Determining differences between critical closing pressure and resistance-area product: responses of the healthy young and old to hypocapnia

Healthy ageing has been associated with lower cerebral blood flow velocities (CBFVs); however, the behaviour of hemodynamic parameters associated with cerebrovascular tone (critical closing pressure, CrCP) and cerebrovascular resistance (resistance-area product, RAP) remains unclear. Specifically, evidence supports ageing being associated with greater cerebrovascular tone and resistance during exercise with elevated CrCP and RAP in older individuals at rest and during exercise. Comprehensive hemodynamic assessment of CrCP and RAP during hyperventilation-induced hypocapnia in two distinct age groups (young ≤ 49 and old > 50) has not been described. CBFV in the middle cerebral artery (CBFV, transcranial Doppler), blood pressure (BP, Finometer) and end-tidal CO2 (EtCO2, capnography) were recorded in 104 healthy individuals (43 young [age 33.8 (9.3) years], 61 old [age 64.1 (8.5) years]) during a minimum of 60 s of metronome-driven hyperventilation-induced hypocapnia. Autoregulation index was calculated as a function of time, using a moving window autoregressive-moving average model. CBFV was reduced in response to age (p < 0.0001) and hypocapnia (p = 0.023) (young 57.3 (14.4) vs. 44.9 cm s−1 (11.1), old 51.7 (12.9) vs. 37.8 cm s−1 (9.6)). Critical closing pressure (CrCP) increased significantly in response to hypocapnia (young 37.6 (18.5) vs. 39.7 mmHg (16.0), old 33.9 (13.5) vs. 39.3 mmHg (11.4); p < 0.0001). Resistance-area product was increased in response to age (p = 0.001) and hypocapnia (p = 0.004) (young 1.02 (0.40) vs. 1.09 mmHg cm s−1 (11.07), old 1.16 (0.34) vs. 1.34 mmHg cm s−1 (0.39)). RAP and not CrCP mediates differences in cerebrovascular resistance responses to hypocapnia between the healthy young and old individuals.


Introduction
Carbon dioxide (CO 2 )-induced changes in vasomotor tone are influenced by several interactors including blood pressure, hypoxia, neuronal and autonomic activity [1]. However, the literature surrounding cerebrovascular reactivity (CVR) changes during healthy ageing remains an area of controversy [2,3]. Cerebral hemodynamic parameter behaviours over the physiological range of arterial CO 2 (PaCO 2 ) and improvem e n t s i n c e r e b r a l a u t o r e g u l a t i o n ( C A ) d u r i n g hyperventilation-induced hypocapnia have been well described [4][5][6]. During disease states, namely carotid stenosis and stroke, impaired CVR or CA are associated with increased risk for ischaemic events or worse stroke outcome, respectively [7,8]. The lack of capability of cerebral vessels to modify their calibre in response to CO 2 stimulus represents a crucial biomarker to differentiate health and disease [7]. Unfortunately, in order to progress our understanding, factors influencing CVR and CA including age and sex need to be taken into consideration. Zhu and colleagues [9] demonstrated advanced age is associated with lower resting CBFV and increased CVR during hypocapnia. Interestingly, they found increased CVR during hypercapnia, which contravenes findings from several other studies [10,11]. The association of ageing with lower CBFV appears to be well accepted [10]. Though others have found no age-associated differences in CVR, with suggestions that arterial stiffness explains agerelated differences in cerebrovascular conductance [12]. Importantly, the behaviour of hemodynamic parameters associated with cerebrovascular tone (critical closing pressure, CrCP) and cerebrovascular resistance (resistance-area product, RAP) with respect to ageing remains unclear. Specifically, evidence supports ageing being associated with greater cerebrovascular tone and resistance during exercise with elevated CrCP and RAP in older individuals at rest and during exercise. In 2019, a large study demonstrated once more that advancing age is associated with decreases in CBFV, increases in CVR and reduced vasoconstriction during hypocapnia, though no reference to standard hemodynamic parameters was provided [13]. This aligns with the lack of data on the separate effects of the interaction of age with hypocapnia using RAP and CrCP, instead of CVR.
There is a distinct lack of studies assessing vasoconstrictive stimuli as compared to vasodilative studies [14]. McKetton and colleagues [14] described the limitations of using blood oxygenation level-dependent (BOLD) magnetic resonance imaging (MRI) to assess hypocapnic conditions, including inability to hyperventilate without motion artefacts and elevated inter-subject variability. Longitudinal assessment of CVR using transcranial Doppler over a time course of 1 to 3 years has shown no significant differences from baseline CVR, demonstrating robust reproducibility and acceptable intersubject variability [11]. Importantly, Galvin and colleagues [15] demonstrated elevated CVR during hypocapnia in those with coronary artery disease and age-matched controls, suggesting the improved CVR and lower CBFV may provide insight into important mechanisms underlying neurological risk with ageing. Changes in CVR are considered to occur beyond the young-old age category (> 50 years), with veryold (> 80 years) exhibiting similar responses to the young-old [12].
This study aimed to identify if healthy ageing is associated with differences in response to CO 2 change. Specifically, to understand the relationship between key CA metrics, the autoregulation index (ARI) calculated using the linear autoregressive-moving average (ARMA) model [6] to quantify the influence of CA or EtCO 2 on CBFV, as well as assessing resistance-area product (RAP, primary determinant of cerebrovascular resistance) and critical closing pressure (CrCP, measure of cerebral arterial tonus and intracranial pressure). We hypothesise that no differences exist in key hemodynamic parameters (CBFV, HR (heart rate), ABP (arterial blood pressure), CrCP, RAP and ARI) between the young (< 50 years) and old (> 50 years) during hypocapnic challenge. By understanding the differences and interactions between these central and peripheral parameters, there exists an opportunity to determine the mechanisms governing the regulation of cerebrovascular tone and dynamic CA processes during healthy ageing.

Subjects and measurements
The study was conducted in accordance with the Declaration of Helsinki (2000). Ethical approval was obtained from the University of Leicester Ethics Committee (Reference: jm591-c033) and the Northampton Research Ethics Committee (11/ EM/0369). Healthy volunteers were recruited from a variety of settings including the local community, University departmental staff, students and their relatives. Participants aged above 18 years were included. Exclusion criteria were physical disease in the upper limb, poor insonation of both temporal bone windows and any significant history of cardiovascular, neurological or respiratory disease. Smokers were excluded. All participants provided written, informed consent. The dataset generated from the younger individuals has been used to inform related publications [5,6,16].

Experimental protocol
The research was undertaken in the University of Leicester's Cerebral Hemodynamics in Ageing and Stroke Medicine (CHiASM) research laboratory, maintained at a constant ambient temperature of approximately 24°C and free of distraction. For the purposes of the study, participants were asked to refrain from caffeine and alcohol for a minimum period of 4 h prior to measurements being undertaken. Beat-to-beat BP was recorded continuously using the Finometer® device (FMS, Finapres Measurement Systems, Arnhem, Netherlands), which was attached to the middle finger. The servocorrecting mechanism of the Finometer® was switched on and then off prior to measurements. The hand bearing the finger cuff was at the level of the heart to negate any hydrostatic pressure bias. HR was recorded using a standard 3-lead electrocardiogram (ECG).
EtCO 2 was measured throughout using small nasal cannulae (Salter Labs) connected to a capnograph (Capnocheck Plus). Bilateral insonation of the middle cerebral arteries (MCAs) was performed using transcranial Doppler (TCD) ultrasound (Viasys Companion III; Viasys Healthcare) with a 2 MHz probe. The probes were secured in place with a head-frame that was adjusted to ensure comfort at the outset. The MCAs were identified according to two main characteristics: signal depth and velocities.
Measurements were continuously recorded at a rate of 500 samples/s in the PHYSIDAS data acquisition system (Department of Medical Physics, University Hospitals of Leicester NHS Trust). Systolic and diastolic brachial BP readings (OMRON Model 705IT) were performed at each stage of the measurements (normocapnia and hypocapnia) with a minimum of three recordings per individual. These values were then used to calibrate the Finometer recordings.

Hyperventilation induction strategy
Following a 20-min supine stabilisation period, a 5-min supine baseline recording was taken of the subject breathing spontaneously at rest. Hyperventilation strategies were conducted at least once, with repeated assessments conducted where possible, with 5-min intervals between each to allow stabilisation of all parameters and return to normocapnia. The hyperventilation induction strategies involved 60 s of rest, with hyperventilation being maintained for a minimum of 90 s whilst supine. Use of a continuous metronome (KORG Metronome MA-30) started at a rate analogous to that of the subject's baseline resting rate. After 30 s to 1 min of baseline recording, the rate was increased gradually over a period of 60 s to reach a hyperventilation rate 40% greater than baseline (around 25 breaths per minute). This was maintained for a further minimum of 60 s and followed by a minimum of 90 s rest.

Data analysis
Data collected corresponded to individual recordings for each participant at baseline and during hypocapnia. First, recordings were inspected visually and calibrated to measure systolic and diastolic OMRON BP. Narrow spikes (< 100 ms) were removed using linear interpolation and the CBFV recording was then passed through a median filter. All signals were then low pass filtered with a zero-phase Butterworth filter with cutoff frequency of 20 Hz. Automatic detection of the QRS complex of the ECG, to mark the R-R interval, was used, but visual inspection was also undertaken with manual correction whenever necessary. This allowed HR, mean ABP and mean CBFV to be calculated for each cardiac cycle. The peak of the EtCO 2 signal was detected, and breath-by-breath values were linearly interpolated and resampled in synchrony with the cardiac cycle.
Baseline files were analysed using a moving window autoregressive-moving average (MW-ARMA) model as described by Dineen and colleagues [17]. Initially, an ARMA model is adopted to estimate the CBFV response to a step change in BP and the autoregulation index (ARI) was estimated by comparison with the 10 template CBFV step responses proposed by Tiecks and colleagues [18] using the first 60 s of data. The 60-s window was then shifted by 0.6 s and a new estimate of ARI was calculated. This process was repeated until the end of the signal was reached thus generating ARI estimates at each 0.6-s intervals [17]. This produced multiple estimates of ARI, which were then averaged to produce a single baseline ARI value for each file. Having estimates of ARI every 0.6 s is sufficient to represent changes that can take place due to hypocapnia, even at very high respiratory rates caused by hyperventilation, which produce estimates of EtCO 2 with time intervals always longer than 2 s. The critical closing pressure (CrCP) and resistance-area product (RAP) were estimated using the first harmonic method [19] as demonstrated by the two equations provided by Panerai [19]: CrCP can also be estimated using a frequency-domain approach and application of the Fourier transform (Eq. 1): where V(f), P a (f) and CrCP(f) are the Fourier transforms of CBFV, ABP and CrCP, respectively. RAP is assumed to be constant. If CrCP is also assumed to be constant, it will be zero for all values of f > 0. Applying this rule to the first harmonic (f = 1) leads to For the hyperventilation strategy, continuous estimates of ARI were produced for each file using the same MW-ARMA model. These were then digitally marked at the point of EtCO 2 increase (signifying the end of hyperventilation) as this proved to be the most recognisable and reproducible point. Marked files were synchronised at 90 s.

Statistical analysis
Data normality was confirmed with the Kolmogorov-Smirnov test. Baseline measurements were assessed for differences between values derived for right and left hemispheres using a paired Student's t test. These were averaged when no significant differences were found. All peripheral and cerebral hemodynamic values were compared using multiple independent t tests with Bonferroni correction to counteract multiple comparisons. Values of p < 0.05 were considered significant. Pearson's correlation coefficient analysis was used to assess for specific relationships between variables with positive interactions and differences between age groups.

Results
One hundred and four healthy individuals, 43 young (≤ 49) and 61 old (≥ 50), were studied. The mean age of the younger group was 33.8 (9.3) and older group 64.1 years (8.5) ( Table 1). The younger group had 24 females (56%) and the older group had 28 (46%) females. Figure 1 shows representative recordings of the CBFV response to hyperventilation in a single subject.

Effect of hypocapnia on cerebral and peripheral hemodynamics
As anticipated, there was a significant reduction in EtCO 2 with hyperventilation which was not age dependent (p < 0.0001). In addition, this was associated with increased HR (p = 0.016) and MAP (p = 0.026) ( Table 1). The population response to the hypocapnic challenge is demonstrated in

Main findings
The four important findings of this study are (1) demonstration for the first time that young and old healthy individuals have different cerebrovascular resistance mechanisms during hypocapnia; (2) older individuals mount a significantly greater HR response to hypocapnia (3) demonstration that dynamic response to hypocapnia, as expressed by the ARI, is CO 2 -dependent; and (4) this study supports previous work demonstrating increases in CrCP and RAP in response to hypocapnia. Importantly, this study distinguishes differences between RAP and CrCP with reference to healthy ageing (Fig. 4).

Effect of age and hypocapnia on cerebral hemodynamics
This study does not demonstrate an age-related elevation in cerebrovascular response to hypocapnic challenge as seen in a prior study [15]. CrCP has previously been shown to increase with hypocapnia though age was not considered within this particular study [4]. Importantly in this study, ARI improved with CO 2 change (p = 0.008) but did not vary across age groups, demonstrating comparable ability in older and younger individuals to autoregulate during hypocapnia. This finding concurs with previous work demonstrating a lack of an association between increasing age and ARI change during normocapnia [20]. However, most marked is the dramatic rise in RAP in the older individuals beyond a non-significant rise Values are mean (SD). CBFV, CrCP, RAP and ARI were averaged for the right and left MCAs CBFV cerebral blood velocity, HR heart rate, MAP mean arterial blood pressure; EtCO 2 end-tidal carbon dioxide, CrCP critical closing pressure, RAP resistance-area product, ARI Autoregulation Index in CrCP between the two age groups. RAP characterises vascular resistance according to the slope of the linear ABP-CBF relationship, and CrCP refers to the theoretical pressure at which CBF falls to zero and is thought to be a marker of cerebrovascular transmural wall tension (therefore incorporating intracranial pressure effects) [21]. The rise of RAP in older individuals has been demonstrated at baseline as shown in this study, though not during hypocapnia [21]. This study provides evidence that older normotensive individuals have greater RAP to younger individuals during normocapnia and hypocapnia [19]. Crucially, as has been shown during hypotension, the responses of CrCP and RAP are similar, as was demonstrated in this study, albeit with very mild hypotension induced during hypocapnia. Ogoh and colleagues [22] previously showed older normotensive adults had an elevated RAP but not CrCP, and reassuringly, this has been confirmed within this study [22]. Prior work has demonstrated a greater relative change in CrCP in younger adults during upright posture with a suggestion CrCP response in younger adults maybe more sensitive [21]. This study provides for the first time an arterial gas paradigm to refute inferences from prior studies assessing metabolic demand, which highlight that CrCP is crucial in healthy ageing [21]. Overall, the study provides a more detailed perspective on the influence of CrCP and RAP on governance of mechanisms involved in vasomotor control during healthy ageing and hypertensive states [22]. The results highlight RAP as a key differentiator during which accentuates cerebrovascular resistance during hypocapnia as individuals age.

Effect of age and hypocapnia on peripheral hemodynamics
Nowak and colleagues [23,24] demonstrated that hyperventilation-induced cerebral vasoconstriction led to a greater HR increment in those with orthostatic intolerance. Cerebral vasomotor response to hypocapnia was increased during this hypocapnia period with an expected lower CBFV. Despite a lack of data supporting this phenomenon in older as opposed to younger individuals, this study confirms the presence of a greater HR response in circumstances of hypocapnia and improved CVR. Brown and colleagues demonstrated that hypercapnia increases minute ventilation with little effect on HR, and prior work has shown that mild hyperventilation does not affect HR variability (HRV) indices [25]. However, this study, in a large cohort of individuals across a wide spectrum of ages, provides a different perspective to previously accepted HRV literature. At rest, during normocapnia, older age individuals have consistently lower HRV than younger people [26]; however, under experimental hypocapnic challenge, there is a paradoxical response. This paradoxical response has been documented during hypoxaemia with older men having smaller percentage increases in sympathetic nervous system activity from their elevated baselines, though demonstrating an attenuated tachycardia during acute hypoxaemia [26]. This study provides novel data highlighting the presence of a similar attenuated tachycardia during hypocapnia in older adults. The behaviour of RAP with reference to HR during hypocapnia is of interest. This relationship between RAP and posture change has been previously assessed in the young and old [21]. This study showed that during change from supine to sit to stand (likely precipitating a HR response) was not associated with a significant change in RAP or indeed CrCP [21]. However, the older had greater RAP during changes in posture with a positive trend. Our study supports the findings for RAP, though refutes the importance of CrCP demonstrated during posture change, for hypocapnia conditions. However, our study does support the lack of significant differences in the behaviour of CrCP between age groups, as shown with posture change. In addition, our study shows an interaction effect between age and CO 2 change for HR response, though we cannot draw comparisons as to the behaviour of RAP and CrCP with this work as this study is the first study to assess such variables across the adult lifespan during hypocapnic conditions [13].

Clinical considerations
The presence of raised RAP and CrCP in older adults as compared to younger adults raises the importance of cerebrovascular resistance as rest and during hypocapnia as an important Corresponding shaded areas represent the ±SD boundaries marker of ageing vascularity. In addition, the importance of standardising PaCO 2 operating points for clinical studies comparing data with patient data with healthy controls is paramount [6,27]. This study does not support a HR response being associated with the cerebrovascular resistance index, RAP. This therefore highlights the potential relationship between cerebrovascular disease and cardiac disease with worsening cerebrovascular resistance possibly propagating effects on HR and overall cardiac demand. Importantly, with prior work demonstrating raised RAP and similar CrCP during hypertensive states, there is a suggestion that chronic hypertension may affect the ability of vessels to respond to CO 2 stimulus as opposed to alterations in tone. A number of previous studies assessing CrCP in health and disease states have shown that CrCP is influenced by structural and physiological parameters and responds to changes in pressure within the cranial cavity [19]. This phenomenon was highlighted by Robertson and colleagues [21] during their reference to Bstructural vs functional^, with the former being more closely related to adaptation to chronic hypertension [21]. The key question is the potential for hypocapnic challenge to be used as a biomarker of healthy ageing and feasibility of such assessments. With a well-tolerated response across a wide age range, there is scope to extend this assessment to those with   Fig. 2 (continued) disease states for which a vasoconstrictive response would not be considered adverse (i.e. not in acute ischaemic stroke but perhaps in vascular dementia syndromes for which a spectrum of small vessel disease severity exists).

Limitations
Several limitations are to be considered within this study setup. Firstly, when considering TCD studies, in order to estimate CBF from CBFV, it is presumed the MCA diameter must remain constant. Prior work has shown changes in MCA diameter occur at extreme hypercapnia; however, changes during hypocapnia are less understood [28,29]. Secondly, the authors have previously demonstrated sex differences across the physiological range of PaCO 2 , with a downward parallel shift of the RAP dependency on EtCO 2 . This demonstrated males have higher RAP values than females across the range of PaCO 2 . Therefore, with a similar male and female split in both groups, these effects are unlikely to have been significant, though should be acknowledged. Importantly, recent work has shown that autoregulation is able to withstand the effects of female sex hormones and therefore, the pre-and post-menopausal effects influences are less likely to have affected the results [30]. Thirdly, sampling expired CO 2 with nasal prongs during hyperventilation can underestimate EtCO 2 ; however, unlike the face mask, they do not precipitate a sympathetic response and therefore, it is considered an acceptable pragmatic methodological setup for this cerebral hemodynamic study [31]. Fourthly, the potential influence of current or historical cardiorespiratory fitness is not considered within this study, and this has previously been shown to influence cerebrovascular reactivity to CO 2 [32] and dynamic cerebral autoregulation [33,34]. Lastly, decline in hypercapnic reactivity was shown in the Rotterdam cohort in individuals aged between 75 and 90 years [35]. Although they did not include hypocapnia, this physiological phenomenon, and different behaviour exhibited by the real extremes of age, is beyond the considered age groups within this study.

Future directions
This study provides further support for vascular responses to PaCO 2 as a biomarker of ageing and describes mechanistic changes associated with ageing in greater detail than prior studies. However, further work is required to elucidate this phenomenon in disease states and to characterise variation between different cerebrovascular pathologies. Specifically, assessing RAP response during exercise whereby HRV and  response is broader and reliable correlations between the heart-brain axis can be investigated. In a large group of young and old healthy individuals, we show resistance-area product (RAP) and not critical closing pressure (CrCP) mediates differences in cerebrovascular resistance responses to hypocapnia between the healthy young and old individuals. These novel findings underline the importance of assessing the separate contributions of RAP and CrCP. In addition, our findings support previous studies demonstrating exacerbated heart rate responses in the old.