APPLICATION OF COMPLEMENTARY METHODS BEYOND STANDARD PATHOLOGY TO INVESTIGATE KETAMINE NEUROTOXICITY IN MODELS OF PEDIATRIC ANESTHESIA

Ketamine is widely used as a pediatric anesthetic; however, growing evidence suggests that exposure during critical windows of brain development may induce neurotoxicity. This thesis aimed to preclinically characterize ketamine-induced neurotoxicity in the rat neonatal brain, elucidate the affected cell populations and mechanisms, and refine both in vivo methodology and human-relevant new approach methods (NAMs). A standardized in vivo model was established using Sprague-Dawley rats at postnatal day 7, a period of intense synaptogenesis and high vulnerability to anesthetic injury. Repeated intraperitoneal ketamine administrations (5 x 20 mg/kg at 90-minute intervals) produced a prolonged anesthetic state and neurotoxic effects were evaluated in cortex, hippocampus, and corpus callosum using a panel of histological and immunohistochemical markers (H&E, Cleaved Caspase-3, γH2AX, GFAP, Iba-1, Olig2, NeuN). A preliminary study compared whole-area analysis with standardized microscopic field sampling, demonstrating that 12 fields per region provided quantitative results comparable to whole-area assessment. A subsequent methodological study systematically tested progressive field reduction using correlation, agreement, and group-comparison statistics, confirming that this level of sampling remains necessary for robust quantification of apoptosis and DNA damage, whereas some glial and neuronal markers can be reliably assessed with fewer fields. Across in vivo studies, ketamine consistently induced transient but marked apoptosis and DNA damage, peaking 2 hours after the last injection and returning to baseline by 16 hours, with no sex-dependent differences. γH2AX emerged as a sensitive marker of ketamine-induced DNA damage, paralleling apoptosis, detected section and Cleaved Caspase-3. The inclusion of both vehicle-treated and unmanipulated control groups demonstrated that neither handling nor vehicle administration significantly influenced apoptotic or cellular endpoints, excluding a procedure-related effect. Double immunofluorescence and confocal microscopy confirmed that neurons and oligodendrocytes are the primary apoptotic population, while microglia and, to a lesser extent, astrocytes predominantly contribute to phagocytosis of neuronal apoptotic debris. Detection of Cleaved Caspase-9 in affected cells supported the activation of the intrinsic, mitochondria-dependent apoptotic pathway, consistent with a mechanism involving oxidative stress, mitochondrial dysfunction, and downstream caspase activation. A pharmacokinetic study following a single ketamine injection confirmed rapid brain penetration and clearance of the parent drug, supporting the rapid onset of observed effects and guiding to selection of relevant concentrations for in vitro testing. Finally, ketamine effects were investigated in a human iPSC-derived BrainSphere model cultured on microelectrode arrays. Ketamine produced a rapid, concentration-dependent reduction in spikes, bursts, and network synchronization, followed by partial recovery and rebound hyperexcitability after washout, differentially affecting glutamatergic, GABAergic, and dopaminergic components. These findings confirmed NMDA receptor-mediated modulation of human neuronal networks and established a pharmacologically relevant NAM that captures key functional aspects of ketamine action. Overall, this work provides a rigorously characterized neonatal rat model, an optimized quantitative method of digital image analysis, and a complementary human in vitro system, advancing understanding of the temporal and cellular mechanisms underlying ketamine-induced neurotoxicity in the developing brain.