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

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Abstract

Purpose

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.

Methods

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.

Results

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.

Conclusion

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.

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taken from the adjacent areas of the single nerve piece in B are shown, respectively (C, D). The length (mm) of the intervals as well as the whole course of the nerved are marked (B). Boxed areas on the nerve course, marked as 9A-E are the portions that were transversely vibratome cut and visualized on confocal microscope (D, please see Fig. 9). The graph presented on the right bottom corner of the figure, shows the fascicular DiI labeling per section presented with ratio of fluorescent intensity measured in fascicules corresponding to the boxed areas, indicated as 9A-E in Fig. 8D. Data in the graph are presented in mean ± standard deviation of fluorescent intensity ratio per boxed area. CFN common fibular nerve, SN sciatic nerve, TN tibial nerve. Markings of 9A-E seen in D are the region of interests that processed for vibratome sections and confocal microscope evaluation of these are represented in Fig. 9

Fig. 9

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Acknowledgements

We thank to Assistant Prof. Maria Veldhuizen and Prof. A. Hakan Öztürk, from the Anatomy Department of Mersin University, Faculty of Medicine for their precious discussions with us on our work and comments on the latest version of the manuscript. We greatly appreciate the guidance and support of Professors Reha Erzurumlu and Feng C. Zhou, from the Anatomy and Neurobiology Department of Maryland University School of Medicine and Anatomy and Cell Biology Department of Indiana University School of Medicine, respectively.

Funding

The authors have no financial or personal relationship with any third party whose interests could be positively or negatively influenced by the article’s content. Most of the research consumables used in this study was belong to the core facility of the Anatomy Department of Mersin University, Faculty of Medicine and the rest were supported by Scientific Research Projects Unit of Mersin University (2020–1-AP5-4104) to ÖNC.

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Authors and Affiliations

  1. Department of Anatomy, Faculty of Medicine, Mersin University, Mersin, Turkey

    Nail Can Öztürk & Turan Koç

  2. Biotechnology Research Center, Mersin University, Mersin, Turkey

    Nail Can Öztürk

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  1. Nail Can Öztürk
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  2. Turan Koç
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ÖNC—project development, data collection and analysis, supervision, and manuscript writing. KT—data collection and management, and help to organize the manuscript.

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Correspondence to Nail Can Öztürk.

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This material has not been published in whole or in part elsewhere. The manuscript is not currently being considered for publication in another journal. The anatomical protocol of the study was accepted by the by Mersin University, Ethics Board of Clinical Research (Approval # 2019/513). Postmortem human fetuses used in this study are legally registered in the educational and research gross anatomical archive of the Anatomy Department of Mersin University, Faculty of Medicine. This study conforms to recognized standards of Helsinki Declaration.

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Öztürk, N.C., Koç, T. Testing the suitability of neuroanatomical tracing method in human fetuses with long years of postmortem delay. Surg Radiol Anat 44, 769–783 (2022). https://doi.org/10.1007/s00276-022-02942-7

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