This study was supported by the NIH (NIDCR R00- DE025053, PI:Naveh). We would like to thank Harvard Center for Biological Imaging for infrastructure and support. All figures are generated with biorender.com.

All animal experiments were performed in compliance with NIH&#39;s Guidelines for the Care and Use of Laboratory Animals and guidelines from the Harvard University Institutional Animal Care and Use Committee (Protocol no. 01840).
1. Orthodontic Tooth Movement
<ol>
	<li>To generate a mouse bed, use a flat plastic platform with a wedge shaped, 45&#176; angled headrest. The headrest can be generated by cutting a plastic box.
	<ol>
		<li>Elevate the head end of the platform to generate an approximately 30&#176; angle between the headrest and the table plane. Attach a bent thick paper clip (0.036&#34; in diameter) to the head side end to hold the upper incisors.</li>
		<li>On the tail end, generate an elevated surface to which an orthodontic power chain can be attached to hold the lower incisors. See Figure 1 for an example platform.</li>
	</ol>
	</li>
	<li>Anesthetize mouse by&#160;intraperitoneal injection of xylazine at 10 mg/kg and ketamine 100mg/kg using 1 mL syringe and a 27 Gauge needle.</li>
	<li>Place anesthetized mouse on custom-made platform and immobilize upper jaw by hooking the upper incisors on the paperclip loop. Open the mouse lower jaw with the orthodontic power chain hooked on the lower incisors. Keep the cheeks retracted with the mini-colibri mouth retractor.</li>
	<li>Place the platform under a surgical microscope or any other stereoscope that can reach to 5-6x magnification.</li>
	<li>Apply 50 &#181;L of saline (roughly 1 drop) on the mouse eyes to prevent corneal dehydration. Replenish saline every 20 min.</li>
	<li>Cut a piece of an aluminum wire (0.08 mm diameter) 1 cm in length. Slide the wire from the buccal side lingually in the interproximal area bellow the contact point between first and second molars using a microsurgical needle holder. Leave 2 mm free edge in front of the first molar in order to be threaded into the spring end.</li>
	<li>Cut a piece of nickel titanium (NiTi) coil, around 7 to 9 threads in length. 
	NOTE: The elastic properties of the coil will provide constant force for orthodontic movement. The total unstrained length of the coil should be shorter than the gap between the incisor and the molar. Keep in mind that an extra 2 threads are needed on each end to anchor coil to the tooth. Aluminum wire is selected in order to reduce scanning artefacts such as beam hardening during the micro-CT scan.</li>
	<li>Insert NiTi coil spring (0.15 mm wire diameter, 0.9 mm inner coil diameter; delivers a force of 10 g) between lower first molar and lower incisor. Use the wire ligature inserted around the first molar in step 1.6, twist the wire tightly around 2 threads of the coil spring to fix the coil on the molar side.</li>
	<li>To ensure uniform force level, use exactly 3 active threads between the first molar and the incisor. Temporarily remove the power chain from the incisor and loop 2 to 3 unstrained threads over the incisor to anchor the coil. Slide the threads down to the incisor free gingival margin.</li>
	<li>Place a layer of flowable composite resin on the incisal border of the coil and cure it with dental curing light. Replace the power chain after curing the resin.</li>
	<li>Using the same curing light, heat up the NiTi coil for 20 s. This will tighten the NiTi coil. The finished placement is shown Figure 1C.</li>
	<li>Either leave the contralateral side intact or insert a sham such as the wire between the first and&#160;second molars.</li>
	<li>Place the anaesthetized mouse under a heated light to keep the mouse warm until recovery.</li>
	<li>Place the mouse back into an individual cage and monitor daily. No diet change is necessary during orthodontic movement. 
	NOTE: OTM device on one side causes some discomfort but does not impair feeding. However, inserting devices on both sides are not advised due to the added amount of discomfort. Pain medication is not necessary unless outward signs of pain are seen.</li>
</ol>
2. Micro-CT scan of PDL fibers in fresh hemi-mandibles
<ol>
	<li>Mounting the hemi-mandible (Figure 2)
	<ol>
		<li>After the desired duration of orthodontic movement, sacrifice the mouse via cervical dislocation. Remove the mandible and separate into hemi-mandibles. 
		&#8203;NOTE: Since the sample will not be fixed, it is critical to perform the dissection of the jaw and mounting as soon as possible, ideally within 30 min.</li>
		<li>Remove the surrounding soft tissue gently with a clean lint-free wipe.</li>
		<li>Remove the orthodontic device using microsurgical scissors and tweezers under a stereo microscope with at least 4x magnification.</li>
		<li>Keep sample moist in a 1.5 mL volume, micro-centrifuge tube along with a piece of lint-free wipe moistened with water.</li>
		<li>Place packable dental composite resin into the sample slot on the stage, then place the fresh hemi-mandible into the composite. Before mounting make sure that the bone surface in contact with the dental composite is free from any soft tissues and dry, otherwise dental composite will not cure properly.</li>
		<li>Adjust the position of the hemi-mandible until the first molar is centered at the midline groove of the stage. Make sure the occlusal surface is horizontal. Cure the composite when satisfied with the positioning. 
		NOTE: Additional small amounts of dental composites can be placed on the sides of the hemi-mandible and/or across the incisor to aid in stabilizing the sample.</li>
		<li>Place dampened lint-free wipe inside the humidity pools in the sample stage. Place dental composite on the occlusal surface of the first molar. Before closing the chamber, make sure nothing blocks the x-ray path at the specimen level.</li>
		<li>Affix the chamber in the micro-CT. Screw the chamber into the micro-CT sample stage so that movement during imaging is minimized.</li>
		<li>Turn on the x-rays and take 2D images while lowering the anvil vertically, until the tip of the anvil is surrounded by the composite but no increase in the force is detected.</li>
		<li>Once anvil is embedded in the composite, close the x-ray source. Then, open the micro-CT chamber and cure the composite through the clear Plexiglass window.</li>
	</ol>
	</li>
	<li>Micro-CT settings
	<ol>
		<li>Set the source voltage to 40 kV and current to 200 &#181;A. Using a 10x magnification detector, position the sample within the frame of view. Use binning of 2 for the captured images. 
		NOTE: Since the PDL is significantly less dense than the bone and tooth, visualizing the PDL requires higher power and exposure time. This protocol will provide settings for visualizing the PDL.</li>
		<li>Set single image exposure time to 25 s. Set rotation of the sample stage to a range of 183 degrees or more. Set the scan for 2500 projections. Do not use any x-ray source filter, the resulting scans have a voxel size of 0.76 &#181;m on each side.</li>
		<li>Collect a reference scan for proper reconstruction according to the micro-CT guidelines. Use 1/3 number of reference images as the total projections. Reconstruct the volume without additional binning, using a back projection filtered algorithm.</li>
	</ol>
	</li>
</ol>
3. Clearing method (Figure 3)
<ol>
	<li>Prepare five 1.5 mL micro-centrifuge tubes.</li>
	<li>Prepare 1.4 mL of the following solutions in 1.5 mL micro-centrifuge tubes: 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS), 50% ethanol (EtOH) in deionized (DI) water, 70% EtOH in DI water, and two tubes of 100% EtOH. 
	NOTE: PFA is used for fixation of the sample. ECi clearing will also work on unfixed samples. To clear unfixed samples, simply skip the PFA step.</li>
	<li>Place dissected hemi-mandible in 4% PFA. Cover with aluminum foil and place on the rocker on gentle setting at room temperature for 6 h.</li>
	<li>Move the hemi-mandible to 50% EtOH. Place it on the rocker covered from light for 16 h.</li>
	<li>Move the hemi-mandible to 70% EtOH. Place it on the rocker covered from light for 16 h.</li>
	<li>Move the hemi-mandible to 100% EtOH. Place it on the rocker covered from light for 16 h.</li>
	<li>Repeat 3.6. in the second 100% EtOH tube.</li>
	<li>Prepare 5 mL of ECi in a glass or polypropylene tube. 
	NOTE: ECi dissolves polystyrene, but not polypropylene. Also, if not using a tissue with fluorescent proteins the sample can be exposed to light during the clearing procedure.</li>
	<li>Move the hemi-mandible to ECi tube. Cover the tube with aluminum foil and place on the rocker on gentle setting for a minimum of 12 h. 
	NOTE: The protocol can be paused here. Dehydrated sample can be stored in ECi at room temperature. The freezing or melting point of ECi is 6.5 to 8.0 &#176;C. Do not store at 4 &#176;C.</li>
	<li>The hemi-mandible is ready for imaging with fluorescence microscope. 
	NOTE: During imaging, the sample must be immersed in the ECi to remain optically transparent.</li>
</ol>

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The authors have nothing to disclose.

Generating OTM in mice is highly desired due to the size, genetics and handling advantages. Using the mandible provides an easy handling both in terms of tissue dissection as well as sample preparation and imaging. Here we presented a method to generate OTM with translational movement of the tooth inside the bone within 7 days of OTM. Using this protocol, the overall duration of the tooth movement can be extended, since the activated coil delivers a constant force level&#160;for movement of up to approximately 1 mm. Howe...

The basic underlying biological process in orthodontic tooth movement (OTM) is bone remodeling. The trigger for this remodeling process is attributed to changes in the structure of the periodontal ligament (PDL) such as extracellular matrix (ECM) stress, necrosis as well as blood vessel destruction and formation1,2,3. Other possible triggers for alveolar bone remodeling are related to force sensing by osteocytes in the bone, as well as mechanical deformation of the alveolar bone itself; however their role in OTM is still not fully elucidated4,5.
Despite many studies aimed at revealing structure-function relations of the PDL during OTM, a clear functional mechanism is yet to be defined6,7. The major reason for this is the challenge in retrieving data of a soft tissue (PDL) located between two hard tissues (cementum and alveolar bone). The accepted methods to collect structural information usually necessitate fixation and sectioning that disrupt and modify the PDL structure. Moreover, most of these methods yield 2D data that even if not distorted, give only partial and localized information. Since the PDL is not uniform in its structure and function, an approach that addresses the intact 3D structure of the entire tooth-PDL-bone complex is warranted.
This paper will describe a method for generating an OTM in mice and two methods that enable 3D visualization of the collagen fibers in the PDL without any sectioning of the sample.
Murine models are widely used for in-vivo experiments in medicine, developmental biology, drug delivery and structural studies. They can be genetically modified to eliminate or enhance specific proteins and function; they provide fast, repeatable and predictable developmental control; they are also easy to image due to their small size8. Despite their many advantages, mouse models in dental research are not used frequently, especially when clinical manipulations are warranted, mostly due to the small sized teeth. Animal models such as rats9,10,11, dogs12,13, pigs14,15,16 and monkeys17 are used more often than mice. With the recent development of high-resolution imaging techniques, the advantages of utilizing a mouse model to decipher the convoluted processes in OTM are numerous. This paper presents a method to generate a mesial movement of the molar tooth in the mandible with constant force levels that trigger&#160;bone remodeling. Most of the OTM experiments in rodents are done in the maxilla, since the mobility of the mandible and the presence of the tongue add another complexity level. However, the mandible has many advantages when 3D structural integrity is desired. It can be easily dissected as a whole bone; in some species it can be separated into two hemi-mandibles through the fibrous symphysis;&#160;it is compact, flat and contains only the teeth without any sinus spaces. In contrast, the maxilla is a part of the skull and closely related to other organs and structures, thus extensive sectioning is needed in order to dissect the alveolar bone with the associated teeth.
Using an in house humidity chamber coupled to a loading system inside a high resolution micro-CT that enables phase enhancement, we developed a method to visualize fresh fibrous tissues in 3D as previously described9,18,19,20,21,22,23. Fresh tissues are scanned immediately after the animal is sacrificed without any staining or fixation, which reduces tissue artefacts as well as&#160; alterations of biomechanical properties. These 3D data can be utilized for distribution and direction analyses of the fibers as described elsewhere19.
The second 3D whole tissue imaging method presented here is based on optical clearing of the mandible which enables imaging of the PDL fibers through the bone without any sectioning. Interestingly it also enables visualization of the collagen fibers of the bone itself, however this will not be discussed here. In general, there are two methods for tissue clearing. The first is aqueous-based clearing where the sample is immersed in an aqueous solution with a refractive index greater than 1.4 either through a simple immersion, hyperhydration or hydrogel embedding. However, this method is limited in the level of transparency as well as the structural preservation of the tissue and therefore necessitates fixation of the tissue. The second method which yields highly transparent samples and does not require fixation is the solvent-based clearing method24,25. We generated a modified solvent-based clearing method based on ethyl-3-phenylprop-2-enoate (ethyl cinnamate, ECi) for the mandibular samples. This method has the advantages of using non-toxic food-grade clearing agent, minimal tissue shrinkage, and preservation of fluorescent proteins.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1-mL BD Luer-Lok syringe</td>
			<td>BD</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr>
			<td>1X phosphate buffered saline</td>
			<td>VWR Life Sciences</td>
			<td>0780-10L</td>
			<td></td>
		</tr>
		<tr>
			<td>200 proof ethanol</td>
			<td>VWR Life Sciences</td>
			<td>V1016</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum alloy 5019 wire</td>
			<td>Sigma-aldrich</td>
			<td>GF15828813</td>
			<td>0.08 mm diameter wire, length 100th, temper hard. Used as wire ligature around molar.</td>
		</tr>
		<tr>
			<td>Avizo 9.7</td>
			<td>Thermo Fisher Scientific</td>
			<td>N/A</td>
			<td>Used to analyze microCT scans</td>
		</tr>
		<tr>
			<td>Castroviejo Micro Needle Holders</td>
			<td>Fine Science Tools</td>
			<td>12060-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Clr Plan-Apochromat 20x/1.0,CorrVIS-IR M27 85mm</td>
			<td>Zeiss</td>
			<td>N/A</td>
			<td>Used for second harmonic generation imaging</td>
		</tr>
		<tr>
			<td>Cone socket handle, single ended, hand-form</td>
			<td>G.Hartzell and son</td>
			<td>126-CSH3</td>
			<td>Handle of the inspection mirror</td>
		</tr>
		<tr>
			<td>EC Plan-Neofluar 5x/0.16</td>
			<td>Zeiss</td>
			<td>440321-9902</td>
			<td>Used for light-sheet imaging</td>
		</tr>
		<tr>
			<td>Elipar DeepCure-S LED curing light</td>
			<td>3M ESPE</td>
			<td>76985</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf safe-lock tubes, 1.5mL</td>
			<td>Eppendorf</td>
			<td>22363204</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl cinnamate, &#62;= 98%</td>
			<td>Sigma-aldrich</td>
			<td>W243000-1KG-K</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypodermic Needle, 27G x 1/2&#39;&#39;</td>
			<td>BD</td>
			<td>305109</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketathesia 100mg/ml</td>
			<td>Henry Schein Animal Health</td>
			<td>NDC:11695-0702-1</td>
			<td></td>
		</tr>
		<tr>
			<td>KIMWIPES delicate task wipers</td>
			<td>Kimberly-Clark</td>
			<td>21905-026 (VWR Catalog number)</td>
			<td>Purchased from VWR</td>
		</tr>
		<tr>
			<td>LightSheet Z.1 dual illumination microscope system</td>
			<td>Zeiss</td>
			<td>LightSheet Z.1/LightSheet 7</td>
			<td>Used for lightsheet imaging</td>
		</tr>
		<tr>
			<td>LSM 880 NLO multi-photon microscope</td>
			<td>Zeiss</td>
			<td>LSM 880 NLO</td>
			<td>Used for two-photon imaging</td>
		</tr>
		<tr>
			<td>MEGAmicro, plane, 5mm dia, SS-Thread</td>
			<td>Hahnenkratt</td>
			<td>6220</td>
			<td>Front surface inspectrio mirror</td>
		</tr>
		<tr>
			<td>MicroCT machine, MicroXCT-200</td>
			<td>Xradia</td>
			<td>MICRO XCT-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-Colibri</td>
			<td>Fine Science Tools</td>
			<td>17000-01</td>
			<td></td>
		</tr>
		<tr>
			<td>PermaFlo Flowable Composite</td>
			<td>Ultradent</td>
			<td>948</td>
			<td></td>
		</tr>
		<tr>
			<td>Procedure platform</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Custom-made from lab materials</td>
		</tr>
		<tr>
			<td>Routine stereo micscope M80</td>
			<td>Leica Micosystems</td>
			<td>M80</td>
			<td></td>
		</tr>
		<tr>
			<td>Sentalloy NiTi open coil spring</td>
			<td>TOMY Inc.</td>
			<td></td>
			<td>A 0.15mm diameter closed NiTi coil with an inner coil diameter of 0.9mm delivers a force of 10g. Similar products can be purchased from Dentsply Sirona.&#160;</td>
		</tr>
		<tr>
			<td>T-304 stainless steel ligature wire, 0.009&#39;&#39; diameter</td>
			<td>Orthodontics</td>
			<td>SBLW109</td>
			<td>0.009&#39;&#39;(.23mm) diameter, Soft temper</td>
		</tr>
		<tr>
			<td>X-Ject E (Xylazine) 100mg/ml</td>
			<td>Henry Schein Animal Health</td>
			<td>NDC:11695-7085-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Z100 Restorative, A2 shade</td>
			<td>3M ESPE</td>
			<td>5904A2</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

This paper presents a method to produce OTM as well as two methods for 3D imaging of collagen fibers inside the PDL without any sectioning. For animal research purposes, when alignment of the teeth is not necessary, a tooth movement is considered orthodontic if it generates remodeling of the alveolar bone at all root levels. Constant force level applied on teeth is required in order to generate a reliable OTM. Here, an activated shape-memory NiTi coil is used to generate a consistent force of 10 g throughout the experime...

Watch this Scientific Journal Video about 3D Imaging of PDL Collagen Fibers during Orthodontic Tooth Movement in Mandibular Murine Model at JoVE.com

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

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 ...

3d-imaging-pdl-collagen-fibers-during-orthodontic-tooth-movement

Research

JoVE Journal

Medicine

4.8K Views.  School of Dental Medicine, Harvard University. 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. 

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

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The cell line was originally induced by intracranial injection of 3-methyl-cholantrene into a C57BL/6 syngeneic mouse strain 1; therefore, immunologically competent C57BL/6 mice can be used. While we use GL261, the following protocol can be used for the implantation and monitoring of any intracranial mouse tumor model. GL261 cells were engineered to stably express firefly luciferase (GL261-luc). We also created the brighter GL261-luc2 cell line by stable transfection of the luc2 gene expressed from the CMV promoter. C57BL/6-cBrd/cBrd/Cr mice (albino variant of C57BL/6) from the National Cancer Institute, Frederick, MD were used to eliminate the light attenuation caused by black skin and fur. With the use of albino C57BL/6 mice; in vivo imaging using the IVIS Spectrum in vivo imaging system is possible from the day of implantation (Caliper Life Sciences, Hopkinton, MA). The GL261-luc and GL261-luc2 cell lines showed the same in vivo behavior as the parental GL261 cells. Some of the shared histological features present in human GBMs and this mouse model include: tumor necrosis, pseudopalisades, neovascularization, invasion, hypercellularity, and inflammation 1.
Prior to implantation animals were anesthetized by an intraperitoneal injection of ketamine (50 mg/kg), xylazine (5 mg/kg) and buprenorphine (0.05 mg/kg), placed in a stereotactic apparatus and an incision was made with a scalpel over the cranial midline. A burrhole was made 0.1mm posterior to the bregma and 2.3mm to the right of the midline. A needle was inserted to a depth of 3mm and withdrawn 0.4mm to a depth of 2.6mm. Two &mu;l of GL261-luc or GL261-luc2 cells (107 cells/ml) were infused over the course of 3 minutes. The burrhole was closed with bonewax and the incision was sutured.
Following stereotactic implantation the bioluminescent cells are detectable from the day of implantation and the tumor can be analyzed using the 3D image reconstruction feature of the IVIS Spectrum instrument. Animals receive a subcutaneous injection of 150&mu;g luciferin /kg body weight 20 min prior to imaging. Tumor burden is quantified using mean tumor bioluminescence over time. Tumor-bearing mice were observed daily to assess morbidity and were euthanized when one or more of the following symptoms are present: lethargy, failure to ambulate, hunched posture, failure to groom, anorexia resulting in &gt;10% loss of weight. Tumors were evident in all of the animals on necropsy.

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

Intracranial implantation of GL261 cells into C57BL/6 mice produces malignant gliomas that recapitulate many of the hallmarks of human glioblastoma multiforme. We used GL261 cells stably expressing luciferase to allow us to use in vivo imaging to follow tumor progression. The surgery and 3D in vivo imaging are demonstrated.

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The ...

Three-dimensional Imaging of Nociceptive Intraepidermal Nerve Fibers in Human Skin Biopsies

A punch biopsy of the skin is commonly used to quantify intraepidermal nerve fiber densities (IENFD) for the diagnosis of peripheral polyneuropathy 1,2. At present, it is common practice to collect 3 mm skin biopsies from the distal leg (DL) and the proximal thigh (PT) for the evaluation of length-dependent polyneuropathies 3. However, due to the multidirectional nature of IENFs, it is challenging to examine overlapping nerve structures through the analysis of two-dimensional (2D) imaging. Alternatively, three-dimensional (3D) imaging could provide a better solution for this dilemma.
In the current report, we present methods for applying 3D imaging to study painful neuropathy (PN). In order to identify IENFs, skin samples are processed for immunofluorescent analysis of protein gene product 9.5 (PGP), a pan neuronal marker. At present, it is standard practice to diagnose small fiber neuropathies using IENFD determined by PGP immunohistochemistry using brightfield microscopy 4. In the current study, we applied double immunofluorescent analysis to identify total IENFD, using PGP, and nociceptive IENF, through the use of antibodies that recognize tropomyosin-receptor-kinase A (Trk A), the high affinity receptor for nerve growth factor 5. The advantages of co-staining IENF with PGP and Trk A antibodies benefits the study of PN by clearly staining PGP-positive, nociceptive fibers. These fluorescent signals can be quantified to determine nociceptive IENFD and morphological changes of IENF associated with PN. The fluorescent images are acquired by confocal microscopy and processed for 3D analysis. 3D-imaging provides rotational abilities to further analyze morphological changes associated with PN. Taken together, fluorescent co-staining, confocal imaging, and 3D analysis clearly benefit the study of PN.

In order to study the changes of nociceptive intraepidermal nerve fibers (IENFs) in painful neuropathies (PN), we developed protocols that could directly examine three-dimensional morphological changes observed in nociceptive IENFs. Three-dimensional analysis of IENFs has the potential to evaluate the morphological changes of IENF in PN.

A punch biopsy of the skin is commonly used to quantify intraepidermal nerve fiber densities (IENFD) for the diagnosis of peripheral polyneuropathy 1,2. ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

3D Ultrasound Imaging: Fast and Cost-effective Morphometry of Musculoskeletal Tissue

The developmental goal of 3D ultrasound imaging (3DUS) is to engineer a modality to perform 3D morphological ultrasound analysis of human muscles. 3DUS images are constructed from calibrated freehand 2D B-mode ultrasound images, which are positioned into a voxel array. Ultrasound (US) imaging allows quantification of muscle size, fascicle length, and angle of pennation. These morphological variables are important determinants of muscle force and length range of force exertion. The presented protocol describes an approach to determine volume and fascicle length of m.&#160;vastus lateralis and m.&#160;gastrocnemius medialis. 3DUS facilitates standardization using 3D anatomical references. This approach provides a fast and cost-effective approach for quantifying 3D morphology in skeletal muscles. In healthcare and sports, information on the morphometry of muscles is very valuable in diagnostics and/or follow-up evaluations after treatment or training.

3D ultrasound imaging (3DUS) allows fast and cost-effective morphometry of musculoskeletal tissues. We present a protocol to measure muscle volume and fascicle length using 3DUS.

The developmental goal of 3D ultrasound imaging (3DUS) is to engineer a modality to perform 3D morphological ultrasound analysis of human muscles. 3DUS ...

Determining and Controlling External Power Output During Regular Handrim Wheelchair Propulsion

The use of a manual wheelchair is critical to 1% of the world&#39;s population. Human powered wheeled mobility research has considerably matured, which has led to improved research techniques becoming available over the last decades. To increase the understanding of wheeled mobility performance, monitoring, training, skill acquisition, and optimization of the wheelchair-user interface in rehabilitation, daily life, and sports, further standardization of measurement set-ups and analyses is required. A crucial stepping-stone is the accurate measurement and standardization of external power output (measured in Watts), which is pivotal for the interpretation and comparison of experiments aiming to improve rehabilitation practice, activities of daily living, and adaptive sports. The different methodologies and advantages of accurate power output determination during overground, treadmill, and ergometer-based testing are presented and discussed in detail. Overground propulsion provides the most externally valid mode for testing, but standardization can be troublesome. Treadmill propulsion is mechanically similar to overground propulsion, but turning and accelerating is not possible. An ergometer is the most constrained and standardization is relatively easy. The goal is to stimulate good practice and standardization to facilitate the further development of theory and its application among research facilities and applied clinical and sports sciences around the world.

Accurate and standardized assessment of external power output is crucial in the evaluation of physiological, biomechanical, and perceived stress, strain, and capacity in manual wheelchair propulsion. The current article presents various methods to determine and control power output during wheelchair propulsion studies in the laboratory and beyond.

The use of a manual wheelchair is critical to 1% of the world&#39;s population. Human powered wheeled mobility research has considerably matured, which ...

Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, cardiac stem cell therapy is studied by delivering SCs concurrently with the induction of myocardial injury. However, this approach presents two significant limitations: the early hostile pro-inflammatory ischemic environment may affect the survival of transplanted SCs, and it does not represent the subacute infarction scenario where SCs will likely be used. Here we describe a two-part series of surgical procedures for the induction of ischemia-reperfusion injury and delivery of mesenchymal stem cells (MSCs). This method of stem cell administration may allow for the longer viability and retention around damaged tissue by circumventing the initial immune response. A model of ischemia reperfusion injury was induced in mice accompanied by the delivery of mesenchymal stem cells (3.0 x 105), stably expressing the reporter gene firefly luciferase under the constitutively expressed CMV promoter, intramyocardially 7 days later. The animals were imaged via ultrasound and bioluminescent imaging for confirmation of injury and injection of cells, respectively. Importantly, there was no added complication rate when performing this two-procedure approach for SC delivery. This method of stem cell administration, collectively with the utilization of state-of-the-art reporter genes, may allow for the in vivo study of viability and retention of transplanted SCs in a situation of chronic ischemia commonly seen clinically, while also circumventing the initial pro-inflammatory response. In summary, we established a protocol for the delayed delivery of stem cells into the myocardium, which can be used as a potential new approach in promoting regeneration of the damaged tissue.

Stems cells are continuously investigated as potential treatments for individuals with myocardial damage, however, their decreased viability and retention within injured tissue can impact their long-term efficacy. In this manuscript we describe an alternative method for stem cell delivery in a murine model of ischemia reperfusion injury.

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, ...

Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for analyzing collateral arteries and leukocytes in arteriogenesis is ex vivo (immuno-) histological methodology. However, this technique does not allow the measurement of dynamic processes such as blood flow, shear stress, cell-cell interactions, and particle velocity. This paper presents a protocol to monitor in vivo processes in growing collateral arteries during arteriogenesis utilizing intravital imaging. The method described here is a reliable tool for dynamics measurement and offers a high-contrast analysis with minimal photo-cytotoxicity, provided by multiphoton excitation microscopy. Prior to analyzing growing collateral arteries, arteriogenesis was induced in the adductor muscle of mice by unilateral ligation of the femoral artery.
After the ligation, the preexisting collateral arteries started to grow due to increased shear stress. Twenty-four hours after surgery, the skin and subcutaneous fat above the collateral arteries were removed, constructing a pocket for further analyses. To visualize blood flow and immune cells during in vivo imaging, CD41-fluorescein isothiocyanate (FITC) (platelets) and CD45-phycoerythrin (PE) (leukocytes) antibodies were injected intravenously (i.v.) via a catheter placed in the tail vein of a mouse. This article introduces intravital multiphoton imaging as an alternative or in vivo complementation to the commonly used static ex vivo (immuno-) histological analyses to study processes relevant for arteriogenesis. In summary, this paper describes a novel and dynamic in vivo method to investigate immune cell trafficking, blood flow, and shear stress in a hindlimb model of arteriogenesis, which enhances evaluation possibilities notably.

The recruitment of leukocytes and platelets constitutes an essential component necessary for the effective growth of collateral arteries during arteriogenesis. Multiphoton microscopy is an efficient tool for tracking cell dynamics with high spatio-temporal resolution in vivo and less photo-toxicity to study leukocyte recruitment and extravasation during arteriogenesis.

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for ...

3D Imaging of the Liver Extracellular Matrix in a Mouse Model of Non-Alcoholic Steatohepatitis

Non-alcoholic steatohepatitis (NASH) is the most common chronic liver disease in the United States, affecting more than 70 million Americans. NASH can progress to fibrosis and eventually to cirrhosis, a significant risk factor for hepatocellular carcinoma. The extracellular matrix (ECM) provides structural support and maintains liver homeostasis via matricellular signals. Liver fibrosis results from an imbalance in the dynamic ECM remodeling process and is characterized by excessive accumulation of structural elements and associated changes in glycosaminoglycans. The typical fibrosis pattern of NASH is called &#34;chicken wire,&#34; which usually consists of zone 3 perisinusoidal/pericellular fibrosis, based on features observed by Masson&#39;s trichrome stain and Picrosirius Red stains. However, these traditional thin two-dimensional (2D) tissue slide-based imaging techniques cannot demonstrate the detailed three-dimensional (3D) ECM structural changes, limiting the understanding of the dynamic ECM remodeling in liver fibrosis.
The current work optimized a fast and efficient protocol to image the native ECM structure in the liver via decellularization to address the above challenges. Mice were fed either with chow or fast-food diet for 14 weeks. Decellularization was performed after in situ portal vein perfusion, and the two-photon microscopy techniques were applied to image and analyze changes in the native ECM. The 3D images of the normal and NASH livers were reconstituted and analyzed. Performing in situ perfusion decellularization and analyzing the scaffold by two-photon microscopy provided a practical and reliable platform to visualize the dynamic ECM remodeling in the liver.

The present protocol optimizes the liver in situ perfusion/decellularization and two-photon microscopy methods to establish a reliable platform to visualize the dynamics of extracellular matrix (ECM) remodeling during non-alcoholic steatohepatitis (NASH).

Non-alcoholic steatohepatitis (NASH) is the most common chronic liver disease in the United States, affecting more than 70 million Americans. NASH can ...

Analyzing Alveolar Bone Remodeling to Explore Skeletal Mechanical Biology

Using Inducible Osteoblastic Lineage-Specific Stat3 Knockout Mice to Study Alveolar Bone Remodeling During Orthodontic Tooth Movement

The alveolar bone, with a high turnover rate, is the most actively-remodeling bone in the body. Orthodontic tooth movement (OTM) is a common artificial process of alveolar bone remodeling in response to mechanical force, but the underlying mechanism remains elusive. Previous studies have been unable to reveal the precise mechanism of bone remodeling in any time and space due to animal model-related restrictions. The signal transducer and activator of transcription 3 (STAT3) is important in bone metabolism, but its role in osteoblasts during OTM is unclear. To provide in vivo evidence that STAT3 participates in OTM at specific time points and in particular cells during OTM, we generated a tamoxifen-inducible osteoblast lineage-specific Stat3 knockout mouse model, applied orthodontic force, and analyzed the alveolar bone phenotype. 
Micro-computed tomography (Micro-CT) and stereo microscopy were used to access OTM distance. Histological analysis selected the area located within three roots of the first molar (M1) in the cross-section of the maxillary bone as the region of interest (ROI) to evaluate the metabolic activity of osteoblasts and osteoclasts, indicating the effect of orthodontic force on alveolar bone. In short, we provide a protocol for using inducible osteoblast lineage-specific Stat3 knockout mice to study bone remodeling under orthodontic force and describe methods for analyzing alveolar bone remodeling during OTM, thus shedding new light on skeletal mechanical biology.

Author Spotlight: Comparing Alveolar and Long Bone Remodeling to Explore OTM Model Potential 

This study provides a protocol for using inducible osteoblast lineage-specific Stat3 knockout mice to study bone remodeling under orthodontic force and describes methods for analyzing alveolar bone remodeling during orthodontic tooth movement, thus shedding light on skeletal mechanical biology.

The alveolar bone, with a high turnover rate, is the most actively-remodeling bone in the body. Orthodontic tooth movement (OTM) is a common artificial ...

A Murine Maxillary Orthodontic Model Guide to Bone Remodeling Analysis

The Establishment of a Murine Maxillary Orthodontic Model

Due to the lack of reproducible protocols for establishing a murine maxillary orthodontic model, we present a reliable and reproducible protocol to provide researchers with a feasible tool to analyze mechanical loading-associated bone remodeling. This study presents a detailed flowchart in addition to different types of schematic diagrams, operation photos, and videos. We performed this protocol on 11 adult wide-type C57/B6J mice and harvested samples on postoperative days 3, 8, and 14. The micro-CT and histopathological data have proven the success of tooth movements coupled with bone remodeling using this protocol. Furthermore, according to the micro-CT results on days 3, 8, and 14, we have divided bone modeling into three stages: preparation stage, bone resorption stage, and bone formation stage. These stages are expected to help researchers concerned with different stages to set sample collection time reasonably. This protocol can equip researchers with a tool to carry out regenerative analysis of bone remodeling.

Author Spotlight: Insights into an Efficient Murine Maxillary Orthodontic Model Protocol

Here we demonstrate step-by-step a manageable, orthodontic tooth movement protocol operated on a murine maxillary model. With the explicit explanation of each step and visual demonstration, researchers can master this model and apply it to their experimental needs with a few modifications.

Due to the lack of reproducible protocols for establishing a murine maxillary orthodontic model, we present a reliable and reproducible protocol to ...

Methods Article

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