Feasibility of Detecting Brain Areas Involved in Extreme Breath-Hold Diving

We report Blood Oxygen Level Dependent (BOLD) signal changes recorded in the brain of an elite breath-hold diver during voluntary dry long breath-hold by means of fMRI. An independent component analysis (ICA) method was applied to extract brain areas that are putatively involved in the apnea process network. We discuss the hypothesis that these BOLD signal variations express the functional adaptive diving response under long apnea at rest. This is a preliminary report, which results are promising for large series investigations. DOI: 10.14302/issn.2470-5020.jnrt-21-3699 Corresponding author: Danilo Cialoni, DAN Europe, Contrada Padune 11, 64026 Roseto degli Abruzzi (TE), Italy, Phone: 39.085.8930333, Fax: 39.085.8930050, Email: dcialoni@daneurope.org


Introduction
Breath-hold diving (BH-diving) triggers a complex adapting mechanism called "diving response" that is the result of several psycho-physiological components [1]. The major physiological components of the diving response that occur during BH-diving are peripheral vasoconstriction, bradycardia and decreased cardiac output, on the other hand we have an increase of cerebral and myocardial blood flow, an increase of blood pressure and splenic constriction that ensure adequate oxygen delivery to the brain and to the heart [2,3]. The increase of CO 2 level (hypercapnia) contributes to dyspnea sensation, which also leads to others physiological responses and adaptation mechanisms [4]. Some authors focused on the significant increase of cerebral blood flow (CBF) in elite divers, as compared to non-Breath Hold divers (BH-divers), and showed that even if hypoxia and hypercapnia occur at the end of long BH-diving, some oxygen-conserving mechanisms can occur [5]. Cardiovascular magnetic resonance imaging (CMR), performed to investigate changes in the cardiovascular system during BH-diving, showed that prolonged BH-diving caused stress in the cardiovascular system, however with no sign of acute myocardial injury [6]. The effect of prolonged BH-diving on the brain is poorly understood field of investigation. During BH-diving, the brain is rapidly subject to an increase of the hypoxia, which is responsible for the loss of consciousness that can occur at the end of a prolonged BH-diving [7].
Cerebral Decompression illness (DCI) mechanism in BH-divers is still controversial and many possible pathogenic mechanisms have been considered as caused of neurological symptoms in BH-diving [8], a recent case report described high bubble formation, recorded by echocardiography, in a BH-diver consolidating the hypothesis that, at least in some cases, bubbles formation could be involved in cerebral injures [9]. Finally brain MRI with fluid-attenuated inversion recovery (FLAIR) has been used to evaluate the lesions of two breath-hold divers [10].
Recent developments in MRI techniques allow today the functional brain mapping using venous blood oxygenation level-dependent (BOLD) MRI which relies on changes in deoxyhemoglobin, with a higher sensitivity at high magnetic fields [11]. This in-depth technique that permits a functional MRI (fMRI) has been introduced to evaluate brain activity by detecting changes associated with the blood flow [12].
The fMRI technique has developed in different variants that can be classified into two main types: resting state (RS-FMRI) versus action/event-related fMRI [13]. RS-fMRI aims at recording the BOLD signals all over the brain while the patient is at rest. Then, robust statistical analyses enable to detect concordant areas of the brain that resonate at low frequencies, resulting in network nodes for areas that resonate at similar frequencies [14]. Despite all this knowledge, there aren't data about fMRI applications to underwater activities. The aim of our study was to detect the concordant functioning areas of the brain during prolonged breath-holds in a world-class apnea diver, by means of RS-fMRI.

Materials and Methods
Subject and Breath-Hold Protocol

Results
We found the dorsal pons, cerebellar hemispheres (superior aspect) and whole vermis,   The eloquent areas disclosed during both experiments are mapped on a brain mesh as nodes of both networks ( Figure 3).
In BH-diving (as in our experiment) the extended time without breathing exposed the subject to brain hypoxia/ hypercapnia associated with a decrease of cardiac output and peripheral HbO2. The figure 4 show the hypoxia and hypercapnia BH-diving related obtained the same day of fRNM test but during a performance even longer (9 min, 7 sec) which illustrates this aspect.

Discussion
To our knowledge, our study is the first to investigate BOLD signals changes in the brain under voluntary static dry long breath-hold. The first challenge in this study was the self-control start/stop apnea of diver. Indeed, as in wet conditions, the subject was free to start and stop breath-holding, and the scan time was set to 765 s in order to record enough scans during the breath-hold period.
Our main findings encompass the brain eloquent areas likely involved to the first and second apnea, namely the brainstem and cerebellum, prefrontal, parietal, occipital and insular cortices. We also found a typical pattern of BOLD signals variation, consistent all apnea long, with some variations during apnea depending upon the eloquent regions of the brain. We assume that the eloquent areas were activated in response to apnea as well as in the control of apnea.
In the brain, variations of oxygenation at the arterial venous junction generate BOLD signals. The signal changes observed are closely related to the changes in arterial oxygen saturation during hypoxia [18]. In such conditions, brain oxygenation is preserved by compensatory mechanisms called the "diving response" and brain arterial autoregulation [2,19].
This response includes systemic changes, notably vagal reaction with bradycardia, peripheral vasoconstriction related to sympathetic nervous system stimulation and spleen contraction, to reduce tissue oxygen uptake, as well as brain vasodilatation, increased cerebral blood flow and fluctuations of the hemoglobin concentration [20]. These mechanisms, including both vascular and metabolic changes [21], are presumably responsible for the BOLD signals recorded in our subject.
Independent component analysis (ICA) is one of the most popular methods proven efficient, consistent and reliable, to identify low-frequency resting-state patterns and to show temporal and spatial correlations in the brain [22,23]. We used this method to extract  occur [28]. The decision of stopping the breath-hold might be based on these neurological disturbances. Our patient described an out of the body experience and a "Samba" feeling (myoclonic agitation, presenting usually as agonist/antagonist muscular activities recalling cerebellar activation) during the second apnea. These movements are considered to be likely due to cerebral hypoxia; in our data, metabolites build up is more prone to explain this feeling, happening just before he decided to stop the apnea. This seems to be in line with some experiences reported during altered states of consciousness such as recovery after narcotic states or presyncopal situations [29,30]. Also, it is difficult to comment on the significance of the lateralization of activated areas. We found visual activation in the left hemisphere, inferior parietal and supramarginal gyri in the right hemisphere, while other activation areas were observed bilaterally. This lateralization might be related to the dominant hemisphere, which was likely the right in our left-handed subject.
Our results are limited to one individual.
Moreover, they are likely incomplete due to several technical considerations. This was the very first experiment in a challenging situation and the design of the study as well as the data analysis methods will benefit from the present findings to a larger study. We cannot yet hypothesize on the functional circuitry existing between these areas in the induction and sustaining of apnea and the way these nodes interact in the networks. We conclude that under hypoxic conditions, the mechanism for sustaining brain function in response to/ control of long breath-holding likely involves different areas of the central nervous system (the cerebrum, the brainstem and the cerebellum) implicated in a complex network. Still, more studies are needed to establish a specific relationship between those areas and dry voluntary long breath hold. Our data may stimulate the use of fMRI to better understand brain adaptations strategies during breath-hold diving.