Name: precision measurements and parametric models of vertebral endplates
Uploaded: 2019-09-17T12:30:00
Description: Watch this Scientific Journal Video about Precision Measurements and Parametric Models of Vertebral Endplates at JoVE.com

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 (<stron...

<ol>
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Z., Subburaj, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+3D+Kinematic+Model+of+the+Spine+for+Idiopathic+Scoliosis+Simulation.">Development of 3D Kinematic Model of the Spine for Idiopathic Scoliosis Simulation.</a> Computer-Aided Design and Applications. 7, 153-161 (2010).</li><li>Cukovic, S., Devedzic, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 3D modeling and simulation of scoliosis: An integrated knowledgeware approach. , 411-415 (2015).</li><li>&#262;ukovi&#263;, 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=.">.</a> Non-Ionizing Three-Dimensional Estimation of Axial Vertebral Rotations in Adolescents Suffering from Idiopathic Scoliosis. , (2018).</li><li>Panjabi, M. M., Duranceau, J., Goel, V., Oxland, T., Takata, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cervical+human+vertebrae.+Quantitative+three-dimensional+anatomy+of+the+middle+and+lower+regions.">Cervical human vertebrae. Quantitative three-dimensional anatomy of the middle and lower regions.</a> Spine (Phila Pa 1976). 16, 861-869 (1991).</li><li>Panjabi, M. M., 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=Thoracic+human+vertebrae.+Quantitative+three-dimensional+anatomy.">Thoracic human vertebrae. Quantitative three-dimensional anatomy.</a> Spine (Phila Pa 1976). 16, 888-901 (1991).</li><li>Ravi, B., Rampersaud, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+magnification+error+in+lateral+spinal+digital+radiographs.">Clinical magnification error in lateral spinal digital radiographs.</a> Spine (Phila Pa 1976). 33, E311-E316 (2008).</li><li>Silva, M. J., Wang, C., Keaveny, T. M., Hayes, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+and+computed+tomography+thickness+measurements+of+the+human,+lumbar+vertebral+shell+and+endplate.">Direct and computed tomography thickness measurements of the human, lumbar vertebral shell and endplate.</a> Bone. 15, 409-414 (1994).</li><li>Langrana, N. A., Kale, S. P., Edwards, W. T., Lee, C. K., Kopacz, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+and+analyses+of+the+effects+of+adjacent+end+plate+curvatures+on+vertebral+stresses.">Measurement and analyses of the effects of adjacent end plate curvatures on vertebral stresses.</a> The Spine Journal. 6, 267-278 (2006).</li><li>Chrzan, 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=Cranioplasty+prosthesis+manufacturing+based+on+reverse+engineering+technology.">Cranioplasty prosthesis manufacturing based on reverse engineering technology.</a> Medical Science Monitor. 18, (2012).</li><li>De Santis, 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=Reverse+engineering+of+mandible+and+prosthetic+framework:+Effect+of+titanium+implants+in+conjunction+with+titanium+milled+full+arch+bridge+prostheses+on+the+biomechanics+of+the+mandible.">Reverse engineering of mandible and prosthetic framework: Effect of titanium implants in conjunction with titanium milled full arch bridge prostheses on the biomechanics of the mandible.</a> Journal of Biomechanics. 47, 3825-3829 (2014).</li><li>Keating, A. P., Knox, J., Bibb, R., Zhurov, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+plaster,+digital+and+reconstructed+study+model+accuracy.">A comparison of plaster, digital and reconstructed study model accuracy.</a> Journal of Orthodontics. 35, 191-201 (2008).</li><li>Numajiri, T., 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=Designing+CAD/CAM+Surgical+Guides+for+Maxillary+Reconstruction+Using+an+In-house+Approach.">Designing CAD/CAM Surgical Guides for Maxillary Reconstruction Using an In-house Approach.</a> Journal of Visualized Experiments. , (2018).</li></ol>

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...

Watch this Scientific Journal Video about Precision Measurements and Parametric Models of Vertebral Endplates at JoVE.com

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, ...

This accurate and reproducible method can be used to obtain detailed and comprehensive geometry data from vertebral endplates and to develop a parametrical model without digitizing too many landmarks. This reverse engineering system can be used to efficiently develop an accurate representation of the anatomical character of sophisticated vertebrae surfaces with good reliability and reproducibility. This protocol can contribute to the design of personalized spinal implants, surgical planning, clinical diagnoses, and the development of accurate finite element models.The method can be used for the sophisticated morphological studies and the parametric model can apply to other imaging modalities such as computed tomography and MRI. Although a beginner may need extra time to complete the procedure, the technique requires a short learning curve overall, to master. Demonstrating the procedure will be Hang Feng, a clinician from my laboratory.Before beginning the procedure, place a dry cervical vertebra, without pathologic deformation or broken parts, vertically onto the platform of the scanner, with the endplate facing the camera lens. Start the scanning process to obtain CloudPoint data and open the software for processing point clouds. Click Import to import the point cloud data and generate a digital graphic of the vertebra.Click Wrap to package the imaging data into a STL format file, to transform the point cloud into Mesh, which will convert a point object into a polygon object and open an appropriate 3D reconstruction and data processing software program. Under the New sub-menu, Click File and select Part in the list of types. Click Start and Shape and Digitized Shape Editor.Then click the Import icon, select the STL format file and click Apply and Okay. To define the endplate 3D co-ordinate system, Mark one anatomic landmark on each of the left and right endpoints of the epiphysial rim and mark landmark on anterior median point. Click the Line icon and select the two trailing edge endpoints to define a posterior frontal line.Click the Plane icon to set the plane type to be normal to the curve and select the posterior frontal line in anterior median point to define the midsagittal plane. Click Start, Shape, and Quick Surface Reconstruction. To generate an intersecting curve, Click the Planar Section icon enter 1 in the number option and select the end plate image and midsagittal plane.Click Curve from the Scan icon and select the intersection of the intersecting curve and posterior epiphysial rim, defining the intersection as the as the posterior median point. Click the Point icon, Points and Planes Repetition. Then, select the midsagittal diameter and enter 1 in the Instances option, to define of the midpoint of the midsagittal diameter.Click the Axis System icon and select the midpoint of the midsagittal diameter, as the origin. The line parallel to the posterior frontal line as the x-axis, the midsagittal diameter as the y-axis and the line pointing forward and perpendicular to the X-Y plane as the z-axis The appropriateness of the coordinate system can be determined according to whether the intersection of the defined midsagittal plane line and the coronal plane is perpendicular to the endplate section. To fit the characteristic curves, and points on the endplate surface, select the midsagittal diameter and enter 3 in the Instances option to divide the midsagittal diameter equally into four parts.Click the Planar Section icon, enter 1 in the number option, and select the endplate image and the 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. Define the line between the two intersections as the mid frontal diameter and divide the mid frontal diameter equally into four parts.To measure the length of a quarter of the midsagittal diameter, click the Measure Between icon. To generate two fitting curves, on one side of the frontal part, click the Planar Section icon, enter 2 in the number option, and enter the measured value in the Step option, and select the endplate image and X-Z plane. Then, click Swap to generate two fitting curves on the other side.Next, select 11 equidistant points in each curve, and obtain nine fitting curves on the endplate surface, as demonstrated. Then, click Curve from the Scan icon and select the intersection of the fitting curves and the midsagittal curve, to obtain a total of 66 points on each endplate. To measure the length of the line parameter that is the distance between two measured points, click the Measure Between icon.To measure the concavity parameters, click Start, Shape, and Generative Shape Design, and click the Sketch icon and the X-Y plane. Click the Circle icon and origin and drag the cursor of the mouse to an appropriate distance before clicking. Then, click the Exit Workbench icon.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. Zoom in and click Start, Shape and Quick Surface Reconstruction, followed by the 3D Curve icon, to find and create the most concave point. Then, click the Measure Between icon, and select the most concave point and X-Y plane, to measure the whole endplate concavity depth.To quantify the surface area parameters, click the Measure Inertia icon, and click endplate surface, to measure the surface area. To determine the fit order of the parametric equation, open the data analysis and visualization software, and input X the corresponding data in the command window. Click Enter and input Z the corresponding data.Then input the code as indicated. For parameter equation fitting, input cftool"and click Enter to bring up the curve fitting tool. Then, input the co-ordinates of a curve in the command window as demonstrated.Select Polynomial and enter the fit order to gain the parametric equation of the endplate surface. To obtain the geometric data, input the X and Y co-ordinate values of any point on the endplate in the command window, and input the parameters of the equation that have been fitted, using the Polynomial tool. Then, input the equation and click Enter to obtain the result.To obtain the 3D simulation graphics, enter the polynomial parameters into the command window, and input the code as indicated. Input the indicated code and equation. Then, enter the indicated code.Using a highly accurate optical 3D range flatbed scanner, as demonstrated, these representative endplates were converted into more than 4500 digital points, to adequately characterize their morphology. Using the measurement protocol as demonstrated, a spatial analysis of the endplate surfaces was conducted, and representative curves were fitted and quantified on the surface to characterize each endplate morphology. The measurements included the concavity depth and concavity apex location in the midsagittal plane.In addition to those of the whole endplate concavity and any specific sections of interest. Next, the components of the endplates, epiphyseal rim, and central endplate were separated and their lengths and areas were obtained. The parametric equation of each curve was then deduced, based on the co-ordinates of 11 points.Defining a 3D co-ordinated system for each endplate, is critical for the success of the protocol. The uncovertebral joint can be distinguished from the endplate by defining a best-fit plane, in terms of the anterior-most and posterior-most points of the bilateral uncinate processes. This protocol provides an accurate and reproducible method for performing a morphological study of complex surfaces.

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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 ...

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 ...

Contractility Measurements of Human Uterine Smooth Muscle to Aid Drug Development

Discovery and characterization of novel pharmaceutical compounds or biochemical probes rely on robust and physiologically relevant assay systems. We describe methods to measure ex vivo myometrium contractility. This assay can be used to investigate factors and molecules involved in the modulation of myometrial contraction and to determine their excitatory or inhibitory actions, and hence their therapeutic potential in vivo. Biopsies are obtained from women undergoing cesarean section delivery with informed consent. Fine strips of myometrium are dissected, clipped and attached to a force transducer within 1 mL organ baths superfused with physiological saline solution at 37 &#176;C. Strips develop spontaneous contractions within 2&#8211;3 h under set tension and remain stable for many hours (&#62;6 h). Strips can also be stimulated to contract such as by the endogenous hormones, oxytocin and vasopressin, which cause concentration-dependent modulation of contraction frequency, force and duration, to more closely resemble contractions in labor. Hence, the effect of known and novel drug leads can be tested on spontaneous and agonist-induced contractions.
This protocol specifically details how this assay can be used to determine the potency of known and novel agents by measuring their effects on various parameters of human myometrial contraction. We use the oxytocin- and V1a receptor antagonists, atosiban and SR49059 as examples of known compounds which inhibit oxytocin- and vasopressin-induced contractions, and demonstrate how this method can be used to complement and validate pharmacological data obtained from cell-based assays to aid drug development. The effects of novel agonists in comparison to oxytocin and vasopressin can also be characterized. Whilst we use the example of the oxytocin/ vasopressin system, this method can also be used to study other receptors and ion channels that play a role in uterine contraction and relaxation to advance the understanding of human uterine physiology and pathophysiology.

This article describes experimental protocols to study ex vivo contractions of human myometrium and their application in drug discovery. This technique is used to improve the understanding of myometrial physiology and pathophysiology as well as to validate pharmacological data from novel research probes or drug leads.

Discovery and characterization of novel pharmaceutical compounds or biochemical probes rely on robust and physiologically relevant assay systems. We ...