Elsevier

Experimental Eye Research

Volume 139, October 2015, Pages 136-143
Experimental Eye Research

Methods in eye research
Combined 3DISCO clearing method, retrograde tracer and ultramicroscopy to map corneal neurons in a whole adult mouse trigeminal ganglion

https://doi.org/10.1016/j.exer.2015.06.008Get rights and content

Highlights

  • 3DISCO method: a rapid, reliable method for clearing adult mouse trigeminal ganglion.

  • Ultramicroscopic images of cleared ganglion containing retrogradely labelled neurons.

  • 3D reconstruction of the distribution of corneal neurons.

  • Automated counting of corneal neurons.

Abstract

Tissue clearing and subsequent imaging of intact transparent tissues have provided an innovative way to analyze anatomical pathways in the nervous system. In this study, we combined a recent 3-dimensional imaging of solvent cleared organ (3DISCO) procedure, light-sheet microscopy, fluorescent retrograde tracer, and Imaris software to 3D map corneal sensory neurons within a whole adult mouse trigeminal ganglion (TG). We first established the optimized steps to easily and rapidly clear a fixed TG. We found that the 3DISCO procedure gave excellent results and took less than 3 h to clear the TG. In a second set of experiments, a retrograde tracer (cholera toxin B Alexa 594-conjugated) was applied to de-epithelialized cornea to retrograde-labeled corneal sensory neurons. Two days later, TGs were cleared by the 3DISCO method and serial imaging was performed using light-sheet ultramicroscopic technology. High-resolution images of labeled neurons can be easily and rapidly obtained from a 3D reconstructed whole mouse TG. We then provided a 3D reconstruction of corneal afferent neurons and analyzed their precise localization in the TG. Thus, we showed that neurons supplying corneal sensory innervation exhibit a highly specific limited dorsomedial localization within the TG. We report that our combined method offers the possibility to perform manual (on 20 μm sections) and automated (on 3D reconstructed TG) counting of labeled cells in a cleared mouse TG. To conclude, we illustrate that the combination of the 3DISCO clearing method with light-sheet microscopy, retrograde tracer, and automatic counting represents a rapid and reliable method to analyze a subpopulation of neurons within the peripheral and central nervous system.

Introduction

The cornea is the most densely innervated tissue of the body (Muller et al., 2003, Muller et al., 1996, Muller et al., 1997). The corneal sensory nerve fibers originate from neuronal cell bodies located in the ipsilateral trigeminal ganglion (TG) (Felipe et al., 1999, Marfurt and Del Toro, 1987, Marfurt et al., 1989). The TG is composed of three nerve branches: (i) the ophthalmic branch, V1, (ii) the maxillary branch, V2, and (iii) the mandibular branch, V3, each branch having its own primary sensory neurons organized inside the ganglion (Marfurt et al., 1989).

Until now, published studies in the literature have reported the distribution of TG neurons supplying the corneal innervation by means of classical histological approaches. All of these studies used serial TG tissue sections from animals whose corneas were treated with various retrograde tracers (such as Cholera toxin subunit B (CTB), Fast blue, Fluorogold, horseradish peroxidase (HRP), wheat germ agglutinin (WGA), etc) (Bron et al., 2014, Ivanusic et al., 2013, Marfurt and Del Toro, 1987). However, these procedures required serial sections, microscopic analysis, and manual cell counting, all of which are time consuming (Bron et al., 2014, Ivanusic et al., 2013, Marfurt and Del Toro, 1987). The only way to obtain spatial organization of cells in tissues was to obtain sections and image the cells by epifluorescence, confocal, or two-photon microscopy. Unfortunately, these methods are not suitable to perform spatial reconstruction in large and thick tissues (up to 500 μm of thickness). Nowadays, new technologies have arisen to help scientists overcome this optical limit. For the last three years, several groups have developed new tissue clearing methods for enhanced imaging of entire tissues: ClearT (Kuwajima et al., 2013), SeeDB (Ke et al., 2013, Ke and Imai, 2014), ScaleA (Hama et al., 2011), CLARITY (Chung et al., 2013, Tomer et al., 2014), PACT/PARS (Yang et al., 2014), CUBIC (Susaki et al., 2014, Tainaka et al., 2014), and 3-dimensional imaging of solvent cleared organ (3DISCO) (Erturk and Bradke, 2013, Erturk et al., 2014). When combined with subsequent light-sheet laser scanning microscopy (Dodt et al., 2007), 3DISCO, offers the possibility of imaging entire transparent organs without the need for sectioning (Dodt et al., 2007, Erturk et al., 2012, Erturk and Bradke, 2013).

In this study, we report the 3D mapping of corneal afferent neurons within the adult mouse TG using the 3DISCO clearing method. More interestingly, we combined this procedure with a fluorescent retrograde tracer, light-sheet microscopy, and automated counting using Imaris software to rapidly obtain a 3D somatotopy of corneal neurons in the entire TG. Here, we provide evidence that clearing a whole mouse TG (containing retrograde labeled neurons) with 3DISCO and light-sheet microscopy imaging allows to obtain, in one day, a precise somatotopy of a specific neuronal population in solvent-cleared tissue.

Section snippets

Supplies

Surgical blade (Swann-Morton Ref. S0201).

70% ethanol solution (Ethanol 100% Sigma–Aldrich Ref. 32221, v70EtOH100%/v30H20).

Ophthalmic gel (Dechra, Lubrithal Eye Gel, 10 g).

Cholera toxin subunit B (CTB)-Alexa Fluor 594 conjugate (CTB-594, Cell Signaling, Danvers, MA, USA, Ref. C34777).

1× PBS (Gibco, Ref. 10010-023).

Paraformaldehyde (Sigma–Aldrich, Ref. 441244).

Stuart tube rotator (model SB3).

Tetrahydrofuran absolute, over molecular sieve, containing ∼ 0.025% 2,6-di-tert-butyl-4-methylphenol as

Retrograde tracing experiments and tissue preparation

To identify the corneal afferent neurons in the TG, we used CTB-Alexa Fluor 594 conjugate as a tracer. Animals were anesthetized intraperitoneally with a mixture of Ketamine 1000 U (100 mg/kg body weight) and xylazine (10 mg/kg body weight, Virbac, France). Only the left eye was processed in these experiments. The right eye was maintained hydrated during the surgery by placing a drop of ophthalmic gel.

Under a binocular microscope, a filter paper disc (3 mm in diameter) soaked with 70% ethanol

Results

In this study, we report the 3D distribution of retrograde-labeled corneal neurons in a whole adult mouse TG using a combination of the 3DISCO clearing method, light-sheet microscopy, and fluorescent retrograde tracer.

Discussion

Recently, clearing and imaging intact transparent tissues have offered a novel approach to visualize a specific tissue in its entirety. Several important new clearing methods have been developed and used: ScaleA2 (Hama et al., 2011), 3DISCO (Belle et al., 2014, Erturk et al., 2012, Erturk and Bradke, 2013, Erturk et al., 2014), SeeDB (Ke et al., 2013, Ke and Imai, 2014), CLARITY (Chung et al., 2013, Tomer et al., 2014), PACT/PARS (Yang et al., 2014) and CUBIC (Susaki et al., 2014, Tainaka

Conflicts of interest

The authors declare no potential conflicts of interest.

Acknowledgments

This study was supported by the Agence Nationale pour la Recherche Grant R06282DS, Université Pierre and Marie Curie (Paris VI), the Institut National de la Santé et de la Recherche Médicale LABEX (ANR-10-LABX-65) and the Rotary Club programme “Espoir en tête”. The authors thank R. N. Cooper for editorial assistance with this manuscript. We thank Stéphane Fouquet (imagery platform) and the staff of the animal house facilities at the Institut de la Vision for their help.

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