1: Evaluate the effects of microgravity on ocular artery structure/function. • In vivo Measurements (anesthetized mice): • VisualSonics Vevo 2100 ultrasound system was used for blood velocity measurement. The central retinal artery was located by color Doppler. • A TonoLab tonometer was used to measure intraocular pressure (IOP). • The Heidelberg Spectralis HRA + OCT system was used to acquire fundus and cross-sectional images of the mouse retina. • In vitro Functional Studies of the Ophthalmic Artery: • Isolated ophthalmic arteries were cannulated with glass micropipettes and pressurized to 75 cmH2O. Vessel diameter changes/contractile status to acetylcholine, sodium nitroprusside, and endothelin-1 were monitored and recorded.

2: Evaluate the effects of microgravity on ocular vein structure/function. • In vitro Functional Studies of the Angular Vein: • The mouse angular vein, which drains the supratrochlear and supraorbital vein, was isolated and mounted in a wire myograph. • Assessment of Retinal Microvascular Barrier Function: • Intravital microscopy (multiphoton laser scanning microscope) was used to acquire real-time images of FITC-dextran extravasation in anesthetized mice to measure microvascular permeability in the mouse retina. • Mice were injected with FITC-dextran (10 kDa) via the jugular vein, followed by intravital imaging of the retinal microcirculation every 5 min for 40 min. Retinal microvascular permeability/barrier integrity was measured based on extravasation fluorescence intensity of FITC-dextran.

3: Evaluate the effects of microgravity on ocular lymphatic structure/function. • Assessment of Ocular Lymphatic Vessel Function: • Isolated ocular collecting lymphatics were cannulated with glass micropipettes and pressurized from 0.5 to 4 cmH2O. Diameter changes were tracked and recorded to measure phasic and tonic contractile/pump function. • Assessment of Ocular Lymphatic Tissue Structure: • Perilymphatic tissue was collected for immunohistochemical analysis of local immune cells (mast cells, macrophages, dendritic cells, and lymphocytes) and for multiplex analysis of cytokines.

In addition to conducting experiments to address these primary missions we also0used a team of scientists to conduct the dissection and specimen storage for NASA's Biospecimen Sharing Program, since this occurred during the COVID pandemic and NASA did not allow their normal BSP team top travel. As a result of our BSP efforts we were granted permission to acquire some additional tissues for study.

A brief summary of the results from these novel studies (as seen below) provided the first comprehensive analysis of the effects of microgravity on ocular vascular function where the predominant changes associated with SANS in astronauts occur. These results will help define the roles the ocular vascular function may play in the etiology of SANS and could lead to the development of countermeasures for SANS.

• Aim 1: Effects of microgravity on ocular artery structure/function • Central retinal artery blood flow velocity was lower in the flight mice. • Trends towards higher IOP in the flight mice. • Trends towards increased total retinal thickness in the flight mice. • Ophthalmic arteries from flight mice exhibited normal vasodilator function but diminished basal vessel tone and endothelin-1-induced vasoconstriction.

• Aim 2: Effects of microgravity on ocular vein structure/function • Increase in retinal microvascular permeability. • The trends observed in isolated angular veins were attenuated vasoconstriction and vasodilation to adrenergic stimulus and exogenous nitric oxide donors, respectively.

• Aim 3: Effects of microgravity on ocular lymphatic structure/function • There was a large decrease (~40%) in ocular lymphatics from flight that had pumping activity. • Trends towards decreased lymph pump strength and increased pump frequency in those that exhibited intrinsic pumping activity. • Complete loss of shear-dependent dilation and trends towards impaired shear-dependent reductions in pumping

Specific Aim 1: Evaluate the effects of microgravity on ocular artery structure/function. Aim 1A: We completed the in situ studies by performing intraocular pressure (IOP) measurements, optical coherence tomography (OCT) /retinal fundus imaging, and Doppler ultrasound measurements in the flight, habitat ground control (HGC), and vivarium ground control (VGC) cohorts of mice. We found that central retinal artery blood flow velocity was lower in the flight mice compared to the HGC and VGC mice. IOP was about 20% higher in the spaceflight mice than in the VGC mice. Data analysis of the OCT images showed trends toward increased total retinal thickness in the flight mice. Aim 1B: We completed the in vitro studies for assessment of vasomotor function of isolated and pressurized ophthalmic arteries in all 3 cohorts of mice. We found that the dilations of isolated ophthalmic arteries to endothelium-dependent agonist acetylcholine and endothelium-independent agent sodium nitroprusside were not significantly different among cohorts. By contrast, basal tone and constriction to endothelin-1 were lower in ophthalmic arteries from flight mice. We collected the eyes and plasma from all 3 cohorts of mice and attempted to perform assays to determine the cytokine/inflammatory protein levels in these samples.

Specific Aim 2: Evaluate the effects of microgravity on ocular vein structure/function. Aim 2A: Following the mission to the International Space Station (ISS), vasomotor responses were measured in isolated angular veins from nine C57BL/6J mice using wire myography. The angular vein was mounted on the jaw of a Danish Myotech wire myograph on 15µm gold-plated tungsten wire. Following a normalization procedure, veins were exposed to agonists that allowed for the examination of endothelial cell and smooth muscle cell function. Vivarium controls – consisting of mice of the same cohort housed in conventional vivarium cages; and habitat controls – consisting of mice housed in specially designed habitats exposed to similar CO2 levels, temperatures, etc., as on the ISS; were used for comparison. Preliminary analysis demonstrates that vasoconstriction to the adrenergic receptor agonist was reduced following spaceflight as compared to vivarium controls, with ground controls demonstrating an intermediate response. Vasodilation to an exogenous nitric oxide donor was reduced following spaceflight as compared to both vivarium and ground controls. These data suggest that angular vein function is altered following exposure to the space environment. Aim 2B: We performed experiments to assess the function of ocular veins after 37 days of spaceflight (RR23) in male mice. Retinal microvascular permeability was assessed in vivarium, ground control, and space mice. The mice were anesthetized with isoflurane and injected (iv) with FITC-dextran-10kDa as an indicator of vascular permeability. This was followed by the imaging of the retinal venules for FITC-dextran extravasation under a multi-photon microscope. We observed significant increases in venular permeability in the flight group compared to the vivarium and ground control groups after FITC-dextran injection. The ocular tissue from another set of mice from each group were collected and processed for immunohistochemistry and molecular biology studies. The work demonstrated trends towards reductions in the expression of the blood-retinal barrier (BRB) tight junction proteins (zonula occludens-1, claudin-5, and occludin).

Specific Aim 3: Aim 3A: Evaluate the effects of microgravity on isolated ocular lymphatic structure/function. ~40% of the ocular lymphatics in the flight mice had no phasic pumping activity, whereas ~80% of all of the control groups did. There was no significant change in the resting tone of the lymphatics in flight versus controls, but there was a complete loss of the shear-induced inhibition of tone seen in normal/control ocular lymphatics. There were trends toward: decreased pump amplitude, increased lymph pump contraction frequency, and impaired flow/shear-dependent impact on the lymph pump amplitude and flow in flight versus controls. Aim 3B: In a subset of the mice (n= 5 side per group) we performed ocular lymph flow tracer studies by injecting trace amounts of a large molecular weight dextran into the tissues of both eyes (left and right) associated with the primary draining ocular lymphatics followed by acquisition and tracer analysis of both of the superficial cervical lymph node 15 minutes after injection to get an assessment of lymph transport in the 3 cohort groups. Ocular lymph transport was greatly decreased (> 300%) in the flight group compared to either control or the lumped controls (that were not statistically different from each other). This direct measure of ocular lymph transport shows significant declines in ocular lymph flow from the flight mice compared to both control groups and provides a direct correlation to the impaired lymphatic pump function observed in isolated ocular lymphatics from the flight group.

1: Evaluate the effects of microgravity on ocular artery structure/function.

2: Evaluate the effects of microgravity on ocular vein structure/function.

3: Evaluate the effects of microgravity on ocular lymphatic structure/function.

Information from these novel studies will provide the first comprehensive analysis of the effects of microgravity on ocular vascular function where the predominant changes associated with SANS in astronauts occur. It will also help define the roles these may play in the etiology of SANS and could lead to the development of countermeasures for SANS.

Spaceflight Associated Neuro-ocular Syndrome (SANS) develops in astronauts completing long-duration spaceflights and is diagnosed based on one or more findings: optic disc edema, hyperopic shifts, globe flattening, or choroidal folds. Prolonged exposure to the headward fluid shift that occurs in weightlessness is regarded as the primary instigating factor for ocular changes, but the pathophysiology of SANS remains unclear. Also, there has been no systematic analysis of the ocular vascular changes in microgravity. The central hypothesis of this project is that microgravity/spaceflight-induced changes in the structure/function of the ocular vasculature, at the level of arteries, veins, and lymphatics, lead to alterations in ocular hydrodynamics and promote signs of SANS.

Specific Aim 1: Evaluate the effects of microgravity on ocular artery structure/function. Aim 1A: We completed the in situ studies by performing intraocular pressure (IOP) measurements, optical coherence tomography (OCT) / retinal fundus imaging and Doppler ultrasound measurements in the flight, habitat ground control (HGC) and vivarium ground control (VGC) cohorts of mice. We found that central retinal artery blood flow velocity was lower in the flight mice compared to the HGC and VGC mice. IOP was about 20% higher in the spaceflight mice than in the VGC mice. Data analysis of the OCT images is currently ongoing. Aim 1B: We completed the in vitro studies for assessment of vasomotor function of isolated and pressurized ophthalmic arteries in all 3 cohorts of mice. We found that the dilations of isolated ophthalmic arteries to endothelium-dependent agonist acetylcholine and endothelium-independent agent sodium nitroprusside were not different among cohorts. By contrast, constriction to endothelin-1 was lower in ophthalmic arteries from flight mice. We collected the eyes and plasma from all 3 cohorts of mice and will perform assays to determine the water content/edema (wet-dry ratio) and cytokine/inflammatory protein levels in these samples.

Specific Aim 2: Evaluate the effects of microgravity on ocular vein structure/function. Aim 2A: Following the mission to the International Space Station (ISS), vasomotor responses were observed in isolated angular veins from nine C57BL/6J mice using wire myography. The angular vein was mounted on the jaw of a Danish Myotech wire myograph on 15µm gold-plated tungsten wire. Following a normalization procedure, veins were exposed to agonists that allowed for the examination of endothelial cell and smooth muscle cell function. Vivarium controls – consisting of mice of the same cohort housed in conventional vivarium cages; and habitat controls – consisting of mice housed in specially designed habitats exposed to similar CO2 levels, temperatures, etc., as on the ISS; were used for comparison. Preliminary analysis demonstrates that vasoconstriction to the adrenergic receptor agonist was reduced following spaceflight as compared to vivarium controls, with ground controls demonstrating an intermediate response. Vasodilation to an exogenous nitric oxide donor was reduced following spaceflight as compared to both vivarium and ground controls. These data suggest that angular vein function is altered following exposure to the space environment. Aim 2B: We performed experiments to assess the function of ocular veins after 39 days of spaceflight (RR23) in male mice. Retinal microvascular permeability was assessed in vivarium, ground control, and space mice. The mice were anesthetized with isoflurane and injected (iv) with FITC-dextran-10kDa as an indicator of vascular permeability. This was followed by the imaging of the retinal venules for FITC-dextran extravasation under a multi-photon microscope. We observed significant increase in vascular permeability in the flight group compared to the vivarium group after FITC-dextran injection. We also observed an increase in permeability in the flight group compared to the ground control group that was not significant statistically. The ocular tissue from another set of mice from each group were collected and processed for immunohistochemistry and molecular biology studies. The work is currently in progress for evaluating the changes in localization and expression of blood-retinal barrier (BRB) tight junction proteins (zonula occludens-1, claudin-5, and occludin).

Specific Aim 3: Evaluate the effects of microgravity on ocular lymphatic structure/function. ~40% of the ocular lymphatics in the flight mice had no phasic pumping activity, whereas ~80% of all of the control groups did.

There was no significant change in the resting tone of the lymphatics in flight versus controls, but there was a complete loss of the shear-induced inhibition of tone seen in normal/controls.

There were trends towards: increased lymph pump contractions frequency, decreased pump amplitude, and impaired flow/shear-dependent impact on the lymph pump amplitude and flow in flight versus controls.

1: Evaluate the effects of microgravity on ocular artery structure/function.

2: Evaluate the effects of microgravity on ocular vein structure/function.

3: Evaluate the effects of microgravity on ocular lymphatic structure/function.

Information from these novel studies will provide the first comprehensive analysis of the effects of microgravity on ocular vascular function where the predominant changes associated with SANS in astronauts occur. It will also help define the roles these may play in the etiology of SANS and could lead to the development of countermeasures for SANS.