We wish to express our sincere appreciation to Miss Beatriz M. Szawka for her contribution to the schematic diagram and to Mrs. Ilma Mar&#231;al de Souza for her technical support. J.A.A.A. is the recipient of a fellowship granted by Funda&#231;&#227;o Carlos Chagas Filho de Amparo &#224; Pesquisa do Estado do Rio de Janeiro (FAPERJ, E-26/200.331/2024), Brazil. This study was supported by Conselho Nacional de Desenvolvimento Cient&#237;fico e Tecnol&#243;gico (406928/2023-1), Funda&#231;&#227;o de Amparo a Pesquisa do Estado de Minas Gerais and Coordena&#231;&#227;o de Aperfei&#231;oamento de Pessoal de N&#237;vel Superior (Finance code 001), Brazil.&#160;The authors thank Prof. Dr. Eduardo H. M. Nunes from LabBio/UFMG for the X-ray microtomography analysis.

All procedures strictly adhered to the ethical standards established by the Universidade Federal de Minas Gerais Ethics Committee (No. 166/2022). Before each experiment, a sample size calculation is mandatory. Use 8-10-week-old male C57BL6/J wild-type mice weighing approximately 20-30 g. The mice must be housed in a cage within a room maintained at 25 &#176;C, adhering to a 12 h light/12 h dark cycle. Following coil attachment, the animal should be fed with a soft diet. Daily monitoring should include assessments of body weight and overall health.
1. Mechanically-induced alveolar bone remodeling
<ol>
	<li>Using distal cut pliers, cut the 0.25 x 0.76 inches NiTi open-coil spring to the following dimensions of six loops and two loop-shaped ends positioned perpendicular to the spring using orthodontic Weingart pliers.</li>
	<li>Shape the 0.20 mm diameter round chrome-nickel (CrNi) wire to the desired configuration with loop-shaped ends using Mathieu tweezers and a round-shaped instrument as a size reference.</li>
	<li>Put together the loop-shaped ends of the coil and the 0.20 mm round CrNi wire.</li>
	<li>Anesthetize the animal with an intraperitoneal injection of 0.2 mL of solution containing xylazine (10 mg/Kg) and ketamine (100 mg/Kg). Before starting the procedure, evaluate the depth of anesthesia using the pedal reflex. Carefully pinch one of the animal&#39;s toes using tweezers. The absence of a reflex indicates an adequate plane of general anesthesia. To avoid corneal injuries and post-operative pain, apply an ocular lubricant after the animal is anesthetized.</li>
	<li>Position the animal in dorsal decubitus on a surgical table, immobilizing its limbs to restrict movement and enable intraoral access.</li>
	<li>Utilize a mouth-opener, fashioned from 0.50 mm diameter wire and secured with a 0.08 mm wire, to facilitate full visualization while preventing head movement. Use the right side as the experimental side (OTM side) and the left side as the control without an orthodontic coil (Control side). 
	NOTE: Enhanced visualization of intraoral structures must be achieved using a stereomicroscope and an optical light system.</li>
	<li>Clean and etch the right first molar and incisor surfaces using acetone and a self-etching primer, respectively. The system is self-etching, meaning it does not require prior acid conditioning.
	<ol>
		<li>Apply the primer in a single step, which also functions simultaneously as an acid and an adhesive. Using a microbrush, collect a small amount of self-etching primer and apply it to the occlusal surface of the upper first molar and incisors. Care must be taken in this step to ensure that the self-etching primer does not reach the proximal surface between the first and second molars, as this could cause the dental elements to stick together, preventing tooth movement.&#160;Light cure the primer at the occlusal surface of the&#160;molars and incisors for 30 s.</li>
	</ol>
	</li>
	<li>Bond the distal end of a six-loop NiTi open-coil spring to the occlusal surface of the right first maxillary molar with light-cured resin&#160;and light cure for 30 s. Add additional resin increment&#160;to the edge of the wire to avoid harm to the mice and light cure for 30 s.</li>
	<li>Activate the coil using a specifically designed apparatus consisting of a rail and crank mechanism attached to the surgical table. This enables longitudinal movement to slide back and forth.</li>
	<li>Connect the free loop-shaped end of the 0.20 mm round wire to the hook of the tension gauge.</li>
	<li>Upon activation of the crank, move the surgical table along the rail until the dynamometer registers a force of 0.35 N.</li>
	<li>Bond the 0.20 mm round wire to both upper incisors to anchor the coil. No further reactivation is performed during the experimental period.&#160;Cut the wire to detach the animal from the dynamometer.&#160; Add another increment of resin so the metal edge of the device is not exposed and does not hurt&#160;the animal. Light cure for 30 s. Disassemble the animal from the table.</li>
	<li>The upper right first molar with a device imposing a force of 0.35 N in the mesial direction consists of the experimental side. Use the left side of the maxilla (without an orthodontic appliance) as a control4,16.</li>
	<li>Maintain this device for a period of 12 days without any activation being required. Do not use any pain control medication. Orthodontic movement occurs through an inflammatory process, and pain-relieving medication may negatively affect the arachidonic acid cascade, impacting the rate of bone remodeling and potentially invalidating the results.</li>
	<li>After the end of the surgery, treat the animals with a subcutaneous injection of&#160;saline solution to avoid dehydration during the adaptation period with the device. Keep the animal in an individual cage with heating until it is fully recovered and only after this period place the animal in collective cages.</li>
	<li>Euthanize the mouse on the 12th&#160;day performed by sedation with intraperitoneal injection of a 0.2 mL solution containing xylazine (10 mg/Kg) and ketamine (100 mg/Kg) followed by decapitation with sharp scissors.</li>
</ol>
2. Micro-CT measurements
<ol>
	<li>Harvest the maxillary bone with sharp scissors cutting all soft tissue, the zygomatic bone in the sagittal plane, and the frontonasal suture and the spheno-occipital synchondrosis in the coronal plane. Immerse the maxillary bone in 10% neutral buffered formalin (pH=7.4) for a fixation period of 48 h. After this period, change the formaldehyde solution to 70% alcohol.</li>
	<li>Perform micro-CT scanning of the maxillary bone with the following parameters for high-resolution scans: isotropic voxel size of 9 to 18 &#181;m, X-ray settings of 50 kV, 0.5 mm aluminum filter, and rotation angle of 0.5&#176;.&#160;More than one jaw can be fit during the microCT scanning.</li>
	<li>Reconstruct the acquired images using the microtomography reconstruction program indicated by the manufacturer of the micro-computed tomography (microCT) used17.</li>
	<li>Position the reconstructed images using the 3D Inspection program indicated by the manufacturer of the microtomography used.</li>
	<li>Quantify OTM by measuring the difference in linear distance between the cement-enamel junction (CEJ) of the first and second molars of the right hemi-maxilla (OTM side) relative to the left hemi-maxilla (control side). Utilize the proper microCT analyzer software with the line tool for this measurement17,18,19,20.</li>
	<li>Check the samples for the presence of orthodontic-induced inflammatory root resorption (OIIRR). Select the region of interest (ROI) of the disto-vestibular root of the first maxillary molar by using an irregular, anatomic region of interest drawn manually contouring method. Measure the following parameters: root mineral density (RMD; g/cm3) and percentage of root volume per total volume (RV/TV; %). Utilize the proper microCT analyzer software with the 3D volumetric tool for this measurement16,21.</li>
	<li>Conduct the reconstruction of the first maxillary molar using Mimics software and analyze the obtained data to draw conclusions regarding OTM and OIIRR in the experimental model16,21.</li>
</ol>

Investigating Orthodontic Tooth Movement and Bone Adaptation Mechanisms in Mice

<ol>
	<li>Kohli, N., 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=Bone+remodelling+in+vitro:+Where+are+we+headed?:+-A+review+on+the+current+understanding+of+physiological+bone+remodelling+and+inflammation+and+the+strategies+for+testing+biomaterials+in+vitro.">Bone remodelling in vitro: Where are we headed?: -A review on the current understanding of physiological bone remodelling and inflammation and the strategies for testing biomaterials in vitro.</a> Bone. 110, 38-46 (2018).</li><li>Epsley, 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=The+effect+of+inflammation+on+bone.">The effect of inflammation on bone.</a> Front Physiol. 11, 511799 (2021).</li><li>Bolamperti, S., Villa, I., Rubinacci, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+remodeling:+an+operational+process+ensuring+survival+and+bone+mechanical+competence.">Bone remodeling: an operational process ensuring survival and bone mechanical competence.</a> Bone Res. 10 (1), 48 (2022).</li><li>Taddei, S. R., 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=Experimental+model+of+tooth+movement+in+mice:+a+standardized+protocol+for+studying+bone+remodeling+under+compression+and+tensile+strains.">Experimental model of tooth movement in mice: a standardized protocol for studying bone remodeling under compression and tensile strains.</a> J Biomech. 45 (16), 2729-2735 (2012).</li><li>Guerrero, J. A., 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=Maxillary+suture+expansion:+A+mouse+model+to+explore+the+molecular+effects+of+mechanically-induced+bone+remodeling.">Maxillary suture expansion: A mouse model to explore the molecular effects of mechanically-induced bone remodeling.</a> J Biomech. 108, 109880 (2020).</li><li>Ibrahim, A. Y., Gudhimella, S., Pandruvada, S. N., Huja, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolving+differences+between+animal+models+for+expedited+orthodontic+tooth+movement.">Resolving differences between animal models for expedited orthodontic tooth movement.</a> Orthod Craniofac Res. 20, 72-76 (2017).</li><li>Kirschneck, C., Bauer, M., Gubernator, J., Proff, P., Schr&#246;der, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+assessment+of+mouse+models+for+experimental+orthodontic+tooth+movement.">Comparative assessment of mouse models for experimental orthodontic tooth movement.</a> Sci Rep. 10 (1), 12154 (2020).</li><li>Huang, H., Williams, R. C., Kyrkanides, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+orthodontic+tooth+movement:+molecular+mechanisms.">Accelerated orthodontic tooth movement: molecular mechanisms.</a> Am J Orthod Dentofac Ortho. 146 (5), 620-632 (2014).</li><li>Ariffin, S. H. Z., Yamamoto, Z., Abidin, I. Z., Wahab, R. A. M., Ariffin, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+molecular+changes+in+orthodontic+tooth+movement.">Cellular and molecular changes in orthodontic tooth movement.</a> Sci World J. 11, 1788-1803 (2011).</li><li>Alhasyimi, A. A., Pudyani, P. P., Asmara, W., Ana, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+post-orthodontic+tooth+stability+by+carbonated+hydroxyapatite-incorporated+advanced+platelet-rich+fibrin+in+rabbits.">Enhancement of post-orthodontic tooth stability by carbonated hydroxyapatite-incorporated advanced platelet-rich fibrin in rabbits.</a> Orthod Craniofac Res. 21, 112-118 (2018).</li><li>Klein, Y., 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=Immunorthodontics:+in+vivo+gene+expression+of+orthodontic+tooth+movement.">Immunorthodontics: in vivo gene expression of orthodontic tooth movement.</a> Sci Rep. 10 (1), 8172 (2020).</li><li>Fujimura, Y., 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=Influence+of+bisphosphonates+on+orthodontic+tooth+movement+in+mice.">Influence of bisphosphonates on orthodontic tooth movement in mice.</a> Eur J Orthod. 31 (6), 572-577 (2009).</li><li>Waldo, C. M., Rothblatt, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histologic+response+to+tooth+movement+in+the+laboratory+rat;+procedure+and+preliminary+observations.">Histologic response to tooth movement in the laboratory rat; procedure and preliminary observations.</a> J Dental Res. 33 (4), 481-486 (1954).</li><li>Chavez, M. B., 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=Guidelines+for+micro-computed+tomography+analysis+of+rodent+dentoalveolar+tissues.">Guidelines for micro-computed tomography analysis of rodent dentoalveolar tissues.</a> JBMR Plus. 5 (3), e10474 (2021).</li><li>Trelenberg-Stoll, V., Wolf, M., Busch, C., Drescher, D., Becker, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardized+assessment+of+bone+micromorphometry+around+teeth+following+orthodontic+tooth+movement:+A+&#181;CT+split-mouth+study+in+mice.">Standardized assessment of bone micromorphometry around teeth following orthodontic tooth movement: A &#181;CT split-mouth study in mice.</a> J Orofac Ortho. 83 (6), 403-411 (2022).</li><li>Santos, M. 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=Targeting+phosphatidylinositol-3-kinase+for+inhibiting+maxillary+bone+resorption.">Targeting phosphatidylinositol-3-kinase for inhibiting maxillary bone resorption.</a> J Cell Physiol. 238 (11), 2651-2667 (2023).</li><li>Macari, 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=Oestrogen+regulates+bone+resorption+and+cytokine+production+in+the+maxillae+of+female+mice.">Oestrogen regulates bone resorption and cytokine production in the maxillae of female mice.</a> Arch Oral Biol. 60 (2), 333-341 (2015).</li><li>Macari, 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=Lactation+induces+increases+in+the+RANK/RANKL/OPG+system+in+maxillary+bone.">Lactation induces increases in the RANK/RANKL/OPG system in maxillary bone.</a> Bone. 110, 160-169 (2018).</li><li>Pereira, L. J., 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=Aerobic+and+resistance+training+improve+alveolar+bone+quality+and+interferes+with+bone-remodeling+during+orthodontic+tooth+movement+in+mice.">Aerobic and resistance training improve alveolar bone quality and interferes with bone-remodeling during orthodontic tooth movement in mice.</a> Bone. 138, 115496 (2020).</li><li>Silva, F. R. F., 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=Protective+effect+of+bovine+milk+extracellular+vesicles+on+alveolar+bone+loss.">Protective effect of bovine milk extracellular vesicles on alveolar bone loss.</a> Mol Nutri Food Res. 68 (3), 2300445 (2024).</li><li>Amaro, E. R. 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=Estrogen+protects+dental+roots+from+orthodontic-induced+inflammatory+resorption.">Estrogen protects dental roots from orthodontic-induced inflammatory resorption.</a> Arch Oral Biol. 117, 104820 (2020).</li><li>Xu, X., Zhou, J., Yang, F., Wei, S., Dai, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+micro-computed+tomography+to+evaluate+the+dynamics+of+orthodontically+induced+root+resorption+repair+in+a+rat+model.">Using micro-computed tomography to evaluate the dynamics of orthodontically induced root resorption repair in a rat model.</a> PLoS One. 11 (3), e0150135 (2016).</li><li>Bouxsein, M. 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=Guidelines+for+assessment+of+bone+microstructure+in+rodents+using+micro-computed+tomography.">Guidelines for assessment of bone microstructure in rodents using micro-computed tomography.</a> J Bone Mineral Res. 25 (7), 1468-1486 (2010).</li><li>Kanou, 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=Effect+of+age+on+orthodontic+tooth+movement+in+mice.">Effect of age on orthodontic tooth movement in mice.</a> J Dent Sci. 19 (2), 828-836 (2024).</li></ol>

The authors have no conflicts of interest to declare.

Here, we describe a standardized protocol designed to elucidate the cellular and molecular mechanisms underlying bone remodeling during OTM. A thorough understanding of these mechanisms in mice requires a meticulously planned protocol to ensure accuracy and reliability7,11. Studies conducted by our research group have shown that this protocol effectively reduces operator variability by incorporating a tension gauge and a specially designed apparatus, establishing...

Bone remodeling is an ongoing process orchestrated by osteoclasts, osteoblasts, bone lining cells, and osteocytes, essential for maintaining the integrity of the adult skeleton1,2. Primarily driven by the differentiation and activity of osteoclasts and osteoblasts, this dynamic process involves the resorption and deposition of bone, triggered by mechanical stress and loading3,4,5.
Animal experiments play a pivotal role in elucidating the intricate biological and cellular mechanisms underpinning orthodontic tooth movement (OTM)6,7. This process involves a diverse array of cell types, such as osteoblasts, osteoclasts, osteocytes, fibroblasts, and immune cells like macrophages and T cells, situated within the jawbone and periodontal ligament7,8. These cells dynamically respond to mechanical stimuli and changes in the local milieu, influencing the composition and architecture of the surrounding bone7,8. Moreover, they also trigger an inflammatory response at a cellular level, even though there are no pathogens present. This inflammatory response plays a role in increasing the turnover of bone tissue9.
Various animal models, including mice, rats, rabbits, dogs, and monkeys, have been utilized in experimental studies of OTM7,8,10. Among these, rodents, particularly mice, are favored for investigating the initial phases of tooth movement and bone remodeling6. Previous research has emphasized the advantages of using mouse models over rat models, primarily due to the widespread availability of genetically modified strains, enabling detailed exploration of genetic influences in OTM7,11. Currently, two main models are employed to induce tooth movement in mice. The first method entails inserting a nickel-titanium (NiTi) coil spring between the first upper molar and upper incisors4,12. The second approach involves placing an elastic band within the interdental space between the first and second upper molars13. The primary outcomes analyzed typically include the magnitude of the tooth movement and bone microarchitecture, preferably evaluated using micro-computed tomography (micro-CT)14.&#160;Ideally, assessing the integrity of dental roots is important to ensure that appropriate forces are employed to produce OTM4.
While micro-CT is widely acknowledged as the gold standard for evaluating the microarchitecture of mineralized tissues14, the absence of standardized methodologies and protocols for scanning, analyzing, and reporting data often presents challenges in discerning the precise procedures employed, interpreting results, and facilitating comparisons between different OTM models14,15.
Here, we present a step-by-step guide to the OTM mouse model, including micro-CT acquisition and analysis of OTM, bone microstructure, and dental roots. This method entails applying controlled mechanical force to the first molar to induce movement within the jawbone. The selection of this method stems from several factors, including feasibility, relevance, and precision. Such an approach enables detailed quantitative analysis, providing valuable insights into the biological processes underlying orthodontic tooth movement and facilitating the development of improved orthodontic treatment strategies in the future.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetone</td>
			<td>Sigma-Aldrich</td>
			<td>67-64-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Distal cut pliers</td>
			<td>Quinelato</td>
			<td>QO.700.00</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynamometer</td>
			<td>SHIMPO</td>
			<td>FGE-5XY</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiber Optic Illuminator</td>
			<td>Cole-Parmer</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>ketamine</td>
			<td>Syntec</td>
			<td>100477-72-3</td>
			<td></td>
		</tr>
		<tr>
			<td>NiTi open-coil spring 0.25 x 0.76</td>
			<td>Lancer Orthodontics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#216; 0.20 mm round chrome-nickel (CrNi)</td>
			<td>Morelli</td>
			<td>55.01.208</td>
			<td></td>
		</tr>
		<tr>
			<td>Round CrNi Hard Elastic Orthodontic Wire &#216;0.50 mm (.020 inch)</td>
			<td>Morelli</td>
			<td>55.01.050</td>
			<td></td>
		</tr>
		<tr>
			<td>Round CrNi Tie Wire &#216;0.20 mm (.008 inch)</td>
			<td>Morelli</td>
			<td>55.01.208</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Quimis</td>
			<td>Q7740SZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Transbond Plus Self Etching Primer</td>
			<td>3M</td>
			<td>LE-Q100-1004-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Weingart Plier</td>
			<td>Quinelato</td>
			<td>QO.120.00</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Syntec</td>
			<td>23076-35-9</td>
			<td></td>
		</tr>
		<tr>
			<td>MicroCT Analysis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Skyscan 1174v2</td>
			<td>Bruker</td>
			<td>1174v2</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NRecon</td>
			<td>Skyscan</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>DataViewer</td>
			<td>Skyscan</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>CTAn</td>
			<td>Skyscan</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Mimics</td>
			<td>Materialise</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

studying orthodontic tooth movement in mice

Orthodontic tooth movement (OTM) represents a dynamic process in which the alveolar bone undergoes resorption at compression sites and deposition at tension sites, orchestrated by osteoclasts and osteoblasts, respectively. This mechanism serves as a valuable model for studying various aspects of bone adaptation, including root resorption and the cellular response to mechanical force stimuli. The protocol outlined here offers a straightforward approach to investigate OTM, establishing 0.35 N as the optimal force in a mouse model employing a nickel-titanium (NiTi) coil spring. Utilizing micro-computed tomography analysis, we quantified OTM by assessing the discrepancy in the linear distance at the cement-enamel junction. The evaluation also included an analysis of orthodontic-induced inflammatory root resorption, assessing parameters such as root mineral density and the percentage of root volume per total volume. This comprehensive protocol contributes to advancing our understanding of bone remodeling processes and enhancing the ability to develop effective orthodontic treatment strategies.

This protocol enables the investigation of an OTM mouse model using a NiTi coil spring. With a force of 0.35 N applied, the mean CEJ distance on the control side between the first and second molars was 243.69 &#181;m (Figure 1A, line A), whereas on the OTM side was measured at 284.66 &#181;m (Figure 1A, line B). The difference between the OTM and control sides was 40.97 &#181;m (Figure 1B). The linear distance betwe...

Studying Orthodontic Tooth Movement in Mice

Here, we present a protocol for studying orthodontic tooth movement (OTM), serving as a suitable model for investigating the mechanisms of bone adaptation, root resorption, and the response of bone cells to mechanical stimuli. This comprehensive guide provides detailed information on the OTM model, micro-computed tomography acquisition, and subsequent analysis.

Orthodontic tooth movement (OTM) represents a dynamic process in which the alveolar bone undergoes resorption at compression sites and deposition at ...

studying-orthodontic-tooth-movement-in-mice

Research

JoVE Journal

Biology

492 Views.  Universidade Federal do Rio de Janeiro. Here, we present a protocol for studying orthodontic tooth movement (OTM), serving as a suitable model for investigating the mechanisms of bone adaptation, root resorption, and the response of bone cells to mechanical stimuli. This comprehensive guide provides detailed information on the OTM model, micro-computed tomography acquisition, and subsequent analysis.

Video: Studying Orthodontic Tooth Movement in Mice

Studying Aggression in Drosophila (fruit flies)

Aggression is an innate behavior that evolved in the framework of defending or obtaining resources. This complex social behavior is influenced by genetic, hormonal and environmental factors. In many organisms, aggression is critical to survival but controlling and suppressing aggression in distinct contexts also has become increasingly important. In recent years, invertebrates have become increasingly useful as model systems for investigating the genetic and systems biological basis of complex social behavior. This is in part due to the diverse repertoire of behaviors exhibited by these organisms. In the accompanying video, we outline a method for analyzing aggression in Drosophila whose design encompasses important eco-ethological constraints. Details include steps for: making a fighting chamber; isolating and painting flies; adding flies to the fight chamber; and video taping fights. This approach is currently being used to identify candidate genes important in aggression and in elaborating the neuronal circuitry that underlies the output of aggression and other social behaviors.

Studying Aggression in Drosophila fruit flies

Aggression is an innate behavior that evolved in the framework of defending or obtaining resources.  This complex social behavior is influenced by ...

The Structure of Skilled Forelimb Reaching in the Rat: A Movement Rating Scale

Skilled reaching for food is an evolutionary ancient act and is displayed by many animal species, including those in the sister clades of rodents and primates. The video describes a test situation that allows filming of repeated acts of reaching for food by the rat that has been mildly food deprived. A rat is trained to reach through a slot in a holding box for food pellet that it grasps and then places in its mouth for eating. Reaching is accomplished in the main by proximally driven movements of the limb but distal limb movements are used for pronating the paw, grasping the food, and releasing the food into the mouth. Each reach is divided into at least 10 movements of the forelimb and the reaching act is facilitated by postural adjustments. Each of the movements is described and examples of the movements are given from a number of viewing perspectives. By rating each movement element on a 3-point scale, the reach can be quantified. A number of studies have demonstrated that the movement elements are altered by motor system damage, including damage to the motor cortex, basal ganglia, brainstem, and spinal cord. The movements are also altered in neurological conditions that can be modeled in the rat, including Parkinson's disease and Huntington's disease. Thus, the rating scale is useful for quantifying motor impairments and the effectiveness of neural restoration and rehabilitation. Because the reaching act for the rat is very similar to that displayed by humans and nonhuman primates, the scale can be used for comparative purposes. from a number of viewing perspectives. By rating each movement element on a 3-point scale, the reach can be quantified. A number of studies have demonstrated that the movement elements are altered by motor system damage, including damage to the motor cortex, basal ganglia, brainstem, and spinal cord. The movements are also altered in neurological conditions that can be modeled in the rat, including Parkinson's disease and Huntington's disease. Thus, the rating scale is useful for quantifying motor impairments and the effectiveness of neural restoration and rehabilitation.

Experiments on animals were performed in accordance with the guidelines and regulations set forth by the University of Lethbridge Animal Care Committee in accordance with the regulations of the Canadian Council on Animal Care.

The skilled reaching scale divides the movement by a forelimb in a reach for food act into composite elements each of which are evaluated with a three-point scale. The rating scale is described for a normal rat and can be applied toward evaluating neurological motor disorders.

Skilled reaching for food is an evolutionary ancient act and is displayed by many animal species, including those in the sister clades of rodents and ...

Using an EEG-Based Brain-Computer Interface for Virtual Cursor Movement with BCI2000

A brain-computer interface (BCI) functions by translating a neural signal, such as the electroencephalogram (EEG), into a signal that can be used to control a computer or other device. The amplitude of the EEG signals in selected frequency bins are measured and translated into a device command, in this case the horizontal and vertical velocity of a computer cursor. First, the EEG electrodes are applied to the user s scalp using a cap to record brain activity. Next, a calibration procedure is used to find the EEG electrodes and features that the user will learn to voluntarily modulate to use the BCI. In humans, the power in the mu (8-12 Hz) and beta (18-28 Hz) frequency bands decrease in amplitude during a real or imagined movement. These changes can be detected in the EEG in real-time, and used to control a BCI ([1],[2]). Therefore, during a screening test, the user is asked to make several different imagined movements with their hands and feet to determine the unique EEG features that change with the imagined movements. The results from this calibration will show the best channels to use, which are configured so that amplitude changes in the mu and beta frequency bands move the cursor either horizontally or vertically. In this experiment, the general purpose BCI system BCI2000 is used to control signal acquisition, signal processing, and feedback to the user [3].

In this video, we demonstrate the steps required to run a brain-computer interface experiment, including setting up the EEG cap, calibrating the system, and training the user to move a cursor in two dimensions using imagined movements.

A brain-computer interface (BCI) functions by translating a neural signal, such as the electroencephalogram (EEG), into a signal that can be used to ...

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

Transverse Aortic Constriction in Mice

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart failure.1 TAC initially leads to compensated hypertrophy of the heart, which often is associated with a temporary enhancement of cardiac contractility. Over time, however, the response to the chronic hemodynamic overload becomes maladaptive, resulting in cardiac dilatation and heart failure.2 The murine TAC model was first validated by Rockman et al.1, and has since been extensively used as a valuable tool to mimic human cardiovascular diseases and elucidate fundamental signaling processes involved in the cardiac hypertrophic response and heart failure development. When compared to other experimental models of heart failure, such as complete occlusion of the left anterior descending (LAD) coronary artery, TAC provides a more reproducible model of cardiac hypertrophy and a more gradual time course in the development of heart failure. Here, we describe a step-by-step procedure to perform surgical TAC in mice. To determine the level of pressure overload produced by the aortic ligation, a high frequency Doppler probe is used to measure the ratio between blood flow velocities in the right and left carotid arteries.3, 4 With surgical survival rates of 80-90%, transverse aortic banding is an effective technique of inducing left ventricular hypertrophy and heart failure in mice.

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model to study mechanisms underlying cardiac hypertrophy and heart failure development. Here, we describe procedures to constrict the aorta to create a reproducible degree of cardiac hypertrophy in mice.

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart ...

Ambulatory ECG Recording in Mice

Telemetric ECG recording in mice is essential to understanding the mechanisms behind arrhythmias, conduction disorders, and sudden cardiac death. Although the surface ECG is utilized for short-term measurements of waveform intervals, it is not practical for long-term studies of heart rate variability or the capture of rare episodes of arrhythmias. Implantable ECG telemeters offer the advantages of simple surgical implantation, long-term recording of electrograms in ambulatory mice, and scalability with simultaneous recordings of multiple animals. Here, we present a step-by-step guide to the implantation of telemeters for ambulatory ECG recording in mice. Careful adherence to aseptic technique is required for favorable survival results with the possibility of implantation and recording from weeks to months. Thus, implantable ECG telemetry is a valuable tool for detection of critical information on cardiac electrophysiology in ambulatory animal models such as the mouse. 

Telemetric ECG has emerged as an essential tool in evaluating animal models for cardiac arrhythmias and sudden cardiac death. Here, we present a stepwise guide to telemetric ECG recordings for application in long-term ambulatory ECG monitoring in mice.

Telemetric ECG recording in mice is essential to understanding the mechanisms behind arrhythmias, conduction disorders, and sudden cardiac death. Although ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.
In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel&#39;s cartilage, nasal glands, tongue, and ear.

Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods.

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental ...

Nanopodia - Thin, Fragile Membrane Projections with Roles in Cell Movement and Intercellular Interactions

Adherent cells in culture maintain a polarized state to support movement and intercellular interactions. Nanopodia are thin, elongated, largely F-actin-negative membrane projections in endothelial and cancer cells that can be visualized through TM4SF1 (Transmembrane-4-L-six-family-1) immunofluorescence staining. TM4SF1 clusters in 100-300 &#956;m diameter TMED (TM4SF1 enriched microdomains) containing 3 to as many as 14 individual TM4SF1 molecules. TMED are arranged intermittently along nanopodia at a regular spacing of 1 to 3 TMED per &#956;m and firmly anchor nanopodia to matrix. This enables nanopodia to extend more than 100 &#956;m from the leading front or trailing rear of polarized endothelial or tumor cells, and causes membrane residues to be left behind on matrix when the cell moves away. TMED and nanopodia have been overlooked because of their extreme fragility and sensitivity to temperature. Routine washing and fixation disrupt the structure. Nanopodia are preserved by direct fixation in paraformaldehyde (PFA) at 37 &#176;C, followed by brief exposure to 0.01% Triton X-100 before staining. Nanopodia open new vistas in cell biology: they promise to reshape our understanding of how cells sense their environment, detect and identify other cells at a distance, initiate intercellular interactions at close contact, and of the signaling mechanisms involved in movement, proliferation, and cell-cell communications. The methods that are developed for studying TM4SF1-derived nanopodia may be useful for studies of nanopodia that form in other cell types through the agency of classic tetraspanins, notably the ubiquitously expressed CD9, CD81, and CD151.

Nanopodia are thin but fragile membrane channels that extend up to 100 &#956;m from a cell's leading front or trailing rear and sense the cellular environment. Direct fixation at 37 &#176;C, gentle washing, and avoidance of organic solvents like ethanol, methanol, or acetone and of higher Triton X-100 concentrations are required to observe these cellular structures.

Adherent cells in culture maintain a polarized state to support movement and intercellular interactions. Nanopodia are thin, elongated, largely ...

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Fluorescence microscopy is an indispensable tool for the direct assessment of mitochondrial structure and function in live cells and for studying the mitochondrial structure-function relationship, which is primarily modulated by the molecules governing fission and fusion events between mitochondria. This paper describes and demonstrates specific methods for studying mitochondrial structure and function in live as well as in fixed tissue in the model organism Drosophila melanogaster. The tissue of choice here is the Drosophila ovary, which can be isolated and made amenable for ex vivo live confocal microscopy. Furthermore, the paper describes how to genetically manipulate the mitochondrial fission protein, Drp1, in Drosophila ovaries to study the involvement of Drp1-driven mitochondrial fission in modulating the mitochondrial structure-function relationship. The broad use of such methods is demonstrated in already-published as well as in novel data. The described methods can be further extended towards understanding the direct impact of nutrients and/or growth factors on the mitochondrial properties ex vivo. Given that mitochondrial dysregulation underlies the etiology of various diseases, the described innovative methods developed in a genetically tractable model organism, Drosophila, are anticipated to contribute significantly to the understanding of the mechanistic details of the mitochondrial structure-function relationship and to the development of mitochondria-directed therapeutic strategies.

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Specific methods for studying mitochondrial structure and function in live and fixed Drosophila ovaries are described and demonstrated in this paper.

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial ...