Relationship between oxygen supply and cerebral blood flow assessed by transcranial Doppler and near – infrared spectroscopy in healthy subjects during breath – holding
© Molinari et al; licensee BioMed Central Ltd. 2006
Received: 20 July 2005
Accepted: 19 July 2006
Published: 19 July 2006
Breath – holding (BH) is a suitable method for inducing cerebral vasomotor reactivity (VMR). The assessment of VMR is of clinical importance for the early detection of risk conditions and for the follow-up of disabled patients. Transcranial Doppler ultrasonography (TCD) is used to measure cerebral blood flow velocity (CBFV) during BH, whereas near-infrared spectroscopy (NIRS) measures the concentrations of the oxygenated (O2Hb) and reduced (CO2Hb) hemoglobin. The two techniques provide circulatory and functional-related parameters. The aim of the study is the analysis of the relationship between oxygen supply and CBFV as detected by TCD and NIRS in healthy subjects performing BH.
20 healthy subjects (15 males and 5 females, age 33 ± 4.5 years) underwent TCD and NIRS examination during voluntary breath – holding. VMR was quantified by means of the breath-holding index (BHI). We evaluated the BHI based on mean CBFV, O2Hb and CO2Hb concentrations, relating the baseline to post-stimulus values. To quantify VMR we also computed the slope of the linear regression line of the concentration signals during BH. From the NIRS signals we also derived the bidimensional representation of VMR, plotting the instantaneous O2Hb concentration vs the CO2Hb concentration during the BH phase. Two subjects, a 30 years old current smoker female and a 63 years old male with a ischemic stroke event at the left middle cerebral artery, were tested as case studies.
The BHI for the CBFV was equal to 1.28 ± 0.71 %/s, the BHI for the O2Hb to 0.055 ± 0.037 μ mol/l/s and the BHI for CO2Hb to 0.0006 ± 0.0019 μ mol/l/s, the O2Hb slope was equal to 0.15 ± 0.09 μ mol/l/s and the CO2Hb slope to 0.09 ± 0.04 μ mol/l/s. There was a positive correlation between the CBFV and the O2Hb increments during BH (r = 0.865). The bidimensional VMR pattern shows common features among healthy subjects that are lost in the control studies.
We show that healthy subjects present a common VMR pattern when counteracting cerebral blood flow perturbations induced by voluntary BH. The proposed methodology allows for the monitoring of changes in the VMR pattern, hence it could be used for assessing the efficacy of neurorehabilitation protocols.
Unlike the other organs, human brain needs a constant oxygen supply in order to maintain its functional and structural integrity. The local amount of oxygen stored in the brain tissues is small compared to the metabolic needs, hence a specific mechanism is necessary in order to ensure the correct oxygenation levels. This mechanism has to provide oxygen during both resting condition and focal cortical activity. The strict coupling existing between "activation", local oxygen consumption, and increased regional cerebral blood flow constitutes the basis of the so called BOLD effect (Blood Oxygenation Level Dependent) and, hence, of the functional magnetic resonance . Thus, the assessment of cerebral hemodynamics is of paramount importance for determining the response of a subject to an external stimulus or for quantifying cortical activation.
Among the methods allowing a non – invasive and low – cost assessment of cerebral hemodynamics, transcranial Doppler ultrasonography (TCD) plays a fundamental role [2, 3]. By means of TCD it is possible to measure the cerebral arteries blood flow velocity (CBFV) and, hence, analyze the variation of the CBF. However, the limited spatial resolution of this technique allows for the quantification of CBFV only in the macro – vessels (essentially the arteries constituting the Willis circle plus the middle cerebral arteries), whereas a cortical localized modification of blood velocity is impossible to track. Moreover, in about 25% of the patients, it is impossible to perform a TCD examination due to poor skull acoustic windows.
By means of near – infrared spectroscopy (NIRS) it is possible to continuously monitor the local concentrations of oxygenated (O2Hb) and reduced (CO2Hb) in the adult brain. TCD provides a direct measurement of circulatory parameters, whereas NIRS provides more functional and activation-dependent informations. Specifically, it has been demonstrated that NIRS can proficiently measure cerebrovascular reactivity .
In clinical practice, cerebral autoregulation is usually assessed during a CO2 reactivity test . It is known that baroreceptors react to an increased partial pressure of CO2 by inducing vasodilatation in the resistance vessels; hence, the mean CBFV increases and the resistance of the vessels drops . This mechanism is often indicated as vasomotor reactivity (VMR). CO2 reactivity can be induced by means of acetazolamide injection, by means of direct CO2inhalation (usually at the 5% – 7% concentration), or by means of simple breath – holding (BH).
In the last five years, a great variety of studies combining TCD and/or NIRS have been devoted to the assessment of VMR in subjects affected by acute and chronic pathologies: microangiopathy , migraine , carotid artery occlusion  and depression . Recently, NIRS has been also used for the cerebral activity quantification during motion tasks . From a rehabilitation point of view, NIRS proved successful in monitoring motor reorganization in hemiparetic stroke patients .
Traditionally, in response to a CO2 test, VMR is quantified by relating baseline values (these values can be the mean CBFV as well as the concentrations of O2Hb and CO2Hb) to post – stimulus values ; while the stimulus phase is not taken into consideration. Since VMR determines a continuous modification of such values during time, omitting the analysis of the stimulus phase may lead to uncertainties and poor comprehension of the VMR itself.
The aim of the study is the analysis of the relationship between oxygen supply and CBFV as detected by TCD and NIRS in healthy subjects performing BH. We studied a population consisting of 20 healthy volunteers and we showed the vasoreactivity patterns the subjects had during BH. We introduced a bidimensional representation of VMR based on the O2Hb and CO2Hb concentration changes that we consider useful to gain a better comprehension of VMR. Finally, we showed that this methodology could be used for assessing a subject's VMR condition, comparing the data of two case studies to those of the normal population.
Currently, we enrolled in this study 20 (15 males and 5 females) healthy non-smokers volunteers (age, mean ± sd = 33 ± 4.5 years). Before being included in this study, all the subjects underwent clinical examinations intended to exclude cerebral, cardiac, and circulatory diseases. According to the rules of the local Hospital in which the tests were hold, the subjects were asked to sign an informed consent.
We also tested several healthy current smokers subjects and some pathologic subjects. Due to the great variability of our sample population of smokers and pathologic subjects, we decided to present in this paper only two case reports which we found indicative of their category. The first subject was a healthy current smoker 30 years old female. She had been smoking for 12 years and she smoked an average of 15 cigarettes/day. The subject (indicated as subject A in the following) underwent the same clinical examinations of the normal controls and did not show any sign of cerebral, cardiac, and circulatory diseases. The second subject was a post-stroke, 63 years old, man. He had suffered from a ischemic stroke to the left middle cerebral artery (MCA) about 2 years before being enrolled in the study, when he was tested for the first time. He showed aphasia, motor impairment, and poor scores in fluency and verbal tests. After a year of drug therapy (antihypertensive and antiaggregating agents) and logopedic therapy, this subject was tested for the second time. He reported an improvement in motor control and reaching tasks, and increased his AAT (Aachener Aphasie Test) score from 52/60 to 56/60.
We applied TCD and NIRS during baseline conditions and during CO2 reactivity. To trigger CO2reactivity, we chose the voluntary breath – holding technique. A major advantage of this choice is simplicity, since, to induce hypercapnia, there is no need for further devices (i.e. a capnograph with a breathing mask). This technique, however, is subject dependent: it is impossible, in experimental conditions, to establish a BH duration equal for all the subjects. To cope with this difficulty, we preliminary instructed the subjects on how to perform the BH and we let them test the procedure once before starting the recordings. In particular, we instructed the subjects to hold the breath after a normal breathing, in order to avoid an increase of the thoracic pressure, and we controlled they could hold the breath for a minimum time of 20 s. According to previously published experimental protocols, we instructed the subjects to end breath – holding when they felt comfortable .
The experimental protocol was the following:
• to derive baseline conditions, the subjects were allowed to rest for about 10 minutes in a dimmed and quiet room, laying comfortably in a supine position with eyes closed and breathing room air;
• when we observed stable signals (i.e. when the concentrations of O2Hb and CO2Hb and the CBFV did not show remarkable variations from their mean values), the subjects were instructed to perform a breath – holding after a normal inspiration;
• at the end of the apnea, the subjects were asked to rest for 5 minutes and we collected signals related to the post – stimulus conditions.
Changes in the concentrations of O2Hb and CO2Hb were measured by means of a near – infrared spectroscopy device (NIRO 300, Hammamatsu Photonics, Australia). The emitting probe of the NIRS equipment was placed on the left frontal side of the subjects, 2 cm beside the midline and about 3 cm above the supraorbital ridge. We chose this positioning in order to avoid the sinuses and to place the probes on a poorly perfused and very thin skin layer. BH is supposed to induce a perturbation in cerebral cortex that is systemic and not regional or localized, hence the frontal lobe was a suitable location also for the absence of hairs. The receiving sensor was fixed laterally to the emitter at a distance of about 5 cm. According to previous studies and theoretical models already developed , we set a differential pathlength factor equal to 5.97. Previous works [15, 16] demonstrated that with a source – detector distance equal to approximately 5 cm the NIRS equipment is capable of detecting effectively the chromophores concentration changes on the surface of the cerebral cortex.
During the test, we also monitored the end-tidal CO2 and the mean arterial blood pressure by means of a specific monitor equipped with a capnographic module.
According to previous studies , we used the breath – holding index (BHI) to quantify vascular reactivity. This index can be defined for any quantity related to the cerebral circulation, since it simply relates post – stimulus quantities to pre-stimulus quantities.
From the TCD data, we derived a BHI based on the mean blood flow velocity (MV). MV can approximately be defined as :
• PV is the peak systolic blood flow velocity;
• EDV is the end – diastolic blood flow velocity.
The BHI derived from the MV (which is indicated as BHI V in the following) was then defined according to the following expression:
• V BASE represents the MV averaged on a 10s time window when in baseline conditions;
• V BH represents the MV averaged on a 10s time window after the offset of the apnea;
• D BH is the time duration of the BH.
This index is expressed in %/s.
From the TCD data, we also calculated the Gosling's pulsatility index (PI) of the MCA in baseline conditions and in correspondence of the maximum CBFV increase during the apnea. The PI is defined according to the following expression:
This parameter indicates how the ratio between the extreme velocities in the artery modifies as consequence of vasoreactivity and it is often used in VMR studies as a complement to the BHI . To quantify VMR from the NIRS data, we estimated the chromophores concentration changes with respect to the BH duration :
As in equation 2, O2Hb BASE is the oxygenated hemoglobin concentration in baseline conditions, averaged on the same 10s time window during which the V BASE is evaluated, and O2Hb BH is the average concentration after the release of the BH. We calculated the same index also for the CO2Hb ( ).
These reactivity indexes are expressed in μmol/l/s.
VMR bidimensional representation
Results and discussion
Carbon dioxide reactivity triggered by breath – holding
As already pointed out, the three major techniques adopted for triggering CO2 reactivity are: hypercapnia, acetazolamide injection, and breath – holding . We decided to carry on this study using BH as reactivity trigger, since we planned to develop an experimental protocol that could be suitable for any subject, including patients suffering from cerebrovascular, neurological, and chronic diseases.
Breath – holding is obviously subject dependent; while this poses the problem of dealing with different BH durations, we believe this technique is suitable for assessing VMR as response to a sudden and abrupt change in the oxygenation levels, which is a major risk condition for cerebral autoregulation.
BHI and PI indexes derived from TCD signals. Population averaged values of the BHI and of the PIs derived from the TCD measurements. The first row depicts the percentage increment of the CBFV (BHI V ), whereas the second and third rows depict the PI during baseline and after BH respectively. All the values are expressed as mean/sd.
BHI V (%/s)
BHIs derived from NIRS signals. Population averaged values of the BHI and of the slope of the O2Hb and CO2Hb concentration signals derived from the NIRS data (all the values are expressed in μ mol/l/s). The first and the second rows report the BHIs derived from the concentration changes of oxygenated and reduced hemoglobin, the third and fourth rows report the slopes of the time course of the concentration signals during the BH phase (all the values are expressed as mean/sd). The second column reports the first species probability error of a Student's t – test to test the BHI and the slope values against zero (i.e. against no modification induced by the BH) with a confidence level equal to 95%.
We believe that the quantification of VMR by means of the BHIs derived by NIRS signals could be questioned. According to literature, vasomotor reactivity is quantified as the variation of a given physiological parameter as consequence of an external stimulus (usually a CO2increase). As a matter of fact, however, the above defined indices only depends on the baseline and on the post-BH conditions, but what happens during the BH phase is not taken into consideration.
Mean CBFV increases during CO2 reactivity tests as consequence of a pial arteries vasodilation, but then it remains almost constant for periods lasting several seconds . Hence, the quantification of vasomotor reactivity based on pre-apnea and post-apnea values is appropriate. Conversely, as our experimental results clearly show, the local concentration of oxygenated hemoglobin measured by NIRS is a more rapidly evolving quantity, since it depends on the CBFV, on the perfusion pressure, on the degree of artery dilation and on the tissues oxygen extraction rate. Moreover, vasoreactivity is triggered by a CO2increase, but the quantification of VMR itself is usually done by taking into account the increases in both oxygenated and reduced hemoglobin; this because VMR is a functional physiological process aiming at maintaining a proper chromophores concentration in brain tissues. Hence, we believe that for a proper interpretation and evaluation of the VMR during BH it is necessary to observe the reactivity pattern during the apnea phase. We propose to measure the slopes of the O2Hb and of the CO2Hb concentration signals and to use them for quantifying VMR during voluntary breath-holding. This quantity, in fact, is strictly related to the time course of the hemoglobin concentration signal. This index is also implicitly normalized with respect to the BH duration; this enables direct a comparison of the results among different subjects.
NIRS vasoreactivity patterns
As pointed out above, the BHI is a measure of VMR that relates the baseline to the post-stimulus values. Cerebral concentrations of O2Hb and CO2Hb, however, strongly vary during BH as consequence of vasodilation and of the local oxygen demand; thus, a more complete evaluation of VMR should be made by taking into account what happens during the BH phase.
Figure 2 reports an example of the changes occurring in the O2Hb (red line) and CO2Hb (blue line) concentrations during BH of a single healthy subject. Three main features can be observed on the time course of the two concentrations:
1. an initial phase, similar to the the baseline, in which the two chromophores concentrations do not significantly change;
2. the VMR phase, in which there is a strong increase of the O2Hb (and, hence, of the total hemoglobin, that roughly corresponds to the regional cerebral blood volume) while the CO2Hb is kept at a baseline level;
3. a plateau phase when the vasodilation has already reached its maximum, characterized by an almost constant level of O2Hb and a progressive increase of the CO2Hb level.
At the end of the BH, a recovery phase takes the concentration signals to baseline values. Despite the great variability affecting the NIRS signals, we found these common features in all the subjects we tested, provided that the BH duration was at least of 20 seconds. Figure 5 reports the population averaged O2Hb (left diagram) and CO2Hb (right diagram) concentration signals during BH. In order to make the signals comparable, we normalized the BH duration of each subject and set the initial concentrations (i.e., at the BH onset) equal to zero. The superimposed vertical bars represent the instantaneous standard error. Starting from 20% of the BH duration, the O2Hb signal depicts an increase in the variability that is due to the fact that, by that time, VMR had its onset. The linear increase of the O2Hb continues until 80% of the BH duration, then variability reduces and a region of plateau can be observed. Conversely, the CO2Hb shows a more variable behavior, but its average concentration remains at baseline values almost until the 90% the BH, when an increase, which cannot be further compensated, determines the end of the BH.
Bidimensional VMR representation
Vasoreactivity is a physiological mechanism that ensures the correct brain oxygenation both in baseline conditions and dynamically in consequence of perturbations to the blood oxygenation level. Specifically, during hypoxaemia, the decrease of the arterial partial pressure of oxygen, and the consequent increase of the arterial partial pressure of carbon dioxide, triggers VMR. The mechanisms that determine the onset of vasoreactivity are still debated .
If TCD is useful to document the increased CBFV as a physiological response to an increased oxygen demand by the brain tissue and to estimate the drop of the pial arteries resistance, NIRS could be proficiently used to monitor VMR in relation to the local amount of oxygen consumption and extraction. To this purpose, we propose to observe the VMR pattern in a two-dimensional plane, where it is possible to monitor the instantaneous balancing of the two types of hemoglobin and to determine how autoregulation varies the concentration of the two chromophores.
A validation of these result is not straightforward: there are no studies, that we are aware of, that derived such bidimensional patterns from NIRS signals. However, the highly repeatable pattern we found in normal subjects suggests that cerebral autoregulation shows common features when counteracting the effects of BH. From a methodological point of view, we believe that the observation of the bidimensional pattern may be of help in interpreting more complex practical situations where autoregulation is impaired: in these conditions, a different balancing of the two chromophore concentrations could be expected. The following section reports two case studies, whose TCD and NIRS data are compared to our normative data.
Subject A – current smoker
BHIs derived from TCD and NIRS signals for the case studies. Values of the BHI and of the slope of the O2Hb and CO2Hb concentration signals derived from the NIRS data for the two case studies. The first row reports the BH indicators for subject A, the second row reports the same indicators for the first test of subject B, and the third row reports the same indicators for the second test of subject B.
BHI V (%/s)
Subject B – 1st test
Subject B – 2nd test
Subject B – post-stroke subject
Even though further studies are required, we believe this analysis methodology could be useful for monitoring and quantifying the effects of neurorehabilitation trials.
In this paper we proposed a methodology for the assessment of VMR during voluntary BH. This methodology relates oxygen supply to cerebral blood flow by calculating BHIs based on TCD and NIRS data. We introduced a bidimensional representation of VMR during BH that we consider important to monitor the unbalancing between O2Hb and CO2Hb as consequence to a varied local oxygen demand.
On a population of 20 healthy subjects, we showed that the increment of the cerebral blood flow velocity in the middle cerebral artery is linearly correlated to the increment of the O2Hb when vasoreactivity is triggered by voluntary breath holding. Moreover, we provided normative BHI values on this sample population.
We observed that the vasoreactivity pattern of healthy subjects is characterized by common features that are not present if autoregulation is impaired: as an example we presented two case studies (a current smoker healthy subject and a post-stroke subject) and reported their BHIs and their bidimensional VMR patterns.
We believe these normative data could be useful when assessing vasoreactivity of subjects suffering both from chronic than acute pathologies with a direct impact on cerebral circulation.
From a methodological point of view, this joint analysis of TCD and NIRS signals could be used as a low-cost procedure for the bedside assessment of patients. Even though further studies are required in order to test the technique's performances, we consider this methodology as promising and we are planning protocols to monitor the effects of neurorehabilitation protocols in post-stroke patients.
The Authors would like to thank Dr. Silvia Delsanto (Biolab, Dipartimento di Elettronica, Politecnico di Torino) who revised the final draft of the manuscript and who suggested technical improvements, and Dr. Pierangela Giustetto (visiting scientist at the Presidio Sanitario Gradenigo, Torino) who helped in the interpretation of early studies and in the experimental protocol refinement.
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