Dorsal horn mapping: a Fiji toolbox for standardized laminar registration and transparent reporting of spinal cord imaging data from adult C57Bl/6 mice.

Introduction: Rexed's laminae provide the anatomical framework for interpreting the spatial organization of neuronal populations within the spinal dorsal horn and are therefore widely used to describe immunohistochemical, Methods: We developed Dorsal Horn Mapping, an open-source Fiji toolbox that implements a standardized workflow for laminar registration in transverse mouse spinal cord sections. The toolbox aligns segment-specific reference maps to experimental images using user-guided rigid transformations and independent scaling, assigns labeled cells to Rexed's laminae, and transforms their positions into a common spatial reference frame. In addition, it provides tools for quantitative laminar analysis and visualization of pooled datasets as composite reference maps. Results: Validation experiments demonstrated high inter-user reproducibility of template alignment and laminar assignment across users with different levels of anatomical expertise. Registrations showed good agreement with established laminar markers and resulted in highly consistent assignment of neuronal populations to individual laminae. Thus, 81% of IB4-pos pixels were mapped to lamina II (95% CI: 68.4-86.2%) and the estimated difference in cell counts averaged across all users, segments and laminae was 0.08 cells (95% CI: -0.68 to 0.83; Conclusion: Dorsal Horn Mapping provides an accessible framework for standardized laminar registration and transparent spatial reporting of spinal cord imaging data from adult C57BL/6 mice. By introducing a common spatial reference system for dorsal horn anatomy, the toolbox facilitates direct comparison of cellular localization across experiments and laboratories and has the potential to improve reproducibility, comparability, and reporting standards in pain and spinal cord research.