting and injecting CSF in small animals is technically challenging and often results in high mortality rates. Here, we present a cost-effective and efficient method for accessing the CSF in live rodents for fluid collection and infusion purposes. The key element of this protocol is a metal needle tool bent at a unique angle and length, allowing the successful access of the CSF through the foramen magnum. With this method, we can collect 5–10 µL of the CSF from mice and 70–100 µL from rats for downstream analyses, including mass spectrometry. Moreover, our minimally-invasive procedure enables iterative CSF collection from the same animal every few days, representing a significant improvement over prior protocols. Additionally, our method can be used to inject solutions into mice cisterna magna with high success rates and high postoperative recovery rates. In summary, we provide an efficient and minimally-invasive protocol for collecting and infusing reagents into the CSF in live rodents. We envision this protocol will facilitate biomarker discovery and drug development for diseases in the central nervous system.