Here we propose a method for assessment of global ocular structure following spaceflight using micro-computer tomography imaging method. The spaceflight environment, which includes paragravity and radiation exposure, causes unique changes to human physiology, including fluid shifts. These fluid shifts lead to elevated intracranial pressure and has been attributed as a major cause of spaceflight associated neuro-ocular syndrome.
The physiological impact of SANS impacts multiple components of the eye and includes optic disc edema, globe flattening, choroidal and retinal folds, hyperopic and refractive error shifts, and nerve fiber layer infracts. While the physiological characteristics of SANS are well documented, the mechanisms that drive SANS are still poorly understood. In order to have a better understanding of SANS, rodent models are being used for non-invasive imaging techniques.
One of these techniques is micro-CT, which has been successfully used for the evaluation of anatomical structures and pathological processes in animals as small as mice. Micro-CT can achieve a micro-sized resolution, and through the combination with a contrast agent, it can provide a good contrast for the soft tissues. Micro-CT provides a clear advantage compared to traditional methods, including gross anatomy, light microscopy, and histology examination to not cause damage to the geometric profile of the specimens and the spatial relationships among the structures.
In our current study, mice were exposed to the space environment for 35 days aboard the International Space Station to determine whether the space environment induces ocular damage by quantifying the microstructure of the retina, RPE, and choroid layers using Micro-CT. Here we have demonstrated how we have prepared samples for Micro-CT analysis. Mice were used and eyes within 38 plus or minus four hours after splash down.
Eyes were enucleated, and the left eyes were fixed in four percent paraformaldehyde and phosphate buffers saline for 24 hours. After fixation, the eyes were dehydrated in ethanol. To prevent a further and abrupt shrinkage of the fixed sample, a graded series of ethanoic solutions were used.
First, the samples were transferred to 50%ethanol for one hour and then increasing concentrations for one hour each, 70, 80, 90, 96, and 100%Afterwards, the eyes were stained with 10%weight per volume phospholipid acid for six days. Finally, samples were washed in absolute ethanol and then placed in individual two mill plastic containers filled with absolute ethanol for scanning. A cotton pad was also added to stabilize the sample during scanning.
Next, the sample was placed inside a desktop x-ray Micro-CT system SkyScan 1272 scanner to evaluate the retinal damage. For scanning, we used the SkyScan 1272 software. After opening the software, we centered our sample in the frame.
In our protocol, we use no filter and set the matrix to increase the pixel size to four microns. Micro-positioning is used to keep the sample centered in the frame. Next, we check the parameters to maximize the contrast agent within our sample and to calibrate the machine.
To perform the calibration, we remove the sample from the scanner, and we set the scanning parameters. During the calibration, a flat-filled correction is also checked. It should be greater than 80%After calibrations, the sample is reinserted into the scanning chamber.
Before scanning, the sample files are named. Scanning parameters are as follows, rotation step 0.4, frames four, random movement 30, double-check that the pixel size is set to 4 microns. Then the scan is started.
After scanning, NRecon is used to reconstruct the sample. First, we open the files from the scan and view the raw scanning images. We adjust the bounce of the histogram to fit within the curve.
Next, we correct for the beam hardening artifact. Then we adjust the smoothing artifact. Finally, we correct for the ring artifact reduction.
After all of the corrections, we confirmed that our samples fit within our region of interest. Then we can start the reconstruction. We use the DataViewer software to visualize the reconstructed images in all three fields.
For analysis, the CTAn software is used. The reconstructed images are loaded into the software. The optical nerve was used to deliminate the region of interest for analysis.
By calculation, we used the middle slice of the region of interest to perform all of the measurements for analysis. Three repeated measurements were taken to calculate the linear measurement of the area. The following measurements were taken for ocular tissues, retina, RPE, choroid, and Our Micro-CT analysis showed that the cross-section areas of retina, RPE, and choroid layer were significantly lower in spaceflight samples compared to ground controls.
Micro-CT provides an efficient and non-destructive techniques to calculate the size of changes without any manipulation. By using contrast agent, it enhance quality of Micro-CT images, which help us to get successful clear graphic image of the reconstruction without the inter-structure of the specimen. Additionally, it shows in the original data is digitally, thereby increase in accessibility and reproducibility of the findings.
Moreover, basic purpose, it is acceptable to use three-dimensional measurements, but segmentation of the gross three-dimensional structure can be beneficial to provide specific outline of the entire specimen. Our results indicate that spaceflight conditions, especially gravitational changes, may induce acute and short-term response in the eye. Moreover, future work should use volumetric data to perform other analyses that take advantage of the Micro-CT imaging capabilities, since it has been used successfully for studying many normal and pathologic tissues.
