Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

This procedure demonstrates a novel method that is used to detect and quantify the time dependent loss of hind limb bone density in the mouse following spinal cord injury. This is accomplished by first performing a spinal transection injury in an anesthetized mouse under sterile and alac approved conditions. Then after a recovery period, the animal is re anesthetized and X-ray images are obtained of the tibia and fibia of both right and left hind limbs.

This is performed weekly out to 40 days post-injury. Next, an image analysis of the x-ray scans is performed. The final step is to perform a post-mortem assessment of bone density on excised hind limb bones.

Ultimately, results show how spinal cord injury induces a rapid and progressive loss of bone density in the hind limbs of the mouse. This novel method provides strong spatial and temporal resolution using lower doses of x-rays than those employed by other imaging modalities. Osteoporosis is a life-threatening condition that afflicts those living with spinal cord injury.

What we are proposing is a novel method to be used in animal studies of spinal cord injury that allows a researcher to follow longitudinally over large periods of time, the loss of bone associated with osteoporosis. This is important for two reasons. One, this will provide an opportunity to study the mechanisms that shape the development of osteoporosis.

The second is it provides us a manner of testing new interventions designed to either prevent or perhaps reverse the development of osteoporosis in patients who are living with spinal cord injury. Although this method can provide insight into spinal cord injury induced osteoporosis, it can also be applied to other systems such as age-related bone loss that occurs in postmenopausal women, as well as other trauma induced reductions in bone density limb disuses resulting from isolation, following fracture or other damage can result in substantial bone loss during the healing process. Applying whole animal longitudinal low dose X-ray imaging may provide new avenues to the development of therapies designed to minimize or prevent these problems.

Perform the spinal cord injury surgery according to sander procedures and in accordance with local institutional animal care and use committee guidelines. Then beginning on day 10, post spinal cord surgery and continuing at 10 day intervals until day 40. Assess the right and left femurs and tibiae of living anesthetized transection spinal cord injury animals and uninjured aged match controls by scanning using the ivus Lumina XR system before scanning, initialize the Lumina XR device to allow the internal camera to reach the operational temperature of minus 90 degrees Celsius.

Place an anesthetized animal onto the platform within the Lumina xr. Then insert a high magnification lens to allow focus on both the femur and tibial regions. Check that the subject is properly positioned in the field of view.

If necessary, open the door and reposition the animal. Proper positioning of the animal is shown here. Once properly positioned, the x-ray function can be executed.

Make a selection from the energy dropdown list suitable for animal. Then open the control panel. Enable the x-ray feature by check marking x-ray, and acquire the x-ray image.

The entire femur and tibia should be visible as seen here. Since the living image software displays transformed x-ray images by default, ensure that raw x-ray images are displayed by entering the corrections filtering tools menu, and removing the check mark next to x-ray absorption. Raw image data is automatically saved to the hard drive.

Representative TIFF files are also saved. After imaging, the mouse is then returned to its home cage and allowed to recover under investigator observation. The process is then repeated with the next mouse.

First, open the software by double clicking on the living image icon in the dialogue box that appears. Select the folder of interest and click okay. Load an x-ray image by clicking the browse button.

The living image browser displays the selected data along with the user id. Label information and camera configuration information To open data, double click the data row. The image and tool palette are displayed.

The open data is highlighted in blue in the browser. Next, click on ROI tools. In the tool palette in ROI tools, open the type dropdown list and select measurement ROI.

To load the three ROIs used in this experiment, click the square icon and load squares. Using a ruler, measure the length of the femur, adjust the length of the two squares to be one eighth of the total femur length. Adjust the width of the squares to be one 24th of the total femur length.

Now with the ruler, measure one eighth of the distance from the proximal femur end and situate one rectangle so that it is centered at that position. Move the second rectangle to the distal femur end and situate it so that it lies centered within the trabecular region of the knee. Then measure the length of the tibia.

Adjust the length of the box to one eighth of the total tibia length. Adjust the width to one 30th of the total tibia length. Finally, situate the box so that it is centered, and one eighth of the total length of the tibia distance from the proximal tibia end as seen here.

Next, click on the measure icon, the ROI Intensity measurements appear in the x-ray image and the ROI measurements table appears. Export this table to the desired location as a dot CSV file. This will allow the table to be opened using Excel.

Repeat this step with all of the saved images to be analyzed. Finally, consolidate all of the data into one Excel sheet. Statistical significance is determined by a student's T-test using software such as Microsoft Excel or Sigma Plot 11.0 software if desired, the results may be compared to those obtained from postmortem analysis of bone density using dual energy x-ray absorb geometry by utilizing standard protocols for this method.

The relative bone density loss of a mouse, tibia and femur after spinal cord injury when compared to naive mice is detectable using the above method. This figure shows bone density loss after spinal cord injury in the proximal tibia, 10, 20, 30, and 40 days after injury. Compared to age matched naives, there is a significant 12%decrease in bone density of the tibia after just 10 days with up to 15%bone density loss at 40 days.

Here we see bone density loss after spinal cord injury in the proximal femur, 10, 20, 30, and 40 days after injury. Compared to age match naives, a 7%decrease in bone density loss in the proximal femur was observed at 40 days post-injury. This graph shows bone density loss after spinal cord injury in the distal femur 10, 20, 30, and 40 days after the injury.

Compared to aged matched naive controls, loss of bone density in the distal femur could be observed as early as 10 days post-injury and was sustained throughout the remainder of the experiment. This representative image of dual energy x-ray abor geometry data shows bone mineral content and bone mineral density output. The excised femur were analyzed 40 days after spinal cord injury.

To compare the efficacy of the method demonstrated in this protocol to other currently available methods, this figure shows dual energy x-ray abor geometry analysis of bone mineral content in grams in the femur of spinal cord injury. Mice 40 days post surgery versus age match naives. A significant 12%loss of bone mineral content in the spinal cord injury mice when compared to naive was observed.

Here we see dual energy, extra abor geometry analysis of bone mineral density in milligrams, centimeter squared in the femur of spinal cord in injury, mice 40 days post-injury versus H match naive mice. Bone mineral density loss did not change significantly, but did follow a similar trend to bone mineral content Once mastered. This could be accomplished in 20 to 30 minutes, but remember, it's important to position the hind limbs properly.

After observing this video, you should have an idea of how to perform a live animal longitudinal assessment of bone density following spinal cord injury using the Caliper Instruments Lumina XR device.