Preserving In Vivo Tissue Complexity in a Mice Olfactory Nervous System Model Using Whole-Mount Analysis for Studying Neural Regeneration

Summary

This protocol outlines a complete workflow for preserving the anatomical complexity of the mouse olfactory nervous system. Here we have detailed the complete workflow, including dissection, tissue preparation, and imaging. This protocol can be used to study the olfactory system under normal or abnormal conditions, including its natural repair conditions.

Abstract

In the era of three-dimensional (3D) imaging, standard immunohistochemistry has been recognized as highly limited in studies of pathology and complex human anatomy. By analyzing only narrow slices, larger structures and essential aspects of tissue architecture may lose critical contextual details, leading to incomplete or misleading interpretations. While two-dimensional (2D) sections have supported a basic understanding of histoarchitecture, they are insufficient for capturing the true three-dimensional reality of biological tissues.

Increasing numbers of studies across multiple anatomical regions now adopt techniques that isolate intact anatomical units - flat mount, whole-mount, or en face preparations - combined with high-resolution fluorescent microscopy. These approaches offer a more comprehensive visualization and interpretation of structural organization. This is particularly significant in the olfactory nervous system, where defining complex cellular mechanisms underlying its remarkable regenerative ability requires a full-thickness context. Thin sections risk missing key pathophysiological features that remain poorly defined.

Given these limitations and the knowledge gap surrounding the cellular interactions driving olfactory regeneration, developing a refined whole-mount technique capable of preserving full-thickness tissue complexity was essential. Entire olfactory mucosae were isolated as full-thickness samples, accompanied by an optimized antibody-penetration protocol. High-resolution imaging (EVIDENT SpinSR) and 3D reconstruction enabled accurate visualization of cellular interactions in vivo.

Introduction

The olfactory nervous system (ONS) represents a unique and well‑established model for studying mechanisms of neural regeneration, as it retains the unique capacity for neuronal turnover throughout life¹. However, the potential for clinical translation of this neuro-reparative ability remains limited due to significant gaps in our understanding of the behaviors of the cells in the olfactory system^2,3,4. The cellular responses and anatomical organization of ONS, including primary olfactory neurons, olfactory ensheathing cells (OECs), and perineural....

Protocol

All experiments containing animals and transgenically modified cells were conducted with the approval of the Griffith University Biosafety Committee (NLRD/003/2020_var3) and the Griffith University Animal Ethics Committee (MHIQ/04/23/AEC) in accordance with guidelines of the Australian Commonwealth Office of Gene Technology regulator (OGTR).

NOTE: A total of 15 adult laboratory mice (8–9 weeks) from both sexes were used in the study. Specifically, S100β-DsRed transgenic mice in which all OECs express the bright red protein DsRed under control of the S100β promoter. S100β is part of a wider calcium-binding protein family....

Results

Our whole-mount approach presented here enabled the isolation and preservation of the olfactory mucosa in its entirety. In our case, our focus was on understanding cellular and anatomical changes on both macro (full anatomical view) and micro (cellular level) scales.

The whole-mount approach enables us to collect both epithelial and lamina propria compartments and preserve the spatial relationships between axon bundles, olfactory ensheathing cells (OECs), and fibroblasts – our cells of interes.......

Discussion

The last few decades have slowly witnessed the start of the three-dimensional era, with many innovative approaches developing in the scientific field. For instance, three-dimensional reconstruction approaches, made possible by the development of complex whole‑mount imaging methods paired with high‑resolution fluorescent microscopes, have facilitated a better understanding of several full‑thickness tissue histoarchitectures. These three‑dimensional rendering approaches have been used to define seve.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bovine Serum Albumin (BSA)	Sigma-Aldrich	A3294-50G	Immunohistochemistry
Adobe Illustrator		version 28.5
Anti-beta III Tubulin antibody - Neuronal Marker	Abcam	ab18207
Anti-PDGFR beta antibody [APB5]	Abcam	ab91066
bisBenzimide H 33342 trihydrochloride	Sigma Aldrich	14533
COVERSLIP 24X50MM NO. 1.5 100PCS	Bio-strategy	EPBRCS245015GP	Immunohistochemistry
Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluo 488	Thermo Fisher	A-21206
Dumont #15A Bone Cutter - Epoxy Coated	Daniels Health Laboratory Products P/L	11215-02	Tissue dissection
Dumont #7 Forceps - Biologie/Dumostar	Daniels Health Laboratory Products P/L	11297-10	Tissue dissection
Goat anti-Rat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 647	Thermo Fisher	A-21247
Imaris software	Imaris	version 9.5.0	Imaging
Mayo dissecting scissors	ProSciTech	420SS	Tissue dissection
Paraformaldehyde	Sigma-Aldrich	P6148-500G	Tissue Processing
Phosphate buffered saline, sterile PBS Tablets (PBS)	Thermo Fisher Scientific	18912014	Tissue Processing
ProLong Glass Antifade Mountant	Thermo Fisher Scientific	P36980	Immunohistochemistry
Scalpel blade #23	Scalpel Blades Swann Morton	COS240B	Tissue dissection
Scalpel handle #4	ProSciTech	T134	Tissue dissection
Sodium Azide	Sigma-Aldrich	S2002-25G	Tissue Processing
Sucrose	Chem-Supply	57-50-1	Tissue Processing
TOMO Adhesion microscope slides	ProSciTech	GEMS63705-09	Immunohistochemistry
Triton X-100	Sigma-Aldrich	X100-100mL	Immunohistochemistry