Reviving lower body negative pressure as a countermeasure to prevent pathological vascular and ocular changes in microgravity

Mitigation of spaceflight-related pathologies such as spaceflight-associated neuro-ocular syndrome (SANS) and the recently discovered risk of venous thrombosis must happen before deep space exploration can occur. Lower body negative pressure (LBNP) can simulate gravitational stress during spaceflight that is likely to counteract SANS and venous thrombosis, but the ideal dose and method of delivery have yet to be determined. We undertook a review of current LBNP literature and conducted a gap analysis to determine the steps needed to adapt LBNP for in-flight use. We found that to use LBNP in flight, it must be adapted to long time duration/low pressure use that should be compatible with crew activities. A lack of understanding of the etiology of the pathologies that LBNP can counteract hinders the application of LBNP as a countermeasure during spaceflight. Future research should aim at filling the knowledge gaps outlined in this review.


INTRODUCTION
Future human missions beyond low Earth orbit (LEO) require space agencies to revise their operational procedures due to increased challenges in effective ground-space communication, down-sizing of space vehicles, and inability to return quickly to Earth during medical emergencies. As a consequence, human deep space exploration missions will require more crew autonomy as well as robust, reliable, and safe procedures to mitigate unwanted effects of prolonged microgravity (µG) exposure.
During exposure to µG, fluid from the legs moves into the thorax and head, causing redistribution of fluid in the tissues and vessels of the body 1 . The body adapts to this fluid shift by increasing urination which decreases plasma volume; however, increased cardiac output and stroke volume, and decreased mean arterial pressure are still observed, among other effects, indicating the limitations of the body's ability to adapt to µG 2,3 . Cephalad fluid shift is an inevitable consequence of weightlessness and likely associated with many negative health outcomes, some of which have been characterized through ground-based analogs such as posture change, bed rest, water (and dry) immersion, and parabolic flight 4-6 . In particular, effects on cardiac system and orthostatic tolerance have been well explored 1,3,7-9 , but existing countermeasures to mitigate these changes have been found to be largely inadequate, either in terms of time, safety, efficacy, or practicality [10][11][12][13] . Orthostatic tolerance decreases after just days in-flight 14 , yet astronauts will have to function autonomously after landing on the Moon or Mars without a safety medical crew for assistance. Therefore, adequate orthostatic tolerance maintenance in-flight is a key factor for successful and safe planetary surface exploration.
Other pathologies are still being discovered and characterized during long duration LEO missions. Spaceflight-associated neuroocular syndrome (SANS) requires mitigation before planetary missions as SANS poses a significant risk both to the individual crew and the overall mission success 15 . Venous thrombosis was recently identified during a long duration mission 16,17 , and highlighted the risks regarding treating emergent medical conditions inflight. Pathologies related to altered hemodynamics, therefore, also require adequate mitigation strategies before planetary missions in deep space (humanresearchroadmap.nasa.gov).
Most importantly, countermeasures that are able to mitigate these effects of spaceflight need to be developed before long duration spaceflight can occur. Lower body negative pressure (LBNP) is a promising candidate. By simulating gravitational stress during prolonged periods of µG exposure, the body systems that have evolved under 1 G can be maintained and adaptations to µG that are maladaptive upon return to Earth, such as cerebral and ocular restructuring, can be avoided. Pathologies that are specifically linked to fluid shifting in µG are particularly likely to be mitigated by regular exposure to simulated gravitational stress through LBNP. In order of strongest evidence, the possible pathologies that may be mitigated by LBNP are: The aim of this review is therefore to highlight potential benefits of LBNP related to risks associated with long duration spaceflight, to reveal knowledge gaps, and provide research recommendations to fill these knowledge gaps. Considering their operational relevance, the focus of this review will be on SANS, deep vein thrombosis (DVT) and venous pooling, both as its own risk and as a possible causative factor for SANS.

REVIEW METHODOLOGY
A comprehensive narrative review was undertaken to survey the papers relevant to the use of LBNP as a long duration countermeasure for SANS and venous flow pathologies. This methodology was chosen specifically to review the recent changes in the study of SANS and venous flow pathologies within the theoretical context of the applicability of LBNP. Although a full systematic review was outside the scope of this project, survey tools from the Cochrane handbook for systematic reviews (https://training. cochrane.org/handbook) were used to increase the scientific rigor and to standardize the assessment of the included studies.
The search strategy involved keyword searches in PubMed and Google Scholar. Key words included "Lower Body Negative Pressure", "Countermeasures", "Venous Flow", "Thrombosis", "Long Duration Spaceflight", "SANS", "VIIP", "Venous Hemodynamics", and "Cerebral remodeling" combined in Boolean search format. Relevant space actors' repositories were also searched, such as NASA's Technical Reports Server (https://ntrs.nasa.gov/search.jsp? R=20050189209), and ESA's experiment archive (http://eea. spaceflight.esa.int/portal/). Manuscripts were limited to fully accessible peer reviewed papers that were published in or translated into English, which involved studies using human participants or relevant reviews of technology or pathologies.
Distribution of the source country of the included papers is shown in Fig. 1, and the type of studies included in this review are shown in Fig. 2. Further breakdown of included studies is shown in Table 1 below, using the PICOS system.

PATHOLOGIES THAT MAY BE MITIGATED BY LBNP
Venous hemodynamics in µG: what do we currently know? Venous hemodynamics have been found to be particularly sensitive to µG 31 . Fluid shifting to the cephalad region leads to the distension of cardiac chambers, increase in stroke volume, and accumulation of blood in the veins of the head and neck 2,32 . Increase in jugular vein, portal vein, and femoral vein diameter have been noted during spaceflight and µG analogs, which indicates venous pooling in cephalic, splanchnic, and pelvic regions 33 .
Beyond bulk fluid movement, the endothelial effects of venous stasis and pooling during spaceflight must be considered. It is known that the direction of flow and hence the direction of shear stress acting on the endothelium plays a crucial role for arterial wall remodeling processes and arterial thrombogenesis [34][35][36][37] ; however, the role of the direction of venous of flow (reverse flow and stasis) and its potential impact on venous endothelial dysfunction is somewhat understudied. Considering the mechanosensitivity of endothelial cells, it is possible that altered venous flow-mediated shear stress, as a result of altered venous hemodynamics in µG, may also play a role for inflammatory processes preceding venous wall remodeling and thrombogenesis 38 , as seen recently in an International Space Station (ISS) crewmember 17 .
The random discovery of an asymptomatic DVT in the jugular vein during an in-flight science experiment provides many causes for concern 16,17 . So far, there have been no known reports of this pathology in ground-based studies. It is unknown if DVT is not observed in ground-based studies because of lack of imaging/ testing, or if there is a fundamental difference between fluid macro-and micro-circulation during spaceflight that increases the risk of blood clot formation.
SANS: what do we currently know? SANS is characterized by ocular changes and associated changes in the brain. The cardinal finding and current diagnostic criteria is uni-or bilateral optic disc edema of Frisen Grade 1 or more 39 . Additionally flattening of the globe, choroidal and retinal folds, cotton wool spots, and hyperoptic refractive error are often observed in those diagnosed with SANS 40 . Although SANS has only been identified in astronauts after spaceflight and has yet to be convincingly reproduced in a terrestrial analog, it bears some resemblance to terrestrial pathologies such as idiopathic intracranial hypertension (IIH) and normal pressure hydrocephalus (NPH) 39,[41][42][43][44] . However, the exact etiology of SANS remains unknown, which makes terrestrial reproduction for the purposes of studying SANS countermeasures particularly difficult. Multiple hypotheses for the etiology of SANS exist and are explained in great detail in other work 15,39,[45][46][47] . Here we present a summary of the hypothesized etiologies that may be mitigated by the application of LBNP.
The prevailing hypothesis on etiology of SANS is based on a slow but chronic cerebral and ocular overload of fluid and pressure associated with the systemic cephalad fluid shift in weightlessness. This in theory would constitute an increase in intracranial pressure (ICP). During bulk fluid movement, cerebrospinal fluid (CSF) may flow into orbital optic nerve space, and return flow could be impaired as a result of subarachnoid space remodeling, the body's response to this overload of CSF 46 . Reduction of reabsorption of CSF and lymphatic drainage could cause cerebral edema leading to increased ICP 48 .
Alternatively, venous pooling/backflow could be a causative factor for SANS 46 . ICP seems to be governed by pressure in draining veins 49 . Internal jugular vein (IJV) diameter and blood flow decrease when moving from the supine to seated position under 1 G, while vertebral vein blood flow increases to maintain cerebral drainage 16,49 . This indicates that variation in posture (which lost in µG) has significant effects on cerebral drainage and therefore ICP. During spaceflight, the IJV is constantly distended due to the µG-induced headward fluid shift, which may have serious consequences for maintenance of cerebral drainage and ICP 16 . A recent in-flight study of 11 astronauts reported that 55% of the crew had stagnant and retrograde blood flow in their IJV 47 .
Some µG studies have documented that cerebral arterial diameters and blood flow velocity are auto-regulated and do not change significantly during spaceflight 39,50 , but, µG fluid shifts have been documented to cause jugular vein distension, also seen in head down tilt (HDT) 39 . The observed distension of the jugular vein and presumed increased jugular venous pressure during weightlessness could alter Starling forces and favor filtration of fluid into the interstitial space, further increasing ICP 15 .
The difficulty in determining the role of ICP in the complex pathology of SANS and cerebral perfusion and drainage is largely due to the invasive nature of this measurement making it unsuitable for spaceflight 39 . Despite enthusiasm from both the scientific and clinical communities, there are no reliable noninvasive ICP methods available at the time of writing. Invasive measurements have been performed during very short-term weightlessness in parabolic flight 51 , postural manipulation of the gravitational vector 52 , and simulated gravitational stress 21 . Based on this work, it has been hypothesized that ICP is not elevated to pathological levels in weightlessness, but rather stabilizes at levels between that of upright and supine terrestrial levels, thus never reaching normal upright levels to unload the brain 51,52 . Nonpathological but above normal upright levels of ICP without concomitant changes in intraocular pressure (IOP) causes a differential pressure increase across the lamina cribrosa that pushes on the eye, explaining the globe flattening observed in those diagnosed with SANS 15 .
How could LBNP act as a simulation of gravitational stress to mitigate these pathologies? When in an upright position under 1 G, gravity stresses the cardiovascular system by displacing blood and fluid toward the feet. To maintain blood pressure and perfusion of cranial structures, compensatory reflexes are elicited 3 . LBNP simulates the effect of gravitational stress in upright postures, but unlike true gravitational stress, it provokes an abrupt and uniform increase over the body, as opposed to the linear gradient of gravitational stress provided by upright posture in 1 G 53 . Despite these differences, LBNP may be a useful cardiovascular countermeasure; by reversing fluid shift and reducing venous return to the heart, compensatory arterial and cardiopulmonary baroreflexes are activated and maintained 8 .
Given the hypotheses for the etiology of SANS presented above, the use of LBNP to mitigate SANS relies on LBNP's ability to combat cephalad fluid shifting, which has the potential to mitigate increases in ICP, IOP/ICP mismatch, and/or cerebral remodeling. One study found that ICP and internal jugular vein cross-sectional area (IJV CSA) increased during a transition from seated upright to 15°HDT, and LBNP was able to mitigate these increases: LBNP at −25 mmHg reduced ICP during HDT, whereas −50 mmHg reduced ICP and IJV CSA 21 . LBNP at −20 mmHg comfortably induced mechanical loading and desired fluid displacement, and was found to be ideal to lower ICP without impairing cerebral perfusion pressure 21 . Recently, LBNP used during bed rest was shown to reduce choroidal engouement (Lawley et al., 2018, ISGP Conference, Amsterdam, the Netherlands). In 12°HDT with −20 mmHg, LBNP was able to reduce increases in optic nerve sheath diameter, which is linked to ICP and intracranial CSF, which indicates the ability of LBNP to influence CSF 22 .
Taken together, LBNP can unload cerebral structures 21,22,28,51 , has a noticeable effect on cerebral circulation 7,49 and on systemic cardiovascular adaptations and venous flow 2,7 . LBNP at −10 mmHg for 1 h decreased central venous pressure without affecting blood pressure or heart rate 54 , which indicates that it can activate the normal baroreceptors reflexes without causing undue stress on the body. Therefore, LBNP may maintain habitual endothelial shear stress 36 , counteracting endothelial dysfunction and venous pooling, and may also reduce plasma loss 18 , and thus mitigate orthostatic intolerance. LBNP was shown to increase blood flow in ten of 17 sessions during a recent ISS study, demonstrating its ability to alter hemodynamics during spaceflight 16 .

HOW COULD LBNP BE APPLIED SAFELY AND EFFECTIVELY IN µG?
Feasibility of LBNP for use in space LBNP has been in use by the American and Russian space agencies since the Shuttle and MIR eras, but the difficulty of application of LBNP, in terms of the safety risk of syncope in space when applying high pressures for short durations, and the inefficiency in regard to time usage when applying low pressures for long periods of time, coupled with an unknown dose response led to it falling out of use on the American and European sides 55 . However, the Russian cosmonauts continue to use a version of LBNP, the Chibis suit, to this day on the ISS. Other partners on the ISS use the Chibis infrastructure purely for research, as was done during the Fluid Shifts Experiment, but not as a countermeasure 12,18,56 .
To assuage safety concerns and to make LBNP more user friendly, lower pressures should be used for countermeasure purposes. As described above in Section "How could LBNP act as a simulation of gravitational stress to mitigate these pathologies?", lower pressures (i.e. −10 mmHg to −20 mmHg) are seemingly most effective at mitigating the pathologies associated with loss of orthostatic tolerance and SANS. While higher pressures have been tested and occasionally found to be effective, the risk of syncope in space and loss of cerebral perfusion make high pressure applications both risky and impractical, as astronauts would need to be supervised under these higher pressures to monitor unwanted effects, which effectively negates the time benefit of high intensity/low duration application.
Recently, Dr. Petersen and team supported by NASA, have developed a mobile, wearable LBNP suit 28 . Further development of this effort will potentially facilitate LBNP as a feasible  countermeasure for long duration spaceflight as it does not interfere with daily activities of the crewmembers 28 .
What is the minimal effective dose of LBNP? LBNP research in the early days of spaceflight went down to −100 mmHg 55 , which was likely to be both unnecessary and dangerous, and has likely contributed to the technique being discontinued throughout the ISS era. Recent research shows that smaller magnitudes are effective, though the exact minimal effective dose for each identified fluid-shiftrelated pathology is unknown and likely to differ in what times and pressures are required 21,28 . Given the safety risk associated with bulk fluid redistribution in space, to convince flight surgeons and the crew that LBNP is effective and safe as a countermeasure, there should be a significant safety margin in how much pressure can be applied, and applied pressures and exposure times should be well below levels known to cause (pre-) syncope. High pressures are more likely to have the negative effects of syncope and reduced cerebral perfusion pressure, therefore instead of moving toward a high pressure, small time duration, research has trended in the long applications of low pressures.

RESEARCH GAPS AND RECOMMENDATIONS FOR FUTURE RESEARCH SANS research gaps and recommendations
Over the course of this research, we identified series of knowledge gaps related to SANS that are listed below: 1. There is a lack of knowledge regarding the etiological mechanism and contributing risk factors for the ocular and cerebral changes associated with SANS. Testing sooner after landing, and using longer term follow-up studies to clarify reversibility may help reveal this etiology 39 . 2. There is a lack of appropriate diagnostic tools to measure and monitor changes in relevant parameters in a noninvasive manner. This is especially relevant for ICP, which requires an invasive port for measurements. Although there is currently some indication that in-flight invasive ICP will happen, and pre-and post-flight invasive ICP is already being done (humanresearchroadmap.nasa.gov), invasive investigations of ICP are unlikely to work in a long duration spaceflight scenario due to the high risk of adverse events, and the level of training and expertise needed to execute the gold standard procedures. Unfortunately, non-invasive ICP measurement tools are severely lacking 57 . 3. The reliability of ground-based analogs for simulating SANS etiology is unknown. There is some recent evidence that SANS can be simulated on Earth, as optic disc edema developed in 45% of people after 30 days of strict 6°HDT in 0.5% CO 2 (ref. 58 ). However, this prevalence is higher than the prevalence of~15% of astronauts, indicating a different underlying pathology or that HDT exaggerates fluid shift 47 . 4. There is a lack of countermeasures to prevent SANS or appropriate treatments to mitigate/reverse the changes observed in-and post-flight.
Therefore, the key recommendations for investigating SANS and effectively mitigating it are: 1. Due to the lack of widespread use of LBNP as an active countermeasure, the dose needed to treat is unknown and the safety protocols are yet to be validated. 2. It is currently unknown how LBNP affects venous endothelial shear stress, and without understanding the mechanism at hand it is impossible to know how LBNP may effectively mitigate the potentially negative effects of altered venous flow in µG. 3. It is unknown whether maintaining 1 G gravitational stress is favorable during long duration spaceflight, as opposed to letting adaptive changes occur, especially for partial gravity (ie. lunar, Martian). 4. There is a lack of understanding surrounding the mechanisms of bulk fluid movement during the use of LBNP.
Therefore, the recommendations for the study of LBNP as a countermeasure are as follows: 1. Continue to discern the dose needed to treat each pathology, i.e. SANS, orthostatic intolerance, alterations in venous hemodynamics. It is likely that different pathologies may need different protocols to be mitigated fully, due to the variety of mechanisms at work in each pathology. Although research such as the mobile LBNP suit is beginning to shed some light on these issues, they must be fully investigated before commencing in-flight use 28 . 2. More work needs to be done on LBNP in ground-based studies for SANS and venous thrombosis. Venous hemodynamics need to be studied in-depth in space and in spaceflight analogs to better characterize and quantify venous wall shear stress, endothelial dysfunction, early venous wall remodeling mechanisms, and circulating parameters to assess blood clotting, such as D-Dimer. To date, studies are finding favorable outcomes for the use of various countermeasures without fully understanding the etiology of why those countermeasures are beneficial. For example, LBNP and negative pressure respiration was found to be more favorable than just LBNP, but the exact mechanism for this remains largely unclear 60 . 3. Research into operational impacts in µG are necessary, especially for LBNP protocol development, as is predictive adaptation for various levels of gravity (i.e. lunar, Martian). 4. Microvascular responses show that LBNP redistributes fluids differently from standing under 1 G, therefore there is a critical need for well-developed modeling to understand the bulk fluid shift 53,61 .
CONCLUSION SANS and altered venous hemodynamics pose major threats to the health of astronauts during long duration spaceflight, and both pathologies are intricately linked to the lack of gravitational stress. LBNP may be a way to prevent negative outcomes during long duration spaceflight, by recreating or at least simulating to a reasonable level gravitational stress during spaceflight. However, to know how effective LBNP or other countermeasures are, significant research into the mechanism behind these pathologies, including an understanding of bulk fluid shift under µG, monitoring capabilities that are appropriate for spaceflight, and validation of ground-based analogs for modeling these pathologies are needed. Further research is needed into the cause of SANS and the extent of venous thrombosis risk before the full mitigative capacity of LBNP can be defined, and more robust work needs to be done on the implications of combining LBNP with other countermeasures.

DATA AVAILABILITY
No datasets or codes were generated as a result of this research.