This work was funded by the University Paris-Diderot, the French National Institute of Health and Medical Research (INSERM), and the French Institute for Odontological Research (IFRO).

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
<ol>
	<li>Prior to performing this protocol, obtain necessary institutional approval and be sure to comply with all animal care guidelines.</li>
	<li>Apply the design of the research protocol allowing the constitution of different experimental groups in order to test the impact of t...

<ol>
	<li>Jedeon, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enamel+defects+reflect+perinatal+exposure+to+bisphenol+A.">Enamel defects reflect perinatal exposure to bisphenol A.</a> Am J Pathol. 183, 108-118 (2013).</li><li>Jedeon, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+Exposure+to+Bisphenol+a+Exacerbates+Dental+Fluorosis+in+Growing+Rats.">Chronic Exposure to Bisphenol a Exacerbates Dental Fluorosis in Growing Rats.</a> J Bone Miner Res. 31, 1955-1966 (2016).</li><li>Alaluusua, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+dental+aberrations+after+the+dioxin+accident+in+Seveso.">Developmental dental aberrations after the dioxin accident in Seveso.</a> Environ Health Perspect. 112, 1313-1318 (2004).</li><li>Chapple, I. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+lifestyle,+behaviour+or+systemic+diseases+with+dental+caries+and+periodontal+diseases:+consensus+report+of+group+2+of+the+joint+EFP/ORCA+workshop+on+the+boundaries+between+caries+and+periodontal+diseases.">Interaction of lifestyle, behaviour or systemic diseases with dental caries and periodontal diseases: consensus report of group 2 of the joint EFP/ORCA workshop on the boundaries between caries and periodontal diseases.</a> J Clin Periodontol. 44, S39-S51 (2017).</li><li>Nanci, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enamel:+Composition,+Formation,+and+Structure.">Enamel: Composition, Formation, and Structure.</a> Ten Cate's Oral Histology Development, Structure, and Function. , 122-164 (2012).</li><li>Leite, G. A., Sawan, R. M., Teofilo, J. M., Porto, I. M., Sousa, F. B., Gerlach, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exposure+to+lead+exacerbates+dental+fluorosis.">Exposure to lead exacerbates dental fluorosis.</a> Arch Oral Biol. 56, 695-702 (2011).</li><li>Jedeon, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enamel+hypomineralization+due+to+endocrine+disruptors.">Enamel hypomineralization due to endocrine disruptors.</a> Connect Tiss Res. 55, 1-5 (2014).</li><li>Jedeon, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Androgen+receptor+involvement+in+rat+amelogenesis:+an+additional+way+for+endocrine+disrupting+chemicals+to+affect+enamel+synthesis.">Androgen receptor involvement in rat amelogenesis: an additional way for endocrine disrupting chemicals to affect enamel synthesis.</a> Endocrinology. 157, 4287-4296 (2016).</li><li>Weerheijm, K. L., Jalevik, B., Alaluusua, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molar-incisor+hypomineralisation.">Molar-incisor hypomineralisation.</a> Caries Res. 35, 390-391 (2001).</li><li>J&#228;levik, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+and+Diagnosis+of+Molar-Incisor-+Hypomineralisation+(MIH):+A+systematic+review.">Prevalence and Diagnosis of Molar-Incisor- Hypomineralisation (MIH): A systematic review.</a> Eur Arch Paediatr Dent. 11, 59-64 (2010).</li><li>Alaluusua, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aetiology+of+Molar-Incisor+Hypomineralisation:+A+systematic+review.">Aetiology of Molar-Incisor Hypomineralisation: A systematic review.</a> Eur Arch Paediatr Dent. 11, 53-58 (2010).</li><li>Jedeon, K., Berdal, A., Babajko, S., Gibert, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tooth,+target+organ+of+Bisphenol+A,+could+be+used+as+a+biomarker+of+exposure+to+this+agent.">The tooth, target organ of Bisphenol A, could be used as a biomarker of exposure to this agent.</a> Bisphenol A: Sources, Risks of Environmental Exposure and Human Health Effects. , 205-225 (2015).</li><li>Fejerskov, O., Larsen, M. J., Richards, A., Baelum, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dental+tissue+effects+of+fluoride.">Dental tissue effects of fluoride.</a> Adv Dent Res. 8, 15-31 (1994).</li><li>Robinson, C., Connell, S., Kirkham, J., Brookes, S. J., Shore, R. C., Smith, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+fluoride+on+the+developing+tooth.">The effect of fluoride on the developing tooth.</a> Caries Res. 38, 268-276 (2004).</li><li>Sahlberg, C., Pavlic, A., Ess, A., Lukinmaa, P. 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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...

Many developmental enamel defects may result from exposure to environmental toxicants and/or inappropriate life-style1,2,3,4. 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 synthetized1,2. 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 hypomineralizations1,2,3,6,7,8. 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 unclear9,10 despite many causal factors having been hypothesized9,10,11,12.
Another important enamel hypomineralization pathology due to environmental factors is dental fluorosis (DF), which is the consequence of excessive fluoride absorption (&#62;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 (&#8804;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.

<table>
	<tbody>
		<tr>
			<td>Bisphenol A</td>
			<td>Sigma Aldrich, Saint Louis MO</td>
			<td>239658</td>
			<td></td>
		</tr>
		<tr>
			<td>formalin 10%</td>
			<td>Sigma-Aldrich, Saint Louis, MO</td>
			<td>HT5012</td>
			<td></td>
		</tr>
		<tr>
			<td>Tri-Reagent</td>
			<td>Euromedex, France</td>
			<td>TR118</td>
			<td></td>
		</tr>
		<tr>
			<td>RLT buffer</td>
			<td>Qiagen, Les Ulis, France</td>
			<td>74126</td>
			<td>RNeasy Protect Mini Kit</td>
		</tr>
		<tr>
			<td>Androgen receptor antibody</td>
			<td>Santa Cruz Biotechnology, Santa Cruz, CA)</td>
			<td>sc-816</td>
			<td>rabbit polyclonal antibody</td>
		</tr>
		<tr>
			<td>PBS 10x</td>
			<td>EUOMEDEX</td>
			<td>ET330.A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium fluoride (NaF)</td>
			<td>Sigma-Aldrich, Saint Louis, MO</td>
			<td>S-1504</td>
			<td></td>
		</tr>
		<tr>
			<td>paraplast regular</td>
			<td>Leica microsystems, Nanterre cedex,&#160;France</td>
			<td>39601006</td>
			<td>called was/parafin in the text</td>
		</tr>
		<tr>
			<td>tissue OCT</td>
			<td>VWR, Fontenay-sous-Bois, France</td>
			<td>411243</td>
			<td></td>
		</tr>
		<tr>
			<td>Extra Fine Bonn Scissors - Straight/8.5 cm</td>
			<td>PHYMEP , Paris, France</td>
			<td>14084-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Handle for Scalpel Blades - 12.5 cm</td>
			<td>PHYMEP, Paris, France</td>
			<td>10035-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved Scalpel Blade</td>
			<td>PHYMEP , Paris, France</td>
			<td>10035-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Knife - Fine/Straight Tip</td>
			<td>PHYMEP , Paris, France</td>
			<td>10055-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Circle Knife</td>
			<td>PHYMEP, Paris, France</td>
			<td>10059-15</td>
			<td></td>
		</tr>
		<tr>
			<td>scalpel blades n&#176;11 Swann-Morton</td>
			<td>VWR, Fontenay-sous-Bois, France</td>
			<td>233-0024</td>
			<td></td>
		</tr>
		<tr>
			<td>binocular lens</td>
			<td>Leica biosystems, Nanterre cedex,&#160;France</td>
			<td>MZFLIII</td>
			<td></td>
		</tr>
	</tbody>
</table>

micro-dissection of enamel organ from mandibular incisor of rats exposed to environmental toxicants

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.

Many enamel defects, such as dental fluorosis12,13, may result from environmental conditions due to excessive fluoride absorption or enamel hypomineralization similar to MIH due to exposure to some EDCs1,7,22. These developmental enamel defects may be experimentally reproduced on rats (Figure 1)<sup class="xr...

Watch this Scientific Journal Video about Micro-dissection of Enamel Organ from Mandibular Incisor of Rats Exposed to Environmental Toxicants at JoVE.com

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

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.

Enamel defects resulting from environmental conditions and ways of life are public health concerns because of their high prevalence. These defects result ...

The overall goal of this procedure is to micro-dissect rat enamel organ and separate the dental epithelium according to the main stages of cell differentiation during enamel synthesis. This method can help answer key questions in the field of enamel pathologies such as dental fluorosis, molar incisor hyperenamelization and heredity enamel genesis imperfectum. The main advantage of this technique is to the ability to further analyze the dental cells using quantitative experiments such as immunohistochemistry without any prior treatments, as well as consequentive investigations such as Though this method can provide insight into molecular mechanisofaction of endogenous and exogenous molecules able to disrupt hemogenesis.After euthanizing an experimental rat, remove all the skin from the lower lips using a number 11 scalpel in order to get easy access to the lower incisors. Then, with the same scalpel, make an incision between the two lower incisors. By applying light pressure, the mandible will then split into two halves.Next, cut the temporal-mandibular joint with the scalpel to detach the jaw while holding the hemi-mandible with fine tweezers. Then cut the surrounding soft tissues with a scalpel and remove all of the muscles, tendons, and ligaments using a scraper until the bone is completely clean. To isolate the incisor, carefully shave off the bone by placing the blade parallel to the major longitudinal axis of the incisor.Begin shaving at the boney ridge, near the tips of the incisor, and work toward the other end of the incisor, near the cervical loop. Next, make an incision with the scalpel, distally to the cervical loop to remove the gonion of the hemi-mandible, thus making the incisor easy to remove without damaging the cervical loop or the secretory stage of ameloblasts. Next make a second cut below the second molar and insert the scalpel between the bone and the medial surface of the incisor.When all of the basal bone is lifted, rotate the incisor outwards and carefully remove it using fine tweezers to avoid damaging the enamel organ tissue. Now the entire incisor is isolated. For the dissection, cover the incisor with 200 microliters of PBS in a 60 millimeter dish or plate.To begin, cut the cervical loop, situated immediately at the apical part of the incisor, as previously described. Then take hold of the incisor with fine tweezers, with the labial surface up, and make a scalpel mark at the end of the colorless part of the tissue. This mark corresponds with an underlying white spot that will be observable later.Now, use an excavator or equivalent tool to scrape the cell surface from the mark to the apical end, which corresponds to colorless, secretion stage ameloblasts. Next, make a scalpel mark at the beginning of the orange part, corresponding to the maturation stage ameloblasts and scrape from the scalpel mark to the tip of the incisor. Now remove the two millimeters of tissue that correspond to the transition stage ameloblasts.Do not open the incisor during the enamel organ dissection as this will contaminate the dissected tissues with mesenchyme. Finally, store the tissues in 10%formalin or lysis solution for further investigations. For an enamel matrix protein extraction, remove the incisor from solution briefly until a white spot becomes visible around the middle part of the labial surface.This spot represents the initiation of the enamel mineralization and corresponds to the transition to early stage ameloblasts. Cut away this region of tissue and treat it as needed. If the removed tissue is for cellular RNA and protein extractions use a commercial kit to perform the extraction.Transfer the removed tissue to the lysis solution and grind it into a homogenous mixture. For histological analyses, immunohistochemistry, and NC2 hybridization put the collected tissue in 10%formalin for two hours at room temperature. Then transfer the tissue to PBS and store it at four degrees Celsius.Later the tissue can be embedded in either wax paraffin or tissue OCT. Four groups of male white tail rats were constituted depending on their exposure to fluoride in combination or not with BPA. All animals were observed and dissected on day 65 as described.The quality of the micro-dissected enamel tissues was checked using trichrome Masson staining. Microscopic observation show typical enamel epithelial cells and ameloblast palocide. The absence of mesenchymal contamination was attested by the absence of collagen green staining.Secretion stage ameloblasts express enameline. Maturation stage ameloblasts express kalikine four and also contain high levels of pheratin giving them the ability to store high amounts of iron responsible for the orange color of enamel. These RNAs are preferentially expressed by ameloblasts rather than mesenchymel cells.Collagen one, on the other hand, is expressed by mesenchymel cells and not ameloblasts. Immunohistochemistry was then used to score four specific proteins in the secretion or maturation stage ameloblasts. For example, an androgen receptor, seen in green, is specifically localized to the maturation stage ameloblasts.After watching this video, you should have a good comprehension of how to micro-dissect, the enamel organ of rodents for successful separation between secretion and maturation stages ameloblasts. This approach is very useful for the understanding of in vivo key factor mechanisms responsible for pathological and physiological enamel disease.

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Research

JoVE Journal

Medicine

10.0K Views.  Cordeliers Research Centre. The overall goal of this procedure is to micro-dissect rat enamel organ and separate the dental epithelium according to the main stages of cell differentiation during enamel synthesis. This method can help answer key questions in the field of enamel pathologies such as dental fluorosis, molar incisor hyperenamelization and heredity enamel genesis imperfectum. The main advantage of this technique is to the ability to further analyze the dental cells using quantitative experiments such as immun...