Ocular counter-roll is less affected in experienced versus novice space crew after long-duration spaceflight.

Otoliths are the primary gravity sensors of the vestibular system and are responsible for the ocular counter-roll (OCR). This compensatory eye torsion ensures gaze stabilization and is sensitive to a head roll with respect to gravity and the Gravito-Inertial Acceleration vector during, e.g., centrifugation. To measure the effect of prolonged spaceflight on the otoliths, we quantified the OCR induced by off-axis centrifugation in a group of 27 cosmonauts in an upright position before and after their 6-month space mission to the International Space Station. We observed a significant decrease in OCR early postflight, larger for first-time compared to experienced flyers. We also found a significantly larger torsion for the inner eye, the eye closest to the rotation axis. Our results suggest that experienced cosmonauts have acquired the ability to adapt faster after G-transitions. These data provide a scientific basis for sending experienced cosmonauts on challenging missions that include multiple g-level transitions.

The human vestibular organ: a multisensory system

Humans highly depend on the vestibular organs, located bilaterally in the inner ear, as they detect head movements and transmit this information to the brain to ensure upright posture, gaze stabilization, and spatial orientation[1]. The vestibular organs consist of the semicircular canals (SCCs) that are stimulated by angular accelerations, and the otoliths that detect the sum of linear accelerations acting upon the head. This vector sum is referred to as the Gravito-Inertial Acceleration (GIA) vector.

The otoliths are the primary graviceptors of the vestibular organ by registering linear accelerations on the one hand, including gravitational acceleration, and lateral tilts of the head on the other hand. Otoliths transmit their information to the brain to determine the spatial vertical, which is essential for controlling our eye movements and posture. An important otolith-mediated ocular reflex is the ocular counter-roll (OCR) that is generated when the head is laterally tilted, e.g., while driving around a corner or during centrifugation[2-4]. The OCR tends to rotate the eyes in the opposite direction to the roll tilt and toward the GIA[5] (Fig. 1). Although this reflex is imperfect, since the angle of the rotated eye is smaller than that of the induced tilt, it allows us to maintain gaze stabilization and postural stability, e.g., when making sharp turns during locomotion.

Fig. 1

Visual representation of the ocular counter-roll (OCR).

The OCR tends to rotate the eyes in the opposite direction of the head roll. For example, when a head roll to the right is performed, the eyes will rotate to the left as a compensatory reflex.

To get a better understanding of the importance of otolith-mediated reflexes, we study them in relation to spaceflight. Cosmonauts in the International Space Station (ISS), orbiting around Earth, are subjected to microgravity or <10−6 g. The lack of gravitational input will influence the otolith’s function by decreasing the gain (ratio of eye torsion over head tilt) of otolith-mediated reflexes, indicating a deconditioning of the otoliths[6]. Also, the assessment of the real vertical will be impaired when there is a loss of otolith input to the brain in microgravity[7]. Deconditioning of otolith-mediated reflexes following microgravity exposure has been proposed as one of the multiple causing factors of the postural, locomotor, navigational, and gaze control problems as well as orthostatic intolerance (OI) experienced by returning astronauts[8]. OI is the inability to regulate blood pressure, usually in a standing position, which can induce a (pre)syncope or fainting. These symptoms are generally maintained as long as the otoliths are not yet readapted to Earth’s gravitational level of 1 g[6]. The OCR is therefore an important measure to understand neurovestibular adaptation due to spaceflight since it isolates the effect of otolith adaptations.

The OCR reflex has been used in several studies as a measure of the effect of microgravity on the otoliths[8-14]. It should be noted that there are conflicting results from studies showing a decrease, increase or no change in OCR upon return compared to the preflight OCR[15]. However, these studies often had a small sample size, due to general difficulties and limitations of access to space crew, and due to difficulties in recording torsional eye movements. Moreover, the results were mostly from short-duration spaceflights, making it difficult to generalize these results to prolonged exposure to microgravity. For example, Clément et al. studied seven cosmonauts who went to space for 10–13 days. On Earth, the cosmonauts were rotated along their longitudinal axis and were tilted 10° and 20° off-axis, generating an OCR. However, OCR measurements on the day of landing and up to 4 days postflight did not significantly differ from preflight measurements[15]. Another study, by Kornilova et al., studied a group of 13 cosmonauts with long-duration missions (126–196 days), of whom they obtained OCR measurements by having the cosmonauts tilt their heads sideways 30–35°. They observed a decrease in OCR as measured on the first 2 days after return compared to preflight, which recovered to baseline 8–9 days after landing[16]. Lastly, our group has also demonstrated a decrease in OCR by off-axis centrifugation 3 days after landing, which returned to baseline values as measured 9 days postflight. That study included 25 OCR measurements at each pre- and postflight timepoint, with cosmonauts spending on average 186 days in space[6].

The current study extends our previous research on OCR changes after long-duration spaceflight, of which results have previously been published[6]. The first aim of this study is to report the OCR changes induced by 6-month spaceflights in an extended dataset compared to our previous work. Note that we have now analyzed nearly double the amount of data compared to our previous study and that for the current study, all data were analyzed by the same person to exclude operator bias. The second aim is to determine whether previous experience in space influences the postflight OCR response, which has not yet been investigated. In total, 27 cosmonauts (13 first-time flyers, 14 experienced flyers) took part in this study, several of whom participated in multiple ISS missions (11 cosmonauts participated twice, 1 participated three times, and 1 participated four times). This resulted in a total of 44 longitudinal datasets, where a longitudinal dataset comprises two preflight measurements as a baseline data collection (BDC1 and BDC2), and up to three postflight measurements grouped as 1–3 days (R + 1/3), 4–7 days (R + 4/7), and 8–12 days (R + 8/12) after spaceflight. These 44 longitudinal datasets contained data of 13 first-time, 16 second-time, 8 third-time, 4 fourth-time, and 3 fifth-time flyers. The OCR is induced using an off-axis centrifuge with the cosmonauts seated upright, facing the direction of rotation (right-ear-out during counterclockwise (CCW) and left-ear-out during clockwise (CW) rotation), and with a fixed distance of 0.5 m from the rotation axis. The cosmonauts were first centrifuged for 5 min in a CCW and subsequently in a CW direction. The OCR was recorded during constant angular velocity to purely obtain measurements of otolith function.

Methods

Experiment timeline and subjects

The test used to induce the OCR was the Visual and Vestibular Investigation System (VVIS) located in the Gagarin Cosmonaut Training Centre in Star City near Moscow, Russia. We investigated 27 cosmonauts (N = 13 first-time flyers, N = 14 experienced flyers), several of whom were tested twice or even more times during consecutive spaceflight missions to the ISS (N = 15 were tested once, N = 11 were tested twice, N = 1 was tested three times, N = 1 was tested four times). As a result, 44 cosmonaut experiments were performed, 31 were conducted for frequent flyers, while the other 13 experiments were conducted for the remaining cosmonauts who were first-time flyers (N = 13 first-time flyers, N = 16 second-time flyers, N = 8 third-time flyers, N = 4 fourth-time flyers, and N = 3 fifth-time flyers).

The cosmonauts were tested before and after their 6-month space mission in the ISS between ISS increment mission 16 in October 2007 to increment 61 in April 2020. The preflight experiments consisted of 2 baseline recordings defined as the BDC (BDC1 N = 44, and BDC2 N = 40), and the postflight experiments consisted of 2–3 recordings of the OCR. The first postflight measurement was taken 1–3 days after the return to Earth, defined as R + 1/3 (N = 32). The second postflight measurement, which was not recorded for all cosmonauts, was taken 4–7 days after their return to Earth, defined as R + 4/7 (N = 23). The third postflight measurement was taken 8–12 days after their return to Earth and is therefore defined as R + 8/12 (N = 39). It was impossible to test all the cosmonauts on the same day after their return, due to medical and organizational limitations.

The experiment protocol was designed in accordance with the ethical standards defined in the 1964 Declaration of Helsinki and was accepted by Human Research Multilateral Review Board (HRMRB) and European Space Agency (ESA). Cosmonauts gave their written informed consent prior to their voluntary participation in this study.

Visual and Vestibular Investigation System (VVIS)

The cosmonauts were seated upright on the rotation chair, 0.5 m away from the vertical rotation axis, and securely fastened by a five-point safety harness with a restriction of head movements (Fig. 2). The experimentation room was darkened to avoid any visual motion feedback or fixation during rotation. A visual display, mounted in front of the cosmonaut’s face, was used to project visual targets during parts of the experiment. Binocular three-dimensional video-oculography with infrared video goggles was used to enable continuous recordings of dynamic changes in ocular torsion. 3D video-oculography is a non-invasive method for recording the horizontal, vertical, and torsional components of eye movements.

Fig. 2

Representation of experimental setup.

The net linear acceleration stimulating the otoliths, during off-axis centrifugation, is the vector sum of the gravitational (Ag) and centripetal acceleration (Ac), termed the Gravito-Inertial Acceleration (GIA). When GIA was interpreted as the spatial vertical during centrifugation, the cosmonaut should experience a sensation of 45° tilt (Reprinted from ref. [8] with permission of Macmillan Publishers Ltd, copyright 2015).

At standstill, the calibration of the video goggles and a baseline recording were performed. After an acceleration phase of 30°/s2, the cosmonaut was subjected to a constant angular velocity of 254°/s resulting in an outward centripetal acceleration Ac of 1 g first for 5 min in a CCW direction. The chair was decelerated with a rate of 3°/s2 to standstill. The chair was then manually 180° rotated, and subsequently, the protocol was repeated for 5 min in a CW direction. In between both centrifugation directions, the cosmonaut remains seated while the operator changes the centrifuge configuration to the subsequent (CW) direction. The cosmonaut faced the direction of motion, with the right ear outwards during CCW rotation and the left ear outwards for the CW rotation. The vector sum of the gravitational acceleration Ag and the centripetal acceleration Ac is called the GIA. This GIA was perceived by the subject as the ‘spatial vertical’ and exerted a shear force on the otolith system which caused an illusory or virtually perceived roll tilt of 45° during rotation. As a result, an OCR was induced that tended to orient the eyes toward the GIA and thus away from the direction of the perceived tilt. The virtual tilt was outwards, meaning that the cosmonaut had the impression of tilting to the right when moving CCW and to the left when moving CW. This means that the eyes will rotate toward the left during CCW centrifugation and to the right during the CW centrifugation, always toward the axis of rotation (Fig. 3).

Fig. 3

The rotation axis is placed next to the subject, meaning that both otoliths will be simultaneously stimulated by GIA.

A When the subject was moving according to the counterclockwise (CCW) direction, a virtual tilt of 45° was experienced to the right (right-ear-out, REO). B When the subject was moving according to the clockwise (CW) direction, a virtual tilt of 45° was experienced to the left (left-ear-out, LEO)[6,46] (From Moore, 2001[46] with permission; reproduced from Experimental Brain Research).

OCR measurements

The OCR measurements were taken before, during, and after centrifugation according to a fixed protocol. The first and fourth OCR measurements were respectively taken before and after centrifugation during standstill, where no centripetal force was acting upon the body and thus an OCR of 0° was observed as expected. The second OCR measurement was taken 40 s after the steady-state phase of constant rotational velocity was reached, because we only wanted to assess the contribution of the otolith system to the OCR. During the 40 s, the cupula of the horizontal SCCs returns to its original position and no longer contributes to the OCR. The third OCR measurement was taken 40 s before the start of the deceleration phase. The time interval between the second and third OCR measurements was 3 min and 40 s, during which other protocols were applied during centrifugation. During centrifugation, the second and third OCR measurement, an OCR of on average 5–7°[17] was expected to be measured because of GIA stimulating the otoliths. Each OCR measurement was recorded for 20 s, while the cosmonaut observed a fixation or central dot (CD) on the visual display. The CD was used to cancel out other eye movements, e.g., saccades and nystagmi, during centrifugation. It is important to mention that the CD could prevent horizontal eye motion that may be otolith generated and should therefore be included as a limitation. The OCR was calculated as the difference of the average eye torsion over these 20 s recordings, consisting of 600–1000 frames, between rotation and standstill (Fig. 4).

Fig. 4

Visual overview of conducted measurements.

Before acceleration, at a standstill, the calibration and the first OCR measurement (CD1) were performed. After an acceleration phase of 30°/s, when the maximal rotational velocity of 254°/s was reached during rotation, the second and third measurements of the OCR (CD2 and CD3) were performed. After the cessation of a 3°/s deceleration phase, at standstill, the last OCR measurement was performed (CD4).

The video files obtained during the VVIS experiment contain recordings of the eye movements and were analyzed in a visual programming language (custom made by H.M. in LabVIEW – National Instruments −11500 N Mopac Expwy. Austin, TX, USA) to measure the OCR in degrees. The OCR was calculated for both eyes and centrifugation directions (CCW and CW). As part of quality control, we selectively removed artifactual data arising from eye blinks and eyelashes.

Statistical analysis

The OCR measurements were statistically analyzed in JMP® (version Pro 16. SAS Institute Inc, Cary, NC, 1989–2001), with p < 0.05 as the significance threshold. All sample sizes for our models can be found in Table 1. We built three linear mixed models (LMM) using a stepwise forward approach[18,19]. We first tested our main variables as fixed effects and then systematically tested all interaction terms. The variables included were Timepoint (BDC1, BDC2, R + 1/3, R + 4/7, and R + 8/12), Days After Return (range 1–12), Flight (a cosmonaut’s amount of space missions, range 1–5), Eye (left and right), and Orientation (CCW and CW). The significance threshold used for selecting the fixed effect was set at p = 0.001. Non-significant terms were removed until all combinations were tested and only the significant ones remained. In all models, Cosmonaut was entered as a random intercept to account for the non-independence between observations from the same cosmonaut. Random slope terms Cosmonaut*Flight and Cosmonaut*Flight*Timepoint were added as random effects in case these terms significantly improved the fit of the model, as tested using the Likelihood ratio test. The residuals of all models were checked for normality and homoskedasticity.

Table 1

Overview OCR values.

		CCW left, inner, eye			CCW right, outer, eye			CW left, outer, eye			CW right, inner, eye
		Mean	SEM	Valid N	Mean	SEM	Valid N	Mean	SEM	Valid N	Mean	SEM	Valid N
OCR	BDC1	6.15°	0.08	44	5.53°	0.07	44	5.53°	0.10	44	5.89°	0.11	44
	BDC2	6.02°	0.10	40	5.45°	0.08	40	5.41°	0.10	38	5.94°	0.11	38
	R + 1/3	3.45°	0.17	32	3.05°	0.17	33	2.86°	0.15	32	3.29°	0.17	32
	R + 4/7	4.37°	0.10	23	3.88°	0.10	23	3.63°	0.10	23	4.08°	0.14	23
	R + 8/12	6.03°	0.08	39	5.38°	0.09	40	5.42°	0.08	38	5.88°	0.11	39

This table gives an overview of all OCR values at CD2 for each timepoint (BDC1, BDC2 (two preflight measurements), R + 1/3, R + 4/7, and R + 8/12 (three postflight measurements)), centrifugation direction (CCW and CW), and eye (left and right eye).

Our first model evaluated the factors influencing the OCR measurements pre- and postflight, to investigate the effect of long-duration spaceflight. The categorical variable Timepoint was used as a fixed effect, defining two preflight measurements (BDC1, BDC2) and three postflight measurements (R + 1/3, R + 4/7, R + 8/12). The random slope term (Cosmonaut*Flight*Timepoint) and the random intercept terms (Cosmonaut and Cosmonaut*Flight) were kept in the model according to their Likelihood ratio tests (p < 0.0001 for all). Given the significant term Eye*Orientation, we created a new interaction term Eye_Rotation defining the inner and outer eye with respect to the rotational axis of the centrifuge. Dunnett’s correction for multiple testing was used for the post hoc analysis to compare OCR at the first baseline measurement (BDC1) to each of the following timepoints.

Our second model evaluated how the OCR measurements were evolving in the first week after return from a space mission (selection threshold: Days after return <8), the readaptation to the normal gravitational level of Earth. The random slope term (Cosmonaut*Flight*Timepoint) and the random intercept terms (Cosmonaut and Cosmonaut*Flight) were kept in the model according to their Likelihood ratio tests (p < 0.0001 for all). The continuous variable Days After Return was entered as a fixed effect to model the effect of the days since return from a space mission.

To investigate the effect of the previous spaceflight, or experience, on the otolith-mediated OCR[20], we modeled the impact of the number of previous spaceflights (variable Flight) on the changes observed in OCR at R + 1/3. For this analysis, we entered the percent change in OCR at R + 1/3 compared to BDC1 as a dependent variable in the model. The number of flights was entered as a fixed effect using a piecewise linear model with a breakpoint at Flight = 2. The position of the breakpoint was based on visual inspection and our previous results. The regression lines were fitted using a LMM. the random intercept terms (Cosmonaut and Cosmonaut*Flight) were kept in the model according to their Likelihood ratio tests. The model for the mean is:

Equation 1, where Y is the percentage change in OCR at R + 1/3 and k = 2.

X is the number of flights the cosmonaut has experienced.

The regression line is made of two segments with a changing slope beyond two flights. The first segment (for Flight ≤ 2) has the equation: y = β0 + β1*x, with β1 estimating the change in Y per extra flight.

The second segment (for Flight > 2) has equation: y = β0 + β1*x + β2*(x – k). In this equation, β2 estimates the change in slope beyond Flight = 2. The significance of β2 tests whether this change is significant.

The piecewise linear analysis generates two regression slopes, the first one applicable for the flight variable (from 1 to 5) and the second one for data of cosmonauts with more than two previous spaceflights. We then test if that second slope is different from zero. If not, the second slope does not bring any additional information to the regression and the evolution of the data is the same before and after the inflection point.

Table 2

Overview of the valid number of data points for each flight, rotation, eye, and timepoint.

	CCW
	Right						Left						All CCW
	BDC1	BDC2	R + 1/3	R + 4/7	R + 8/12	All right CCW	BDC1	BDC2	R + 1/3	R + 4/7	R + 8/12	All left CCW
F1	13	12	9	10	13	57	13	12	8	10	12	55	112
F2	15	14	12	5	12	58	15	13	12	5	12	57	115
F3	8	6	5	4	7	30	8	7	5	4	7	31	61
F4	4	4	3	3	4	18	4	4	3	3	4	18	36
F5	3	3	3	1	3	13	3	3	3	1	3	13	26
All	43	39	32	23	39	176	43	39	31	23	38	174	350

This table represents the timepoints as the intervals, for each separate day the valid sample size is: R + 1 N = 4, R + 2 N = 9, R + 3 N = 116, R + 4 N = 16, R + 5 N = 68, R + 6 N = 8, R + 7 N = 0, R + 8 N = 12, R + 9 N = 70, R + 10 N = 44, R + 11 N = 18, R + 12 N = 12.

Fx flight number x, CW clockwise, CCW counterclockwise, BDC baseline data collection, R + X return after X days.

Table 3

Overview of the specifications or estimates of the different linear mixed models (LMM).

Fixed effects	Estimate (°)	Std error (°)	CI 95% (°)
Model 1: the effect of long-duration spaceflight on the otolith-mediated reflex, the OCR
Intercept	5.78	0.12	[5.55; 6.02]
Timepoint [BDC2]	0.07	0.16	[−0.24; 0.40]
Timepoint [R + 1/3]	−3.76	0.18	[−4.12; −3.40]
Timepoint [R + 4/7]	−1.96	0.18	[−2.31; −1.60]
Timepoint [R + 8/12]	0.09	0.16	[−0.22; 0.41]
Orientation [CW]	−0.15	0.04	[−0.23; −0.07]
Flight	0.14	0.06	[0.01; 0.26]
Eye_Rotation [Outer Eye]	−0.49	0.04	[−0.57; −0.41]
Timepoint [BDC2]*Flight	−0.07	0.08	[−0.21; 0.11]
Timepoint [R + 1/3]*Flight	0.50	0.09	[0.32; 0.67]
Timepoint [R + 4/7]*Flight	0.12	0.09	[−0.07; 0.31]
Timepoint [R + 8/12]*Flight	−0.07	0.08	[−0.23; 0.09]
Model 2: the OCR re-adapts to the normal gravitational level of Earth
Intercept	−0.26	0.38	[−1.01; 0.48]
Flight	1.03	0.15	[0.75; 1.32]
Orientation [CW]	−0.23	0.07	[−0.38; −0.09]
Eye_Rotation [Outer Eye]	−0.41	0.07	[−0.56; −0.27]
DaysAfterReturn	0.80	0.09	[0.63; 0.97]
DaysAfterReturn*Flight	−0.14	0.03	[−0.21; −0.07]
Model 3: the effect of previous spaceflight on the otolith-mediated OCR changes
Intercept	0.18	0.05	[0.09; 0.28]
Flight	0.19	0.03	[0.14; 0.25]
Flight >2	−0.16	0.04	[−0.24; −0.08]

BDC1 was taken as the reference value.

BDC baseline data collection, R + X return after X days.