Reduced regional cerebral blood ow measured by 99mTc-HMPAO SPECT in microgravity simulated by 5-day dry immersion

Purpose Neuro-ophthalmological changes dened as spaceight-associated neuro-ocular syndrome have been reported after long duration space ights. The pathophysiology of this syndrome remains unclear, with the possible involvement of elevated intracranial pressure. Changes in blood ow in the brain, evaluated indirectly by Doppler, have been reported in ight. However, the effects of microgravity on regional cerebral blood ow (rCBF) are not known. We therefore investigated changes in rCBF in a 5-day dry immersion (DI) model. Moreover, we tested thigh cuffs as a countermeasure to prevent potential microgravity-induced modications in rCBF. Methods 18 healthy male participants underwent 5-day DI with or without a thigh cuffs countermeasure. They were randomly allocated to a control (n = 9) or cuffs (n = 9) group. rCBF was measured 4 days before DI (Pre-DI) and at the end of the fth day of DI (Post-DI), using single-photon emission computed tomography (SPECT) with radiopharmaceutical 99m Tc-hexamethyl propylene amine oxime (HMPAO). SPECT images were processed using statistical parametric mapping (SPM12) software. Results At DI5, we observed a signicant decrease in rCBF in 32 cortical and subcortical patterns, with greater hypoperfusion in the occipital region (occipital peak level: z = 4.51, p uncorr < 0.001) and basal ganglia (putamen peak level: z = 4.71, p uncorr < 0.001; caudate nuclei peak level: z = 3.80, p uncorr < 0.001). No signicant difference was found between the control and cuffs groups on variations in rCBF at DI5. Conclusion 5-day DI induces a relative decrease in rCBF in cortical and subcortical regions. Nevertheless, the consequences of this decrease for brain function and mechanisms need further investigation.


Introduction
Exposure to microgravity has detrimental effects on human physiology, such as muscle atrophy, bone demineralization, sensorimotor and cardiovascular deconditioning, and immune, hormonal and metabolic changes [1,2]. Body uid redistribution begins in the rst hours of space ight. This so-called cephalad uid shift is responsible for cephalic venous stasis, characterized by dilation of the jugular vein and facial oedema. This phenomenon is mainly due to loss of the cranial-to-caudal ow gradient induced by weightlessness [3,4]. Neuro-ocular symptoms have been observed in astronauts on their return from longduration space ights, such as hyperopia, papillary oedema, choroidal folds, cotton wool spots, and posterior globe attening. These symptoms were recently de ned as space ight associated neuro-ocular syndrome [5], and some have also been reported in intracranial idiopathic hypertension [6]. During longduration space ights, the cephalad uid shift observed in astronauts may increase intracranial pressure (ICP), as suggested by the assessment of optic nerve sheath diameter (ONSD) by ultrasound and MRI [7,8]. However, these mechanisms are not fully understood.
Indirect assessment of cerebral blood ow (CBF) by transcranial Doppler ultrasound of the middle cerebral artery, has revealed a decrease in cerebral vascular resistance (CVR) and an increase in CBF during the rst days of space ight, after which these parameters normalize [3]. Cerebral autoregulation is the mechanism that maintains CBF relatively constant, despite variations in cerebral perfusion pressure (CPP). Short-term studies have shown that cerebral autoregulation is preserved or even improved in microgravity, whereas long-term studies have found that it is impaired [9]. Nevertheless, the mechanisms behind modi cations in CBF, CVR and cerebral autoregulation after exposure to weightlessness have not yet been clearly elucidated. Few studies have measured regional (r) CBF in humans after exposure to simulated microgravity. In a study in head-down bed rest (HDBR), rCBF measured by 133 Xe inhalation method had increased at 6 hours, but returned to the baseline state at 72 hours [10]. No study has so far measured rCBF during both space ight and microgravity analogues such as dry immersion (DI).
The aim of the present study was to investigate possible changes in rCBF using DI as a microgravity simulation model. A second objective was to test whether thigh cuffs can serve as a countermeasure, limiting any changes in rCBF, by restricting the cephalad uid shift and potential increase in ICP.

Participants
Twenty healthy men were recruited. Two of them withdrew before the 4 days of baseline data collection for reasons unrelated to the protocol. A total of 18 participants were therefore included in the study and randomly allocated to either a control or a cuffs group (9/9 split). All participants were informed about the experimental procedures and gave their written consent. The experimental protocol was conducted in accordance with the standards set by the Declaration of Helsinki and approved by the local ethics committee (CPP Est III: 2 October 2018, no. ID RCB 2018-A01470-55) and French health authorities (ANSM: 13 August 2018). ClinicalTrials.gov identi er: NCT03915457.

General protocol
The present study was part of DI5-CUFFS, an experiment carried out at the MEDES Space Clinic in Toulouse (France) from 19/11/2018 to 23/03/2019. The experimental protocol consisted of 4 days of ambulatory baseline data collection before DI (Pre-DI), 5 days (120 hours) of DI (DI1 to DI5), and 2 days of ambulatory recovery (post DI).
A week before the beginning of the protocol, participants went to MEDES for a Pre-DI thigh muscle biopsy and resting metabolic rate measurement.
Participants randomized to the cuffs group wore the thigh cuffs throughout the 5 days of DI, from 10 am to 6 pm on DI1, and from 8 am to 6 pm on DI2-DI5. Thigh cuffs are elastic strips that are designed to have the same effects on lower-limb distensibility as a counterpressure of about 30 mmHg [11] (Fig. 1). Calf plethysmography, performed in the supine position at Pre-DI, was undertaken to adjust the cuffs to each participant. At DI1, thigh cuffs were put on immediately prior to DI onset at 10 am.
The general protocol for DI was implemented according to the methodology described elsewhere [12] (Fig. 2). Two participants, one control and one cuffs, underwent DI simultaneously in the same room, in two separate baths (except for two participants, one cuffs and one control, who were each alone in the room). Thermoneutral water temperature was continuously maintained (32.5-33.5 °C). Lights were switched off from 11 pm to 7 am. Daily hygiene, weighing and some speci c measurements required exit from the bath. During these out-of-bath periods, participants maintained the -6° head-down position. Total out-of-bath supine time for the 120 h of immersion was 9.7 ± 1.3 hr. On DI1-DI4, out-of-bath time was 1.1 ± 0.6 hr/day. On DI5, out-of-bath time was 5.3 ± 1.1 hr, owing to a muscle biopsy in the right thigh and encephalic and spinal MRI. Otherwise, during DI, participants remained immersed in a half-seated position for all activities and were continuously subjected to video monitoring. Bodyweight, blood pressure, heart rate and tympanic body temperature were measured daily. Water intake was xed at 35-60 ml/kg/day. Within these limits, water intake throughout the protocol was ad libitum and quanti ed. The menu for each experimental day was identical for all participants, and dietary intake was individually tailored and controlled during the study. Measurements of heart rate and arterial blood pressure were performed with an automatic device twice a day (morning and evening).
SPECT acquisitions 99m Tc-hexamethyl propylene amine oxime ( 99m Tc-HMPAO) is a lipophilic radiopharmaceutical used for measuring rCBF. The radio-labelled compound was prepared from a commercial kit (Cerestab TM ; GE Healthcare, Norway), mixed with sodium-( 99m Tc)-pertechnetate and diluted in a saline solution (0.9% sodium chloride). Four days before DI (Pre-DI), 261 ± 8 MBq of 99m Tc-HMPAO were intravenously administered, within 3 hours of preparation. Before and after the injection, participants were isolated from sensory stimulations. More speci cally, for 10 minutes before and after the injection, they lay in a dark and quiet room, wearing earplugs and a sleep mask. The 99m Tc-HMPAO injection at Pre-DI was conducted in a half-seated position, so that participants were in a similar position to that at Post-DI when, just before the end of DI, 263 ± 10 MBq were injected while participants were immersed in the bath. Both injections took place in the morning.
SPECT-CT acquisitions were performed on a dual-head hybrid camera (SymbiaT6; Siemens Healthcare, Erlangen, Germany) equipped with a low-energy high-resolution collimator. The energy window was 140 keV ± 7.5% (with additional low energy window for scatter correction). Acquisition parameters for SPECT were: 60 projections over 180°, with 30 seconds per projection (matrix: 128 × 128, zoom 1.78). To perform attenuation correction, a brain CT was also acquired with the following parameters: 110 kV, 50 mAs, collimation 6 × 2 mm. Iterative reconstruction was performed with a ash3D algorithm (12 iterations, 8 subsets, 8-mm Gaussian lter). Images with scatter and CT-attenuation corrections were then generated.
Any decrease in radioactivity was corrected during analysis with statistical parametric mapping (SPM12) software, by applying a weighting factor depending on the radioactivity period of 99m Tc for each acquisition.
Statistical analysis SPECT images were processed using SPM12 software [13], implemented in MATLAB (MathWorks, Sherborn, MA). SPM (statistical parametric mapping) combines the general linear model and theoretical Gaussian elds to make statistical inferences about regional effects. All SPECT images were realigned and normalized to a standard template in MNI space (Montreal neurological institute) using SPM12 [14], then smoothed with a Gaussian kernel lter of 8 mm at full width and half maximum. We compared rCBF at Pre-DI and at Post-DI for each group using a paired t test. We also compared the variations in rCBF during DI between the cuffs and control groups, using a two-sample t test. We tested the null hypothesis that the voxel to voxel contrast is zero. We chose an uncorrected threshold p uncorr < 0.001 with an extended threshold of 100 voxels. General haemodynamic parameters (heart rate, systolic, diastolic and mean arterial blood pressure) were expressed as mean ± SD. A paired Student t test was used for comparisons of data between Pre-DI and Post-DI for each group. An unpaired Student t test was performed to compare data between groups (cuffs vs. control). Differences were considered statistically signi cant when p < 0.05.

Results
Baseline group characteristics are detailed in Table 1. There were no signi cant differences between the two groups (cuffs and control) at baseline. rCBF was signi cantly reduced in cortical and subcortical regions at Post-DI, compared with Pre-DI, with a signi cance threshold of p uncorr < 0.001 and an extended threshold of 100 voxels. 32 cortical and subcortical patterns that were signi cantly less perfused at Post-DI than at Pre-DI were individualized, the decrease in rCBF being greater in bilateral occipital regions (occipital peak level: z = 4.51, p uncorr < 0.001) and basal ganglia (putamen peak level: z = 4.71, p uncorr < 0.001; caudate nuclei peak level: z = 3.80, p uncorr < 0.001) ( Table 2, Fig. 3).
There was no signi cant difference in the variation in rCBF at Post-DI compared with Pre-DI between the cuffs and control groups (p uncorr < 0.001 and extended threshold of 100 voxels).
At post-DI, two participants presented a frank hypersignal on the SPECT images, located in the left thalamus. This hypersignal was not present in the images at pre-DI. Analysis of the images of the 18 participants using SPM12 software did not reveal any increase in thalamic blood ow at post-DI compared with pre-DI, for p uncorr < 0.001 and an extended threshold of 100 voxels (Fig. 4).
There was no signi cant difference in blood pressure and heart rate between the cuffs and control groups at Post-DI compared with Pre-DI. Moreover, there was no signi cant variation in these parameters between the measurements made at Pre-DI and at Post-DI for each group (Table 3).

Discussion
After 5 days of DI, we observed a signi cant decrease in rCBF in 32 cortical and subcortical patterns. No previous study had previously measured rCBF in humans after microgravity simulation by DI. A study in HDBR measuring rCBF with the 133 Xe inhalation method found an initial increase at 6 hours, but no difference at 72 hours [10]. Some studies measuring rCBF have been performed in animals. In a 2-week head-down tail suspension study performed in rats, Wilkerson et al. demonstrated a decrease in rCBF in 21 cortical and subcortical patterns, measured with [ 14 C]-IPIA autoradiography, the decrease being more intense in the basal ganglia [15]. In our study, we found a greater alteration in CBF in the basal ganglia and occipital cortex. The basal ganglia interact with the cortex in a system of cortico-subcortical loops, in order to integrate cortical information and relay it to the cortex via the thalamus and brainstem [16]. As they form the hub of information processing in the brain, these regions may be more intensely affected by variations in CBF. Another explanation concerns the potential modi cation in neurotransmitter metabolism. Until now, to the best of our knowledge, there has not been any research on neurotransmitter metabolism in humans in microgravity. In a study with rats, a change in the binding of neurotransmitters to their receptors was noted after 7 days on board Spacelab 3; 5-HT1 receptors were more numerous, and binding of dopamine D-2 in the striatum was decreased [17]. We measured an alteration in rCBF in some regions involved in the control of movements and equilibrium, in sensorimotor, vegetative, cognitive, and limbic functions. These functions have also been found to be impaired during microgravity exposure [18][19][20][21]. However, because of the semi-quantitative measurement of rCBF with 99m Tc-HMPAO, it is not possible to determine whether the decrease in rCBF was su cient to induce or be a consequence of brain function impairments. Further studies are needed to explore modi cations in CBF and their relation to these impairments.
As the cranial box is rigid and inextensible, and intracranial content is not compressible, ICP depends on three parameters: craniospinal elastance, resistance to cerebrospinal uid ow, and brain blood volume [22]. Although ICP has never been directly measured during long exposure to microgravity in humans, indirect evaluation methods (measurement of ONSD) suggest an increase in ICP favoured by the cephalad uid shift [8,23]. However, the magnitude of a possible increase in ICP during space ights and its precise underlying mechanisms remain unclear.
CPP is the result of mean arterial pressure (MAP) and ICP, according to the equation CPP = MAP -ICP [24]. Studies have shown that MAP does not seem to vary signi cantly in studies in HDBR [3] and after 3-day DI [25], consistent with our nding that blood pressure remained unchanged.
According to Poiseuille's law, CVR depends on cerebral vessel diameter. CBF depends on CPP and CVR, according to the equation CBF = CPP / CVR [22]. Cerebral autoregulation is the process of maintaining CBF relatively constant for CPP ranging from 50 to 150 mmHg. Above these limits, CBF varies proportionally to CPP [22]. Cerebral autoregulation is mainly mediated by small arteries that modify their diameter according to the variation in CPP, in order to maintain constant CBF [26]. There is no direct measurement of ICP in microgravity in humans. However, direct measurements performed in animals [27] and indirect measurements in humans [8,28]  4.64 ± 0.40 mm; DI3: 6.01 ± 0.49 mm; p < 0.001) [28]. These ONSD values are equivalent to an elevation of ICP around 20 mmHg, the normal range being between 7 and 15 mmHg [29]. It therefore seems unlikely that a moderate elevation in ICP during DI would exceed the adjustment capacities of CPP. Indeed, cerebral autoregulation has been shown to be preserved or even improved in short-term studies [9]. Nevertheless, according studies in rats, an increase in ICP may increase CVR through compression of the cerebral blood vessels [30].
During HDBR studies, Doppler measurements show an increase in CVR and a decrease in CBF during the rst week, after which these parameters returned to baseline values [3,31,32]. After 3-day DI, Ogoh et al.
failed to observe any variation in CBF as measured by Doppler ultrasound. However, they did observe an increase in CVR [25]. Compared with the literature, our results showing a decrease in CBF after 5-day DI are consistent with the increase in CVR measured during the rst week in simulated microgravity. According to studies performed in head-down tail suspension (HDT) in rats, the increase in CVR could be a consequence of prolonged vasoconstriction in the rst days, in response to the increased blood ow in the brain, due to the HDT position. After several days, the chronic vasoconstriction induces hypertrophy and modi cations in the wall of cerebral arteries [30]. Researchers have found hypertrophy in the media layer, an increase in thickness, an increase in spontaneous tone, and myogenic vasoconstriction of brain arteries mediated by altered secretion of endothelial NO [15,33,34]. According to the authors, the prolonged vasoconstriction and these histological changes could be responsible for an increase in CVR, thus contributing to the decrease in CBF [15]. During DI5-CUFFS, Robin et al. observed a decrease in plasma volume across the 5 days [35]. Likewise, during 3-day DI, Ogoh et al. demonstrated a correlation between the decrease in plasma volume and the decrease in blood ow velocity and conductance in the internal carotid artery [25], suggesting that the loss of plasma volume also contributes to the vasoconstriction of cerebral arteries. In our study, we hypothesized that the decrease in rCBF after 5-day DI is the consequence of three mechanisms that all contribute to the increase in CVR: vasoconstriction of cerebral arteries in response to increased CBF induced by the cephalad uid shift; the decrease in plasma volume; and a moderate increase in ICP, which may contribute to the increase in CVR through compression of cerebral blood vessels.
We did not nd any signi cant variation in rCBF after 5-day DI between the cuffs and control groups. We hypothesized that, by limiting the cephalad uid shift and its consequences, thigh cuffs limit the increase in CVR. During 5-day DI, Arbeille et al. [36] found a signi cantly attenuated increase in volume in the right jugular vein (measured with ultrasound) at 2 hours post-immersion in the cuffs groups. However, at DI4, there was no longer any signi cant difference between the control and cuffs group. Moreover, the right jugular vein was less dilated than it had been 2 hours post-immersion. Therefore, thigh cuffs seem to be effective in limiting the dilatation of the jugular vein in the rst few hours of DI, but their effectiveness seems to diminish after a few days of DI. Studies suggest that thigh cuffs have an effect on the cephalad uid shift and its consequences only when they are worn, and that there is no signi cant memory effect when they are removed at night [37]. It is worth nothing that rCBF was measured in the morning, after a night without thigh cuffs. Therefore, the absence of a signi cant effect of thigh cuffs on the modi cation of rCBF in our study has many possible explanations, including a lack of statistical power, the fact that thigh cuffs appear to have little effect on the cephalad uid shift after 5-day DI, and the absence of a memory effect on rCBF after a night without thigh cuffs.
We observed a frank hypersignal of the left thalamus in two participants at post-DI that had not been present at pre-DI. During the protocol, participants underwent a muscle biopsy in the right thigh about a week before DI, and a second biopsy at DI5. Although the biopsy was performed under local anaesthesia, pain is often experienced after biopsy, its intensity varying according to the individual [38]. The protocol did not include an assessment of post-biopsy pain, but no speci c events were reported during the muscle biopsies. The questionnaires on back pain and general discomfort only elicited signi cantly higher scores than in the pre-immersion period during the rst two days of immersion for both groups [35]. We believe that the most likely explanation for the increased CBF in the left thalamus at post-DI is the sensory stimulus generated by the muscle biopsy in the right thigh, the day before the brain SPECT.
99m Tc-HMPAO is a tried and tested technique for measuring rCBF [39], but it is a SPECT tracer, which has lower spatial resolution than a PET tracer (e.g., 18 F-FDG, O 15 H 2 ). The choice of this tracer was ideal for our study, as 99m Tc-HMPAO reaches its binding peak 2-3 minutes after being injected. This allowed us, by injecting participants at the end of DI in the bath, to image rCBF while they were still immersed. After the end of DI, participants underwent a lower-body negative pressure test at the MEDES clinic after the injection, and then went to the nuclear medicine department for the SPECT scans. On account of the study design, the interval between the injection and scans was different at Pre-DI than at Post-DI: scans began 20 minutes after injection at Pre-DI, and after 90 min at Post-DI. Because of the irreversible brain binding of 99m Tc-HMPAO after its injection, we were able to make the acquisitions comparable, by correcting for the radioactive decay of 99m Tc by applying a weighting factor for each image in the analysis with SPM12 software.
Our study had several limitations; there was the small sample size (N = 18), which may have weakened the statistical power of our results. This could explain why we did not nd a signi cant result by applying an alpha risk adjustment with the familywise error rate or false discovery rate. However, we did adjust the alpha risk and applied a good extent threshold that made our results more robust. For radioprotection reasons, we injected our volunteers with a less active radiotracer (261 ± 8 MBq), but increased the acquisition time to 30 minutes to limit the deterioration of the signal-to-noise ratio. Moreover, DI immersion is a model of microgravity with a particular environment for the subject, such as physical immobility, that could also in uence the decrease in cerebral perfusion.

Conclusion
Our experiment showed that a 5-day DI induces a decrease in rCBF in cortical and subcortical regions. Prolonged vasoconstriction of cerebral arteries in response to increased CBF resulting from the cephalad uid shift and a moderate increase in ICP may contribute to the increase in CVR, thus inducing a decrease in rCBF. As possible consequences for brain function were not investigated in our study, further studies are needed to better understand the effects and consequences of microgravity on CBF.   Table 3. Measurements for heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP) systolic blood pressure (SBP) and mean blood pressure (MBP), at baseline (Pre-DI) and the end of dry immersion (Post-DI). All measurements were performed in the morning. Mean ± standard deviation.
Signi cance coe cient between the Cuffs and Control groups with unpaired Student t test, signi cance threshold p < 0.05. No signi cant difference was found between control vs. cuffs and between measurements at Pre-DI vs. Post-DI for each group. Statistical parametric map (tscore) of the negative variation in regional cerebral perfusion at Post-DI compared with Pre-DI, paired t test, puncorr < 0.001, extent threshold > 100 voxels.