gms | German Medical Science

72. Jahrestagung der Deutschen Gesellschaft für Neurochirurgie (DGNC)
Joint Meeting mit der Polnischen Gesellschaft für Neurochirurgie

Deutsche Gesellschaft für Neurochirurgie (DGNC) e. V.

06.06. - 09.06.2021

Preparation and visualisation of murine subarachnoid space (SAS) for histological and electron microscopy (EM) analyses

Präparation und Visualisierung des murinen Subarachnoidalraumes (SAR) für histologische und elektronenmikroskopische (EM) Analysen

Meeting Abstract

  • presenting/speaker Katharina Tielking - Charité Universitätsmedizin, Klinik für Neurochirurgie, Berlin, Deutschland
  • Ran Xu - Charité Universitätsmedizin, Klinik für Neurochirurgie, Berlin, Deutschland
  • Sara Timm - Charité Universitätsmedizin, Institute of Vegetative Anatomy, Berlin, Deutschland
  • Petra Schrade - Charité Universitätsmedizin, Institute of Vegetative Anatomy, Berlin, Deutschland
  • Felix Thomas Schoknecht - Charité Universitätsmedizin, Klinik für Neurochirurgie, Berlin, Deutschland
  • Matthias Ochs - Charité Universitätsmedizin, Institute of Vegetative Anatomy, Berlin, Deutschland
  • Peter Vajkoczy - Charité Universitätsmedizin, Klinik für Neurochirurgie, Berlin, Deutschland

Deutsche Gesellschaft für Neurochirurgie. 72. Jahrestagung der Deutschen Gesellschaft für Neurochirurgie (DGNC), Joint Meeting mit der Polnischen Gesellschaft für Neurochirurgie. sine loco [digital], 06.-09.06.2021. Düsseldorf: German Medical Science GMS Publishing House; 2021. DocP168

doi: 10.3205/21dgnc450, urn:nbn:de:0183-21dgnc4504

Published: June 4, 2021

© 2021 Tielking et al.
This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 License. See license information at http://creativecommons.org/licenses/by/4.0/.


Outline

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Objective: Histological analyses and EM visualization of local effects after subarachnoid hemorrhage (SAH) require a preservation of the anatomical structure of the SAS. However, systematical approaches for conserving SAS in animal models of SAH are scarce in the literature. Hence, we aimed at establishing a protocol to preserve and depict the SAS for further immunohistochemical and EM analyses.

Methods: Male C57Bl/6 mice were operated, and they received either sham operation (n=6) or filament-perforation surgery to induce SAH (n=6). Magnetic Resonance Imaging was performed 24 hours post-operatively to confirm the bleeding. For immunohistochemical analyses, mice were perfused with 4% PFA and the whole skull was harvested. After 12 days of dehydration with an ascending sequence of sucrose, skulls were embedded and carefully cut upside-down with the microtome (Figure 1A [Fig. 1]). Confirmation of anatomical orientation was obtained using Haematoxylin and Eosin (H&E) stainings and immunofluorescence with DAPI, the arachnoid cell marker AKAP12 and endothelial marker cd31. For EM imaging of the SAS, mice were perfused with 2,5% glutaraldehyde, and whole skulls were harvested and a 5*5 mm cube with our region of interest (ROI) was carefully dissected. In the next steps, fixation, fine preparation and documentation of the ROI followed to facilitate orientation with high-magnification EM. After postfixation steps and dehydration of the tissue, samples were coated with gold-palladium as a final step before EM imaging (Figure 1B [Fig. 1]).

Results: The anatomical structures of the SAS were well-preserved in IF staining and EM imaging. Immunohistochemical display of SAS allowed specific analyses of intra- and extracellular compartments in the SAS (Figure 1C [Fig. 1]). EM imaging showed maintained subarachnoid ultrastructure in control and SAH animals (Figure 1D [Fig. 1]). In SAH condition, we observed a remarked collection of RBCs (red blood cells) and affinity towards arachnoid granulations.

Conclusion: With this study, we offer a technically feasible and reproducible approach for preservation and illustration of the SAS for IF stainings and EM imaging. These techniques can be potentially utilized for localization-specific analyses to the SAS in SAH.