Micro-dissection of Enamel Organ from Mandibular Incisor of Rats Exposed to Environmental Toxicants

Summary

Understanding enamel formation and possible alterations requires the study of ameloblast activity. Here, we describe a reliable and consistent method to micro-dissect enamel organs containing secretion- and maturation-stage ameloblasts that may be used for further quantitative and qualitative experimental procedures.

Abstract

Enamel defects resulting from environmental conditions and ways of life are public health concerns because of their high prevalence. These defects result from altered activity of cells responsible for enamel synthesis named ameloblasts, which present in enamel organ. During amelogenesis, ameloblasts follow a specific and precise sequence of events of proliferation, differentiation, and death. A rat continually growing incisors is a suitable experimental model to study ameloblast activity and differentiation stages in physiological and pathological conditions. Here, we describe a reliable and consistent method to micro-dissect enamel organ of rats exposed to environmental toxicants. The micro-dissected dental epithelia contain secretion- and maturation-stage ameloblasts that may be used for qualitative experiments, such as immunohistochemistry assays and in situ hybridization, as well as for quantitative analyses such as RT-qPCR, RNA-seq, and Western blotting.

Introduction

Many developmental enamel defects may result from exposure to environmental toxicants and/or inappropriate life-style^1,2,3,4. Characterization of disrupting events and molecules of amelogenesis using the presently described procedure will promote the use of resulting enamel defects as early markers of exposure to several toxicants, and may help to reconstitute the history of health of each patient during the perinatal period when enamel is synthetized^1,2. Enamel synthesis can be divi....

Protocol

All animals used in the present study were maintained in accordance with guidelines for the care and use of laboratory animals from the French Ministry of Agriculture (A-75-06-12).

1. Animal Exposure to Toxicants

1. Prior to performing this protocol, obtain necessary institutional approval and be sure to comply with all animal care guidelines.
2. Apply the design of the research protocol allowing the constitution of different experimental groups in order to test the impact of the molecule(s) investigated on ameloblast secretion stage and the ameloblast maturation stage. Here, four groups of male Wistar rats were constituted ....

Results

Many enamel defects, such as dental fluorosis^12,13, may result from environmental conditions due to excessive fluoride absorption or enamel hypomineralization similar to MIH due to exposure to some EDCs^1,7,22. These developmental enamel defects may be experimentally reproduced on rats (Figure 1)

Discussion

Altered ameloblast activity and/or disrupted ameloblast proliferation, differentiation, and maturation processes lead to irreversible enamel defects and, in turn, the characterization of enamel defects may help develop understanding of the altered ameloblast activity during amelogenesis. Thus, the studies on isolated enamel organ are determinant to elucidate the pathological events leading to enamel defects whatever their origin, environmental or genetic.

This technique has originally been des.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bisphenol A	Sigma Aldrich, Saint Louis MO	239658
formalin 10%	Sigma-Aldrich, Saint Louis, MO	HT5012
Tri-Reagent	Euromedex, France	TR118
RLT buffer	Qiagen, Les Ulis, France	74126	RNeasy Protect Mini Kit
Androgen receptor antibody	Santa Cruz Biotechnology, Santa Cruz, CA)	sc-816	rabbit polyclonal antibody
PBS 10x	EUOMEDEX	ET330.A
Sodium fluoride (NaF)	Sigma-Aldrich, Saint Louis, MO	S-1504
paraplast regular	Leica microsystems, Nanterre cedex, France	39601006	called was/parafin in the text
tissue OCT	VWR, Fontenay-sous-Bois, France	411243
Extra Fine Bonn Scissors - Straight/8.5 cm	PHYMEP , Paris, France	14084-08
Handle for Scalpel Blades - 12.5 cm	PHYMEP, Paris, France	10035-12
Curved Scalpel Blade	PHYMEP , Paris, France	10035-20
Dissecting Knife - Fine/Straight Tip	PHYMEP , Paris, France	10055-12
Circle Knife	PHYMEP, Paris, France	10059-15
scalpel blades n°11 Swann-Morton	VWR, Fontenay-sous-Bois, France	233-0024
binocular lens	Leica biosystems, Nanterre cedex, France	MZFLIII