This work was funded by Key Discipline Construction Project of Pudong Health Bureau of Shanghai (PWZxk2017-08) and the National Natural Science Foundation of China (81672199). The authors would like to thank Wang Lei for his help in proofreading an earlier version and Li Zhaoyang for his help in developing the parametric model.

This study was approved by the health research ethics board of the authors&#8217; institute. As cervical vertebral bones have more intricate shapes19, the protocol uses the cervical vertebrae as an illustration to facilitate relevant research.
1. Preparation of materials, scanning, and image processing
<ol>
	<li>Collect a dry cervical vertebra without pathologic deformation or broken parts.</li>
	<li>Place the vertebra vertically in the platform of the scanner (Figure 1, see Table of Materials), with the endplate facing the camera lens. Use the active light source of the scanner. Then, start the scanning process to obtain point cloud data (.ASC format). 
	NOTE: According to the pre-scan images, adjust the scanner and position of the vertebra to capture as much surface morphology information as possible.</li>
	<li>Open the software specially used for processing point clouds (see Table of Materials). Click Import to import the point cloud data and generate the digital graphic of the vertebra. Set the sample rate to 100%, select Keep Full Data On Sampling, select the unit of data as millimeters, and click Shade Points. Use the Lasso Selection Tool to select redundant points on the graphic, then click Delete to remove them. Click Reduce Noise and set the smoothness level to its maximum to reduce noise and spikes (Figure 2A,B). 
	NOTE: There are basic software operation instructions at the bottom of the GUI (graphical user interface). Noise points with obvious sharp spurs laterally or vertically should be removed to reduce error.</li>
	<li>Click Wrap to package the imaging data into .stl format file to transform the point cloud into mesh, which will convert a point object into a polygon object. 
	NOTE: Reverse engineering software usually accepts .stl-style 3D format.</li>
	<li>Open the software specially used for 3D reconstruction and data processing (see Table of Materials). Click File then New in the submenu. Select Part in the List of Types. Click Start, then Shape in the submenu, then Digitized Shape Editor. Click the Import icon in the toolbar at the right-hand side of the GUI. In the Import window, select the .stl format file, then click Apply &#62; OK. Click Fit All in the icon in the toolbar at the bottom to load the reconstructed image to the main window of the presentation software. 
	NOTE: Steps 1.5&#8211;2.3.3 are performed with the same software.</li>
	<li>Click Activate in the toolbar at the right-hand side. In the Activate window, select Trap Mode &#62; Polygonal Type &#62; Inside Trap. Then, select the vertebral endplate on the 3D image to remove unneeded vertebral components, such as the posterior elements and osteophytes (Figure 2C).</li>
</ol>
2. Quantification of 3D morphology of the endplate
<ol>
	<li>Defining the endplate 3D coordinate system
	<ol>
		<li>Click Start &#62; Shape in the submenu, then Generative Shape Design. Click the Point icon in the toolbar at the right-hand side. Mark three anatomic landmarks on the epiphyseal rim: the first two are the left and right endpoints of the endplate trailing edge, respectively; the third is the anterior median point.</li>
		<li>Click the Line icon in the toolbar at the right-hand side and select the two trailing edge endpoints to define a posterior frontal line. Click the Plane icon, select the plane type to be normal to curve, then select the posterior frontal line and anterior median point to define the mid-sagittal plane.</li>
		<li>Click Start &#62; Shape &#62; Quick Surface Reconstruction. Click the Planar Section icon, enter 1 in the number option, then select the endplate image and mid-sagittal plane to generate an intersecting curve. Click Curve from the Scan icon and select the intersection of the intersecting curve and posterior epiphyseal rim. Define the intersection as the posterior median point.</li>
		<li>Click Start &#62; Shape &#62; Generative Shape Design. Click the Line icon and select the anterior median point and posterior median point to define a mid-sagittal diameter. Click the Point icon, then Points and Planes Repetition in the submenu. Then, select the mid-sagittal diameter and enter 1 in the Instance(s) option to define the midpoint of the mid-sagittal diameter.</li>
		<li>Click the Axis System icon in the toolbar at the bottom. Then, select the midpoint of the mid-sagittal diameter as the origin, the line parallel to the posterior frontal line as the x-axis, the mid-sagittal diameter as the y-axis, and the line pointing forward and perpendicular to the x-y plane as the z-axis (Figure 3). 
		NOTE: The two trailing edge endpoints are chosen as reference points because they are consistent and show minimum variation in the presence of osteophytes10.</li>
	</ol>
	</li>
	<li>Fitting characteristic curves and points on the endplate surface (Figure 4A&#8211;D)
	<ol>
		<li>Click the Point icon, then Points and Planes Repetition in the submenu. Select the mid-sagittal diameter and enter 3 in the Instance(s) option to divide the mid-sagittal diameter equally into four parts.</li>
		<li>Click Start &#62; Shape &#62; Quick Surface Reconstruction. Click the Planar Section icon, enter 1 in the Number option, then select the endplate image and x-z plane to generate an intersecting curve. Click Curve from the Scan icon and select the two intersections of the x-z plane and epiphyseal rim.</li>
		<li>Define the line between the two intersections as the mid-frontal diameter. In the same way, divide the mid-frontal diameter equally into four parts. 
		NOTE: When the endplate is not symmetrical relative to the med-sagittal plane, choose one of the two endpoints of the mid-frontal curve that has a shorter vertical distance to the z-y plane. Then, define the mid-frontal diameter as 2x the length of the shorter, and divide it equally into four parts.</li>
		<li>Click the Measure Between icon in the toolbar at the bottom to measure the length of a quarter of the mid-sagittal diameter. Click the Planar Section icon, enter 2 in the Number option, enter the measured value in the Step option, then select the endplate image and x-z plane to generate two fitting curves on one side of the frontal part. Click Swap to generate two fitting curves on the other side. In the same way, obtain the other three fitting curves in the sagittal plane. 
		NOTE: The two mid-frontal fitting curves overlap with the two mid-sagittal fitting curves.</li>
		<li>Select 11 equidistant points in each curve for subsequent measurements. Specific method is as follows:
		<ol>
			<li>Taking the mid-sagittal curve as an example, divide the mid-sagittal diameter equally into 10 parts, resulting in a sum of 11 points, including nine intermediate points and two endpoints (refer to steps 2.1.3 and 2.2.1).</li>
			<li>Go through each equidistant point, obtain nine fitting curves on the endplate surface (refer to step 2.2.2). Click Curve from the Scan icon and select the intersection of the fitting curves and the mid-sagittal curve. Finally, obtain a total of 66 points on each endplate (11 points per curve multiplied by six curves). Click the Measure Item icon in the toolbar at the bottom to measure the coordinates of each point.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Measurement of endplate morphological parameters
	<ol>
		<li>Line parameter:
		<ol>
			<li>Click the Measure Between icon to measure the length of line parameter that is the distance between two measured points.</li>
		</ol>
		</li>
		<li>Concavity parameters:
		<ol>
			<li>Create a plane parallel to the x-y plane (Figure 5A): click Start &#62; Shape &#62; Generative Shape Design. Click the Sketch icon in the toolbar at the right-hand side, then click the x-y plane. Click the Circle icon, click Origin on the endplate surface, drag the cursor of the mouse to an appropriate distance, then click. Click the Exit Workbench icon, then the Fill icon, and then click.</li>
			<li>Click the Offset icon, select the filled plane, and enter an appropriate value in the offset option until it is tangent to the most concave part, and zoom in. Click Start &#62; Shape &#62; Quick Surface Reconstruction. Then, click the 3D curve icon to find and create the most concave point. Click the Measure Item icon to measure the coordinates of the most concave point (Figure 5B).</li>
			<li>Click the Measure Between icon, then select the most concave point and x-y plane to measure the whole endplate concavity depth. Similarly, find and create the most concave depth on a particular plane and measure its coordinates.</li>
			<li>Click the Projection icon in the toolbar at the right-hand side, then select the most concave point and x-y plane to obtain the projective point. Click the Measure Item icon to measure the coordinates of the projective point, and determine its distribution based on the coordinates.</li>
		</ol>
		</li>
		<li>Surface area parameters:
		<ol>
			<li>Click the Measure Inertia icon in the toolbar at the bottom and click endplate surface to measure its area. Click the Activate icon and select the central endplate along the inner margins of the epiphyseal ring (refer to step 1.6), then click the Measure Inertia icon to measure its area (Figure 5C). Click the Activate icon, then the central endplate, and finally the Swap icon in the Activate window to obtain an epiphyseal rim. Then, measure its area.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Development of endplate surface mathematical model
<ol>
	<li>Determining the fit order of the parametric equation
	<ol>
		<li>Open the data analysis and visualization software (see Table of Materials). Input x = [corresponding data] in the command window. Click Enter. 
		NOTE: The &#8220;corresponding data&#8221; refers to x-coordinate data of the 11 characteristic points in one curve that has been measured in the previous steps. Click Enter after inputting each command, with the same applying to subsequent operations. Steps 3.1&#8211;5.5 are performed uniformly with the same software.</li>
		<li>In the same way, input z = [corresponding data].</li>
		<li>Input the code for i=1:5 z2=polyfit(x,z,i); Z=polyval(z2,x); if sum((Z-z).^2)&#60;0.01 C=i break; end; end&#8203;. 
		NOTE: The protocol sets the error sum of squares below 0.01 to obtain higher precision, the value of which can be readjusted to satisfy various demands.</li>
		<li>Click Enter to obtain a C value that is the desired fit order.</li>
	</ol>
	</li>
	<li>Parameter equation fitting
	<ol>
		<li>Input cftool and click Enter to bring up the Curve Fitting Tool.</li>
		<li>Input the coordinates of a curve in the command window (refer to steps 3.1.1 and 3.1.2). In the Curve Fitting Tool, select x-coordinate data when fitting frontal plane curves and y-coordinate data when fitting sagittal plane curves in the x data option, select z-coordinate data in the y data option, select polynomial, and enter the fit order obtained. Then, the software will output the parametric equation and goodness of fit automatically. 
		NOTE: As the curve is a 2D image, the default work option is the x and y options in the Curve Fitting Tool when fitting a curve.</li>
		<li>In the similar way, input the 3D coordinates of the 66 points and match the coordinate data to the corresponding axis options. Select polynomial and enter the fit order to gain the parametric equation of the endplate surface (Figure 6B).</li>
	</ol>
	</li>
</ol>
4. Acquisition of geometric data based on parametric equation
<ol>
	<li>Input x- and y-coordinate values of any point on the endplate in the command window.</li>
	<li>Input PX1, PX2, PX3.... 
	NOTE: Px is the parameters of the parametric equation that have been fitted using polynomial in the steps above.</li>
	<li>Input the equation and click Enter to obtain the result (i.e., input format: z = P00 + P10*x + P01*y + P20*x^2 + P11*x*y + P02*y^2 + P30*x^3 + P21*x^2*y + P12*x*y^2 + P03*y^3 + P40*x^4 + P31*x^3*y + P22*x^2*y^2 + P13*x*y^3 + P04*y^4).</li>
</ol>
5. Representation of the endplate based on parametric equation
<ol>
	<li>Input PX1, PX2, PX3....in the command window.</li>
	<li>Input the code X=N1:0.01:N2;. 
	NOTE: N1&#8211;N2 is the range of X-axis data (i.e., the values of the two endpoints of the themid-coronal curve).</li>
	<li>Input the code &#8220;Y=N3:0.01:N4;&#8221;.</li>
	<li>Input the equation (i.e., z=@(x,y)P00 + P10.*x + P01.*y + P20.*x.^2 + P11.*x.*y + P02.*y.^2 + P30.*x.^3 + P21.*x.^2.*y+ P12.*x.*y.^2 + P03.*y.^3 + P40.*x.^4 + P31.*x.^3.*y + P22.*x.^2.*y.^2+ P13.*x.*y.^3 + P04.*y.^4;).</li>
	<li>Input the code ezmesh(z, [N1,N2,N3,N4]) to obtain 3D simulation graphics (Figure 6C).</li>
</ol>

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The authors declare no competing financial interests.

Reverse engineering has been increasingly and successfully applied to the field of medicine, such as cranioplasty20, oral21, and maxillofacial implants21. Reverse engineering measurements, namely product surface digitization, refers to the conversion of surface information into point cloud data employing specific measuring equipment and methods. On the basis of such data, complex surface modeling, evaluation, improvements, and manufacturing can be pe...

A vertebral endplate is the superior or inferior shell of the vertebral body and serves as a mechanical interface to transfer stress between the disc and vertebral body1. It consists of the epiphyseal rim, which is a strong and solid bony labrum surrounding the outer rim of the vertebral body, and the central endplate, which is thin and porous2.
The spine is subject to a wide array of degenerative, traumatic, and neoplastic disorders, which may warrant surgical intervention. Recently, spinal devices such as artificial discs and cages have been widely used. Accurate and detailed morphometric parameters of endplates are necessary for the design and amelioration of spinal implants with effective prosthesis-vertebra contact and bone ingrowth potential3. Furthermore, information on the exact shape and geometry of vertebral endplates is important for understanding the biomechanics. Although the finite element modeling allows for simulation of the real vertebrae and has been widely used to study physiological responses of the spine to various loading conditions4, this technique is patient-specific and not generalizable to all vertebrae. It has been suggested that the intrinsic variability of vertebrae geometry among the general population should be considered when developing the finite element model5. Therefore, the geometric parameters of endplates are conducive to the mesh generation and fidelity enhancement in finite element modeling.
Although the importance of the matching of endplate geometry and implant surface has been discussed in previous studies6,7,8, data on the morphology of vertebral endplates is scarce. Most previous studies have failed to reveal the 3D nature of the endplate9,10,11. A spatial analysis is required to better and fully depict endplate morphology12,13,14. In addition, most studies have employed lower precision measurement techniques10,15,16. Moreover, significant magnification has been reported when geometry parameters are measured by employing radiography or computed tomography (CT)17,18. Though magnetic resonance imaging (MRI) is considered non-invasive, it is less accurate in defining the precise margins of osseous structures11. Due to a lack of a standardized measurement protocol, there are large differences among existing geometric data.
In recent years, reverse engineering, which can digitize the existing physical parts into computerized solid models, has been increasingly applied to the field of medicine. The technique makes it feasible to develop an accurate representation of the anatomical character of sophisticated vertebrae surfaces. The reverse engineering system includes two subsystems: the instrumentation system and software system. The instrumentation system adopted in this protocol has a non-contact optical 3D range flatbed scanner, which is high-speed and highly accurate (precision 0.02 mm, 1,628 x 1,236 pixels). The scanner can efficiently (input time 3 s) capture surface morphology information of the target object and convert it into digital point cloud. The software system (i.e., reverse engineering software) is a computer application for point cloud data processing (see Table of Materials), 3D surface model reconstruction, free curve and surface editing, and data processing (see Table of Materials).
The purposes of the present report are to (1) devise a measurement protocol and algorithm to obtain quantitative parameters of vertebral endplates based on a reverse engineering technique, (2) develop a mathematical model that allows for a realistic representation of vertebral endplates without digitizing too many landmarks. These methods will be beneficial to surgical act planning and finite element modeling.

<table>
	<tbody>
		<tr>
			<td>Catia</td>
			<td>Dassault Systemes, Paris, France</td>
			<td>https://www.3ds.com/products-services/catia/</td>
			<td>3D surface model reconstruction, free curve and surface editing and data processing</td>
		</tr>
		<tr>
			<td>Geomagic Studio</td>
			<td>Geomagic Inc., Morrisville, NC</td>
			<td>https://cn.3dsystems.com/software?utm_source=geomagic.com&#38;utm_medium=301</td>
			<td>point cloud data processing</td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>The MathWorks Inc., Natick,USA</td>
			<td>https://www.mathworks.com/</td>
			<td>analyze data, develop algorithms, and create models</td>
		</tr>
		<tr>
			<td>Optical 3D range flatbed scanner</td>
			<td>Xi&#8217;an XinTuo 3D Optical Measurement Technology Co.Ltd., Xi&#8217;an, Shaanxi, China</td>
			<td>http://www.xtop3d.com/</td>
			<td>acquire surface geometric parameters and convert into digital points</td>
		</tr>
	</tbody>
</table>

precision measurements and parametric models of vertebral endplates

Detailed and comprehensive geometric data of vertebrae endplates is important and necessary to improve the fidelity of finite element models of the spine, design and ameliorate spinal implants, and understand degenerative changes and biomechanics. In this protocol, a high-speed and highly accurate scanner is employed to convert morphology data of endplate surfaces into a digital point cloud. In the software system, the point cloud is further processed and reconstructed into three dimensions. Then, a measurement protocol is performed, involving a 3D coordinate system defined to make each point a 3D coordinate, three sagittal and three frontal surface curves that are symmetrically fitted on the endplate surface, and 11 equidistant points that are selected in each curve. Measurement and spatial analyses are finally performed to obtain geometric data of the endplates. Parametric equations representing the morphology of curves and surfaces are fitted based on the characteristic points. The suggested protocol, which is modular, provides an accurate and reproducible method to obtain geometric data of vertebral endplates and may assist in more sophisticated morphological studies in the future. It will also contribute to designing personalized spinal implants, planning surgical acts, making clinical diagnoses, and developing accurate finite element models.

Using the highly accurate optical 3D range flatbed scanner, the endplates were converted into more than 45,000 digital points, which adequately characterize the morphology (Figure 2A,B).
In the measurement protocol, the spatial analysis of endplate surfaces was conducted. Representative curves were fitted and quantified on the surface to characterize morphology (Figure 4B). The line...

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Precision Measurements and Parametric Models of Vertebral Endplates

A reverse engineering system is employed to record and obtain detailed and comprehensive geometry data of vertebral endplates. Parametric models of vertebral endplate are then developed, which are beneficial to designing personalized spinal implants, making clinical diagnoses, and developing accurate finite element models.

Detailed and comprehensive geometric data of vertebrae endplates is important and necessary to improve the fidelity of finite element models of the spine, ...

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6.4K Views.  Tongji University School of Medicine. A reverse engineering system is employed to record and obtain detailed and comprehensive geometry data of vertebral endplates. Parametric models of vertebral endplate are then developed, which are beneficial to designing personalized spinal implants, making clinical diagnoses, and developing accurate finite element models.

Video: Precision Measurements and Parametric Models of Vertebral Endplates

In vitro Measurements of Tracheal Constriction Using Mice

Transgenic and knockout mice have been powerful tools for the investigation of the physiology and pathophysiology of airways1,2. In vitro tensometry of isolated tracheal preparations has proven to be a useful assay of airway smooth muscle (ASM) contractile response in genetically modified mice. These in vitro tracheal preparations are relatively simple, provide a robust response, and retain both functional cholinergic nerve endings and muscle responses, even after long incubations.
Tracheal tensometry also provides a functional assay to study a variety of second messenger signaling pathways that affect contraction of smooth muscle. Contraction in trachea is primarily mediated by parasympathetic, cholinergic nerves that release acetylcholine onto ASM (Figure 1). The major ASM acetylcholine receptors are muscarinic M2 and M3 which are Gi/o and Gq coupled receptors, respectively3,4,5. M3 receptors evoke contraction by coupling to Gq to activate phospholipase C, increase IP3 production and IP3-mediated calcium release from the sarcoplasmic reticulum3,6,7. M2/Gi/o signaling is believed to enhance contractions by inhibition of adenylate cyclase leading to a decrease in cAMP levels5,8,9,10. These pathways constitute the so called "pharmaco-contraction coupling" of airway smooth muscle11. In addition, cholinergic signaling through M2 receptors (and modulated by M3 signaling) involves pathways that depolarize the ASM which in turn activate L-type, voltage-dependent calcium channels (Figure 1) and calcium influx (so called "excitation-contraction coupling")4,7. More detailed reviews on signaling pathways controlling airway constriction can be found4,12. The above pathways appear to be conserved between mice and other species. However, mouse tracheas differ from other species in some signaling pathways. Most prominent is their lack of contractile response to histamine and adenosine13,14, both well-known ASM modulators in humans and other species5,15.
Here we present protocols for the isolation of murine tracheal rings and the in vitro measurement of their contractile output. Included are descriptions of the equipment configuration, trachea ring isolation and contractile measurements. Examples are given for evoking contractions indirectly using high potassium stimulation of nerves and directly by depolarization of ASM muscle to activate voltage-dependent calcium influx (1. high K+, Figure 1). In addition, methods are presented for stimulations of nerves alone using electric field stimulation (2. EFS, Figure 1), or for direct stimulation of ASM muscle using exogenous neurotransmitter applied to the bath (3. exogenous ACH, Figure 1). This flexibility and ease of preparation renders the isolated trachea ring model a robust and functional assay for a number of signaling cascades involved in airway smooth muscle contraction.

In vitro Measurements of Tracheal Constriction Using Mice

Transgenic mice have been extremely useful in ascribing physiological function to genes. As such, research in general, and functional studies of airway, in particular, have undergone a remarkable shift toward murine models. Here we provide protocols for in vitro trachea constriction studies to evaluate smooth muscle function in murine airway.

Transgenic and knockout mice have been powerful tools for the investigation of the physiology and pathophysiology of airways1,2. In vitro tensometry of ...

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

A Novel Vertebral Stabilization Method for Producing  Contusive Spinal Cord Injury

Clinically-relevant animal cervical spinal cord injury (SCI) models are essential for developing and testing potential therapies; however, producing reliable cervical SCI is difficult due to lack of satisfactory methods of vertebral stabilization. The conventional method to stabilize the spine is to suspend the rostral and caudal cervical spine via clamps attached to cervical spinous processes. &#160;However, this method of stabilization fails to prevent tissue yielding during the contusion as the cervical spinal processes are too short to be effectively secured by the clamps (Figure 1). &#160;Here we introduce a new method to completely stabilize the cervical vertebra at the same level of the impact injury. &#160;This method effectively minimizes movement of the spinal column at the site of impact, which greatly improves the production of consistent SCIs. &#160;We provide visual description of the equipment (Figure 2-4), methods, and a step-by-step protocol for the stabilization of the cervical 5 vertebra (C5) of adult rats, to perform laminectomy (Figure 5) and produce a contusive SCI thereafter. &#160;Although we only demonstrate a cervical hemi-contusion using the NYU/MASCIS impactor device, this vertebral stabilization technique can be applied to other regions of the spinal cord, or be adapted to other SCI devices. &#160;Improving spinal cord exposure and fixation through vertebral stabilization may be valuable for producing consistent and reliable injuries to the spinal cord. &#160;This vertebral stabilization method can also be used for stereotactic injections of cells and tracers, and for imaging using two-photon microscopy in various neurobiological studies.

Vertebral stabilization is necessary for minimizing variability, and for producing consistent experimental spinal cord injuries.&#160; Using a customized stabilizing apparatus in conjunction with the NYU/MASCIS impactor device, we have demonstrated here the proper equipment and procedure for generating reproducible hemi-contusive cervical (C5) spinal cord injuries in adult rats.

Clinically-relevant animal cervical spinal cord injury (SCI) models are essential for developing and testing potential therapies; however, producing ...

MRI and PET in Mouse Models of Myocardial Infarction

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ideal model for testing novel therapeutic strategies.
In vivo magnetic resonance imaging (MRI) gives excellent views of the heart noninvasively with clear anatomical detail, which can be used for accurate functional assessment. Contrast agents can provide basic measures of tissue viability but these are nonspecific. Positron emission tomography (PET) is a complementary technique that is highly specific for molecular imaging, but lacks the anatomical detail of MRI. Used together, these techniques offer a sensitive, specific and quantitative tool for the assessment of the heart in disease and recovery following treatment.
In this paper we explain how these methods are carried out in mouse models of acute myocardial infarction. The procedures described here were designed for the assessment of putative protective drug treatments. We used MRI to measure systolic function and infarct size with late gadolinium enhancement, and PET with fluorodeoxyglucose (FDG) to assess metabolic function in the infarcted region. The paper focuses on practical aspects such as slice planning, accurate gating, drug delivery, segmentation of images, and multimodal coregistration. The methods presented here achieve good repeatability and accuracy maintaining a high throughput.

We describe how to perform MRI and PET imaging of the mouse heart. The protocol is tailored to assess treatment efficacy in models of myocardial infarction and heart failure.

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ...

Production of Apolipoprotein C-III Knockout Rabbits using Zinc Finger Nucleases

Apolipoprotein (Apo) C-III (ApoCIII) resides on the surface of plasma chylomicron (CM), very low density lipoprotein (VLDL) and high density lipoproteins (HDL). It has been recognized that high levels of plasma ApoCIII constitutea risk factor for cardiovascular diseases (CVD). Elevated plasma ApoCIII level often correlates with insulin resistance, obesity, and hypertriglyceridemia. Invaluable knowledge on the roles of ApoCIIIin lipid metabolisms and CVD has been obtained from transgenic mouse models including ApoCIII knockout (KO) mice; however, it is noted that the metabolism of lipoprotein in mice is different from that of humans in many aspects. It is not known until now whether elevated plasma ApoCIII is directly atherogenic. We worked to develop ApoCIII KO rabbits in the present study based on the hypothesis that rabbits can serve as a reasonablemodelfor studying human lipid metabolism and atherosclerosis. Zinc finger nuclease (ZFN) sets targeting rabbit ApoCIIIgene were subjected to in vitro validation prior to embryo microinjection. The mRNA was injected to the cytoplasm of 35 rabbit pronuclear stage embryos, and evaluated the mutation rates at the blastocyst state. Of sixteen blastocysts that were assayed, a satisfactory 50% mutation rate (8/16) at the targeting site was achieved, supporting the use of Set 1 for in vivo experiments. Next, we microinjected 145 embryos with Set 1 mRNA, and transferred these embryos to 7 recipient rabbits. After 30 days gestation, 21 kits were born, out of which five were confirmed as ApoCIII KO rabbits after PCR sequencing assays. The KO animal rate (#KO kits/total born) was 23.8%. The overall production efficiency is 3.4% (5 kits/145 embryos transferred). The present work demonstrated that ZFN is a highly efficient method to produce KO rabbits. These ApoCIII KO rabbits are novel resources to study the roles of ApoCIII in lipid metabolisms.

Recent development in gene targeting tools makes production of knockout (KO) rabbits possible. In the present work, we generated five Apolipoprotein (Apo) C-III KO rabbits using Zinc Finger Nucleases (ZFN). This work demonstrated that ZFN is a highly efficient method to produce KO rabbits.

Apolipoprotein (Apo) C-III (ApoCIII) resides on the surface of plasma chylomicron (CM), very low density lipoprotein (VLDL) and high density lipoproteins ...

Videomorphometric Analysis of Hypoxic Pulmonary Vasoconstriction of Intra-pulmonary Arteries Using Murine Precision Cut Lung Slices

Acute alveolar hypoxia causes pulmonary vasoconstriction (HPV) - also known as von Euler-Liljestrand mechanism - which serves to match lung perfusion to ventilation. Up to now, the underlying mechanisms are not fully understood. The major vascular segment contributing to HPV is the intra-acinar artery. This vessel section is responsible for the blood supply of an individual acinus, which is defined as the portion of lung distal to a terminal bronchiole. Intra-acinar arteries are mostly located in that part of the lung that cannot be selectively reached by a number of commonly used techniques such as measurement of the pulmonary artery pressure in isolated perfused lungs or force recordings from dissected proximal pulmonary artery segments1,2. The analysis of subpleural vessels by real-time confocal laser scanning luminescence microscopy is limited to vessels with up to 50 &#181;m in diameter3.
We provide a technique to study HPV of murine intra-pulmonary arteries in the range of 20-100 &#181;m inner diameters. It is based on the videomorphometric analysis of cross-sectioned arteries in precision cut lung slices (PCLS). This method allows the quantitative measurement of vasoreactivity of small intra-acinar arteries with inner diameter between 20-40 &#181;m which are located at gussets of alveolar septa next to alveolar ducts and of larger pre-acinar arteries with inner diameters between 40-100 &#181;m which run adjacent to bronchi and bronchioles. In contrast to real-time imaging of subpleural vessels in anesthetized and ventilated mice, videomorphometric analysis of PCLS occurs under conditions free of shear stress. In our experimental model both arterial segments exhibit a monophasic HPV when exposed to medium gassed with 1% O2 and the response fades after 30-40 min at hypoxia.

Hypoxic pulmonary vasoconstriction (HPV) is an important physiological phenomenon by which at alveolar hypoxia lung perfusion is matched to ventilation. The major vascular segment contributing to HPV is the intra-acinar artery. Here, we describe our protocol for the analysis of HPV of murine pulmonary vessels with diameters of 20-100 &mu;m.

Acute alveolar hypoxia causes pulmonary vasoconstriction (HPV) - also known as von Euler-Liljestrand mechanism - which serves to match lung perfusion to ...

Accuracy in Dental Medicine, A New Way to Measure Trueness and Precision

Reference scanners are used in dental medicine to verify a lot of procedures. The main interest is to verify impression methods as they serve as a base for dental restorations. The current limitation of many reference scanners is the lack of accuracy scanning large objects like full dental arches, or the limited possibility to assess detailed tooth surfaces. A new reference scanner, based on focus variation scanning technique, was evaluated with regards to highest local and general accuracy. A specific scanning protocol was tested to scan original tooth surface from dental impressions. Also, different model materials were verified. The results showed a high scanning accuracy of the reference scanner with a mean deviation of 5.3 &#177; 1.1 &#181;m for trueness and 1.6 &#177; 0.6 &#181;m for precision in case of full arch scans. Current dental impression methods showed much higher deviations (trueness: 20.4 &#177; 2.2 &#181;m, precision: 12.5 &#177; 2.5 &#181;m) than the internal scanning accuracy of the reference scanner. Smaller objects like single tooth surface can be scanned with an even higher accuracy, enabling the system to assess erosive and abrasive tooth surface loss. The reference scanner can be used to measure differences for a lot of dental research fields. The different magnification levels combined with a high local and general accuracy can be used to assess changes of single teeth or restorations up to full arch changes.

Accuracy is a major demand in dental medicine. To verify accuracy, reference scanners are needed. This article presents a new reference scanner with an adjusted scanning method to acquire a broad variety of dental morphologies with high trueness and precision.

Reference scanners are used in dental medicine to verify a lot of procedures. The main interest is to verify impression methods as they serve as a base ...

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal systems. HFUS has the ability to detect structures as small as 30 &#181;m, a property that has been utilized for visualizing superficial lymph nodes in rodents in brightness (B)-mode. Combining power Doppler with B-mode imaging allows for measuring circulatory blood flow within lymph nodes and other organs. While HFUS has been utilized for lymph node imaging in a number of mouse&#160; model systems, a detailed protocol describing HFUS imaging and characterization of the cervical lymph nodes in mice has not been reported. Here, we show that HFUS can be adapted to detect and characterize cervical lymph nodes in mice. Combined B-mode and power Doppler imaging can be used to detect increases in blood flow in immunologically-enlarged cervical nodes. We also describe the use of B-mode imaging to conduct fine needle biopsies of cervical lymph nodes to retrieve lymph tissue for histological&#160; analysis. Finally, software-aided steps are described to calculate changes in lymph node volume and to visualize changes in lymph node morphology following image reconstruction. The ability to visually monitor changes in cervical lymph node biology over time provides a simple and powerful technique for the non-invasive monitoring of cervical lymph node alterations in preclinical mouse models of oral cavity disease.

This protocol describes the application of high-frequency ultrasound (HFUS) for imaging mouse cervical lymph nodes. This technique optimizes visualization and quantification of cervical lymph node morphology, volume and blood flow. Image-guided biopsy of cervical lymph nodes and processing of lymph tissue for histological evaluation is also demonstrated.

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal ...

Spatial Measurements of Perfusion, Interstitial Fluid Pressure and Liposomes Accumulation in Solid Tumors

The heterogeneous intra-tumoral accumulation of liposomes is a critical determinant of their efficacy. Both the chaotic tumor microcirculation and elevated IFP are linked to the heterogeneous intra-tumoral distribution of nanotechnology-based drug delivery systems such as liposomes. In the present study, the relationship between tumor microcirculation, elevated IFP, and accumulation of nanoparticles was investigated through in vivo experimentation. This was accomplished by evaluation of the tumor microcirculation using dynamic contrast enhanced computed tomography (DCE-CT) and measurement of tumor IFP using a novel image-guided robotic needle placement system connected to the micro-CT scanner. The intra-tumoral accumulation of liposomes was determined by CT image-based assessment of a nanoparticle liposomal formulation that stably encapsulate the contrast agent iohexol (CT-liposomes). CT imaging allowed for co-localization of the spatial distribution of tumor hemodynamics, IFP and CT-liposome accumulation in an individual subcutaneous xenograft mouse model of breast cancer. Measurements led to the discovery that perfusion and plasma volume fraction are strong mediators of the intra-tumoral distribution of liposomes. Furthermore, the results suggest that IFP plays an indirect role in mediating liposome distribution through modulating blood flow.

The heterogeneous intra-tumoral accumulation of liposomes has been linked to an abnormal tumor microenvironment. Herein methods are presented to measure tumor microcirculation by perfusion imaging and elevated interstitial fluid pressure (IFP) using an image-guided robotic system. Measurements are compared to the intra-tumoral accumulation of liposomes, determined using volumetric micro-CT imaging.

The heterogeneous intra-tumoral accumulation of liposomes is a critical determinant of their efficacy. Both the chaotic tumor microcirculation and ...

Absorption of Nasal and Bronchial Fluids: Precision Sampling of the Human Respiratory Mucosa and Laboratory Processing of Samples

The methods of nasal absorption (NA) and bronchial absorption (BA) use synthetic absorptive matrices (SAM) to absorb the mucosal lining fluid (MLF) of the human respiratory tract. NA is a non-invasive technique which absorbs fluid from the inferior turbinate, and causes minimal discomfort. NA has yielded reproducible results with the ability to frequently repeat sampling of the upper airway. 
By comparison, alternative methods of sampling the respiratory mucosa, such as nasopharyngeal aspiration (NPA) and conventional swabbing, are more invasive and may result in greater data variability. Other methods have limitations, for instance, biopsies and bronchial procedures are invasive, sputum contains many dead and dying cells and requires liquefaction, exhaled breath condensate (EBC) contains water and saliva, and lavage samples are dilute and variable.
BA can be performed through the working channel of a bronchoscope in clinic. Sampling is well tolerated and can be conducted at multiple sites in the airway. BA results in MLF samples being less dilute than bronchoalveolar lavage (BAL) samples.
This article demonstrates the techniques of NA and BA, as well as the laboratory processing of the resulting samples, which can be tailored to the desired downstream biomarker being measured. These absorption techniques are useful alternatives to the conventional sampling techniques used in clinical respiratory research.

This manuscript describes the use of nasal and bronchial absorption techniques, specifically using synthetic absorptive matrices (SAM) to sample the mucosal lining fluid (MLF) of the upper and lower airway. These methods provide better standardization and tolerability than existing respiratory sampling techniques.

The methods of nasal absorption (NA) and bronchial absorption (BA) use synthetic absorptive matrices (SAM) to absorb the mucosal lining fluid (MLF) of the ...

Precision Measurements and Parametric Models of Vertebral Endplates

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In vitro Measurements of Tracheal Constriction Using Mice

Models of Bone Metastasis

A Novel Vertebral Stabilization Method for Producing Contusive Spinal Cord Injury

MRI and PET in Mouse Models of Myocardial Infarction

Production of Apolipoprotein C-III Knockout Rabbits using Zinc Finger Nucleases

Videomorphometric Analysis of Hypoxic Pulmonary Vasoconstriction of Intra-pulmonary Arteries Using Murine Precision Cut Lung Slices

Accuracy in Dental Medicine, A New Way to Measure Trueness and Precision

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

Spatial Measurements of Perfusion, Interstitial Fluid Pressure and Liposomes Accumulation in Solid Tumors

Absorption of Nasal and Bronchial Fluids: Precision Sampling of the Human Respiratory Mucosa and Laboratory Processing of Samples