SKUF Protocol: Slice, Keep, Unwrap, Fuse-A Pilot Multimodal Approach to Cardiac Innervation Mapping

Background/Objective: Cardiac innervation plays a critical role in regulating myocardial function and enabling the heart to adapt to physiological and pathological conditions. Although the general features of sympathetic and parasympathetic innervation of the myocardium are well described, the spatial organisation of nerve fibres within the cardiac muscle remains incompletely characterised. This study aimed to develop and validate the SKUF (Slice-Keep-Unwrap-Fuse) protocol, a multimodal framework for mapping myocardial innervation through the integration of histological data and magnetic resonance imaging (MRI). Methods: The study was performed on the heart of a 7-year-old patient who died from rupture of a cerebral vascular malformation without evidence of cardiovascular disease. Prior to histological processing, post-mortem MRI was performed to provide a precise anatomical reference. The heart was sectioned into sequential transverse rings of 4 mm thickness, yielding 71 paraffin blocks. Histological sections (3 μm) were immunostained with antibodies against UCHL-1 to visualise nerve fibres and scanned using an Aperio AT2 system (20× magnification). Automated image analysis was conducted using the SVSSlide Processor module, which included tissue segmentation, colour-based nerve fibre detection, and sliding-window density mapping. Heatmaps were assembled into ring-based myocardial reconstructions and co-registered with MRI slices using combined rigid and deformable registration, followed by three-dimensional reconstruction of innervation patterns. Results: A higher density of nerve fibres was observed in the right ventricular myocardium compared with the left ventricle, whereas larger nerve trunks were identified in the epicardium of the left ventricle. Quantitative analysis revealed a pronounced longitudinal gradient of innervation, with minimal density in the apical region and progressive increases towards the mid-ventricular segments, where maximal density and spatial organisation of neural structures were observed. The atrioventricular groove exhibited the greatest heterogeneity of innervation due to the presence of large nerve trunks and ganglionated plexuses. Integration of histological maps with MRI enabled three-dimensional visualisation of spatial clusters of nerve fibres. Conclusions: The SKUF protocol provides a robust framework for integrating histological and MRI data to generate three-dimensional maps of myocardial innervation. This approach may facilitate the development of high-resolution anatomical atlases of cardiac innervation and support future studies of neurocardiac mechanisms of arrhythmogenesis and targeted neuromodulation.