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.
