After 5 days of DI, we observed a significant 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 133Xe 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 [14C]-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 modification 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 [18-21]. However, because of the semi-quantitative measurement of rCBF with 99mTc-HMPAO, it is not possible to determine whether the decrease in rCBF was sufficient to induce or be a consequence of brain function impairments. Further studies are needed to explore modifications 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 fluid flow, 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 fluid shift [8, 23]. However, the magnitude of a possible increase in ICP during space flights 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 significantly in studies in HDBR [3] and after 3-day DI [25], consistent with our finding 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] argue in favour of a moderate elevation of ICP. During 3-day DI, Kermorgant et al. showed an increase in ONSD of about 30%, as measured with ultrasound (Pre-DI: 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 first 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 first 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 first days, in response to the increased blood flow in the brain, due to the HDT position. After several days, the chronic vasoconstriction induces hypertrophy and modifications 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 flow 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 fluid 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 find any significant variation in rCBF after 5-day DI between the cuffs and control groups. We hypothesized that, by limiting the cephalad fluid shift and its consequences, thigh cuffs limit the increase in CVR. During 5-day DI, Arbeille et al. [36] found a significantly 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 significant 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 first 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 fluid shift and its consequences only when they are worn, and that there is no significant 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 significant effect of thigh cuffs on the modification 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 fluid 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 specific events were reported during the muscle biopsies. The questionnaires on back pain and general discomfort only elicited significantly higher scores than in the pre-immersion period during the first 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.
99mTc-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., 18F-FDG, O15H2). The choice of this tracer was ideal for our study, as 99mTc-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 99mTc-HMPAO after its injection, we were able to make the acquisitions comparable, by correcting for the radioactive decay of 99mTc 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 find a significant 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 influence the decrease in cerebral perfusion.