Tags

Type your tag names separated by a space and hit enter

Fluorescent Labeling and 2-Photon Imaging of Mouse Tooth Pulp Nociceptors.
J Dent Res. 2018 04; 97(4):460-466.JD

Abstract

Retrograde fluorescent labeling of dental primary afferent neurons (DPANs) has been described in rats through crystalline fluorescent DiI, while in the mouse, this technique was achieved with only Fluoro-Gold, a neurotoxic fluorescent dye with membrane penetration characteristics superior to the carbocyanine dyes. We reevaluated this technique in the rat with the aim to transfer it to the mouse because comprehensive physiologic studies require access to the mouse as a model organism. Using conventional immunohistochemistry, we assessed in rats and mice the speed of axonal dye transport from the application site to the trigeminal ganglion, the numbers of stained DPANs, and the fluorescence intensity via 1) conventional crystalline DiI and 2) a novel DiI formulation with improved penetration properties and staining efficiency. A 3-dimensional reconstruction of an entire trigeminal ganglion with 2-photon laser scanning fluorescence microscopy permitted visualization of DPANs in all 3 divisions of the trigeminal nerve. We quantified DPANs in mice expressing the farnesylated enhanced green fluorescent protein (EGFPf) from the transient receptor potential cation channel subfamily M member 8 (TRPM8EGFPf/+) locus in the 3 branches. We also evaluated the viability of the labeled DPANs in dissociated trigeminal ganglion cultures using calcium microfluorometry, and we assessed the sensitivity to capsaicin, an agonist of the TRPV1 receptor. Reproducible DiI labeling of DPANs in the mouse is an important tool 1) to investigate the molecular and functional specialization of DPANs within the trigeminal nociceptive system and 2) to recognize exclusive molecular characteristics that differentiate nociception in the trigeminal system from that in the somatic system. A versatile tool to enhance our understanding of the molecular composition and characteristics of DPANs will be essential for the development of mechanism-based therapeutic approaches for dentine hypersensitivity and inflammatory tooth pain.

Authors+Show Affiliations

1 Klinik für Anästhesiologie, Universitätsklinikum Erlangen, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany.1 Klinik für Anästhesiologie, Universitätsklinikum Erlangen, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany. 2 Instituto de Fisiología, Facultad de Medicina, Escuela de Graduados, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile.1 Klinik für Anästhesiologie, Universitätsklinikum Erlangen, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany.3 Optical Imaging Centre Erlangen, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany.3 Optical Imaging Centre Erlangen, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany.1 Klinik für Anästhesiologie, Universitätsklinikum Erlangen, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany.1 Klinik für Anästhesiologie, Universitätsklinikum Erlangen, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany.1 Klinik für Anästhesiologie, Universitätsklinikum Erlangen, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany.2 Instituto de Fisiología, Facultad de Medicina, Escuela de Graduados, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile.4 Klinik für Zahnerhaltung und Parodontologie, Universitätsklinikum Erlangen, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany.5 Institut für Physiologie und Pathophysiologie, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany.6 Center for Integrated Diagnostics, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.1 Klinik für Anästhesiologie, Universitätsklinikum Erlangen, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

29130364

Citation

Kadala, A, et al. "Fluorescent Labeling and 2-Photon Imaging of Mouse Tooth Pulp Nociceptors." Journal of Dental Research, vol. 97, no. 4, 2018, pp. 460-466.
Kadala A, Sotelo-Hitschfeld P, Ahmad Z, et al. Fluorescent Labeling and 2-Photon Imaging of Mouse Tooth Pulp Nociceptors. J Dent Res. 2018;97(4):460-466.
Kadala, A., Sotelo-Hitschfeld, P., Ahmad, Z., Tripal, P., Schmid, B., Mueller, A., Bernal, L., Winter, Z., Brauchi, S., Lohbauer, U., Messlinger, K., Lennerz, J. K., & Zimmermann, K. (2018). Fluorescent Labeling and 2-Photon Imaging of Mouse Tooth Pulp Nociceptors. Journal of Dental Research, 97(4), 460-466. https://doi.org/10.1177/0022034517740577
Kadala A, et al. Fluorescent Labeling and 2-Photon Imaging of Mouse Tooth Pulp Nociceptors. J Dent Res. 2018;97(4):460-466. PubMed PMID: 29130364.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Fluorescent Labeling and 2-Photon Imaging of Mouse Tooth Pulp Nociceptors. AU - Kadala,A, AU - Sotelo-Hitschfeld,P, AU - Ahmad,Z, AU - Tripal,P, AU - Schmid,B, AU - Mueller,A, AU - Bernal,L, AU - Winter,Z, AU - Brauchi,S, AU - Lohbauer,U, AU - Messlinger,K, AU - Lennerz,J K, AU - Zimmermann,K, Y1 - 2017/11/12/ PY - 2017/11/14/pubmed PY - 2019/4/2/medline PY - 2017/11/14/entrez KW - Fluoro-Gold KW - TRPM8 KW - TRPV1 KW - carbocyanine dye KW - dental primary afferent KW - retrograde labeling SP - 460 EP - 466 JF - Journal of dental research JO - J. Dent. Res. VL - 97 IS - 4 N2 - Retrograde fluorescent labeling of dental primary afferent neurons (DPANs) has been described in rats through crystalline fluorescent DiI, while in the mouse, this technique was achieved with only Fluoro-Gold, a neurotoxic fluorescent dye with membrane penetration characteristics superior to the carbocyanine dyes. We reevaluated this technique in the rat with the aim to transfer it to the mouse because comprehensive physiologic studies require access to the mouse as a model organism. Using conventional immunohistochemistry, we assessed in rats and mice the speed of axonal dye transport from the application site to the trigeminal ganglion, the numbers of stained DPANs, and the fluorescence intensity via 1) conventional crystalline DiI and 2) a novel DiI formulation with improved penetration properties and staining efficiency. A 3-dimensional reconstruction of an entire trigeminal ganglion with 2-photon laser scanning fluorescence microscopy permitted visualization of DPANs in all 3 divisions of the trigeminal nerve. We quantified DPANs in mice expressing the farnesylated enhanced green fluorescent protein (EGFPf) from the transient receptor potential cation channel subfamily M member 8 (TRPM8EGFPf/+) locus in the 3 branches. We also evaluated the viability of the labeled DPANs in dissociated trigeminal ganglion cultures using calcium microfluorometry, and we assessed the sensitivity to capsaicin, an agonist of the TRPV1 receptor. Reproducible DiI labeling of DPANs in the mouse is an important tool 1) to investigate the molecular and functional specialization of DPANs within the trigeminal nociceptive system and 2) to recognize exclusive molecular characteristics that differentiate nociception in the trigeminal system from that in the somatic system. A versatile tool to enhance our understanding of the molecular composition and characteristics of DPANs will be essential for the development of mechanism-based therapeutic approaches for dentine hypersensitivity and inflammatory tooth pain. SN - 1544-0591 UR - https://www.unboundmedicine.com/medline/citation/29130364/Fluorescent_Labeling_and_2_Photon_Imaging_of_Mouse_Tooth_Pulp_Nociceptors_ L2 - http://journals.sagepub.com/doi/full/10.1177/0022034517740577?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub=pubmed DB - PRIME DP - Unbound Medicine ER -