This study was supported by NASA Space Biology grant # NNX15AB41G and LLU Department of Basic Sciences. Sungshin Choi, Dennis Leveson and Rebecca Klotz contributed significantly to the success of our spaceflight study and we greatly appreciate their support. The authors would also like to thank the entire NASA Biospecimen Sharing Program group for their great assistance.
The authors would also like to thank the Center for Dental Research for Micro-CT service.

The study followed the recommendations outlined in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH) and was approved by both the Institutional Animal Care and Use Committee (IACUC) of Loma Linda University (LLU) and The National Aeronautics and Space Administration (NASA). More detailed information regarding this flight experiment can be found elsewhere9,15.
1. Flight and control conditions
NOTE: A 12th Commercial Resupply Service (CRS-12) payload was launched By SpaceX at the Kennedy Space Center (KSC) on a 35-day mission in August 2017 which included onboard 10-week-old male C57BL/6 mice (n = 20) for NASA&#8217;s ninth Rodent Research experiment (RR-9).
<ol>
	<li>Prior to returning to Earth via SpaceX&#8217;s Dragon capsule, have the mice live in NASA&#8217;s Rodent Habitats (RH) aboard ISS for 35 days at an ambient temperature of 26&#8211;28 &#176;C with a 12-hour light/dark cycle throughout the flight.</li>
	<li>Place Ground Control (GC) mice into the same housing hardware used in flight and match environmental parameters such as temperature and carbon dioxide (CO2) levels as closely as possible based on telemetry data.</li>
	<li>Feed GC mice the same NASA food bar diet as their space-based counterparts. Provide both the spaceflight and GC mice with same ad libitum access to water and food.</li>
</ol>
2. Post-flight evaluation of the mice
<ol>
	<li>Within 28 hours of splashdown on Earth, transport the mice to Loma Linda University (LLU). Once there, remove the mice from the animal enclosure hardware and assess for survival and health. 
	NOTE: Upon observation, inspecting personnel reported that all the mice had survived the 35-day space mission and were in good condition, i.e. no noticeable deficiencies/abnormalities.</li>
</ol>
3. Dissecting and preservation of mouse eyes after spaceflight
<ol>
	<li>Within 38(&#177;4) hours of splashdown (n=20/group), euthanize the mice in 100% CO2 and collect their eyes.</li>
	<li>Dissect the right eye retinas and place individually in sterile cryovials, snap-freeze in liquid nitrogen, and keep at &#8722;80 &#176;C prior to use.</li>
	<li>Fix the whole left eyes in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 24 h and then rinse with phosphate-buffered saline (PBS) for micro-CT assays.</li>
</ol>
4. Sample preparation for micro-CT scanning
<ol>
	<li>After fixation, dehydrate the mice&#39;s eyes in ethanol. To forestall any further or abrupt shrinkage of the fixed sample, use a graded series of ethanol solutions: beginning with 50% ethanol for 1 hour and then increasing the concentrations of the ethanol solutions as follows for 1 hour each: 70, 80, 90, 96 and 100%. 
	NOTE: The mice&#39;s eyes must be handled in a hood chamber.</li>
	<li>Phosphomolybdic acid (PMA) staining 
	CAUTION: On account of PMA being corrosive, a carcinogenic, and toxic to organs, appropriate protective personal equipment is necessary, including the use of a fume hood.
	<ol>
		<li>Prepare the staining solution: 10 mg of PMA in 100 mL of absolute ethanol.</li>
		<li>Stain the mice&#39;s eyes (10 wt. % phosphomolybdic acid - PMA dissolved in absolute ethanol) for 6 days.</li>
		<li>Prior to scanning, first wash the eye samples in absolute ethanol and then place each eye in individual 2 mL plastic containers that are filled with 100% absolute ethanol. Add a cotton pad to stabilized samples during scan.</li>
	</ol>
	</li>
</ol>
5. Micro-CT scanning and analysis
NOTE: The SkyScan 1272 scanner, a desktop X-ray Micro-CT system, was used for evaluation of retinal damage in the mice&#39;s eyes
<ol>
	<li>Mount the soft-tissue sample to an appropriate sample holder. To prevent any movement during the X-ray CT measurements, ensure a tight fit of the sample on its holder (Figure 1).</li>
	<li>Upon meticulous alignment of each sample, individually scan the sample via X-rays.
	<ol>
		<li>After opening the software, center the sample in the frame. In the protocol, use no filter and set the matrix to increase the pixel at 4 &#181;m. Use micro-positioning to keep the sample center on the frame.</li>
		<li>After that, check the parameter to maximize the contrast agent. To perform the calibration, remove the sample and check that the flat-field correction is greater than 80%.</li>
		<li>After calibration, reinsert the sample into the scanning chamber. For scanning, use a rotation step of 0.400, a frame averaging of 4, a random movement of 30, and rotate the samples 180&#176;.</li>
	</ol>
	</li>
	<li>Use a positioning jig for repeated measurements. Due to phase-contrast enhancement performed as described, object details as small as 4 &#181;m can be detected from x-rays generated by a sealed micro-focus X-ray tube (tungsten anode) at 50 keV and 80 mA with an integration time of 90 minutes. 
	NOTE: The acquisition parameters laid out in this section for selection to produce overview CT scans with the highest image quality.</li>
	<li>After scanning, use software (e.g., NRecon) to reconstruct the data.
	<ol>
		<li>Adjust the histogram and use the same range (0 &#8211; 0.24) for all the samples. Reconstruct region of interest was a circle, and no scales or labels were used.</li>
		<li>To reduce artifacts during the scanning, use a beam-hardening correction of 20, a smoothing correction of 1, a ring artifact reduction of 6, and perform no alteration in the misalignment compensation. After reconstruction, it was confirmed that the sample was within the region of interest.</li>
		<li>Reposition images using a plane parallel to the optic nerve and lens of the eyes.</li>
	</ol>
	</li>
	<li>After scanning, use software (e.g., DataViewer) to visualize the reconstructed images in all three views. 
	NOTE: If necessary, with this software, the images can be re-positioned using a plane parallel to the optic nerve and lens of the eyes to perform a standardized analysis.</li>
	<li>Descriptive analysis
	<ol>
		<li>Measure the structures using a measuring tool in the software (e.g., CTAn). Use the optical nerve to delimit the region of interest for analysis. By calculation, the protocol used the middle slice to perform the measurements. This evaluation was performed by descriptive analysis (Figure 2 and Figure 3).</li>
		<li>Perform measurements of the retina, retinal pigment epithelium (RPE), choroid, and sclera layer in the sagittal (Figure 2) and axial view (Figure 3). Take three measurements of each structure in order to calculate an average.</li>
	</ol>
	</li>
</ol>

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	<li>Dawson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+New+Space+Race.">The New Space Race.</a> The Politics and Perils of Space Exploration. , 1-24 (2017).</li><li>Mao, X. W., 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=Spaceflight+environment+induces+mitochondrial+oxidative+damage+in+ocular+tissue.">Spaceflight environment induces mitochondrial oxidative damage in ocular tissue.</a> Radiation Research. 180, 340-350 (2013).</li><li>Overbey, E. G., 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=Mice+Exposed+to+Combined+Chronic+Low-Dose+Irradiation+and+Modeled+Microgravity+Develop+Long-Term+Neurological+Sequelae.">Mice Exposed to Combined Chronic Low-Dose Irradiation and Modeled Microgravity Develop Long-Term Neurological Sequelae.</a> International Journal of Molecular Sciences. 20 (17), 4094 (2019).</li><li>Nelson, E. S., Mulugeta, L., Myers, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microgravity-induced+fluid+shift+and+ophthalmic+changes.">Microgravity-induced fluid shift and ophthalmic changes.</a> Life. 4, 621-665 (2014).</li><li>Lee, A. G., Mader, T. H., Robert Gibson, C., Brunstetter, T. J., Tarver, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Space+flight-associated+neuro-ocular+syndrome+(SANS).">Space flight-associated neuro-ocular syndrome (SANS).</a> Eye. 32, 1164-1167 (2018).</li><li>Stenger, M. 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=.">.</a> Evidence Report: Risk of Spaceflight Associated Neuro-ocular Syndrome (SANS). , (2017).</li><li>Garrett-Bakelman, F. E., 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=The+NASA+Twins+Study:+A+multidimensional+analysis+of+a+year-long+human+spaceflight.">The NASA Twins Study: A multidimensional analysis of a year-long human spaceflight.</a> Science. 364, (2019).</li><li>Mader, T. H., 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=Optic+disc+edema,+globe+flattening,+choroidal+folds,+and+hyperopic+shifts+observed+in+astronauts+after+long+duration+space+flight.">Optic disc edema, globe flattening, choroidal folds, and hyperopic shifts observed in astronauts after long duration space flight.</a> Ophthalmology. 118, 2058-2069 (2011).</li><li>Mao, X. W., 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=Characterization+of+mouse+ocular+response+to+a+35-day+spaceflight+mission:+Evidence+of+blood-retinal+barrier+disruption+and+ocular+adaptations.">Characterization of mouse ocular response to a 35-day spaceflight mission: Evidence of blood-retinal barrier disruption and ocular adaptations.</a> Science Reports. 9 (1), 8215 (2019).</li><li>Metscher, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroCT+for+developmental+biology:+a+versatile+tool+for+high-contrast+3D+imaging+at+histological+resolutions.">MicroCT for developmental biology: a versatile tool for high-contrast 3D imaging at histological resolutions.</a> Developmental Dynamics. , 632-640 (2009).</li><li>Silva, J. M. S., 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=Three-dimensional+non-destructive+soft-tissue+visualization+with+X-ray+staining+micro-tomography.">Three-dimensional non-destructive soft-tissue visualization with X-ray staining micro-tomography.</a> Science Reports. 5, 14088 (2015).</li><li>Descamps, E., 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=Soft+tissue+discrimination+with+contrast+agents+using+micro-CT+scanning.">Soft tissue discrimination with contrast agents using micro-CT scanning.</a> Belgian Journal of Zoology. , 20-40 (2014).</li><li>Wu, J., Yin, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+research+of+nasolabial+muscle+structure+in+fetus+with+cleft+lip:+an+iodine+staining+technique+based+on+microcomputed+tomography.">Anatomy research of nasolabial muscle structure in fetus with cleft lip: an iodine staining technique based on microcomputed tomography.</a> Journal of Craniofacial Surgery. 25 (3), 1056-1061 (2014).</li><li>Roque-Torres, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Application of Micro-CT in Soft Tissue Specimen Imaging. In: Orhan K. (eds) Micro-computed Tomography (micro-CT) in Medicine and Engineering. , 139-170 (2020).</li><li>Overbey, E. G., 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=Spaceflight+influences+gene+expression,+photoreceptor+integrity,+and+oxidative+stress-related+damage+in+the+murine+retina.">Spaceflight influences gene expression, photoreceptor integrity, and oxidative stress-related damage in the murine retina.</a> Science Reports. 9 (1), 13304 (2019).</li><li>Elkhoury, J. E., Shankar, R., Ramakrishnan, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolution+and+Limitations+of+X-Ray+Micro-CT+with+Applications+to+Sandstones+and+Limestones.">Resolution and Limitations of X-Ray Micro-CT with Applications to Sandstones and Limestones.</a> Transport in Porous Media. 129, 413-425 (2019).</li><li>Sombke, A., Lipke, E., Michalik, P., Uhl, G., Harzsch, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+and+limitations+of+X-Ray+micro-computed+tomography+in+arthropod+neuroanatomy:+A+methodological+and+comparative+survey.">Potential and limitations of X-Ray micro-computed tomography in arthropod neuroanatomy: A methodological and comparative survey.</a> Journal of Comparative Neurology. 523 (8), 1281-1295 (2015).</li><li>Huang, A. S., Stenger, M. B., Macias, B. 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All authors have nothing to disclose.

The results of this study showed that there were structural changes in the spaceflight mouse eye using the micro-CT technique when compared to GC groups, particularly of the retina, the RPE, and the choroid layers of the eye, as evidenced by their decreased thickness. Micro-CT provides an efficient and non-destructive technique to characterize the changes with no need for manipulation. The use of PMA staining enhanced the quality of the micro-CT images to successfully obtain clear 3D tomographic images after reconstruction, foregoing any need to physically alter the structure of the specimen. An added benefit of these images is that they display the entire region of interest digitally, thereby increasing accessibility as well as reproducibility of the findings. Through the micro-CT images produced during this study, the targeted specimen showed differentiation of the multiple structures like the retina, RPE, choroid, and sclera layer for determination of the thickness of each layer.
A critical step within the protocol is the manipulation of the samples due to their size and texture. The handling of the specimen must be carefully done without putting pressure on the specimen during the preparation. The micro-CT has some limitations: resolution and the lack of standardized values for the parameters. During the scanning, the different micro-CT scanners may have diverse image processing algorithms; yet calibration for a grayscale might be pursued to overcome any problem. After scanning, reconstruction of the images should be based on the tissue and the analysis that will be performed. It can be critical since image quality depends on the tomographic system, the settings, the specimen size as well as the preparation methods16,17.
Due to its successful application in studying several types of normal and pathologic tissues, micro-CT imaging capabilities should be used in future research to compile volumetric data for other analyses. Thus, based on the present study&#8217;s purpose, it was acceptable to use bi-dimensional measurements, but segmentation of the gross 3D structure can also be beneficial to provide a precise outline of the entire specimen. Even with all the advantages of a non-destructive technique, micro-CT will not replace other methods such as immunohistochemistry, but will supplement and allow subsequent histology analyses if desired.
A prolonged spaceflight condition produces a series of structural and functional ocular changes in the astronauts during and after the space mission defined as SANS. The findings include hyperopic shifts, globe flattening, choroidal/retinal folds, and cotton wool spots19. In contrast to astronauts&#8217; optical coherence tomography (OCT) finding of retinal nerve fiber layer thickening, thinning of retina and choroidal layer was documented in this animal micro-CT study. These results were unexpected. This discrepancy may be due to confounding factors. Mice have limited cephalad fluid shift compared to human. This lack of fluid shift may have evoked different responses to gravitational changes. Secondly, mice were dissected within 38 hours after splashdown, and an acute response for re-adaptation may also contribute to morphological changes in the retina and choroid. Confirmation of this possibility requires further measurements during spaceflight and long term after the mission.
The results of this study indicate that space flight conditions, especially gravitational changes, may induce an acute and short-term response in the eye. Further investigation is needed to determine the consequences of the acute changes on ocular on retinal function and mechanism of spaceflight&#8211;induced structure changes.

In the microgravity environment of spaceflight, increased intracranial pressure (ICP) caused by fluid shift may have contributed to spaceflight-associated neuro-ocular syndrome (SANS)1,2,3,4,5. Indeed, over 40% of astronauts have experienced SANS during and after an International Space Station (ISS) mission6, including the spaceflight subject of the NASA Twins Study7. The current pathophysiology of SANS includes physiological changes such as optic disc edema, globe flattening, choroidal and retinal folds, hyperopic refractive error shifts, and nerve fiber layer infarcts (i.e., cotton wool spots) and are well documented5,8. However, the underlying mechanisms of the changes and factors contributing to the development of damage are unclear. In order to have a better understanding of SANS, animal models are available for characterizing the spaceflight-associated changes in retinal structure and function.
In a previous investigation on the same animals, we reported the impact of 35 days of spaceflight on the mouse retina. The results elucidate that spaceflight induces significant damage in the retina and retinal vasculature, and some proteins/pathways associated with cell death, inflammation and metabolic stress were significantly altered following spaceflight9.
Currently, there are a variety of noninvasive imaging techniques established to monitor disease development and progression, as well as physiological responses to various environmental stressors, which are also widely used in small rodent models. One of these techniques is micro-CT, which evaluates anatomical structures and pathological processes, and has successfully been used on organisms as small as mice10.
Micro-CT can achieve a microsized resolution, and it can provide high contrast for volumetric analysis of soft tissues with the addition of the appropriate contrast agent10,11,12,13,14. Micro-CT technology is advantageous compared to traditional methods such as gross anatomy, light microscopy, and histology examination, as it minimizes physical damage to the geometric profile of the specimens and does not alter the spatial relationship among structures. In addition, three-dimensional (3D) models of structures can be reconstructed from micro-CT images12,14. To date, despite evidence showing vision impairment following exposure to the space environment, few data in animal models are available for a better understanding of the spaceflight-associated changes in retinal structure and function. In the current study, mice were flown on a 35-day mission aboard the ISS to determine the impact of the spaceflight environment on ocular tissue structures by quantifying the microstructure of the retina, the RPE, and the choroid layers using micro-CT.

<table><tbody><tr><td>10 wt. % phosphomolybdic</td><td>Sigma</td><td>12026-57-2</td><td></td></tr><tr><td>Ethanol absolute by Baker Analyzed</td><td>VWR</td><td>80252500</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Merck</td><td>L1825</td><td></td></tr><tr><td>X-ray micro-CT system SkyScan 1272 scanner</td><td>Bruker</td><td></td><td></td></tr></tbody></table>

assessment of global ocular structure following spaceflight using a micro-computed tomography (micro-ct) imaging method

Reports show that prolonged exposure to a spaceflight environment produces morphologic and functional ophthalmic changes in astronauts during and after an International Space Station (ISS) mission. However, the underlying mechanisms of these spaceflight-induced changes are currently unknown. The purpose of the present study was to determine the impact of the spaceflight environment on ocular structures by evaluating the thickness of the mouse retina, the retinal pigment epithelium (RPE), the choroid and the sclera layer using micro-CT imaging. Ten-week-old C57BL/6 male mice were housed aboard the ISS for a 35-day mission and then returned to Earth alive for tissue analysis. For comparison, ground control (GC) mice on Earth were maintained in identical environmental conditions and hardware. Ocular tissue samples were collected for micro-CT analysis within 38(&#177;4) hours after splashdown. The images of the cross-section of the retina, the RPE, the choroid, and the sclera layer of the fixed eye was recorded in an axial and sagittal view using a micro-CT imaging acquisition method. The micro-CT analysis showed that the cross-section areas of the retina, RPE, and choroid layer thickness were changed in spaceflight samples compared to GC, with spaceflight samples showing significantly thinner cross-sections and layers compared to controls. The findings from this study indicate that micro-CT evaluation is a sensitive and reliable method to characterize ocular structure changes. These results are expected to improve the understanding of the impact of environmental stress on global ocular structures.

The mean thickness of the retina, RPE, choroid, and sclera layer was recorded using the micro-CT scans after following the protocol above (Figure 1). The technique showed a multiplanar reconstruction of the eyes in three different views. During the analysis, the observer was able to scroll through the whole sample to standardize the analysis right in the middle of the sample.
The micro-CT analysis showed the cross-section areas of the eyes in the sagittal and axial view (Figure 2 and Figure 3) in which were performed the linear measurements. RPE and choroid layer were significantly or trend lower in the spaceflight group when compared to the GC group (Figure 3).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61227/61227fig01v2.jpg" /> 
Figure 1: Micro-CT procedure of soft tissue.&#160;(A) Soft tissue sample (mouse eye). (B) Samples were fixed in 4% formaldehyde in phosphate buffer solution (PBS). After fixation, the mice&#39;s eyes were dehydrated in ethanol. To prevent a further and abrupt shrinkage of the fixed sample, a graded series of ethanolic solutions were used, beginning with 50% ethanol for 1 h and the following ethanol solutions in the concentrations listed, for 1 hour each: 70, 80, 90, 96 and 100%. (C) The mice&#39;s eyes were stained in phosphomolybdic acid (PMA) for 6 days, washed in absolute ethanol and then placed in individual 2 mL plastic containers filled with absolute ethanol. (D) A desktop X-ray micro-CT system scanner was used to evaluate the retinal injury in mice eyes. <a href="https://www.jove.com/files/ftp_upload/61227/61227fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61227/61227fig02.jpg" /> 
Figure 2: Sagittal view of a ground control mouse.&#160;Layers of the eye on the right side of the image are annotated, from top-to-bottom, retina (0.077 mm), retina pigment layer (RPE, 0.038 mm), choroid (0.041 mm), sclera (0.059 mm). This figure has been taken from Overbey et al.15. <a href="https://www.jove.com/files/ftp_upload/61227/61227fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61227/61227fig03.jpg" /> 
Figure 3: Axial view of a ground control mouse.&#160;Layers of the eye on the right side of the image are annotated, from top-to-bottom, retina (0.144 mm), retina pigment layer (RPE, 0.051 mm), choroid (0.041 mm), sclera (0.073 mm). This figure has been taken from Overbey et al.15. <a href="https://www.jove.com/files/ftp_upload/61227/61227fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61227/61227fig04.jpg" /> 
Figure 4: Average thickness of the retinal layer, RPE layer, and the choroid layer measured by micro-CT in the spaceflight and control groups.&#160;Counts were averaged across five retinas per group. Values were represented as mean thickness &#177; standard error (SEM). SEM of the mean is marked with error bars. Significantly lower in cross-section thickness in the spaceflight (FLT) group compared to the ground control (GC) group is denoted &#8216;*&#8217; (p &#60; 0.05). This figure has been taken from Overbey et al.15. <a href="https://www.jove.com/files/ftp_upload/61227/61227fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Assessment of Global Ocular Structure Following Spaceflight Using a Micro-Computed Tomography Micro-CT Imaging Method at JoVE.com

Assessment of Global Ocular Structure Following Spaceflight Using a Micro-Computed Tomography Micro-CT Imaging Method

We present a protocol using high-resolution micro-computed tomography imaging to determine whether spaceflight induced damage on ocular structures. The protocol shows the micro-CT-derived measurement of ex vivo rodent ocular structures. We demonstrate the ability to assess ocular morphological changes following spaceflight using a non-destructive tridimensional technique to evaluate ocular damage.

Assessment of Global Ocular Structure Following Spaceflight Using a Micro-Computed Tomography (Micro-CT) Imaging Method

Reports show that prolonged exposure to a spaceflight environment produces morphologic and functional ophthalmic changes in astronauts during and after an ...

assessment-global-ocular-structure-following-spaceflight-using-micro

Research

JoVE Journal

Biology

5.7K Views.  Loma Linda University. Hier stellen we een methode voor voor de beoordeling van de wereldwijde oculaire structuur na de ruimtevaart met behulp van micro-computer tomografie imaging methode. De ruimtevlucht omgeving, die parazwaartekracht en blootstelling aan straling omvat, veroorzaakt unieke veranderingen in de menselijke fysiologie, met inbegrip van vloeistofverschuivingen. Deze vloeistof verschuivingen leiden tot verhoogde intracraniale druk en is toegeschreven als een belangrijke oorzaak van de ruimtevaart geas...

Video: Assessment of Global Ocular Structure Following Spaceflight Using a Micro-Computed Tomography Micro-CT Imaging Method

Trabecular Meshwork Response to Pressure Elevation in the Living Human Eye

The mechanical characteristics of the trabecular meshwork (TM) are linked to outflow resistance and intraocular pressure (IOP) regulation. The rationale behind this technique is the direct observation of the mechanical response of the TM to acute IOP elevation. Prior to scanning, IOP is measured at baseline and during IOP elevation. The limbus is scanned by spectral-domain optical coherence tomography at baseline and during IOP elevation (ophthalmodynamometer (ODM) applied at 30 g force). Scans are processed to enhance visualization of the aqueous humor outflow pathway using ImageJ. Vascular landmarks are used to identify corresponding locations in baseline and IOP elevation scan volumes. Schlemm canal (SC) cross-sectional area (SC-CSA) and SC length from anterior to posterior along its long axis are measured manually at 10 locations within a 1 mm segment of SC. Mean inner to outer wall distance (short axis length) is calculated as the area of SC divided by its long axis length. To examine the contribution of adjacent tissues to the effect IOP elevations, measurements are repeated without and with smooth muscle relaxation with instillation of tropicamide. TM migration into SC is resisted by TM stiffness, but is enhanced by the support of its attachment to adjacent smooth muscle within the ciliary body. This technique is the first to measure the living human TM response to pressure elevation in situ under physiological conditions within the human eye.

Trabecular meshwork (TM) migration into Schlemm&#8217;s canal space can be induced by acute pressure elevation by ophthalmodynamometer, and observed by spectral domain optical coherence tomography. The goal of this method is to quantify the morphometric response of the living outflow tract to acute pressure elevation in living tissues in situ.

The mechanical characteristics of the trabecular meshwork (TM) are linked to outflow resistance and intraocular pressure (IOP) regulation. The rationale ...

Medicine

Evaluating Ocular Surface Squamous Neoplasia Using Anterior Segment HR-OCT

Anterior High-Resolution Optical Coherence Tomography in the Diagnosis and Therapeutic Monitoring of Ocular Surface Squamous Neoplasia

Ocular surface squamous neoplasia (OSSN) is the most common tumor of the ocular surface, ranging from mild dysplasia to invasive squamous carcinoma. Traditionally, the diagnosis of OSSN relies on histopathological confirmation followed by a full-thickness biopsy. However, in the past two decades, the therapeutic approach to OSSN has shifted from surgical intervention to topical chemotherapy regimens in clinical settings. This shift emphasizes the need for less invasive or non-invasive methods to diagnose ocular surface pathologies. Among various imaging devices, commercially available high-resolution optical coherence tomography (HR-OCT) has emerged as a powerful tool for the characterization of OSSN. HR-OCT provides an in vivo, cross-sectional view of ocular surface lesions, offering an "optical biopsy" for OSSN with high sensitivity and specificity. It provides valuable information in differentiating intraepithelial or invasive OSSN from other benign lesions. Additionally, HR-OCT can be used to monitor the response to topical chemotherapy and to detect subclinical OSSN during follow-up visits. In this article, the scanning protocol for image acquisition is presented, and image interpretation for OSSN is outlined. This standardized, practical, and reproducible approach is recommended in clinical workflows and is expected to assist clinicians in the management of OSSN.

Author Spotlight: Anterior HR-OCT as a Non-Invasive Tool for Characterizing Ocular Surface Squamous Neoplasia

Anterior segment high-resolution optical coherence tomography (HR-OCT) is a promising non-invasive modality for the diagnosis and therapeutic evaluation of ocular surface squamous neoplasia (OSSN). Here, the system setup, scanning technique, and representative diagnostic results are presented.

Ocular surface squamous neoplasia (OSSN) is the most common tumor of the ocular surface, ranging from mild dysplasia to invasive squamous carcinoma. ...

Engineering

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

Neuroscience

In vivo Structural Assessments of Ocular Disease in Rodent Models using Optical Coherence Tomography

Spectral-domain optical coherence tomography (SD-OCT) is useful for visualizing retinal and ocular structures in vivo. In research, SD-OCT is a valuable tool to evaluate and characterize changes in a variety of retinal and ocular disease and injury models. In light induced retinal degeneration models, SD-OCT can be used to track thinning of the photoreceptor layer over time. In glaucoma models, SD-OCT can be used to monitor decreased retinal nerve fiber layer and total retinal thickness and to observe optic nerve cupping after inducing ocular hypertension. In diabetic rodents, SD-OCT has helped researchers observe decreased total retinal thickness as well as decreased thickness of specific retinal layers, particularly the retinal nerve fiber layer with disease progression. In mouse models of myopia, SD-OCT can be used to evaluate axial parameters, such as axial length changes. Advantages of SD-OCT include in vivo imaging of ocular structures, the ability to quantitatively track changes in ocular dimensions over time, and its rapid scanning speed and high resolution. Here, we detail the methods of SD-OCT and show examples of its use in our laboratory in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia. Methods include anesthesia, SD-OCT imaging, and processing of the images for thickness measurements.

Here, we describe the use of spectral-domain optical coherence tomography (SD-OCT) to visualize retinal and ocular structures in vivo in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia.

Spectral-domain optical coherence tomography (SD-OCT) is useful for visualizing retinal and ocular structures in vivo. In research, SD-OCT is a valuable ...

Bioengineering

Anterior Segment Organ Culture Platform for Tracking Open Globe Injuries and Therapeutic Performance

Open globe injuries have poor visual outcomes, often resulting in permanent loss of vision. This is partly due to an extended delay between injury and medical intervention in rural environments and military medicine applications where ophthalmic care is not readily available. Untreated injuries are susceptible to infection after the eye has lost its watertight seal, as well as loss of tissue viability due to intraocular hypotension. Therapeutics to temporarily seal open globe injuries, if properly developed, may be able to restore intraocular pressure and prevent infection until proper ophthalmic care is possible. To facilitate product development, detailed here is the use of an anterior segment organ culture open globe injury platform for tracking therapeutic performance for at least 72 h post-injury. Porcine anterior segment tissue can be maintained in custom-designed organ culture dishes and held at physiological intraocular pressure. Puncture injuries can be created with a pneumatic-powered system capable of generating injury sizes up to 4.5 mm in diameter, similar to military-relevant injury sizes. Loss of intraocular pressure can be observed for 72 h post-injury confirming proper injury induction and loss of the eye&#39;s watertight seal. Therapeutic performance can be tracked by application to the eye after injury induction and then tracking intraocular pressure for multiple days. Further, the anterior segment injury model is applicable to widely used methods for functionally and biologically tracking anterior segment physiology, such as assessing transparency, ocular mechanics, corneal epithelium health, and tissue viability. Overall, the method described here is a necessary next step toward developing biomaterial therapeutics for temporarily sealing open globe injuries when ophthalmic care is not readily available.

Open globe eye injuries may go untreated for multiple days in rural or military-relevant scenarios, resulting in blindness. Therapeutics are needed to minimize loss of vision. Here, we detail an organ culture open globe injury model. With this model, potential therapeutics for stabilizing these injuries can be properly evaluated.

Open globe injuries have poor visual outcomes, often resulting in permanent loss of vision. This is partly due to an extended delay between injury and ...

Using Optical Coherence Tomography and Optokinetic Response As Structural and Functional Visual System Readouts in Mice and Rats

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for the investigation of processes of neurodegeneration, neuroprotection and neuro-repair involving the visual system, as these often correlate well with retinal changes. As a functional readout, visually evoked compensatory eye and head movements are commonly used in experimental models involving the visual function. Combining both techniques allows a quantitative in vivo investigation of structure and function, which can be used to investigate the pathological conditions or to evaluate the potential of novel therapeutics. A great benefit of the presented techniques is the possibility to perform longitudinal analyses allowing the investigation of dynamic processes, reducing variability and cuts down the number of animals needed for the experiments. The protocol described aims to provide a manual for acquisition and analysis of high quality retinal scans of mice and rats using a low cost customized holder with an option to deliver inhalational anesthesia. Additionally, the proposed guide is intended as an instructional manual for researchers using optokinetic response (OKR) analysis in rodents, which can be adapted to their specific needs and interests.

A detailed protocol for the assessment of structural and visual readouts in rodents by optical coherence tomography and optokinetic response is presented. The results provide valuable insights for ophthalmologic as well as neurologic research.

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for ...

Full-Field Optical Coherence Microscopy for Histology-Like Analysis of Stromal Features in Corneal Grafts

The quality of donor corneal stroma, which makes up about 90% of total corneal thickness, is likely to be one of the main, if not the major, limiting factor(s) for success of deep anterior lamellar and penetrating keratoplasty. These are surgical procedures that involve replacing part or all of the diseased corneal layers, respectively, by donated tissue, the graft, taken from a recently deceased individual. However, means to evaluate stromal quality of corneal grafts in eye banks are limited and lack the capability of high-resolution quantitative assessment of disease indicators. Full-field optical coherence microscopy (FF-OCM), permitting high-resolution 3D imaging of fresh or fixed ex vivo biological tissue samples, is a non-invasive technique well suited for donor cornea assessment. Here we describe a method for the qualitative and quantitative analysis of corneal stroma using FF-OCM. The protocol has been successfully applied to normal donor corneas and pathological corneal buttons, and can be used to identify healthy and pathologic features on both the macroscopic and microscopic level, thereby facilitating the detection of stromal disorders that could compromise the outcome of keratoplasty. By improving the graft quality control, this protocol has the potential to result in better selection (and rejection) of donor tissues and hence decreased graft failure.

We describe use of full-field optical coherence microscopy as a method for high quality assessment of corneal donor stroma. This protocol can be used to identify features indicative of health or disease, and is aimed at improving the screening and selection of donor tissues, and hence the results of keratoplasty.

The quality of donor corneal stroma, which makes up about 90% of total corneal thickness, is likely to be one of the main, if not the major, limiting ...

Immunology and Infection

Application of Optical Coherence Tomography to a Mouse Model of Retinopathy

Optical coherence tomography (OCT) offers a noninvasive method for the diagnosis of retinopathy. The OCT machine can capture retinal crosssectional images from which the retinal thickness can be calculated. Although OCT is widely used in clinical practice, its application in basic research is not as prevalent, especially in small animals such as mice. Because of the small size of their eyeballs, it is challenging to conduct fundus imaging examinations in mice. Therefore, a specialized retinal imaging system is required to accommodate OCT imaging on small animals. This article demonstrates a small-animal-specific system for OCT examination procedures and a detailed method for image analysis. The results of retinal OCT examination of very-low-density lipoprotein receptor (Vldlr) knockout mice and C57BL/6J mice are presented. The OCT images of C57BL/6J mice showed retinal layers, while those of Vldlr knockout mice showed subretinal neovascularization and retinal thinning. In summary, OCT examination could facilitate the noninvasive detection and measurement of retinopathy in mouse models.

Here, we describe an in vivo imaging technique using optical coherence tomography to facilitate the diagnosis and quantitative measurement of retinopathy in mice.

Optical coherence tomography (OCT) offers a noninvasive method for the diagnosis of retinopathy. The OCT machine can capture retinal crosssectional images ...

Image-Guided Optical Coherence Tomography to Assess Structural Changes in Rodent Retinas

Ocular diseases, such as age-related macular degeneration, glaucoma, retinitis pigmentosa, and uveitis, are always accompanied by retinal structural changes. These diseases affecting the fundus always exhibit typical abnormalities in certain cell types in the retina, including photoreceptor cells, retinal ganglion cells, cells in the retinal blood vessels, and cells in the choroidal vascular cells. Noninvasive, highly efficient, and adaptable imaging techniques are required for both clinical practice and basic research. Image-guided optical coherence tomography (OCT) satisfies these requirements because it combines fundus photography and high-resolution OCT, providing an accurate diagnosis of tiny lesions as well as important changes in the retinal architecture. This study details the procedures of data collection and data analysis for image-guided OCT and demonstrates its application in rodent models of choroidal neovascularization (CNV), optic nerve crush (ONC), light-induced retinal degeneration, and experimental autoimmune uveitis (EAU). This technique helps researchers in the eye field to identify rodent retinal structural changes conveniently, reliably, and tractably.

The protocol presented here details the procedures of data collection and data analysis for image-guided optical coherence tomography (OCT) and demonstrates its application in multiple rodent models of ocular diseases.

Ocular diseases, such as age-related macular degeneration, glaucoma, retinitis pigmentosa, and uveitis, are always accompanied by retinal structural ...

Three-Dimensional Reconstruction of Orbital Fractures

Reconstruction of orbital fractures can be challenging. The limited exposure, involvement of several wall defects, and the variable three-dimensional (3D) anatomy result in difficulty in achieving superior outcomes in complex cases. The use of patient-specific implants for the reconstruction of orbital defects shows great promise. Yet, proper virtual planning is crucial in these cases, and thus, understanding the anatomy and the different options for the planning is essential. This protocol describes three methods for reconstructing the defects and details the indications for each method. Automated computerized reconstruction is the simplest method, yet it can be used mainly for small defects. Repositioning of the fractured segment is simple, resulting in good anatomical continuity of the fractured wall, yet it requires non-comminuted fractures. Mirroring is the method of choice in comminuted fractures. The drawback of this method is the extensive manipulation that follows the mirroring stage, thus requiring a high level of understanding of the anatomy and implications by the planner. Following a detailed description of the methods, the anatomical structures that should be the main focus when reconstructing and those overlooked are demonstrated. In addition, the pitfalls encountered are described and discussed as well as how to avoid them. These methods can be used in-house or outsourced, yet understanding them is crucial to achieving better results, even in cases where the surgeon does not plan himself.

Personalized medicine for orbital reconstruction is developing rapidly. Due to the delicate nature of the orbit, small discrepancies following fracture reconstruction may cause impairment in visual perception. Here, we describe three methods for virtual 3D reconstruction of orbital defects and their indications and potential pitfalls for correct reconstruction.

Reconstruction of orbital fractures can be challenging. The limited exposure, involvement of several wall defects, and the variable three-dimensional (3D) ...

Assessment of Global Ocular Structure Following Spaceflight Using a Micro-Computed Tomography (Micro-CT) Imaging Method

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