Testing the suitability of neuroanatomical tracing method in human fetuses with long years of postmortem delay

Human tissues in gross anatomical archives with long years of postmortem delays are considered suboptimal relative to recently fixed materials for neuroanatomical tracing studies, yet efficacy of neuroanatomical tracing on archival fetal tissues largely unexplored. We aimed to explore the suitability of human archival tissue in neuroanatomical tracing with lipophilic carbocyanine dyes.

We used crystal and paste forms 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) and analogues for neuroanatomical tracing on different peripheral nerves in 15-18-year archival old formalin-fixed human fetuses. We employed bright-field, fluorescent and confocal microscopy to visualize the peripheric nerve traces, spinal cord and vibratome cut sections. Fluorescent signal of the dyes on epineurium and on axonal membranes were visualized under fluorescence and confocal microscopes and performance of the dye diffusion was assessed by semi-quantitative image analysis.

We followed up seven lipophilic dye embeddings in 16-28 gestational week-old human fetuses (n = 4) with 16.75 ± 1.29-year postmortem delay. The mean distance of distally moved carbocyanine dye diffusion measured on epineurium was detected as 25.11 ± 9.1 mm.

Based on the results of 13 distinct studies performed neuroanatomical tracing with human tissues in the immediate postmortem hours or days, average traced distance was 16.32 ± 15.95 mm, and a 95% confidence interval lower limit of 4.9 mm and upper limit of 27.73 mm. The tracing distances we observed in our current study fall entirely within this confidence interval. To our awareness, this is the first report to demonstrate that specific neuroanatomical tracing presented in axonal membrane level on peripheral nerves is possible on gross anatomical repositories.