Name: micro-dissection of enamel organ from mandibular incisor of rats exposed to environmental toxicants
Uploaded: 2018-03-29T13:30:00
Description: Watch this Scientific Journal Video about Micro-dissection of Enamel Organ from Mandibular Incisor of Rats Exposed to Environmental Toxicants at JoVE.com

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. 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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. 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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.

micro-dissection-enamel-organ-from-mandibular-incisor-rats-exposed-to

Research

JoVE Journal

Medicine

Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each year. While several animal models exist for this procedure and mice are the species that is most commonly used. The reasons for using mice are the relative cost of using this species, the existence of many genetically defined strains that allow for the study of immune responses, and the existence of an extensive array of reagents that can be used to further define responses in this species. This model has been used to define factors in the cornea that are responsible for the relative immune privilege status of this tissue that enables corneal allografts to survive acute rejection in the absence of immunosuppressive therapy. It has also been used to define those factors that are most important in rejection of such allografts. Consequently, much of what we know concerning mechanisms of both corneal allograft acceptance and rejection are due to studies using a murine model of corneal transplantation. In addition to describing a model for acute corneal allograft rejection, we also present for the first time a model of late-term corneal allograft rejection.

Mice have been used as a model for studying many forms of transplantation, including corneal transplantation. We describe in this report a murine model for both acute and late-term corneal transplantation.

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each ...

Fundus Photography as a Convenient Tool to Study Microvascular Responses to Cardiovascular Disease Risk Factors in Epidemiological Studies

The microcirculation consists of blood vessels with diameters less than 150 &#181;m. It makes up a large part of the circulatory system and plays an important role in maintaining cardiovascular health. The retina is a tissue that lines the interior of the eye and it is the only tissue that allows for a non-invasive analysis of the microvasculature. Nowadays, high-quality fundus images can be acquired using digital cameras. Retinal images can be collected in 5 min or less, even without dilatation of the pupils. This unobtrusive and fast procedure for visualizing the microcirculation is attractive to apply in epidemiological studies and to monitor cardiovascular health from early age up to old age.
Systemic diseases that affect the circulation can result in progressive morphological changes in the retinal vasculature. For example, changes in the vessel calibers of retinal arteries and veins have been associated with hypertension, atherosclerosis, and increased risk of stroke and myocardial infarction. The vessel widths are derived using image analysis software and the width of the six largest arteries and veins are summarized in the Central Retinal Arteriolar Equivalent (CRAE) and the Central Retinal Venular Equivalent (CRVE). The latter features have been shown useful to study the impact of modifiable lifestyle and environmental cardiovascular disease risk factors.
The procedures to acquire fundus images and the analysis steps to obtain CRAE and CRVE are described. Coefficients of variation of repeated measures of CRAE and CRVE are less than 2% and within-rater reliability is very high. Using a panel study, the rapid response of the retinal vessel calibers to short-term changes in particulate air pollution, a known risk factor for cardiovascular mortality and morbidity, is reported. In conclusion, retinal imaging is proposed as a convenient and instrumental tool for epidemiological studies to study microvascular responses to cardiovascular disease risk factors.

Retinal image analysis is an unobtrusive procedure for visualizing the microcirculation. The impact of cardiovascular disease risk factors can result in changes of retinal vessel calibers. The procedures to acquire fundus images and the steps for calculating the vessel calibers are described.

The microcirculation consists of blood vessels with diameters less than 150 &#181;m. It makes up a large part of the circulatory system and plays an ...

Technique of Porcine Liver Procurement and Orthotopic Transplantation using an Active Porto-Caval Shunt

The success of liver transplantation has resulted in a dramatic organ shortage. Each year, a considerable number of patients on the liver transplantation waiting list die without receiving an organ transplant or are delisted due to disease progression. Even after a successful transplantation, rejection and side effects of immunosuppression remain major concerns for graft survival and patient morbidity. 
Experimental animal research has been essential to the success of liver transplantation and still plays a pivotal role in the development of clinical transplantation practice. In particular, the porcine orthotopic liver transplantation model (OLTx) is optimal for clinically oriented research for its close resemblance to human size, anatomy, and physiology. 
Decompression of intestinal congestion during the anhepatic phase of porcine OLTx is important to guarantee reliable animal survival. The use of an active porto-caval-jugular shunt achieves excellent intestinal decompression. The system can be used for short-term as well as long-term survival experiments. The following protocol contains all technical information for a stable and reproducible liver transplantation model in pigs including post-operative animal care.

Experimental animal research plays a pivotal role in the development of clinical transplantation practice. The porcine orthotopic liver transplantation model (OLTx) closely resembles human conditions and is frequently used in clinically oriented research. The following protocol contains all information for a reliable porcine OLTx model using an active porto-caval-jugular shunt.

The success of liver transplantation has resulted in a dramatic organ shortage. Each year, a considerable number of patients on the liver transplantation ...

Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice

COPD is projected to be the third most common cause of mortality world-wide by 2020(1). Animal models of COPD are used to identify molecules that contribute to the disease process and to test the efficacy of novel therapies for COPD. Researchers use a number of models of COPD employing different species including rodents, guinea-pigs, rabbits, and dogs(2). However, the most widely-used model is that in which mice are exposed to cigarette smoke. Mice are an especially useful species in which to model COPD because their genome can readily be manipulated to generate animals that are either deficient in, or over-express individual proteins. Studies of gene-targeted mice that have been exposed to cigarette smoke have provided valuable information about the contributions of individual molecules to different lung pathologies in COPD(3-5). Most studies have focused on pathways involved in emphysema development which contributes to the airflow obstruction that is characteristic of COPD. However, small airway fibrosis also contributes significantly to airflow obstruction in human COPD patients(6), but much less is known about the pathogenesis of this lesion in smoke-exposed animals. To address this knowledge gap, this protocol quantifies both emphysema development and small airway fibrosis in smoke-exposed mice. This protocol exposes mice to CS using a whole-body exposure technique, then measures respiratory mechanics in the mice, inflates the lungs of mice to a standard pressure, and fixes the lungs in formalin. The researcher then stains the lung sections with either Gill&#8217;s stain to measure the mean alveolar chord length (as a readout of emphysema severity) or Masson&#8217;s trichrome stain to measure deposition of extracellular matrix (ECM) proteins around small airways (as a readout of small airway fibrosis). Studies of the effects of molecular pathways on both of these lung pathologies will lead to a better understanding of the pathogenesis of COPD.

The goal of this protocol is to provide automated methods to quantify chronic lung pathologies in a murine model of COPD. The protocol includes exposing mice to cigarette smoke (CS), measuring pulmonary function, inflating the lungs, and using morphometry methods to measure emphysema and small airway remodeling in mice.

COPD is projected to be the third most common cause of mortality world-wide by 2020(1).  Animal models of COPD are used to identify molecules that ...

Cardiac Catheterization in Mice to Measure the Pressure Volume Relationship: Investigating the Bowditch Effect

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these strategies is to examine their effects on heart function. There are several techniques to measure in vivo cardiac mechanics (e.g., echocardiography, pressure/volume relations, etc.). Compared to echocardiography, real-time left ventricular (LV) pressure/volume analysis via catheterization is more precise and insightful in assessing LV function. Additionally, LV pressure/volume analysis provides the ability to instantaneously record changes during manipulations of contractility (e.g., &#946;-adrenergic stimulation) and pathological insults (e.g., ischemia/reperfusion injury). In addition to the maximum (+dP/dt) and minimum (-dP/dt) rate of pressure change in the LV, an accurate assessment of LV function via several load-independent indexes (e.g., end systolic pressure volume relationship and preload recruitable stroke work) can be attained. Heart rate has a significant effect on LV contractility such that an increase in the heart rate is the primary mechanism to increase cardiac output (i.e., Bowditch effect). Thus, when comparing hemodynamics between experimental groups, it is necessary to have similar heart rates. Furthermore, a hallmark of many cardiomyopathy models is a decrease in contractile reserve (i.e., decreased Bowditch effect). Consequently, vital information can be obtained by determining the effects of increasing heart rate on contractility. Our and others data has demonstrated that the neuronal nitric oxide synthase (NOS1) knockout mouse has decreased contractility. Here we describe the procedure of measuring LV pressure/volume with increasing heart rates using the NOS1 knockout mouse model.

This article describes the measurement of murine left ventricular function via pressure/volume analysis at different heart rates.

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these ...

Murine Prostate Micro-dissection and Surgical Castration

Mouse models are used extensively to study prostate cancer and other diseases. The mouse is an excellent model with which to study the prostate and has been used as a surrogate for discoveries in human prostate development and disease. Prostate micro-dissection allows consistent study of lobe-specific prostate anatomy, histology, and cellular characteristics in the absence of contamination of other tissues. Testosterone affects prostate development and disease. Androgen deprivation therapy is a common treatment for prostate cancer patients, but many prostate tumors become castration-resistant. Surgical castration of mouse models allows for the study of castration resistance and other facets of hormonal biology on the prostate. This procedure can be coupled with testosterone reintroduction, or hormonal regeneration of the prostate, a powerful method to study stem cell lineages in the prostate. Together, prostate micro-dissection and surgical castration opens up a multitude of opportunities for robust and consistent research of prostate development and disease. This manuscript describes the protocols for prostate micro-dissection and surgical castration in the laboratory mouse.

This manuscript describes the protocols for prostate micro-dissection and surgical castration in the laboratory mouse. We also depict representative results produced by these protocols. Finally, we discuss the advantages and utilization of these protocols.

Mouse models are used extensively to study prostate cancer and other diseases. The mouse is an excellent model with which to study the prostate and has ...

Generation of Organ-conditioned Media and Applications for Studying Organ-specific Influences on Breast Cancer Metastatic Behavior

Breast cancer preferentially metastasizes to the lymph node, bone, lung, brain and liver in breast cancer patients. Previous research efforts have focused on identifying factors inherent to breast cancer cells that are responsible for this observed metastatic pattern (termed organ tropism), however much less is known about factors present within specific organs that contribute to this process. This is in part because of a lack of in vitro model systems that accurately recapitulate the organ microenvironment. To address this, an ex vivo model system has been established that allows for the study of soluble factors present within different organ microenvironments. This model consists of generating conditioned media from organs (lymph node, bone, lung, and brain) isolated from normal athymic nude mice. The model system has been validated by demonstrating that different breast cancer cell lines display cell-line specific and organ-specific malignant behavior in response to organ-conditioned media that corresponds to their in vivo metastatic potential. This model system can be used to identify and evaluate specific organ-derived soluble factors that may play a role in the metastatic behavior of breast and other types of cancer cells, including influences on growth, migration, stem-like behavior, and gene expression, as well as the identification of potential new therapeutic targets for cancer. This is the first ex vivo model system that can be used to study organ-specific metastatic behavior in detail and evaluate the role of specific organ-derived soluble factors in driving the process of cancer metastasis.

This manuscript describes an ex vivo model system comprised of organ-conditioned media derived from the lymph node, bone, lung, and brain of mice. This model system can be used to identify and study organ-derived soluble factors and their effects on the organ tropism and metastatic behavior of cancer cells.

Breast cancer preferentially metastasizes to the lymph node, bone, lung, brain and liver in breast cancer patients. Previous research efforts have focused ...

Isolation and Cultivation of Mandibular Bone Marrow Mesenchymal Stem Cells in Rats

Here we present an efficient method for isolating and culturing mandibular bone marrow mesenchymal stem cells (mBMSCs) in vitro to rapidly obtain numerous high-quality cells for experimental requirements. mBMSCs could be widely used in therapeutic applications as tissue engineering cells in case of craniofacial diseases and cranio-maxillofacial regeneration in the future due to the excellent self-renewal ability and multi-lineage differentiation potential. Therefore, it is important to obtain mBMSCs in large numbers.
In this study, bone marrow was flushed from the mandible and primary mBMSCs were isolated through whole bone marrow adherent cultivation. Furthermore, CD29+CD90+CD45&#8722; mBMSCs were purified through fluorescent cell sorting. The second generation of purified mBMSCs were used for further study and displayed potential in differentiating into osteoblasts, adipocytes, and chondrocytes. Utilizing this in vitro model, one can obtain a high number of proliferative mBMSCs, which may facilitate the study of the biological characteristics, the subsequent reaction to the microenvironment, and other applications of mBMSCs.

This article presents a method that combines whole bone marrow adherence and flow cytometry sorting for isolating, cultivating, sorting, and identifying bone marrow mesenchymal stem cells from rat mandibles.

Here we present an efficient method for isolating and culturing mandibular bone marrow mesenchymal stem cells (mBMSCs) in vitro to rapidly obtain numerous ...

3D Imaging of PDL Collagen Fibers during Orthodontic Tooth Movement in Mandibular Murine Model

Orthodontic tooth movement is a complex biological process of altered soft and hard tissue remodeling as a result of external forces. In order to understand these complex remodeling processes, it is critical to study the tooth and periodontal tissues within their 3D context and therefore minimize any sectioning and tissue artefacts. Mouse models are often utilized in developmental and structural biology, as well as in biomechanics due to their small size, high metabolic rate, genetics and ease of handling. In principle this also makes them excellent models for dental related studies. However, a major impediment is their small tooth size, the molars in particular. This paper is aimed at providing a step by step protocol for generating orthodontic tooth movement and two methods for 3D imaging of the periodontal ligament fibrous component of a mouse mandibular molar. The first method presented is based on a micro-CT setup enabling phase enhancement&#160;imaging of fresh collagen tissues. The second method is a bone clearing method using ethyl cinnamate that enables imaging through the bone without sectioning and preserves endogenous fluorescence. Combining this clearing method with reporter mice like Flk1-Cre;TdTomato provided a first of its kind opportunity to image the 3D vasculature in the PDL and alveolar bone.

We present a protocol for generating orthodontic tooth movement in mice and methods for 3D visualization of the collagen fibers and blood vessels of periodontal ligament without sectioning.

Orthodontic tooth movement is a complex biological process of altered soft and hard tissue remodeling as a result of external forces. In order to ...

A Rat Lung Transplantation Model of Warm Ischemia/Reperfusion Injury: Optimizations to Improve Outcomes

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods for choosing appropriate cuff sizes for the pulmonary vein (PV), pulmonary artery (PA), or bronchus (Br) are varied, thus making the determination of proper cuff size during rat lung transplantation an exercise of trial and error. By standardizing the cuffing technique to use the smallest effective cuff appropriate for the size of the vessel or bronchus, one can make the transplantation procedure safer, faster, and more successful. Since diameters of the PV, PA, and Br are related to the body weight of the rat, we present a strategy to choosing an appropriate size using a weight-based guide. Since lung volume is also related to body weight, we recommend that this relationship should also be considered when choosing the proper volume of air for donor lung inflation during warm ischemia as well as for the proper volume of PBS to be instilled during bronchoalveolar lavage (BAL) fluid collection. We also describe methods for 4th intercostal space dissection, wound closure, and sample collection from both the native and transplanted lobes.

Here, we present optimizations to a rat lung transplantation model that serve to improve outcomes. We provide a size guide for cuffs based on body weight, a measurement strategy to ascertain the 4th intercostal space, and methods of wound closure and BAL (bronchoalveolar lavage) fluid and tissue collection.

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods ...