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 divided into four main stages depending on ameloblast activity⁵. The first step regroups precursor cell and pre-ameloblast proliferation. During the second step, differentiated ameloblasts secrete enamel matrix proteins (EMPs), mainly amelogenin, enamelin and ameloblastin, which determine the thickness of the final enamel. Thus, any disruption of EMP synthesis leads to quantitative defects of enamel. After the deposition of the full enamel thickness, the maturation stage begins. During this stage, apatite crystallite growth in width and thickness allows the enamel to reach the highest mineralization ratio found in a biological tissue, with up to 96% by weight. Disrupting events that occur during the maturation stage lead to qualitative enamel defects. Finally, ameloblasts enter a phase of post-maturation, also called pigmentation in rodents, and undergo apoptosis during tooth eruption making enamel defects (if any) irreparable and irreversible, thus defects provide potential retrospective recording of ameloblast stresses. In rodents, amelogenesis follows a similar sequence of events with the particularity that their incisors are continuously growing, which makes them a suitable model to study the general process of amelogenesis. Thus, any disruption of amelogenesis results in alterations of enamel quality and/or quantity, depending on the time-window of the disrupting event. In that sense, exposure to dioxin, lead, and endocrine-disrupting chemicals (EDCs) such as bisphenol A (BPA), genistein, and vinclozolin, have been shown to generate enamel hypomineralizations^1,2,3,6,7,8. Asymmetric white opaque spots were identified on the incisors of rats exposed to a low-dose BPA dose during the fetal period and the first month after birth¹. These enamel defects in rats, and those of human molar incisor hypomineralization (MIH), share similar clinical, structural, and biochemical characteristics. MIH is a recently described dental enamel pathology, for which the etiology still remains unclear^9,10 despite many causal factors having been hypothesized^9,10,11,12.

Another important enamel hypomineralization pathology due to environmental factors is dental fluorosis (DF), which is the consequence of excessive fluoride absorption (>0.1 mg/kg/day)^13,14. The main source of fluoride is drinking water that is either supplemented or naturally enriched with fluoride. Fluoride is also often prescribed to prevent dental caries, but the prophylactic dose is only 50% lower than the toxic one (≤0.05 mg/kg/day). MIH and DF, two frequent pathologies resulting from exposure to environmental factors, may present common features that need to be characterized due to the potentiation of hypomineralizing effects of fluoride combined with other toxicants such as EDCs² or amoxicillin¹⁵.

Micro-dissection of rat enamel organ containing ameloblasts at different differentiation stages will help to understand the mechanism of action of molecules able to disrupt ameloblast activity and cause enamel defects to be diagnosed after tooth eruption. In other words, the characterization of changes of enamel gene expression and enamel matrix composition due to environmental toxicants allows the reconstitution of the history of exposure to toxicants, and facilitates environmental safety monitoring for public health.

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 depending on their exposure to fluoride (NaF) in combination or not with BPA (Figure 1A)². All animals were observed and dissected on day 65 (Figure 1B).

2. Dissection of Hemi-Mandibles from Adult Rats

1. Euthanize rats by CO₂ asphyxiation, optionally followed by decapitation to the separate head from the rest of the body. Expose rats to CO₂ for 5 min in a dedicated box¹⁶.
2. Remove all the skin from the lower lips using a #11 scalpel in order to get easy access to the lower incisors.
3. With the same scalpel, make an incision between the two lower incisors with a slight pressure and the mandible will be split into two halves.
4. Cut the temporal mandibular joint with a scalpel to detach the jaw, and hold the hemi-mandible with fine tweezers.
5. Cut the surrounding soft tissues with a scalpel and remove all muscles, tendons, and ligaments using a scraper until the bone is completely clean.

3. Isolation of the Incisor^17,18

1. Carefully shave off the bone by placing the blade parallel to the major longitudinal axis of the incisor. Start at the bony ridge near the tips of the incisor (proximal end) until the end of the incisor near the cervical loop. The incision motion proceeds from the proximal to the distal direction.
2. Make a first cut distally to the cervical loop with the scalpel to remove the gonion of the hemi-mandible and permit taking off the incisor easily without damaging the cervical loop or the secretory stage of ameloblasts (red line in the Figure 2).
3. Make a second cut below the second molar, and insert the scalpel between the bone and the medial surface of the incisor. When all the basal bone is lifted, rotate outwards the incisor and take it off carefully to avoid enamel organ tissue impair using fine tweezers.

4. Micro-Dissection of Enamel Organ under Binocular Lens

1. Drop 200 µL of Saline Phosphate Buffer (PBS 1X) on the incisor using a pipette. Take the incisor with fine tweezers with the labial surface facing up. Make a scalpel mark between the colorless and the orange parts of the tissue. This mark corresponds to the underlying white spot which is observable afterwards (Figure 2).
2. Scrape, with an excavator or equivalent tool, the cell surface from the scalpel mark to the apical end corresponding to secretion stage (colorless) ameloblasts, and from the scalpel mark to the tip of the incisor for the maturation stage (orange) ameloblasts.
3. Remove the 2-mm tissue corresponding to the transition stage, as earlier described¹⁹. Do not open the incisor during enamel organ dissection to avoid contamination by the mesenchyme.
4. Cut the cervical loop situated immediately at the apical part of the incisor (Figure 2) as previously described²⁰.
5. Collect it in 10% formalin or lysis solution for further investigations (see Table of Materials).

5. Collection of Separated Enamel Organ Tissues for Further Investigations

1. Drop 200 µL PBS 1X buffer on the incisor.
2. With a #11 scalpel blade, carefully detach the dental epithelial cells gradually in the buffer, separately for each stage with the help of the scalpel mark made previously.
3. For cell RNA and protein extractions, put the tissue in a lysis solution that allows the extraction of both proteins and RNAs (see Table of Materials).
   NOTE: Prepare high quality RNAs by turning the tissue-excavator in the tube, and then grinding the cells until obtaining a homogeneous mixture. The mixture is ready for RNA and protein extractions according to the manufacturer's procedure, which was previously described^2,8,17. Usually, about 5-10 µg total RNAs and 150 µg total proteins were obtained for each preparation.
4. For histological analyses, immunohistochemistry (IHC), and in situ hybridization (ISH), put the cell layer in 10% formalin for 2 h, then store at 4 °C in PBS 1X. The tissue can then be embedded in wax/paraffin or tissue OCT for frozen sections.
5. For EMP extractions, take the incisor with fine tweezers. After a short exposure to air (slight dehydration), a white spot will be visible around the middle part of the labial surface of the incisor, which represents the initiation of the enamel mineralization. This white spot corresponds to the transition-/early maturation-stage of ameloblasts, so use it as an indicator for extraction of EMPs²¹.

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

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