Name: in vivo optical imaging of brain tumors and arthritis using fluorescent sapc-dops nanovesicles
Uploaded: 2014-05-02T14:00:00
Duration: 9 min 4 s
Description: Watch this Scientific Journal Video about In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles at JoVE.com

This work was supported in part by NIH/NCI Grants Number 1R01CA158372-01 (to Qi) and New Drug State Key Project Grant Number 009ZX09102-205 (to Qi). Writing assistance was provided by Dr. Judy Racadio, and was funded by the University of Cincinnati Department of Hematology and Oncology. Vontz Core Imaging Lab (VCIL) in the College of Medicine at the University of Cincinnati.

Ethics statement of animal use. All animal studies were approved by the Institutional Animal Care and Use Committee of the University of Cincinnati (IACUC Protocol Number: 11-05-05-02) and the Cincinnati Children&#39;s Hospital Research Foundation (Animal Welfare Assurance Number A3108-01). All experiments involving mice followed the animal care guidelines of the University of Cincinnati and the Cincinnati Children&#39;s Hospital Research Foundation.
1. Prepare Animal Models
Note: Three different animal models outlined below have been used in our prior studies:
<ol>
	<li>Orthotopic brain tumor mouse: Use Nu/Nu athymic female mice that have been intracranially injected with human U87-&#916;EGFR-Luc cells. These mice develop an aggressive tumor showing typical features of human glioblastoma.</li>
	<li>Genetically engineered brain tumor mouse models13: Breed Mut3 (GFAP-cre; Nf1loxP/+; Trp53-/+) male mice with Trp53loxP/loxP; PtenloxP/loxP females to generate Mut6 mice (GFAP-cre; Nf1loxP/+; Trp53-/loxP; PtenloxP/+). Maintain Mut3 mice in B6CBAF1/J strain by breeding male Mut3 mice with female B6CBAF1/J mice. Genotype the mice between P9 and P12 and confirm the genotypes after harvesting their tissues.</li>
	<li>K/BxN arthritis: Use C57Bl/6J mice that have been intraperitoneally administered with 150 &#181;l sera from KRN x NOD F1 mice. These mice develop arthritis 24 to 48 hr&#160;following sera injection. Imaging of the arthritic mice is performed on day 7 following sera administration, a time point at which mice exhibit overt macroscopic arthritis. Mice should be evaluated using the criteria outlined in the next step.
	<ol>
		<li>Evaluation of mice for macroscopic arthritis using an arthritic index macroscopic scoring system as follows: 0 = no detectable arthritis, 1 = swelling and/or redness of paw or one digit, 2 = two joints involved, 3 = three joints involved, and&#160;4 = severe arthritis of the entire paw and digit.&#160;The arthritic scoring system is used to determine the number of joints affected and the severity of arthritis in the mouse paws.&#160;Even mice with the highest possible arthritic score rarely show signs of immobility.&#160; However, arthritis is monitored 3x/week and mice in excessive pain (such as severe immobility from swollen paws that inhibits food and water consumption) are sacrificed.&#160;&#160;</li>
		<li>Note:&#160;Fluids injected IV into the mouse tail vein have sterility maintained throughout the experiment. Clean, sterile, disposable syringes and vials are used for study solution preparation and administration.</li>
	</ol>
	</li>
</ol>
2. Preparation of Fluorescently-labeled SapC-DOPS Nanovesicles
<ol>
	<li>SapC protein production: Recombinant SapC protein with exact human SapC sequence was produced in E. coli cells as previously described with modifications4. SapC was precipitated by ethanol followed by high performance liquid chromatography purifications. After lyophilization, dry SapC was used and its concentration was determined by its weight.</li>
	<li>Mix SapC protein as previously described7,10,11. Mix DOPS (0.18 mg) and CVM (0.03 mg) in a glass tube and use nitrogen gas to evaporate lipid solvents.</li>
	<li>Add SapC protein powder (0.32 mg) to the mixture, suspend the dry mixture in 1 ml of PBS buffer and bath sonicate for about 15 min&#160;as previously described7,10,11. Then pass the suspension through a Sephadex G25 column (PD-10) to remove free CVM dye. Excitation and emission maxima of the final product, i.e. SapC-DOPS-CVM nanovesicles, are 653 nm and 677 nm, respectively.</li>
</ol>
3. Imaging
<ol>
	<li>Use brain tumor and arthritis mouse models (described above in Step 1) to test the MAROI system. Anesthetize mice to effect with 2% isoflurane. 1-2% isoflurane is maintained for the duration of the imaging procedure. Warm air is continuously and gently delivered into the imaging chamber for the duration of imaging. A&#160;small bead of sterile artificial tears ointment is applied to each eye of the mouse so as to cover and lubricate the eye.&#160;&#160;Place mice into the MARS system by positioning the mice in a supine position with their spine initially directed towards the camera (Figure 1). Calibrate the MARS 380&#176; support film and position the mouse using the rotation software in the Bruker MI protocol tab. Obtain baseline images of mice prior to SapC-DOPS-CVM administration in the manner described below.</li>
	<li>Inject 200 &#181;l of SapC-DOPS-CVM intravenously&#160;into the tail vein of the mouse. Administer to control mice and arthritic or brain tumor-bearing mice.</li>
	<li>Image mice 24 hr post injection and again at 7-9 days post injection by taking fluorescence (25 sec exposure time) and X-ray (10 sec exposure time) images at 10&#176; increments over a course of 380&#176;, creating a slight overlap to ensure there are no gaps in the rotational dataset. Using Bruker MI software, superimpose fluorescent onto X-ray images for anatomical localization.</li>
</ol>
4. Image Analysis
<ol>
	<li>Draw a rectangular ROI encompassing the width of the field of view (FOV) of the disease site (tumor and arthritis). The ROI must be large enough to keep the disease feature within the FOV as the animal moves in the course of the 380&#176; rotation. For brain tumor mice (orthotopic and transgenic models), use the same rectangular ROI on each tumor model and its three (3) respective control mice for all time points (baseline, 24 hr, and 9 days). The positioning of the rectangular ROI for each mouse is preserved over all time points by utilizing anatomical landmarks on each animal&#39;s corresponding X-ray images. The anatomical landmark(s) identified on the tumor model must also be used to place identical rectangular ROIs on each model&#39;s respective controls.&#160;Anatomic landmarks identified on the X-ray images allowing for consistent ROI placement include the base of the skull and the posterior aspect of the zygomatic arch. They are visualized on the right and left lateral skull in the posterior-anterior (PA) image.</li>
	<li>After automatic background subtraction, determine mean fluorescence intensity for every image. Convert the fluorescence images to photons/s/mm2 using Bruker MI imaging software. Plot the fluorescence values as a function of the imaging angles, and apply as error bars the standard deviation of the averaged fluorescence values obtained from control mice using Excel or other graphing software.</li>
</ol>

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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+depth+of+cellular+imaging+in+the+intact+lung+using+far-red+and+near-infrared+fluorescent+probes.">Increased depth of cellular imaging in the intact lung using far-red and near-infrared fluorescent probes.</a> Int J Biomed Imaging. , (2006).</li><li>Gertner-Dardenne, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipophilic+fluorochrome+trackers+of+membrane+transfers+between+immune+cells.">Lipophilic fluorochrome trackers of membrane transfers between immune cells.</a> Immunol Invest. 36, 665-685 (2007).</li><li>Qi, X., 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=Functional+human+saposins+expressed+in+Escherichia+coli.+Evidence+for+binding+and+activation+properties+of+saposins+C+with+acid+beta-glucosidase.">Functional human saposins expressed in Escherichia coli. Evidence for binding and activation properties of saposins C with acid beta-glucosidase.</a> J Biol Chem. 269, 16746-16753 (1994).</li><li>Wang, Y., Grabowski, G. A., Qi, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipid+vesicle+fusion+induced+by+saposin.">Phospholipid vesicle fusion induced by saposin.</a> C. Arch Biochem Biophys. 415, 43-53 (2003).</li><li>Qi, X., Chu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fusogenic+domain+and+lysines+in+saposin.">Fusogenic domain and lysines in saposin.</a> C. 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There is nothing to disclose.

Accurate determination of the location and magnitude of solid tumors and inflammatory foci in rheumatic conditions is critical to implement adequate treatment and follow up disease progression or remission. While valuable, current imaging strategies (X-rays; MRI; ultrasound; X-ray computed tomography) provide incomplete assessments of disease status. For instance, arthritic joint damage is commonly assessed by X-rays, which provides information on bone structure but not on soft tissue inflammation and destruction, characteristic of early stages of the disease. The MAROI method presented here combines the advantages of both X-rays and sophisticated soft tissue imaging modalities (e.g. MRI or ultrasound) in an integrated, noninvasive and simpler platform that also allows for a full 3D mapping and reconstruction of the diseased tissue or organ in small animals such as mice.
This method takes advantage of the selective affinity of SapC-DOPS nanovesicles for exposed phosphatidylserine residues, which are abundant in the membranes of cancer and inflammatory cells. The determinant of this binding is SapC, a fusogenic lysosomal protein with a strong affinity for anionic phospholipids such as phosphatidylserine7,10,11. When conjugated to a fluorescent probe (CVM), systemically injected SapC-DOPS can be traced to tumor and arthritic sites by fluorescence imaging.
Limitations of our method are related to its sensitivity, which at present restricts its use to imaging of small animals like mice. As with other imaging methods, optimal fluorescent signal to noise ratio is constrained by the size of the tumor or the extent of arthritis, and may be compromised when imaging tissues or organs with high background (autofluorescence) such as ears (brain imaging), intestines/feces (abdominal imaging) and paws (hind limb imaging). In this respect, we found that a far-red dye such as CVM provides better spectral separation and resolution in the in vivo setting than other fluorescent probes in the visible range.
Other pitfalls include potential movement of the animal during imaging, both while anesthetized and post mortem (rigor mortis). Hind limb positioning, particularly, is often difficult to stabilize to avoid movement during rotation. In its present state the technique is also time consuming, with scan times as long as 60 min needed to complete a full rotation and acquire high quality images.
The MAROI method presents a number of advantages over other imaging modalities. The ability to image diseased tissue from 38 (or more) different angles allows visualization of fluorescence that may be hindered when assessing it from a single plane; this is valuable in animal studies because it may help minimize the number of false negatives that result from imaging at inappropriate angles. By overlying X-ray and fluorescence images, a precise anatomical localization of the diseased site can be determined. Finally, the possibility of live (in vivo) imaging allows for longitudinal studies to be performed.

Whole animal imaging has become a powerful tool in the study of animal physiopathology. Among current imaging systems, the MS FX PRO allows researchers to accurately visualize fluorescently labeled (or luminescent) compounds and/or tissues in living mice, and simultaneously obtain X-ray images. With the recently introduced multi modal animal rotation system (MARS) a complete, automated rotation of the mouse is achieved in order to capture both fluorescent/luminescent and X-ray images at specific angles1. Image acquisition can be programmed such that sequential image series can be captured at specific, incremental angles as small as 1&#176;. This permits one to identify the optimal orientation of the animal, i.e. that in which the distance between the internally generated fluorescent/luminescent signal and the system&#39;s detection device is the shortest. This, in turn, facilitates precise repositioning of the animal for subsequent imaging sessions during longitudinal studies.
In this report, we describe the implementation of a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of fluorescent marker intensity. MAROI signal curve analysis can be used in longitudinal studies for direct correlation of fluorescent signal distribution to precisely map diseased sites or biological processes of interest.
This system was used to monitor the absorption of fluorescently labeled SapC-DOPS nanovesicles by orthotopic and spontaneous tumors, as well as by arthritic foci, in living mice; it provided multispectral and multimodal data sets derived from complete rotational coverage of the animals. Among the numerous fluorescent probes currently available for in vivo imaging, those emitting in the near-infrared and far-red spectral regions confer the lowest interference with skin and tissues, and provide the highest penetration and image resolution. We used CellVue Maroon (CVM)2,3, a far-red fluorescent cell linker (Ex 647/Em 667), to label SapC-DOPS (SapC-DOPS-CVM)4-12.

<table border="0" cellpadding="0" cellspacing="0" width="1246">
	<colgroup>
		<col /> 
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Dulbecco&#39;s modified eagle medium</td>
			<td style="width:423px;">Gibco (Grand Island, NY)</td>
			<td style="width:307px;">11965</td>
			<td style="width:167px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Fetal Bovine Serum</td>
			<td style="width:423px;">Gibco (Grand Island, NY)</td>
			<td style="width:307px;">16000077</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Penicillin-streptomycin</td>
			<td style="width:423px;">Hyclone (Logan, Utah)</td>
			<td style="width:307px;">SV30010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Dioleoylphosphatidylserine</td>
			<td style="width:423px;">Avanti Polar Lipids (Alabaster, AL)</td>
			<td style="width:307px;">840035C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">CellVue Maroon</td>
			<td style="width:423px;">Molecular Targeting Technologies, Inc. (Exton, PA)</td>
			<td style="width:307px;">C-1001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Sephadex G25 column PD-10</td>
			<td style="width:423px;">Amersham Pharmacia Biotech, (Piscataway, NJ)</td>
			<td style="width:307px;">17-0851-01</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:351px;">New Standard Stereotaxic for Rats and Mice</td>
			<td style="width:423px;">Harvard Apparatus (Holliston, MA)</td>
			<td style="width:307px;">726335</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Bransonic Ultrasonic Cleaners Model 1510</td>
			<td style="width:423px;">Branson Ultrasonics (Danbury,CT)</td>
			<td style="width:307px;">CPN-952-118</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Multi-spectral FX system</td>
			<td>Bruker Corporation (Billerica, MA)</td>
			<td style="width:307px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multi-angle Rotational Optical Imaging Device</td>
			<td>Bruker Corporation (Billerica, MA)</td>
			<td style="width:307px;"> </td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo optical imaging of brain tumors and arthritis using fluorescent sapc-dops nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

We demonstrate here that SapC-DOPS nanovesicles labeled with a far-red dye (CVM) specifically accumulate in orthotopic and spontaneous mouse brain tumors, as well as in arthritic joints of K/BxN mice. Serial fluorescence/X-ray images acquired from an ROI placed over each disease site during complete rotations of the mice were subjected to MAROI curve analysis, which revealed the optimal imaging angle with the highest fluorescence intensity.
The primary purpose for using the MARS system is to determine the optimum angle of fluorescence so that the most accurate measurements can be taken. Representative results from three experiments using mice with brain tumors or arthritis are shown. Using SapC-DOPS-CVM and the MARS system (Figure 1), the best possible image angle for observing the tumor or inflammation due to arthritis was determined. Fluorescence images, followed by an X-ray acquisition, were acquired every 10&#176; during a 380&#176; rotation of the mouse. Fluorescence images were overlaid onto the corresponding X-ray images for image display and rotational movie generation.
Results from the orthotopic brain tumor model are demonstrated in Figure 2. The fluorescence image of a representative orthotopic tumor-bearing mouse (Ortho1) is shown in Figure 2A. The optimal image angle for this animal is 10&#176;, the position at which the fluorescence photon intensity is the greatest (Figure 2B). Measurements were taken before injection with SapC-DOPS-CVM (baseline) and 24 hr after injection. Control mice (tumor free) received a similar treatment.
Figure 3 shows comparable data from the genetically engineered brain tumor mouse model. The fluorescence images and photon measurements were taken before injection with SapC-DOPS-CVM (baseline) and 24 hr (Figures 3A and 3B) and 9 days (Figure 3C) after injection. These graphs show that the optimal imaging angle in the tumor-bearing animal (Tumor Mut49) is 20&#176; 24 hr post injection but changes to 10&#176; 9 days post injection. This suggests that fluorescence signal alteration correlated with morphological changes, likely reflecting tumor growth.
As shown in Table 1, the MAROI method clearly demonstrates that the fluorescent signal decreases for projections at increasing rotation away from the optimal imaging angle. In brain tumors, a 7% average decrease in fluorescent signal was obtained if the animal's physical orientation was &#177;10&#176; offset from the optimal imaging angle. An average 21% decrease in fluorescent signal was measured at &#177;20&#176;. Thus relatively small offsets from the optimal angle can result in significant signal attenuation. Utilizing the MAROI technique for image positioning will allow investigators to produce more consistent and reliable data.
The MAROI method was finally used to assess the targeting of arthritic joints by SapC-DOPS-CVM 24 hr after SapC-DOPS-CVM injection. This animal scored 3 with three arthritic joints. Fluorescence images of toe and ankle of the arthritic mouse are shown in Figures 4A and 4B. Corresponding photon measurements at 10&#176; rotation intervals are graphed in Figures 4C and 4D. The optimal imaging angles found for the toe and ankle are 140&#176; and 120&#176;, respectively.
In summary, the combination of the MAROI system with fluorescent SapC-DOPS nanovesicles represents a noninvasive, accurate and highly sensitive strategy for live imaging, which allows for quantitative studies of tumor and arthritis progression in small animals. The possibility of acquiring a 360&#176; multimodal imaging dataset considerably improves data analysis and interpretation, as compared with what is achievable using single angle imaging techniques.
<img alt="Table 1" fo:content-width="6in" fo:src="/files/ftp_upload/51187/51187table1highres.jpg" src="/files/ftp_upload/51187/51187table1.jpg" width="600px" /> 
Table 1. Optimum imaging angles for each mouse model. The differences between the angle of maximum photon fluorescence (FLR optimum angle) and the standard anatomic angle (X-ray) can be seen. When these two angles become increasingly different, the measured signal changes significantly. <a href="https://www.jove.com/files/ftp_upload/51187/51187table1highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 1" fo:content-width="6in" fo:src="/files/ftp_upload/51187/51187fig1highres.jpg" src="/files/ftp_upload/51187/51187fig1.jpg" width="600px" /> 
Figure 1. Multi-angle rotational optical imaging (MAROI) device. <a href="https://www.jove.com/files/ftp_upload/51187/51187fig1highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 2" fo:content-width="6in" fo:src="/files/ftp_upload/51187/51187fig2highres.jpg" src="/files/ftp_upload/51187/51187fig2.jpg" width="600px" /> 
Figure 2. Fluorescence signal vs. image angle in an orthotopic brain tumor mouse model. (A). Image of the peak fluorescent signal at the optimal image angle of 10&#176;. The blue box shows the ROI used to quantify the emitted photons. (B). Graph of the image angle versus photon emission. The representative orthotopic tumor-bearing mouse (Ortho1) is graphed against averaged fluorescence values from identical ROIs in three nontumor mice. Measurements were taken at baseline (before injection) and 24 hr post injection. Error bars represent Standard Deviation. <a href="https://www.jove.com/files/ftp_upload/51187/51187fig2highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 3" fo:content-width="6in" fo:src="/files/ftp_upload/51187/51187fig3highres.jpg" src="/files/ftp_upload/51187/51187fig3.jpg" width="600px" /> 
Figure 3. Fluorescence signal vs. image angle in a spontaneous brain tumor of a genetically engineered mouse model. (A). Image of the peak fluorescent signal of ROI 1 (top blue box) at the optimal image angle of 20&#176;. (B) and (C). Graphs of the image angle versus photon emission from ROI 1. Values from a representative spontaneous brain tumor-bearing mouse, Tumor-Mut 49, are graphed against averaged values from three non tumor mice. Measurements were taken at baseline (before injection) and 24 hr (B) and 9 days (C) post injection. Error bars represent Standard Deviation. <a href="https://www.jove.com/files/ftp_upload/51187/51187fig3highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 4" fo:content-width="6in" fo:src="/files/ftp_upload/51187/51187fig4highres.jpg" src="/files/ftp_upload/51187/51187fig4.jpg" width="600px" /> 
Figure 4. Fluorescence signal vs. image angle in a mouse with arthritis of the toe and ankle joints. (A) and (B). Images showing the peak fluorescent signal for the toes (A) and ankle joints (B), within the ROIs shown in the red box. (C). Graph of angle versus mean photon emission for the toe. Peak photon emission can be seen at an angle of 140&#176;. (D). Graph of angle versus mean photon emission for the ankle; maximum intensity occurs at an angle of 120&#176;. <a href="https://www.jove.com/files/ftp_upload/51187/51187fig4highres.jpg" target="_blank">Click here to view larger image</a>.

Watch this Scientific Journal Video about In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles at JoVE.com

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

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Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&beta; using a Whole Blood Stimulation Assay

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues as well as altered cytokine homeostasis. The innate immune system plays a crucial role in recognizing pathogens and other endogenous danger stimuli. One of the major cytokines released by innate immune cells is Interleukin (IL)-1. Therefore, we utilize a whole blood stimulation assay in order to measure the secretion of inflammatory cytokines and specifically of the pro-inflammatory cytokine IL-1&beta; 1, 2, 3.
Patients with genetic dysfunctions of the innate immune system causing autoinflammatory syndromes show an exaggerated release of mature IL-1&beta; upon stimulation with LPS alone. In order to evaluate the innate immune component of patients who present with inflammatory-associated pathologies, we use a specific immunoassay to detect cellular immune responses to pathogen-associated molecular patterns (PAMPs), such as the gram-negative bacterial endotoxin, lipopolysaccharide (LPS). These PAMPs are recognized by pathogen recognition receptors (PRRs), which are found on the cells of the innate immune system 4, 5, 6, 7. A primary signal, LPS, in conjunction with a secondary signal, ATP, is necessary for the activation of the inflammasome, a multiprotein complex that processes pro-IL-1&beta; to its mature, bioactive form 4, 5, 6, 8, 9, 10.
The whole blood assay requires minimal sample manipulation to assess cytokine production when compared to other methods that require labor intensive isolation and culturing of specific cell populations. This method differs from other whole blood stimulation assays; rather than diluting samples with a ratio of RPMI media, we perform a white blood cell count directly from diluted whole blood and therefore, stimulate a known number of white blood cells in culture 2. The results of this particular whole blood assay demonstrate a novel technique useful in elucidating patient cohorts presenting with autoinflammatory pathophysiologies.

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&#946; using a Whole Blood Stimulation Assay

We describe a simple immunoassay to measure the production of pro-inflammatory cytokines, such as IL-1 beta production, in patients presenting with autoinflammatory phenotypes. By activating cells in whole blood cultures with pathogen-associated molecular patterns, specifically with lipopolysaccharide, cytokine secretion can be conveniently evaluated in whole blood supernatants.

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues ...

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

Probing the Brain in Autism Using fMRI and Diffusion Tensor Imaging

Newly emerging theories suggest that the brain does not function as a cohesive unit in autism, and this discordance is reflected in the behavioral symptoms displayed by individuals with autism. While structural neuroimaging findings have provided some insights into brain abnormalities in autism, the consistency of such findings is questionable. Functional neuroimaging, on the other hand, has been more fruitful in this regard because autism is a disorder of dynamic processing and allows examination of communication between cortical networks, which appears to be where the underlying problem occurs in autism. Functional connectivity is defined as the temporal correlation of spatially separate neurological events1. Findings from a number of recent fMRI studies have supported the idea that there is weaker coordination between different parts of the brain that should be working together to accomplish complex social or language problems2,3,4,5,6. One of the mysteries of autism is the coexistence of deficits in several domains along with relatively intact, sometimes enhanced, abilities. Such complex manifestation of autism calls for a global and comprehensive examination of the disorder at the neural level. A compelling recent account of the brain functioning in autism, the cortical underconnectivity theory,2,7 provides an integrating framework for the neurobiological bases of autism. The cortical underconnectivity theory of autism suggests that any language, social, or psychological function that is dependent on the integration of multiple brain regions is susceptible to disruption as the processing demand increases. In autism, the underfunctioning of integrative circuitry in the brain may cause widespread underconnectivity. In other words, people with autism may interpret information in a piecemeal fashion at the expense of the whole. Since cortical underconnectivity among brain regions, especially the frontal cortex and more posterior areas 3,6, has now been relatively well established, we can begin to further understand brain connectivity as a critical component of autism symptomatology.
A logical next step in this direction is to examine the anatomical connections that may mediate the functional connections mentioned above. Diffusion Tensor Imaging (DTI) is a relatively novel neuroimaging technique that helps probe the diffusion of water in the brain to infer the integrity of white matter fibers. In this technique, water diffusion in the brain is examined in several directions using diffusion gradients. While functional connectivity provides information about the synchronization of brain activation across different brain areas during a task or during rest, DTI helps in understanding the underlying axonal organization which may facilitate the cross-talk among brain areas. This paper will describe these techniques as valuable tools in understanding the brain in autism and the challenges involved in this line of research.

Neuroimaging techniques, such as functional MRI and Diffusion Tensor Imaging have become increasingly useful in characterizing the cognitive and neural deficits in autism. An examination of brain connectivity in autism at a network level along with adaptations for scanning children with developmental disabilities is presented.

Newly emerging theories suggest that the brain does not function as a cohesive unit in autism, and this discordance is reflected in the behavioral ...

Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants

Perinatal brain injury remains a significant cause of infant mortality and morbidity, but there is not yet an effective bedside tool that can accurately screen for brain injury, monitor injury evolution, or assess response to therapy. The energy used by neurons is derived largely from tissue oxidative metabolism, and neural hyperactivity and cell death are reflected by corresponding changes in cerebral oxygen metabolism (CMRO2). Thus, measures of CMRO2 are reflective of neuronal viability and provide critical diagnostic information, making CMRO2 an ideal target for bedside measurement of brain health.
Brain-imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) yield measures of cerebral glucose and oxygen metabolism, but these techniques require the administration of radionucleotides, so they are used in only the most acute cases.
Continuous-wave near-infrared spectroscopy (CWNIRS) provides non-invasive and non-ionizing radiation measures of hemoglobin oxygen saturation (SO2) as a surrogate for cerebral oxygen consumption. However, SO2 is less than ideal as a surrogate for cerebral oxygen metabolism as it is influenced by both oxygen delivery and consumption. Furthermore, measurements of SO2 are not sensitive enough to detect brain injury hours after the insult 1,2, because oxygen consumption and delivery reach equilibrium after acute transients 3. We investigated the possibility of using more sophisticated NIRS optical methods to quantify cerebral oxygen metabolism at the bedside in healthy and brain-injured newborns. More specifically, we combined the frequency-domain NIRS (FDNIRS) measure of SO2 with the diffuse correlation spectroscopy (DCS) measure of blood flow index (CBFi) to yield an index of CMRO2 (CMRO2i) 4,5.
With the combined FDNIRS/DCS system we are able to quantify cerebral metabolism and hemodynamics. This represents an improvement over CWNIRS for detecting brain health, brain development, and response to therapy in neonates. Moreover, this method adheres to all neonatal intensive care unit (NICU) policies on infection control and institutional policies on laser safety. Future work will seek to integrate the two instruments to reduce acquisition time at the bedside and to implement real-time feedback on data quality to reduce the rate of data rejection.

We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.

Perinatal brain injury remains a significant cause of infant mortality  and morbidity, but there is not yet an effective bedside tool that can  accurately ...

In vivo Macrophage Imaging Using MR Targeted Contrast Agent for Longitudinal Evaluation of Septic Arthritis

Macrophages are key-cells in the initiation, the development and the regulation of the inflammatory response to bacterial infection. Macrophages are intensively and increasingly recruited in septic joints from the early phases of infection and the infiltration is supposed to regress once efficient removal of the pathogens is obtained. The ability to identify in vivo macrophage activity in an infected joint can therefore provide two main applications: early detection of acute synovitis and monitoring of therapy.
In vivo noninvasive detection of macrophages can be performed with magnetic resonance imaging using iron nanoparticles such as ultrasmall superparamagnetic iron oxide (USPIO). After intravascular or intraarticular administration, USPIO are specifically phagocytized by activated macrophages, and, due to their magnetic properties, induce signal changes in tissues presenting macrophage infiltration. A quantitative evaluation of the infiltrate is feasible, as the area with signal loss (number of dark pixels) observed on gradient echo MR images after particles injection is correlated with the amount of iron within the tissue and therefore reflects the number of USPIO-loaded cells.
We present here a protocol to perform macrophage imaging using USPIO-enhanced MR imaging in an animal model of septic arthritis, allowing an initial and longitudinal in vivo noninvasive evaluation of macrophages infiltration and an assessment of therapy action.

In vivo Macrophage Imaging Using MR Targeted Contrast Agent for Longitudinal Evaluation of Septic Arthritis

We demonstrate how to perform macrophage MR imaging using ultrasmall superparamagnetic contrast agent (USPIO) in septic arthritis, allowing an initial and longitudinal in vivo non-invasive evaluation of macrophages infiltration and an assessment of therapy efficacy.

Macrophages are key-cells in the initiation, the development and the regulation of the inflammatory response to bacterial infection. Macrophages are ...

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo optical and &#956;CT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, &#956;CT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical &#956;CT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo bioluminescent/fluorescent imaging with &#956;CT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.

Combined In vivo Optical and  &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Combined optical and &#956;CT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

Creating Anatomically Accurate and Reproducible Intracranial Xenografts of Human Brain Tumors

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live animal &#8211;&#160;particularly in sites with unique physiological and architectural qualities such as the brain. In vitro and ectopic models cannot account for features such as vasculature, blood brain barrier, metabolism, drug delivery and toxicity, and a host of other relevant factors. Orthotopic models have their limitations too, but with proper technique tumor cells of interest can be accurately engrafted into tissue that most closely mimics conditions in the human brain. By employing methods that deliver precisely measured volumes to accurately defined locations at a consistent rate and pressure, mouse models of human brain tumors with predictable growth rates can be reproducibly created and are suitable for reliable analysis of various interventions. The protocol described here focuses on the technical details of designing and preparing for an intracranial injection, performing the surgery, and ensuring successful and reproducible tumor growth and provides starting points for a variety of conditions that can be customized for a range of different brain tumor models.

The brain is a unique site with qualities that are not well represented by in vitro or ectopic analyses. Orthotopic mouse models with reproducible location and growth characteristics can be reliably created with intracranial injections using a stereotaxic fixation instrument and a low pressure syringe pump.

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live ...

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. 
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the present protocol, mice expressing green fluorescent protein (gfp) under the promoter of Cx3cr1 (BALB/c-Cx3cr1gfp/gfp) were used to image microglia cells in vivo in the retina. Microglia are resident macrophages of the retina and have been implicated in several retinal diseases1,2,3,4,5,6. This protocol provides a detailed approach for generation of retinal B-scans, with SD-OCT, and imaging of microglia cell distribution in Cx3cr1gfp/gfp mice with SLO in vivo, using an ophthalmic imaging platform system. The protocol can be used in several reporter mouse lines. However, there are some limitations to the protocol presented here. First, both SLO and SD-OCT, when used in the high-resolution mode, collect data with high axial resolution but the lateral resolution is lower (3.5 &#181;m and 6 &#181;m, respectively). Moreover, the focus and saturation level in SLO is highly dependent on parameter selection and correct alignment of the eye. Additionally, using devices designed for human patients in mice is challenging due to the higher total optical power of the mouse eye compared to the human eye; this can lead to lateral magnification inaccuracies7, which are also dependent on the magnification by the mouse lens among others. However, despite that the axial scan position is dependent upon lateral magnification, the axial SD-OCT measurements are accurate8.

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

This protocol describes how high-resolution imaging techniques such as spectral domain optical coherence tomography and scanning laser ophthalmoscopy can be utilized in small rodents, using an ophthalmic imaging platform system, to obtain information on retinal thickness and microglial cell distribution, respectively.

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the ...