The overall goal of this procedure is to model and then visualize a mouse model of bacterial infiltration of an orthopedic femoral nailing. This is accomplished by first surgically implanting a medical grade titanium kirschner wire into the femur of a lyse EGFP mouse. In the second step, the implant is inoculated with bioluminescent bacteria.
Next bioluminescent signal is quantified by sequential in vivo optical imaging, and the implant is visualized by micro CT imaging. Ultimately, quantification of the bioluminescent signals allows monitoring of the bacterial burden throughout the course of infection and micro CT imaging allows the visualization of the changes in the quality and dimensions of the bone that occur during the infection. Generally, individuals new to this method will struggle because the surgical technique is not very forgiving.
In particular, asymmetrically placed implants can fracture the femur, creating increased pain for the animal and unreliable data points. This Mouse model could provide a valuable preclinical model system for evaluating the protective immune responses and mechanisms in septic arthritis, osteomyelitis and periprosthetic osteolysis and bone damage, all of which are hallmarks of orthopedic implant infections in humans. I will now demonstrate the surgical procedures with assistance from Dr.Yew Wang, a research specialist, and Jonathan Ian, a laboratory technician in our laboratory Under sterile conditions.
Using the proper PPE confirms sedation of an anesthetized 12 week old male lye, EGFP mouse by toe pinch, then shaved the operative knee followed by three alternating scrubs with 70%alcohol and Betadine. Next, make a midline incision in the skin overlying the right knee joint, extending the incision so that the extensor mechanism can be well-defined. Then perform a medial para patellar arthrotomy and use an addin forceps to sublux the quadriceps patellar tendon extensor mechanism laterally.
Once the intercondylar notch of the femur is in plain view, use a 25 gauge needle followed by a 23 gauge needle to manually ream the intramedullary canal, taking care to remain parallel to the femoral shaft. To avoid asymmetric reaming and potential femur fracture, then use a pin holder to manually push a 0.8 millimeter diameter medical grade titanium kirschner wire in a retrograde direction into the intramedullary canal. Use pin cutters to cut the end of the wire so that it extends approximately one millimeter into the knee joint space.
Now use a micro pipette to dispense two microliters of one times 10 to the third CFU of bioluminescent S aureus Zen 29 onto the tip of the implant within the knee joint space. Then use forceps to reduce the quadriceps patellar complex back to midline and close the overlying subcutaneous tissue and skin with absorbable subcuticular sutures. To image the animal in 2D.
First place the mice ventral side up into the imaging chamber of an IVUS spectrum optical whole animal in vivo imaging system. Next, confirm that luminescent is checked and that open is selected as the emission filter and select field of view C equal to 13 centimeters. Then scroll the exposure time down until auto shows in the window to choose auto exposure and click acquire to capture the bioluminescent image to perform sequential in vivo fluorescent imaging.
Check the box next to fluorescent and choose the 465 nanometer excitation and 520 nanometer emission filters. Then scroll the exposure time down to auto. Again, select field of view C and click acquire to capture the fluorescence image to quantify the in vivo bioluminescent signals as the total flux in a region of interest or ROI using the living image software.
First, expand the ROI tool section of the tool palette. Next, select the circle icon and the number of ROIs that correspond to the number of subject animals in the field of view and resize the ROIs to encompass the entire area of the bioluminescent diffusion pattern collected. Then select Measure ROIs in ROI tools and choose the all and copy tabs to copy and subsequently paste the ROI values into a spreadsheet.
Then under the ROI tools tab of the tool palette, name the series of ROIs and click save. Next to quantify the in vivo fluorescent signal back in the living image software window. Activate the fluorescent image by clicking in that window.
Expand the ROI tools in the tool palette and select the just saved ROI series from the dropdown menu and click load. Then click measure ROIs again to open the ROI measurement window and copy the values onto a spreadsheet for micro CT image acquisition. Begin by inserting the large bore cover and adapter arm for the imaging shuttle into the instrument.
Place an anesthetize lice EGFP mouse into the mouse imaging shuttle, and then insert the imaging shuttle into the adapter arm. Push the arm into the bore and close the door. Now open the CT software at the top of the acquisition panel.
Select the preset choice 60 millimeter field of view standard dynamic from the dropdown list to automatically select the optimized acquisition parameters, including the voltage and current selections in the control panel. Select the I button to turn on the live mode and use the X and yxi controls to move the subject to the zero and 90 degree gantry position to center the animal in the X capture window. Then click the I button to turn the live mode off and click the CT scan button to acquire a dynamic scan image with the 60 millimeter field of view.
Then export the acquired image in a DICOM format and save it for later access for 3D optical image acquisition. First, remove the solid metal plate from the surface of the instrument and insert the imaging shuttle plate with a cutout for accepting the shuttle. Transfer the mouse imaging shuttle from the micro CT into the cutout plate and snap it into place.
Then in living image in the acquisition control panel, select the imaging wizard, choose bioluminescence, then delight and select the bacteria reporter from the dropdown menu. The appropriate emission for the model will be chosen automatically in this case, 500 to 620 nanometers. Then select next and designate the imaging subject as mouse auto settings will be selected, allowing the auto exposure to maximize the signal quality while avoiding saturation field of view will be set to C equals 13 centimeters.
Click finish. The sequence window of the acquisition panel will then be automatically populated with the delight sequence to acquire the delight data, select acquire sequence. One image will be acquired per emission filter chosen.
The auto exposure will select the optimal settings at each wavelength as per the imaging wizard selections. After the image acquisition is complete, expand the surface topography tab under the tool palette. Next, select the orientation that accurately reflects the side of the animal facing the camera or top of the ivus instrument.
Click generate surface and crop the region of the field of view that contains the animal. Use the purple mask to define the boundary of the animal and then select finish. The surface will appear automatically.
Save the result under the surface topography tab and then close the tab. Then to reconstruct the 3D optical source position, expand the delight 3D reconstruction tab. To use the diffuse optical reconstruction algorithms implemented in living image, the images acquired for the delight sequence will be listed by a mission wavelength.
Select start and the data preview window will open displaying the sequence to be analyzed, set to the automatically calculated optimized optical threshold. Then select reconstruct to automatically run the reconstruction algorithms and to generate a 3D representation of the optical source. Next, click the 3D icon in the toolbar to open the DICOM browser and double click on the previously acquired Quantum FX dataset to load it into the living image 3D view.
Expand the 3D optical tools in the tool palette and deselect the display subject surface box in the surface tab to deselect the surface topography. Then use the histogram under the volume tab of the 3D multimodality tool section of the tool palette to manually create a lookup table for visualizing the skeleton and the K-wire implant visible in the micro CT image 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. Then right click in the histogram to generate the points and form a curve to isolate that area of the histogram.
After saving both the skeleton and the K-wire implant data, double click any generated point in the histogram to color code any desired components. For micro CT image analysis, use the quantum FX software to select the image of interest and then launch the viewer. Then select the rotate tool and reorient the image.
To visualize the longitudinal axis of the femur, select the measurement tool and measure the length of the femur. Next, launch the 3D viewer to generate the 3D renderings and adjust the threshold to show the changes in the bone anatomy associated with the implant infection. Apply the clipping planes so that the 3D rendering is limited to the desired cross section of the area of interest in the distal femur and launch the analyze 11.0 software package.
Load the star vox file that was used to create the 3D rendering and launch the image calculator tool. Use the region pad tool to crop the image, removing any planes that do not include the femur. Then open the oblique sections tool and use the three points option to locate points at the middle of the femur, greater trocanter and the end of the pin.
Use these points to create an oblique plane and then generate an image with the new slices. Open the ROI tool and display the trans axial slices, adjusting the minimum and maximum settings to display the cortical bone. Create contours for the slices corresponding to the distal 25%of the femur, and use the propagate regions tool to interpolate between these contours and create a 3D region of interest.
Save this region of interest as an object map, and then open the sample options tool. Next, select the checkbox for the object map that was just created, and click the configure log stats button to confirm that the volume checkbox and the sample images button are selected. Then export the volume measurements into a data analysis program and normalize the outer bone volumes from the later time points to the first image time point using this formula where X represents the time point of interest.
To visualize the 3D region of interest on top of the bone load the CT image in the volume render tool and load the object map containing the 3D region of interest. Then go to view objects and set the original to on open the preview window and launch the render types menu and select object compositing. Then click the threshold button and adjust the thresholds to show the bone and object map using the same fixed threshold range for all time points.
Finally, click the rotation button and set the orientation to a true anterolateral view. Click render to generate the final rendering and save the rendering from the main volume render window. In this study, the bioluminescent signals of Zen 36 infected mice remained above the background signals of sham infected mice for the duration of the experiment.
In addition, the EGFP fluorescent signals were higher than the sham infected mice at early time points, but approached the background levels during the course of infection to visualize the optical signals in the anatomical context of the post-surgical knee joints in 3D, the optical images generated using the IVUS spectrum imaging system. For this representative experiment were coregistered with micro CT images generated using the quantum fx micro CT imaging system. As just demonstrated the optical data was then mapped onto the micro CT images in 3D using a diffuse optical tomography reconstruction algorithm as illustrated in this movie, micro CT imaging also allows the visualization and quantification of consequential changes in the quality and dimensions of the bone that occur during the infection.
As previously reported, the outer bone volume of the distal femur substantially increases over time. To quantify these changes, 3D volumetric image analysis can be performed and changes in the bone volume over time can be normalized to the initial bone volume. As expected from the micro CT images, the outer bone volume substantially increases in infected mice compared to sham infected mice After its development.
This technique paved the way for our laboratory to use this model to evaluate the efficacy of antibiotic loaded implant coatings to determine the role of interleukin one beta in promoting neutrophil recruitment to the infected post-surgical joint and to evaluate and compare prophylactic and treatment antibiotic regimens. Following these procedures, other methods like photoacoustic imaging can be performed to detect near infrared fluorescent signals, which may be important for the future development of fluorescent probes designed to diagnose the presence of an infection.
