Enhancing Experimental Rigor and Reproducibility in Laboratory Animal Research on the gut–brain–immune Axis

Laboratory animal models are essential for investigating neuroimmune and systemic biological processes; however, experimental outcomes are frequently shaped by unrecognized biological and methodological variability. Increasing evidence indicates that coordinated dysfunction across multiple epithelial interfaces—including gastrointestinal, dermal, and respiratory barriers—represents an underappreciated determinant of experimental variability in laboratory animal research. In standardized rodent studies, such barrier interdependence may contribute to systemic inflammation, neuroimmune priming, and inconsistent experimental outcomes. This narrative review synthesizes evidence from laboratory animal science and comparative pathology to propose a unified multi-barrier framework for interpreting experimental validity in gut–brain–immune (GBI) axis research. We examine how epithelial barrier disruption, metabolic and mitochondrial stress, and innate immune activation—particularly sterile inflammatory pathways involving NLRP3 signaling—interact to influence central nervous system homeostasis and commonly measured neuroimmune and behavioral readouts. Emerging concepts, including epigenetic programming and glymphatic clearance, are discussed as integrative mechanisms linking peripheral physiological stress to central outcomes. The review further highlights environmental stressors, housing conditions, species- and strain-specific susceptibility, and perimortem tissue handling as major yet frequently overlooked confounders in laboratory animal studies. Comparative observations from wild and captive species are incorporated as sentinel indicators of barrier vulnerability within a One Health perspective. Finally, methodological refinements, including nano-enabled delivery platforms, are discussed as experimental tools to improve dosing consistency and mechanistic interpretability. By framing experimental variability through a laboratory animal–centered, multi-barrier perspective, this review aims to support improved experimental design, enhance reproducibility, and strengthen translational integrity while reinforcing principles of ethical refinement.