Name: combined in vivo optical and &#181;ct imaging to monitor infection, inflammation, and bone anatomy in an orthopaedic implant infection in mice
Uploaded: 2014-10-16T13:00:00
Duration: 18 min 40 s
Description: Watch this Scientific Journal Video about Combined In vivo Optical and ÂµCT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice at JoVE.com

This work was supported by an H &#38; H Lee Surgical Resident Research Scholars Program (to J.A.N.), an AO Foundation Start-Up grant S-12-03M (to L.S.M.) and a National Institutes of Health grant R01-AI078910 (to L.S.M.).

Ethics statement: All animals were handled in strict accordance with good animal practice as defined in the federal regulations as set forth in the Animal Welfare Act (AWA), the 1996 Guide for the Care and Use of Laboratory Animals and the PHS Policy for the Humane Care and Use of Laboratory Animals and all animal work was approved by the Johns Hopkins Animal Care and Use Committee (Protocol #: MO12M465).
1. Preparing the Inoculum of Mid-logarithmic Bioluminescent Bacteria
<ol>
	<li>Streak bioluminescent S. aureus strain Xen29 (or another bioluminscent strain such as Xen36) onto tryptic soy agar plates (tryptic soy broth in agar [1.5%]). 
	NOTE: S. aureus Xen2921 is a genetically engineered S. aureus strain that contains a modified lux operon derived from Photorhabdus luminescens, which is integrated into a stable native plasmid found in this bacterial strain. These engineered bacteria constitutively emit light from live and metabolically active cells.</li>
	<li>Grow the colonies on the plates by incubating them at 37 &#176;C for approximately 16 hr (O/N).</li>
	<li>Select single bacterial CFU and culture in shaking liquid TSB (240 rpm) for approximately 16 hr (O/N).</li>
	<li>Perform a sub-culture with 1/50 dilution of the O/N culture to obtain mid-logarithmic growth phase bacteria (approximately 2 hr duration).</li>
	<li>Pellet, resuspend and wash the bacteria 3x in PBS.</li>
	<li>Estimate the bacterial inocula (1 x 103 CFU in 2 &#956;l PBS) by determining the optical density absorbance at 600 nm.</li>
	<li>Verify the CFU in the inoculum after culturing the bacteria O/N on plates.</li>
</ol>
2. Mouse Surgical Procedures
NOTE: For these experiment, use a twelve-week old male LysEGFP mice. These mice possess enhanced green fluorescent protein (EGFP) expressing myeloid cells (which consist of mostly neutrophils)20. Maintain sterile conditions during surgery and after surgical prep with betadine and 70% alcohol by placing each mouse on a sterile drape on top of a hard surface water circulating heating pad. Use gown, sterile gloves, mask and sterilize instruments.
<ol>
	<li>Anesthetize the mouse using a 2% inhalation of isoflurane. Use vet ointment on eyes to prevent dryness while under anesthesia. Assess the appropriate level of anesthesia by observing the respiratory rate, muscle tone, toe pinch, corneal reflex and color of mucous membranes. Cover the mice with a sterile surgical drape with a hole at the surgery site on the right knee.&#160;The mouse should get supportive heating measures to maintain body temperature while under anesthesia. Warm 37 &#176;C water circulating in a warm water jacketed blanket or a water circulating hard plastic heated workstation (ProStation, Patterson, Scientific) are good ways to prevent hypothermia.</li>
	<li>Inject buprenorphine (sustained-release formulation) (2.5 mg/kg) subcutaneously just prior to surgery. Additional doses of sustained-release buprenorphine may be administered at 3 day intervals as needed for analgesia.</li>
	<li>Shave the operative knee and prep using three alternating scrubs using betadine and 70% alcohol.</li>
	<li>Perform a midline incision in the skin overlying the right knee joint. Extend the skin incision so that the extensor mechanism can be well defined.</li>
	<li>Perform a medial parapatellar arthrotomy and sublux the quadriceps-patellar tendon extensor mechanism laterally with an Adson forceps. 
	NOTE: This brings the intercondylar notch of the femur into plain view.</li>
	<li>Manually ream the intramedullary canal using a 25 G needle followed by a 23 G needle. 
	Note: To avoid damage to femur, a stabilizing platform can be used. This should be important to the technique to minimize an accidental fracture of the femur.</li>
	<li>Insert a medical-grade titanium Kirschner-wire (0.8 mm diameter) by using a press-fit technique, which entails manually pushing it using a pin holder, in a retrograde direction into the intramedullary canal. 
	NOTE: Titanium K-wires were used as there were fewer artifacts seen on the &#956;CT images with titanium K-wires compared with stainless steel K-wires 16.</li>
	<li>Cut the end of the Kirschner-wire with pin cutters so that the cut end of the K-wire extends approximately 1 mm into the knee joint space.</li>
	<li>Using a micropipette, pipette 2 &#181;l of 1 x 103 CFU of bioluminescent S. aureus Xen29 onto the tip of the implant within the knee joint space. 
	NOTE: More volume leads to wider tissue contamination and less discrete imaging. 
	NOTE: In control uninfected mice, add 2 &#181;l of sterile saline without any bacteria.</li>
	<li>Reduce the quadriceps-patellar complex back to midline using forceps and close the overlying subcutaneous tissue and skin using absorbable subcuticular sutures. 
	NOTE: Do not leave an animal unattended until it has regained sufficient consciousness to maintain sternal recumbency. Do not return an animal that has undergone surgery to the company of other animals until fully recovered.</li>
	<li>At the end of the experiments, euthanized all animals using carbon dioxide inhalation according the Johns Hopkins Animal Care and Use Committee guidelines. Verify death by observing the animal fails to recover within 10 min after carbon dioxide exposure ends and cervical dislocation.</li>
</ol>
3. 2D Optical Imaging (In vivo Bioluminescent and Fluorescent Imaging)
<ol>
	<li>Anesthetize LysEGFP mice (e.g. 2% inhalation isoflurane) and place them with ventral side up into an imaging chamber.</li>
	<li>Perform in vivo bioluminescent imaging using the IVIS Spectrum optical whole animal in vivo imaging system. First, check Luminescent and confirm the choice of an open filter selection, field of view (FOV) C - 13 cm, and scroll exposure time down to Auto (autoexposure setting). Autoexposure will automatically adjust acquisition time (shutter speed), binning (digital pixel binning), and f-stop (aperture) of the instrument to optimize signal intensity while avoiding saturation. Then click Acquire to capture the bioluminescent image. 
	NOTE: For in vivo bioluminescent imaging, image mice between 1 - 5 min.</li>
	<li>Perform sequential in vivo fluorescent imaging by checking the box next to Fluorescent. Choose the 465 nm excitation filter and 520 nm emission filter. Scroll exposure time down to Auto and select FOV C (Step 3.2.1). Then click Acquire to capture the fluorescence image. 
	NOTE: For in vivo fluorescent imaging, image mice between 0.5 sec.</li>
	<li>Quantify the in vivo bioluminescent signals as total flux (photons/sec) in a region of interest (ROI) using Living Image software by first expanding the ROI Tools section of the Tool Palette.
	<ol>
		<li>Select the Circle Icon and the number of ROIs which correspond to the number of subject animals in the FOV. Resize the ROI to encompass the region of interest i.e., the bioluminescent diffusion pattern collected.</li>
		<li>Select Measure ROIs in ROI Tools in the Tool Palette and the ROI Measurement Window will appear. Total Radiance (photons/sec) values represent the sum of bioluminescent pixels within the generated ROI.</li>
		<li>Choose the SELECT ALL and COPY tabs in the bottom right hand corner of this window will transfer the information to the clipboard and allow pasting into subsequent programs for analysis.</li>
	</ol>
	</li>
	<li>Quantify the in vivo fluorescent signals as total radiant efficiency ([photons/sec] / [&#181;W/cm2]) within a circular region of interest (ROI) using Living Image software.
	<ol>
		<li>Within the Living Image software window, expand ROI Tools in the Tool Palette. Select the Circle Icon and the number of ROIs which corresponds to the number of subject animals in the FOV.</li>
		<li>Resize the ROI to encompass the region of interest corresponding closely to the bioluminescent signal from the previous image acquisition.</li>
		<li>Select Measure ROIs in ROI Tools in the Tool Palette and the ROI Measurement Window will appear. 
		NOTE: Total Radiant Efficiency ([photons/s] / [&#181;W/cm2]) represents the sums of fluorescent pixels within the ROI.</li>
		<li>Select All and Copy tabs in the bottom right hand corner of this window will transfer the information to the clipboard and allow pasting into subsequent programs for analysis.</li>
	</ol>
	</li>
</ol>
4. &#956;CT Image Acquisition
<ol>
	<li>Place the anesthetized LysEGFP mice into an imaging chamber. 
	NOTE: This imaging chamber is designed to fit in both the IVIS Spectrum imaging system and the Quantum FX in vivo &#956;CT imaging system to allow co-registration of the optical and &#956;CT images.</li>
	<li>Open the CT software and select the Menu preset 60 mm FOV std dynamic from the dropdown.</li>
	<li>Insert the large bore cover and the adapter arm for the imaging shuttle into the instrument.</li>
	<li>Place the mouse imaging shuttle into the adapter arm then push the arm into the bore and close the door. Turn on Live Mode (the Eye Button on the Control Panel) and position the subject at the 0&#176; and 90&#176; gantry position using the X-axis and Y-axis controls to center the animal in the X capture window. Then turn off Live Mode by clicking the Eye Button.</li>
	<li>Acquire a dynamic scan image with the 60 mm FOV by clicking the CT Scan button (next to the Live Mode Button). Export the acquired image in the DICOM format and store in a location that can be accessed later. 
	NOTE: The approximate dose will be 26 mGy per scan. A 30 mm FOV can be used if better resolution is desired.</li>
</ol>
5. 3D Optical Image Acquisition, Formation and &#181;CT Co-registration
<ol>
	<li>Place the mouse imaging shuttle insert into the Spectrum by positioning the imaging shuttle containing the mouse into this insert and ensure the mouse does not move.</li>
	<li>Using Living Image, select Imaging Wizard in the Acquisition Control Panel to begin the wizard setup. To start, choose Bioluminescence, then DLIT, and then select the reporter &#8220;Bacteria&#8221; from the dropdown menu and the appropriate emission filters for the model will be chosen automatically, in this case, 500 - 620 nm.
	<ol>
		<li>Select Next, then designate the acquisition parameters and subject information in the final window. Specifically, Imaging Subject will be Mouse, Auto Settings will be selected allowing autoexposure to maximize signal quality while avoiding saturation, and Field of View will be set to C - 13cm.</li>
		<li>Select Finish in the final window and the sequence window of the Acquisition Panel will be automatically populated with the DLIT sequence. There will be one image acquired per emission filter chosen and autoexposure will choose optimal settings at each wavelength as per the Imaging Wizard selections. The generated sequence also includes a structured light image needed for subject surface generation via the Surface Topography tool detailed below.</li>
	</ol>
	</li>
	<li>Select Acquire Sequence to acquire the DLIT data.</li>
	<li>After the image acquisition is complete, generate the surface topography. Start by expanding the Surface Topography tab under the Tool Palette.
	<ol>
		<li>Next, select the orientation that accurately reflects the side of the animal facing the camera or top of the IVIS instrument. Then click Generate Surface. Crop the region of the FOV that contains the animal.</li>
		<li>Then use the purple mask to define the bounds of the animal. 
		NOTE: The masking tool uses color contrast so animals with dark fur or skin will not mask appropriately from the stage.</li>
		<li>Select Finish and the surface will appear automatically. Save the result under the Surface Topography tab then close the tab as we will no longer need it.</li>
	</ol>
	</li>
	<li>Reconstruct the 3D optical source position using the diffuse optical reconstruction algorithms implemented in Living Image22 by expanding the DLIT 3D Reconstruction tab.
	<ol>
		<li>Images acquired for the DLIT sequence are shown. 
		NOTE: The software automatically verifies quality of the acquired data and will deselect images deemed too dim or where saturation is present. Select Start on the bottom right-hand side.</li>
		<li>If required, one can adjust the threshold for each bioluminescent image by double clicking and using the thresholding slider on the bottom left hand side. 
		NOTE: this is mainly to include lower intensity signal and caution should be practiced when thresholding higher as this may adjust overall intensity of the final reconstructed source.</li>
		<li>When satisfied with the threshold, select Reconstruct to automatically run the reconstruction algorithms which will result in a 3D representation of the optical source.</li>
	</ol>
	</li>
	<li>Open the DICOM browser by clicking the 3D icon in the Tool Bar at the top of the software (third from left) and search for the Quantum FX image acquired previously.
	<ol>
		<li>Load this image into the Living Image 3D View tab by double clicking on the file for import. 
		NOTE: The fiducial should be automatically detected and result in the &#956;CT image being registered with the 3D optical image.</li>
	</ol>
	</li>
	<li>Deselect the surface topography visualization map by expanding 3D Optical Tools in the Tool Palette and deselecting the check box labeled Display Subject Surface in the Surface tab.</li>
	<li>Manually create a lookup table to visualize the skeleton and the K-wire implant that are visible in the &#956;CT image using the histogram under the Volume tab of the 3D Multi-Modality Tools section of the Tool Palette.
	<ol>
		<li>The histogram represents the distribution of voxel intensities in the 3D volumetric data and their color opacity. To determine where the particular tissue of interest is in the histogram, use the slider tool to threshold the rendering until the tissue or structure is visible.</li>
		<li>Then right click in the histogram to generate points and form a curve to isolate that area of the histogram. This will be repeated for each structure &#8211; skeleton followed by the K-wire implant and can be saved as a lookup table for future analyses.</li>
		<li>Components can be color coded if so desired by double clicking any generated point in the histogram and selecting the desired color from the pop up window.</li>
	</ol>
	</li>
</ol>
6. &#956;CT Image Visualization and Analysis
<ol>
	<li>Using the Quantum FX software, select the image of interest and launch Viewer. Select the rotate tool and reorient the image to visualize the longitudinal axis of the femur. Select the measurement tool and measure the femur length.</li>
	<li>Launch the 3D Viewer to generate 3D renderings. Adjust the threshold to show the changes in bone anatomy associated with implant infection.</li>
	<li>Apply clipping planes so that the 3D rendering is limited to the desired cross-sectional section of the area of interest in the distal femur.</li>
	<li>Launch the Analyze 11.0 software package. Load the *.VOX file that was used to create the 3D rendering.</li>
	<li>Launch the Image Calculator tool. Use the &#8216;Region Pad&#8217; tool to crop the image (remove planes that don&#8217;t include the femur).</li>
	<li>Launch the Oblique Sections tool. Use the 3 points option to find points at the middle of the femur, greater trochanter and the end of the pin. Make these points an oblique plane and generate an image with new slices.</li>
	<li>Launch the Region of Interest tool. Display the transaxial slices. Adjust the min and max settings to display the cortical bone. Create contours for the slices corresponding for a several slices (with an approximate interval of 5 slices) for the slices corresponding to the distal 25% of the femur. Use the &#8216;Propagate Regions&#8217; tool to interpolate between these contours and create a 3D region of interest. Save this region of interest as an object map.</li>
	<li>Launch the &#8216;Sample Options&#8217; tool. Select the checkbox for the object map that was just created and select the radio buttons for the appropriate options. Click the &#8216;Configure Log Stats&#8217; button to confirm that the &#8216;Volume&#8217; checkbox is selected. Click the &#8216;Sample Images&#8217; button to make the actual measurements.</li>
	<li>Export the volume measurements into a data analysis program. Normalize the outer bone volumes from later time points to the first imaged time point using the formula: &#916; Volume (%) = ([Volume(day X) &#8211; Volume(day 2)]/[Volume(day 2)]) x 100. 
	NOTE: In this formula, the variable &#8220;X&#8221; represents the time point of interest. The resultant number will represent the change in the size of outer bone volume of the distal femur over time.</li>
	<li>To visualize the 3D region of interest on top of the bone, load the CT image in the &#8216;Volume Render&#8217; tool. Load the object map containing the 3D region of interest. Go to &#8216;View&#8217;&#8217;Objects&#8217; and set the &#8216;Original&#8217; to be &#8216;On&#8217;. Open the &#8216;Preview&#8217; window. Launch the &#8216;Render Types&#8217; menu and select &#8216;Object compositing&#8217;.</li>
	<li>Click the &#8216;Threshold&#8217; button and tool and adjust the thresholds to show the bone and object map. Use the same fixed threshold range for all time points. Click the &#8216;Rotation&#8217; button and set the orientation to be a true anterolateral view. Click &#8216;Render&#8217; to generate the final rendering. Save the rendering from the main &#8216;Volume Render&#8217; window.</li>
</ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What's+new+in+adult+reconstructive+knee+surgery.">What's new in adult reconstructive knee surgery.</a> J Bone Joint Surg Am. 94, 182-188 (2012).</li><li>Ning, 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=Maltodextrin-based+imaging+probes+detect+bacteria+in+vivo+with+high+sensitivity+and+specificity.">Maltodextrin-based imaging probes detect bacteria in vivo with high sensitivity and specificity.</a> Nat Mater. 10, 602-607 (2011).</li><li>Panizzi, P., 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=In+vivo+detection+of+Staphylococcus+aureus+endocarditis+by+targeting+pathogen-specific+prothrombin+activation.">In vivo detection of Staphylococcus aureus endocarditis by targeting pathogen-specific prothrombin activation.</a> Nat Med. 17, 1142-1146 (2011).</li><li>van Oosten, 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=Realtime+in+vivo+imaging+of+invasive+and+biomaterial+associated+bacterial+infections+using+fluorescently+labelled+vancomycin.">Realtime in vivo imaging of invasive and biomaterial associated bacterial infections using fluorescently labelled vancomycin.</a> Nat Commun. 4, 2584 (2013).</li><li>Kong, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+tuberculosis+with+endogenous+beta+lactamase+reporter+enzyme+fluorescence+in+live+mice.">Imaging tuberculosis with endogenous beta lactamase reporter enzyme fluorescence in live mice.</a> Proc Natl Acad Sci USA. 107, 12239-12244 (2010).</li></ol>

J.A.M., B.N.T., E.L., N.Z., K.P.F. are paid employees of PerkinElmer, which manufactures the imaging instruments, provided the Xen29 bioluminescent S. aureus strain, and paid for the publication costs of this video-article. The remaining authors have nothing to disclose.

Multimodality imaging such as imaging techniques that utilize in vivo optical imaging in conjunction with &#956;CT imaging provides a new technological approach that allows the 3D visualization, quantification and longitudinal monitoring of biologic processes in an anatomical context1-4. The protocols in the present study provide detailed information of how in vivo bioluminescent and fluorescent imaging can be combined with &#956;CT imaging in an orthopaedic prosthetic implant infection model in mice to monitor the bacterial burden, neutrophilic inflammation and anatomical changes in the bone noninvasively and longitudinally over time. Taken together, the information obtained by combining optical and structural imaging represents a major technological advance, which may be particularly well-suited to study biological processes and pathological conditions that affect the musculoskeletal system.
One interesting finding that should be pointed out is that we observed that the EGFP-neutrophil fluorescent signals decreased to background levels by 14-21 days and remained at background levels for the duration of the experiment despite the presence of bioluminescent bacteria. It is unlikely that the X-ray irradiation impacted neutrophil survival as we observed similar kinetics of the neutrophil signals in non-irradiated mice19. In our previous work involving a model of S. aureus infected wounds, neutrophil infiltration involved a combination of robust neutrophil recruitment from the circulation, prolonged neutrophil survival at the site of infection and the homing of KIT+ progenitor cells to the abscess, where they locally give rise to mature neutrophils23. It is likely that similar processes contributed to neutrophil infiltration in the orthopaedic implant S. aureus infection model. Although it is unknown why the neutrophil signals decreased in the orthopaedic infection model, it could be that the immune response changed over time as this infection progressed from an acute to chronic infection and this is a subject of future investigation.
There are limitations with this mouse model of orthopaedic prosthetic joint infection and the in vivo multimodality imaging that should be noted. First, this mouse model is an oversimplification of the actual procedures and materials used in orthopaedic surgery in humans24. Nonetheless, this model does recapitulate the chronic infection and ensuing inflammation in the bone and joint tissue that is seen in human orthopaedic implant infections8,9. In addition, to obtain the &#956;CT images, relatively low doses of X-ray irradiation were used to minimize any adverse effects on the health of the animals during the course of infection. For better resolution of bone, higher doses of X-ray radiation could be used for &#956;CT imaging on euthanized animals. However, this would eliminate the capability to noninvasively and longitudinally monitor the bone changes over the duration of the experiments.
In conclusion, multimodality imaging involving the combination of in vivo whole animal optical imaging with anatomical &#956;CT imaging has permitted more comprehensive information about the infection and inflammatory response. In addition, these techniques have permitted the evaluation of the consequences of the infection and inflammation on the bone and joint tissue. Future work could take advantage of multimodality imaging to evaluate the efficacy of antimicrobial therapies, immune responses, pathogenesis of disease and the reactive changes in the bone as we have begun to investigate14-18. In addition, multimodality imaging could evaluate probes and tracers to diagnose the presence of an infection as previously described in animal models a thigh infection, endocarditis, pulmonary infections and biomaterial infections25-28. Finally, the use of the multimodality imaging could be expanded beyond infectious diseases and used across disciplines, including orthopaedics, rheumatology and oncology, to investigate other conditions that impact the musculoskeletal system, such as skeletal cancer, metastatic disease, fractures and arthritis5-7.

Multimodality preclinical imaging techniques that involve the combination of optical and anatomical information allow the visualization and monitoring of biologic phenomena in 3D1-4. Since &#956;CT imaging permits the exquisite visualization of bone anatomy, using &#956;CT imaging in conjunction of with optical imaging represents a unique combination that might be especially useful for investigating processes that involve bone biology5-7. An example would be to use these techniques to study orthopaedic implant infections, which represent a disastrous complication following orthopaedic surgical procedures8,9. Bacteria biofilms form on the implanted foreign objects that promote survival of the bacteria by serving as a physical barrier that prevents immune cells from sensing the infection and blocks antibiotics from accessing the bacteria10,11. The chronic and persistent infection of the joint tissue (septic arthritis) and bone (osteomyelitis) induces bone resorption that leads to loosening of the prosthesis and eventual failure8,9. This resulting periprosthetic osteolysis is associated with increased morbidity and mortality12,13.
In our prior work, in vivo bioluminescent and fluorescent imaging was used together with X-ray and micro-computed tomography imaging (&#181;CT) in an orthopaedic prosthetic joint infection model in mice14-19. This model involved placing a titanium Kirschner-wire (K-wire) in such a manner that the cut end of the implant extended in the knee joint from the femurs of mice14-19. An inoculum of Staphylococcus aureus (bioluminescent strain Xen29 or Xen36) was then pipetted onto the surface of the implant in the knee joint before the surgical site was closed14-19. In vivo optical imaging was used to detect and quantify the bioluminescent signals, which corresponded to the number of bacteria in the infected joint and bone tissue14-19. In addition, in vivo fluorescence imaging of LysEGFP mice, which possess fluorescent neutrophils20, was used to quantify the numbers of neutrophils that emigrated to the infected knee joints containing the K-wire implants14,19. Finally, anatomical imaging modalities, including high-resolution X-ray imaging and &#181;CT imaging, permitted respective 2D and 3D anatomic imaging of the affected bone over the entire duration of chronic infection, which we would arbitrarily end typically between 2 and 6 postoperative weeks16,18. Using this model, the efficacy of local and systemic antimicrobial therapy, protective immune responses and pathologic anatomical changes in bone could be evaluated14-18. In this manuscript, the detailed protocols for the optical and &#181;CT imaging modalities in this orthopaedic prosthetic joint infection model were provided as a representative system to study biological processes in the anatomic context of the bone. These include the surgical procedures to model an orthopaedic prosthetic joint infection in mice, 2D and 3D in vivo optical imaging procedures (to detect bacterial bioluminescent signals and fluorescent neutrophil signals), &#181;CT imaging acquisition and analysis and co-registration of 3D optical images with the &#181;CT images.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Xen36 bioluminescent Staphylococcus aureus strain</td>
			<td>PerkinElmer, Hopkinton, MA</td>
			<td></td>
			<td>Bioluminescent Staphylococcus aureus strain derived from ATCC 49525 (Wright), a clinical isolate from a bacteremia patient</td>
		</tr>
		<tr>
			<td>Tryptic soy broth</td>
			<td>BD Biosciences, Franklin Lakes, NJ</td>
			<td>211825</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Soy Agar</td>
			<td>BD Biosciences, Franklin Lakes, NJ</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>LysEGFP knockin mouse strain</td>
			<td></td>
			<td></td>
			<td>Not commercially available. This strain contains a knockin of enhanced green fluorescence protein (EGFP) into the lysozyme M gene</td>
		</tr>
		<tr>
			<td>Betadine</td>
			<td>Purdue Products, Stamford, CT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Kirschner-wire (titanium, 0.8 mm diameter)</td>
			<td>Synthes, West Chester, PA</td>
			<td>492.08</td>
			<td></td>
		</tr>
		<tr>
			<td>Wire Cutter - Duracut T.C.</td>
			<td>H&#38;H Company, Ontario, Canada</td>
			<td>83-7002</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter, Deerfield, IL</td>
			<td>118718</td>
			<td></td>
		</tr>
		<tr>
			<td>Vicryl 5-0 sutures (P-3 Reverse cutting)</td>
			<td>Ethicon, Summerville, NJ. Purchased through VWR International.</td>
			<td>95056-936</td>
			<td></td>
		</tr>
		<tr>
			<td>Sustained-release Buprenorphine (5 ml - 1 mg/ml)</td>
			<td>Zoopharm, Windsor, CO</td>
			<td></td>
			<td>analgesic</td>
		</tr>
		<tr>
			<td><a href="http://www.perkinelmer.com/Catalog/Product/ID/IVISSPE?utm_source=JoVE&amp;utm_medium=display&amp;utm_content=LST-IDT2014-GLO-Q414-JoVE&amp;utm_campaign=LST-IDT2014-GLO">IVIS Spectrum Imaging System</a></td>
			<td>PerkinElmer, Hopkinton, MA</td>
			<td></td>
			<td>optical in vivo imaging system</td>
		</tr>
		<tr>
			<td><a href="http://www.perkinelmer.com/Catalog/Category/ID/Quantum%20FX%20%20microCT?utm_source=JoVE&amp;utm_medium=display&amp;utm_content=LST-IDT2014-GLO-Q414-JoVE&amp;utm_campaign=LST-IDT2014-GLO">Quantum FX in vivo &#956;CT system</a></td>
			<td>PerkinElmer, Hopkinton, MA</td>
			<td></td>
			<td>&#956;CT in vivo imaging system</td>
		</tr>
		<tr>
			<td><a href="http://www.perkinelmer.com/Catalog/Category/ID/Living%20Image%20Software?utm_source=JoVE&amp;utm_medium=display&amp;utm_content=LST-IDT2014-GLO-Q414-JoVE&amp;utm_campaign=LST-IDT2014-GLO">IVIS SpectrumCT Imaging System</a></td>
			<td>PerkinElmer, Hopkinton, MA</td>
			<td></td>
			<td>combined optical and &#956;CT in vivo imaging system</td>
		</tr>
		<tr>
			<td><a href="http://www.perkinelmer.com/Catalog/Product/ID/119240?utm_source=JoVE&amp;utm_medium=display&amp;utm_content=LST-IDT2014-GLO-Q414-JoVE&amp;utm_campaign=LST-IDT2014-GLO">Living Image Software</a></td>
			<td>PerkinElmer, Hopkinton, MA</td>
			<td></td>
			<td>Image analysis software for in vivo optical imaging</td>
		</tr>
	</tbody>
</table>

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.

In vivo bioluminescent and fluorescent imaging
In the present study, the protocol is described for this previously published model of an orthopaedic prosthetic joint infection in mice14-19, which involves the surgical placement of a titanium K-wire implant that extends from an intramedullary canal in the femur into the joint space14-19. S.&#160;aureus bioluminescent strain Xen29 (1 x 103 CFU in 2 &#956;l PBS) was pipetted directly on top of the end titanium implant in the knee joint prior to closing the surgical site16. To visualize and quantify the bacterial burden and neutrophil influx noninvasively in anesthetized LysEGFP mice, in vivo whole animal optical imaging was performed to sequentially image the bioluminescent signals from the bacteria and the EGFP fluorescent signals from the infiltrating neutrophils using the IVIS Spectrum optical whole animal in vivo imaging system on three postoperative days (i.e., days 2, 14 and 28). The bioluminescent signals of Xen29-infected mice remained above background signals of sham-infected mice for the duration of the experiment (Figure 1A,C)16. Our previous work demonstrated that the in vivo bioluminescent signals closely approximated the numbers of ex vivo CFU isolated from the joint/bone tissue and adherent to the implants17,18. In addition, the EGFP fluorescent signals were higher than sham-infected mice at early time points but approached background levels during the course of infection (Figure 2B,C)16.
3D co-registration of in vivo optical signals with &#956;CT images
To visualize the optical signals (i.e., bacterial bioluminescent and EGFP fluorescent signals) in the anatomical context of the post-surgical knee joints in 3D, the optical images generated using the IVIS Spectrum imaging system were co-registered with &#956;CT images generated using the Quantum FX &#956;CT imaging system. This co-registration could be accomplished because the mouse imaging chamber could be inserted into either machine to ensure that the mice were in the exact same orientation. To verify this accuracy, the results were compared with an image acquisition performed using the IVIS Spectrum-CT&#160;in vivo imaging system that integrates both modalities into one instrument without requiring physical relocation of the animal. To map the optical data onto the &#956;CT images in 3D, we utilized a diffuse optical tomography reconstruction algorithm16. The resultant 3D reconstruction is shown (Movie 1).
In addition, &#956;CT imaging allowed the visualization and quantification of the consequential changes in the quality and dimensions of the bone that occurred during the infection (Figure 2)16. As previously reported, the outer bone volume of the distal femur substantially increased over time (Figure 2A) 16. To quantify these changes, 3D volumetric image analysis was performed on the distal 25% of the boney surface of the femur and the changes in the bone volume over time were normalized to the initial bone volume. The outer bone volume substantially increased in infected mice compared to sham-infected mice (Figure 2B)16. The increase in the distal femur outer bone volume was likely due to bone damage caused by the infection of the joint tissue and bone, which were observed using &#956;CT imaging and histologic analysis16.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51612/51612fig1highres.jpg" width="500" /> 
Figure 1. 2D in vivo bioluminescent and fluorescent signals. S.&#160;aureus Xen29 or no bacteria (uninfected) were inoculated into the knee joint after K-wire placement and LysEGFP mice were imaged using the IVIS Spectrum imaging system16. (A) Mean in vivo bioluminescent signals as measured by total flux (photons/sec) &#177; sem. (B) Mean in vivo EGFP fluorescent signals as measured by total radiant efficiency (photons/sec) / (&#181;W/cm2) &#177; sem. (C) Representative in vivo bioluminescent and fluorescent signals overlaid onto a black and white photographic image of the mice. The limit of detection of the bacterial burden using in vivo bioluminescent imaging is between 1 x 102 and 1 x 103 CFU. *p &#60; 0.05, &#8224;p &#60; 0.01, &#8225;p &#60; 0.001 Xen29-infected mice versus sham-infected mice (Student&#8217;s t-test [two-tailed]). Please note this is a representative figure that includes previously published data generated using Xen29 and imaged with the IVIS Lumina XR imaging system16. <a href="https://www.jove.com/files/ftp_upload/51612/51612fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51612/51612fig2highres.jpg" width="500" /> 
Figure 2. 3D &#956;CT imaging. S.&#160;aureus Xen29 or no bacteria (uninfected) were inoculated into the knee joint after K-wire placement and mice were imaged using the Quantum FX in vivo &#956;CT system. (A) Representative 3D &#956;CT renderings of Xen29-infected mice (upper panels) and sham-infected mice (lower panels). (B) Percentage of outer bone volume change (distal 25% of the femurs) normalized to the initial time point (mean &#177; sem). *p &#60; 0.05, &#8224;p &#60; 0.01, &#8225;p &#60; 0.001 Xen29-infected mice versus sham-infected mice (Student&#8217;s t-test [two-tailed]). Please note this is a representative figure that includes previously published data generated using the bioluminescent strain S. aureus Xen29 and imaged with the Quantum FX in vivo &#181;CT imaging system16. <a href="https://www.jove.com/files/ftp_upload/51612/51612fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<a href="https://www.jove.com/files/ftp_upload/51612/Movie_1.mpg" target="_blank">Movie 1</a>.&#160;Representative 3D anatomical co-registration of the Xen29 bioluminescent signals and the EGFP-neutrophil fluorescent signals in combination with the &#956;CT images. The images are rotated on the vertical axis.

Watch this Scientific Journal Video about Combined In vivo Optical and ÂµCT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice at JoVE.com

Combined In vivo Optical and  &amp;#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.

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 ...

combined-vivo-optical-ct-imaging-to-monitor-infection-inflammation

Research

JoVE Journal

Medicine

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Bronchoalveolar Lavage (BAL) for Research; Obtaining Adequate Sample Yield

We describe a research technique for fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) using manual hand held suction in order to remove nonadherent cells and lung lining fluid from the mucosal surface. In research environments, BAL allows sampling of innate (lung macrophage), cellular (B- and T- cells), and humoral (immunoglobulin) responses within the lung.
BAL is internationally accepted for research purposes and since 1999 the technique has been performed in &#62; 1,000 subjects in the UK and Malawi by our group.
Our technique uses gentle hand-held suction of instilled fluid; this is designed to maximize BAL volume returned and apply minimum shear force on ciliated epithelia in order to preserve the structure and function of cells within the BAL fluid and to preserve viability to facilitate the growth of cells in ex vivo culture. The research technique therefore uses a larger volume instillate (typically in the order of 200 ml) and employs manual suction to reduce cell damage.
Patients are given local anesthetic, offered conscious sedation (midazolam), and tolerate the procedure well with minimal side effects. Verbal and written subject information improves tolerance and written informed consent is mandatory. Safety of the subject is paramount. Subjects are carefully selected using clear inclusion and exclusion criteria.
This protocol includes a description of the potential risks, and the steps taken to mitigate them, a list of contraindications, pre- and post-procedure checks, as well as precise bronchoscopy and laboratory techniques.

Bronchoalveolar Lavage BAL for Research; Obtaining Adequate Sample Yield

We describe a research technique for fiberoptic bronchoscopy and bronchoalveolar lavage using low pressure suction. The technique is used to harvest immune cells from the lung bronchoalveolar surfaces. Local anesthetic and mild conscious sedation (midazolam) is used. Subjects tolerate the procedure well and experience minimal side effects.

We describe a research technique for fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) using manual hand held suction in order to remove ...

Experimental Human Pneumococcal Carriage

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major source of transmission and the prerequisite of invasive disease. A model of carriage will allow accurate determination of the immunological correlates of protection, the immunizing effect of carriage and the effect of host pressure on the pathogen in the nasopharyngeal niche. Further, methods of carriage detection useful in epidemiologic studies, including vaccine studies, can be compared.
Aim
We aim to develop an EHPC platform that is a safe and useful reproducible method that could be used to down-select candidate novel pneumococcal vaccines with prevention of carriage as a surrogate of vaccine induced immunity. It will work towards testing of candidate vaccines and descriptions of the mechanisms underlying EHPC and vaccine protection from carriage1. Current conjugate vaccines against pneumococcus protect children from invasive disease although new vaccines are urgently needed as the current vaccine does not confer optimal protection against non-bacteraemic pneumonia and there has been evidence of serotype replacement with non-vaccine serotypes2-4.
Method
We inoculate with S. pneumoniae suspended in 100 &mu;l of saline. Safety is a major factor in the development of the EHPC model and is achieved through intensive volunteer screening and monitoring. A safety committee consisting of clinicians and scientists that are independent from the study provides objective feedback on a weekly basis.
The bacterial inoculum is standardized and requires that no animal products are inoculated into volunteers (vegetable-based media and saline). The doses required for colonization (104-105) are much lower than those used in animal models (107)5. Detecting pneumococcal carriage is enhanced by a high volume (ideally &gt;10 ml) nasal wash that is relatively mucus free. This protocol will deal with the most important parts of the protocol in turn. These are (a) volunteer selection, (b) pneumococcal inoculum preparation, (c) inoculation, (d) follow-up and (e) carriage detection.
Results
Our current protocol has been safe in over 100 volunteers at a range of doses using two different bacterial serotypes6. A dose ranging study using S. pneumoniae 6B and 23F is currently being conducted to determine the optimal inoculation dose for 50% carriage. A predicted 50% rate of carriage will allow the EHPC model to have high sensitivity for vaccine efficacy with small study numbers.

Experimental human pneumococcal carriage offers a natural model of carriage and a potential model for use in vaccine development. This technique is valuable yet complex and involves clinical risk by introducing a pathogen into a human. We have developed a detailed protocol.

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of cerebral malaria can be difficult. We present an experimental mouse model of cerebral malaria that shares multiple features of the human disease, including edema and microvascular pathology. Using magnetic resonance imaging (MRI), we can detect and track the blood-brain barrier disruption, edema development, and subsequent brain swelling. We describe multiple MRI techniques that can visualize these pertinent pathological changes. Thus, we show that MRI represents a valuable tool to visualize and track pathological changes, such as edema, brain swelling, and microvascular pathology, in vivo.

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

We describe a mouse model of experimental cerebral malaria and show how inflammatory and microvascular pathology can be tracked in vivo using magnetic resonance imaging.

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of ...

Treatment with Vancomycin Loaded Calcium Sulphate and Autogenous Bone in an Improved Rabbit Model of Bone Infection

Bone infection results from bacterial invasion, which is extremely difficult to treat in clinical, orthopedic, and traumatic surgery. The bone infection may result in sustained inflammation, osteomyelitis, and eventual bone non-union. Establishment of a feasible, reproducible animal model is important to bone infection research and antibiotic treatment. As an in vivo model, the rabbit model is widely used in bone infection research. However, previous studies on rabbit bone infection models showed that the infection status was inconsistent, as the amount of bacteria was variable. This study presents an improved surgical method for inducing bone infection on a rabbit, by blocking the bacteria in the bone marrow. Then, multi-level evaluations can be carried out to verify the modelling method.
In general, debriding necrotic tissue and implantation of vancomycin-loaded calcium sulphate (VCS) are predominant in antibiotic treatment. Although calcium sulphate in VCS benefits osteocyte crawling and new bone growth, massive bone defects occur after debriding. Autogenous bone (AB) is an appealing strategy to overcome bone defects for the treatment of massive bone defects after debriding necrotic bone.
In this study, we used the tail bone as an autogenous bone implanted in the bone defect. Bone repair was measured using micro-computed-tomography (micro-CT) and histological analysis after animal sacrifice. As a result, in the VCS group, bone non-union was consistently obtained. In contrast, the bone defect areas in the VCS-AB group were decreased significantly. The present modeling method described a reproducible, feasible, stable method to prepare a bone infection model. The VCS-AB treatment resulted in lower bone non-union rates after antibiotic treatment. The improved bone infection model and the combination treatment of VCS and autogenous bone could be helpful in studying the underlying mechanisms in bone infection and bone regeneration pertinent to traumatology orthopedic applications.

This study presents an improved rabbit model infected with Staphylococcus aureus by blocking the same amount of bacteria in bone marrow. Vancomycin loaded calcium sulphate and autogenous bone are used for antibiotic and bone repair treatment. The protocol could be helpful for studying bone infection and regeneration.

Bone infection results from bacterial invasion, which is extremely difficult to treat in clinical, orthopedic, and traumatic surgery. The bone infection ...

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. Therefore, adjuvants with both humoral and cellular immuno-stimulatory capabilities that are able to promote cytotoxic T lymphocytes (CTL) responses are urgently needed. Thus, faithful monitoring of adjuvant candidates that induce cross-priming and subsequently enhance CTL generation represents a crucial step in vaccine development. In here we present an application for a method that uses SIINFEKL-specific (OT-I) T cells to monitor the cross-presentation of the model antigen ovalbumin (OVA) in vivo in the presence of different adjuvant candidates. This method represents a rapid test to select adjuvants with the best cross-priming capabilities. The proliferation of CD8+ T cells is the most valuable indication of cross-priming and it is also regarded as a correlate of adjuvant-induced cross-presentation. This feature can be evaluated in different immune organs like lymph nodes and spleen. The extent of the CTL generation can also be monitored, thereby giving insights on the nature of a local (draining lymph node mainly) or a systemic response (distant lymph nodes and/or spleen). This technique further allows multiple modifications for testing drugs that can inhibit specific cross-presentation pathways and also offers the possibility to be used in different strains of conventional and genetically modified mice. In summary, the application that we present here will be useful for vaccine laboratories in industry or academia that develop or modify chemical adjuvants for vaccine research and development.

Rapid In Vivo Assessment of Adjuvant&#039;s Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

We present here an application for a standard immunological technique (CFSE stained OT-I proliferation) intended to rapidly monitor adjuvant-mediated cytotoxic T lymphocyte (CTL) generation in vivo. This fast estimation of CTL capacities is useful for the development of prophylactic vaccines against intracellular pathogens as well as therapeutic cancer vaccines.

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. ...

The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies that promote efficient bone healing are non-existent and in high demand. Effective and reproducible animal models of fractures healing are needed to understand the complex biological processes associated with bone regeneration. Many animal models of fracture healing have been generated over the years; however, murine fracture models have recently emerged as powerful tools to study bone healing. A variety of open and closed models have been developed, but the closed femoral fracture model stands out as a simple method for generating rapid and reproducible results in a physiologically relevant manner. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice and facilitate a post-fracture stabilization of the femur by inserting an intramedullary steel rod. Although devices such as a nail or a screw offer greater axial and rotational stability, the use of an intramedullary rod provides a sufficient stabilization for consistent healing outcomes without producing new defects in the bone tissue or damaging nearby soft tissue. Radiographic imaging is used to monitor the progression of callus formation, bony union, and subsequent remodeling of the bony callus. Bone healing outcomes are typically associated with the strength of the healed bone and measured with torsional testing. Still, understanding the early cellular and molecular events associated with fracture repair is critical in the study of bone tissue regeneration. The closed femoral fracture model in mice with intramedullary fixation serves as an attractive platform to study bone fracture healing and evaluate therapeutic strategies to accelerate healing.

The murine closed femoral fracture model is a powerful platform to study fracture healing and novel therapeutic strategies to accelerate bone regeneration. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice using an intramedullary steel rod to stabilize the femur.

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies ...

High-Throughput Analysis of Optical Mapping Data Using ElectroMap

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we present, in a step-by-step guide, the use of ElectroMap for analysis, quantification, and mapping of high-resolution voltage and calcium datasets acquired by optical mapping. ElectroMap analysis options cover a wide variety of key electrophysiological parameters, and the graphical user interface allows straightforward modification of pre-processing and parameter definitions, making ElectroMap applicable to a wide range of experimental models. We show how built-in pacing frequency detection and signal segmentation allows high-throughput analysis of entire experimental recordings, acute responses, and single beat-to-beat variability. Additionally, ElectroMap incorporates automated multi-beat averaging to improve signal quality of noisy datasets, and here we demonstrate how this feature can help elucidate electrophysiological changes that might otherwise go undetected when using single beat analysis. Custom modules are included within the software for detailed investigation of conduction, single file analysis, and alternans, as demonstrated here. This software platform can be used to enable and accelerate the processing, analysis, and mapping of complex cardiac electrophysiology.

This protocol describes the setup and use of ElectroMap, a MATLAB-based open-source software platform for analysis of cardiac optical mapping data. ElectroMap provides a versatile high-throughput tool for analysis of optical mapping voltage and calcium datasets across a wide range of cardiac experimental models.

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we ...

Immunofluorescence Labelling of Human and Murine Neutrophil Extracellular Traps in Paraffin-Embedded Tissue

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They mature in the bone marrow and are released into the peripheral blood, where they circulate for about 6&#8722;8 h; however, in tissue, they can survive for days. By diapedesis through the endothelium, they leave the blood stream, enter tissues, and migrate towards the site of an infection following chemotactic gradients.&#160;Neutrophils can combat invading microorganisms by phagocytosis,&#160;degranulation, and generation of neutrophil extracellular traps (NETs). This protocol will help to detect NETs in paraffin-embedded tissue. NETs are the result of a process called NETosis, which leads to the release of nuclear, granular, and cytoplasmic components either from living (vital NETosis) or dying (suicidal NETosis) neutrophils. In vitro, NETs form cloud-like structures, which occupy a space several times larger than that of the cells from which they descended. The backbone of NETs is chromatin, to which a selection of proteins and peptides originating from granules and cytoplasm are bound. Thereby, a high local concentration of toxic compounds is maintained so that NETs can capture and inactivate a variety of pathogens including bacteria, fungi, viruses, and parasites, while diffusion of the highly active NET components leading to damage in neighboring tissue is limited. Nevertheless, in recent years it has become apparent that NETs, if generated in abundance or cleared insufficiently, do have pathological potential ranging from autoimmune diseases to cancer. Thus, detection of NETs in tissue samples may have diagnostic significance, and the detection of NETs in diseased tissue can influence the treatment of patients. Since paraffin-embedded tissue samples are the standard specimen used for pathological analysis, it was sought to establish a protocol for fluorescent staining of NET components in paraffin-embedded tissue using commercially available antibodies.

Neutrophil extracellular traps (NETs) are three-dimensional structures generated by stimulated neutrophil granulocytes. It has become clear in recent years that NETs are involved in a wide variety of diseases. Detection of NETs in tissue may have diagnostic relevance, so standardized protocols for labelling NET components are required.

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They ...