Cerebrovascular reactivity alterations due to subconcussive repetitive head trauma in asymptomatic high school football players

Chronic neurological damage as a result of chronic repetitive head trauma is a major concern for football athletes today. Repetitive concussions have been linked to many neurological disorders. Recently, it has been reported that repetitive subconcussive events can contribute to long-term neurodegeneration. For these reasons, it is important to understand the effect repetitive subconcussive head trauma has on brain health in young athletes. Past research has demonstrated that cerebrovascular reactivity (CVR), an important mediator of cerebrovascular regulation, is impaired following mild traumatic brain injury (mTBI). This impairment increases susceptibility to secondary injury following mTBI. In this study, Breath-Hold (BH) task based functional Magnetic Resonance Imaging (fMRI) was used to track CVR changes in asymptomatic high school football athletes across three competition seasons. Athletes in the first competition season had higher exposure to head impacts than the athletes during the second and the third seasons. Baseline scans were acquired before the start of the season, and follow-up scans were obtained during and after the season to track the potential changes in CVR as a result of experienced trauma. Noncollision-sport athletes were scanned over two sessions as controls during the first and third competition season. CVR decreased significantly in football athletes during the first half of their season in the first completion season but not in any other competition seasons. Controls did not show any significant changes in CVR. The results suggest that athletes getting higher exposure to head impacts in short duration of time drives cerebrovascular changes that may place athletes at higher risk of getting injured. These results also indicate that the brain may not be able to adapt quickly to abrupt increases in contact activity (as associated with the beginning of practice and competition), transiently increasing risk for symptomatic injury

BOLD ФМРТ КАРТИРОВАНИЕ ФУНКЦИОНАЛЬНО ЗНАЧИМЫХ ЗОН КОРЫ У ПАЦИЕНТОВ С ОПУХОЛЯМИ ГОЛОВНОГО МОЗГА С ПРИМЕНЕНИЕМ ДОПОЛНИТЕЛЬНЫХ ФИЗИОЛОГИЧЕСКИХ ПАРАМЕТРОВ

Introduction. Preoperative fMRI often shows a reduced BOLD response in the areas located perifocal to brain tumors caused by pathological vasoreactivity as a result of neurovascular uncoupling.The aim of this study was an accurate identification of the eloquent cortical areas near brain tumors by measuring vasoreactivity using the breath holding test.Material and methods. 23 patients with brain tumors located near eloquent cortical areas underwent fMRI mapping of the language and / or motor cortical areas depending on the location of the tumor. Breath hold test was  also included, the design of which coincided with motor and language block paradigms.Results. A statistical dependency between motor, speech tasks and breath-hold BOLD-response was included into the BOLD analysis using coherence. fMRI activation maps generated by this method showed activation in the eloquent areas adjacent to brain tumors which were not detected by the standard analysis. This study shows that neurovascular uncoupling affects the accuracy of BOLD fMRI in brain tumors.Conclusion. The results of fMRI mapping can be partially improved by the incorporating vasoreactivity measurements into a standard analysis.Введение. Предоперационные BOLD фМРТ часто показывают понижение BOLD-ответа, вызванное патологической вазореактивностью вследствие нейроваскулярного разобщения в перифокальных опухолям зонах головного мозга.Цель исследования: разработать алгоритм получения карт BOLD-активации, учитывающий измененную гемодинамику у пациентов с внутримозговыми и внемозговыми опухолями, при сопоставлении карт BOLD-ответа на моторные и речевые задания с/без включения задачи задержки дыхания.Материалы и методы. В исследование включено 23 пациента с опухолями головного мозга, расположенными вблизи функционально значимых зон коры. Всем пациентам осуществлялось фМРТ-картирование речевых и/или двигательных зон коры головного мозга в зависимости от локализации опухоли, а также проводился тест с задержкой дыхания, дизайн которого совпадал с моторными и речевыми блоковыми парадигмами.Результаты. Полученная зависимость между моторными или речевыми заданиями и ответом на задержку дыхания была включена в постобработку данных фМРТ с использованием когерентного анализа. Карты, построенные этим методом, показали клинически значимые области активации, прилегающие к опухолям головного мозга, не выявлявшиеся с помощью стандартного метода анализа.Заключение. Результаты фМРТ-картирования можно частично улучшить включением данных измерения вазореактивности в стандартный анализ

Separating vascular and neuronal effects of age on fMRI BOLD signals.

Accurate identification of brain function is necessary to understand the neurobiology of cognitive ageing, and thereby promote well-being across the lifespan. A common tool used to investigate neurocognitive ageing is functional magnetic resonance imaging (fMRI). However, although fMRI data are often interpreted in terms of neuronal activity, the blood oxygenation level-dependent (BOLD) signal measured by fMRI includes contributions of both vascular and neuronal factors, which change differentially with age. While some studies investigate vascular ageing factors, the results of these studies are not well known within the field of neurocognitive ageing and therefore vascular confounds in neurocognitive fMRI studies are common. Despite over 10 000 BOLD-fMRI papers on ageing, fewer than 20 have applied techniques to correct for vascular effects. However, neurovascular ageing is not only a confound in fMRI, but an important feature in its own right, to be assessed alongside measures of neuronal ageing. We review current approaches to dissociate neuronal and vascular components of BOLD-fMRI of regional activity and functional connectivity. We highlight emerging evidence that vascular mechanisms in the brain do not simply control blood flow to support the metabolic needs of neurons, but form complex neurovascular interactions that influence neuronal function in health and disease. This article is part of the theme issue 'Key relationships between non-invasive functional neuroimaging and the underlying neuronal activity'.This work is supported by the British Academy (PF160048), the Guarantors of Brain (G101149), the Wellcome Trust (103838), the Medical Research Council (SUAG/051 G101400; and SUAG/046 G101400), European Union’s Horizon 2020 (732592) and the Cambridge NIHR Biomedical Research Centre

Reproducibility of BOLD signal change induced by breath holding.

Blood oxygen level dependent (BOLD) contrast is influenced by some physiological factors such as blood flow and blood volume that can be a source of variability in fMRI analysis. Previous studies proposed to use the cerebrovascular response data to normalize or calibrate BOLD maps in order to reduce variability of fMRI data both among brain areas in single subject analysis and across subjects. Breath holding is one of the most widely used methods to investigate the vascular reactivity. However, little is known about the robustness and reproducibility of this procedure. In this study we investigated three different breath holding periods. Subjects were asked to hold their breath for 9, 15 or 21 s in three separate runs and the fMRI protocol was repeated after 15 to 20 days. Our data show that the BOLD response to breath holding after inspiration results in a complex shape due to physiological factors that influence the signal variation with a timing that is highly reproducible. Nevertheless, the reproducibility of the magnitude of the cerebrovascular response to CO2, expressed as amplitude of BOLD signal and number of responding voxels, strongly depends on duration of breath holding periods. Breath holding period of 9 s results in high variability of the magnitude of the response while longer breath holding durations produce more robust and reproducible BOLD responses

Neuropharmacological Investigation Of Stress And Nicotine Self-Administration Among Current Cigarette Smokers

ABSTRACT NEUROPHARMACOLOGICAL INVESTIGATION OF STRESS AND NICOTINE SELF-ADMINISTRATION AMONG CURRENT CIGARETTE SMOKERS by ERIC ANDREW WOODCOCK August 2017 Advisor: Dr. Mark K. Greenwald Major: Neuroscience (Translational) Degree: Doctor of Philosophy Nicotine use, especially cigarette smoking, is a significant public health problem. Existing pharmacotherapies attenuate nicotine craving and withdrawal symptoms. However, the majority of patients relapse within the first year of treatment. Treatment studies indicate a commonly cited precipitant to smoking relapse is stress. Pharmacotherapies do not attenuate, and may exacerbate, the effects of acute stress. Experimental studies (preclinical and clinical) indicate that acute stress potentiates drug-seeking behavior across drugs of abuse. Despite a robust literature linking acute stress and substance use, neurobiological mechanisms remain poorly understood. A more complete understanding of the neurobiological effects of acute stress on brain function may facilitate development of novel interventions. Adjunctive stress-blunting medications may improve the effectiveness of existing pharmacotherapies. The present study investigated the effects of pharmacological stress-induction among cigarette smokers. Non-treatment-seeking cigarette smokers were recruited locally and screened for psychiatric, medical, and neuroimaging contraindications. Using a double-blind, placebo-controlled within-subject random cross-over design, participants (N = 21) completed two oral-dosing experimental sessions: active (yohimbine [YOH] 54mg + hydrocortisone [HYD] 10mg) and placebo (YOH 0mg + HYD 0mg) stress. Prior research indicated that YOH+HYD is a robust pharmacological stress-induction technique that stimulates the Autonomic Nervous System (ANS) and Hypothalamic-Pituitary-Adrenal (HPA) axis systems, increases circulating levels of noradrenaline and cortisol (two primary stress hormones), and potentiates drug-seeking behavior. Throughout each experimental session, subjective and physiological effects were measured. In addition, participants completed a 60min magnetic resonance imaging (MRI) scan which consisted of three task paradigms: 1) letter 2-back, 2) smoking cued letter N-back, and 3) breath-hold challenge. Participants completed a working memory paradigm (letter 2-back) during proton functional magnetic resonance spectroscopy (1H fMRS). Left dorsolateral prefrontal cortex (dlPFC) neurochemistry was evaluated during letter 2-back task performance. Next, participants completed a cued N-back paradigm that consisted of images (cigarette smoking or neutral) centered behind capitalized letters across three levels of N-back task difficulty: 0-, 1-, and 2-back. Finally, participants were instructed (visually) to control their breathing across three phases: ‘normal’ breathing, paced breathing (3s in/3s out), and breath-hold challenge (11s). After the MRI scan, participants completed a choice progressive ratio task. Across 11 independent choice trials, participants could earn one cigarette puff (preferred brand) or money ($0.25) via behavioral responding. Each successive unit earned (puffs or money, independently) was associated with a higher response requirement (progressive ratio schedule). At the end of the 30min task, participants smoked the exact number of cigarette puffs earned and/or were provided the amount of money earned. Number of puffs earned and smoked was a direct measure of nicotine-seeking and self-administration behavior (nicotine motivation). Participants were compensated for their time. Results indicated that oral pretreatment with YOH+HYD increased biomarkers of a physiological stress response: systolic and diastolic blood pressure, heart rate, saliva cortisol and α-amylase (indirect biomarker of noradrenaline levels), relative to placebo. YOH+HYD potentiated nicotine-seeking and self-administration behavior (controlling for nicotine dependence level), relative to placebo. Appetitive and relief-motivated cigarette craving, nicotine withdrawal symptoms, negative affect, and anxiety levels increased throughout each session, but did not differ by experimental session (active vs. placebo stress). Similarly, positive affect decreased throughout each session, but did not as a function of stress. 1H fMRS indicated that letter 2-back performance increased left dlPFC glutamate (GLU) levels relative to interleaved fixation cross rest (indicative of task engagement) during the placebo, but not active stress, session. Further, YOH+HYD impaired letter 2-back response accuracy, relative to placebo. Across N-back levels (0-, 1-, and 2-back), fMRI indicated more robust neural activation across ‘reward’-associated brain regions in response to smoking images (\u3e neutral images) during placebo, relative to active stress. Results demonstrated YOH+HYD induced a sustained physiological stress response (ANS and HPA axis) and potentiated nicotine-seeking and self-administration. YOH+HYD attenuated dlPFC task engagement and impaired response accuracy during a well-established working memory task. These findings provide experimental support for a plausible neurobiological mechanism through which acute stress may potentiate nicotine self-administration. Acute stress-impaired dlPFC function may potentiate nicotine self-administration and, among abstinence-motivated individuals, precipitate smoking relapse. Prior research demonstrated dlPFC function is associated with a host of cognitive processes (e.g. delayed gratification, self-control, decision making, etc.) associated with prolonged smoking abstinence. Future studies are needed to confirm this hypothesis, investigate dose-response relationships, and evaluate the efficacy of stress-blunting medications in combination with existing pharmacotherapies for smoking cessation

Instrumentation and methods for frequency-domain and multimodal near-infrared spectroscopy

In this thesis, instrumentation for a frequency-domain (FD) near-infrared spectroscopy (NIRS) device and for multimodal brain measurements was implemented. Different techniques were applied to human and newborn lamb brain studies. A method to detect light leakage in NIRS measurements was developed. The FD instrument, developed at the Helsinki University of Technology was extended, by implementing 16 pseudo-differential preamplifiers for the analog-to-digital converters with a low noise and excellent interchannel isolation. An instrumentation for a digital signal-processor based lock-in amplifier was also developed. Methods for increasing the number of wavelengths and source positions in the imaging instrument were studied. A second-generation source system with a fast fiber-optic switch and four high-power laser diodes with a low noise temperature-stabilizing electronics was implemented. The imaging device was placed into a cabinet to enable its portability. New detection and source fiber terminals were developed for multimodal brain studies. The different versions of the imaging instrument were applied to four human brain measurements. In a breath-holding and hyperventilation study, the effects of source-detector distance (SDD) and measurement wavelength on the contrast of NIRS responses and the frequency content of signals were studied. Hemodynamic changes in the human brain related to the changes in sleep stages were detected. The multimodal NIRS and electroencephalography measurement setup was implemented and used to study the effects of baseline blood flow changes on the visually evoked hemodynamic and neuronal responses. The feasibility of NIRS as a part of multimodal monitoring setup to detect cerebral hemodynamic changes induced by iloprost and nitric oxide in the preterm lamb brain was also demonstrated. The linearity of the FD measurement parameters as a function of SDD on the human forehead was studied. The regression of phase measurement was observed to be sensitive to light leakage from source to detectors, much more than the regression of modulation amplitude or average intensity measurement. Utilizing this observation, a method to detect light leakage based on the pathlength measurement was developed. The contrast and depth sensitivity of NIRS signals were shown to decrease in measurements where light leakage occurs

Optimization of Functional MRI methods for olfactory interventional studies at 3T

Functional MRI technique is vital in investigating the effect of an intervention on cortical activation in normal and patient population. In many such investigations, block stimulation paradigms are still the preferred method of inducing brain activation during functional imaging sessions because of the high BOLD response, ease in implementation and subject compliance especially in patient population. However, effect of an intervention can be validly interpreted only after reproducibility of a detectable BOLD response evoked by the stimulation paradigm is first verified in the absence of the intervention. Detecting a large BOLD response that is also reproducible is a difficult task particularly in olfactory Functional MRI studies due to the factors such as (a) susceptibility-induced signal loss in olfactory related brain areas and (b) desensitization to odors due to prolonged odor stimulation, which is typical when block paradigms are used. Therefore, when block paradigms are used in olfactory interventional Functional MRI studies, the effect of the intervention may not be easily interpretable due to the factors mentioned above. The first task of this thesis was to select a block stimulation paradigm that would produce a large and reproducible BOLD response. It was hypothesized that a BOLD response of this nature could be produced if within-block and across-session desensitization could be minimized and further, that desensitization could be minimized by reducing the amount of odor by pulsing the odor stimulus within a block instead of providing a continuous odor throughout the block duration. Once the best paradigm was selected, the second task of the thesis was to select the best model for use in general linear model (GLM) analysis of the functional data, so that robust activation is detected in olfactory related brain areas. Finally, the third task was to apply the paradigm and model that were selected as the best among the ones tested in this thesis, to an olfactory interventional Functional MRI study investigating the effect of food (bananas) eaten to satiety on the brain activation to the odor related to that food. The methods used in this thesis to ensure valid interpretation of an interventional effect, can serve as a template for the experimental design of future interventional Functional MRI studies

Cerebrovascular influence on brain and cognitive aging

A dysfunctional cerebrovascular system can result in severe adverse effects on brain health and cognitive aging. Recently, Fabiani et al., (2014) introduced a novel non-invasive approach of quantifying cerebrovascular health using diffuse optical imaging in a sample of older adults. This method is based on the estimation of the arterial pulse across the whole scalp. From these estimates, three indices reflecting arterial health can be extracted: pulse amplitude, arterial compliance and pulse transit time. In their initial paper, Fabiani et al. (2014) showed that, in older adults, these indices are correlated with important variables, including volumetric changes in the brain and in psychometric measures. In this thesis, Chapter 1 discusses the importance of cerebrovascular health in brain and cognitive aging followed by a brief introduction to diffusive optical methods and its advantages in quantifying cerebrovascular health. Chapter 2 contains a series of two experiments examining how changes in pulse amplitude reflect changes in cerebrovascular tone (i.e. vasodilation and vasoconstriction) of cerebral blood vessels. We used both a physiological voluntary breath holding task to track generalized changes and a cognitive Sternberg task to track localized changes in cerebrovascular tone. Further, we found that an index of cerebrovascular reactivity derived from the breath holding task was associated with age and cognitive functioning. These results indicate that cerebral pulse amplitude works well as a proxy measure of blood pressure in the brain. Chapter 3 contains a replication and extension of the work presented in Fabiani et al., (2014) to investigate changes in pulse amplitude and arterial compliance in a group of younger and older adults. The study also contains methodological improvements whereby we employed a denser optical recording array and increased data collection time substantially in order improve signal to noise ratio. The results indicate strong reliability for both pulse amplitude and arterial compliance measures. We replicated the initial findings, demonstrating that associations with age, cardiorespiratory fitness, brain anatomy and cognition can also be found across the adult lifespan. Further, we found new evidence supporting the value of regional arterial compliance in predicting working memory performance on the operation word span (OSPAN) task. Chapter 4 contains a study investigating the relationships of arterial compliance with measures of cerebral white matter lesion (manifested as white matter signal abnormalities (WMSA) on T1 weighted images) and white matter microstructure integrity (measured using DTI indices of fractional anisotropy and mean diffusivity). Using hierarchical regression, we found that arterial compliance predicts variance in WMSA over and above age and systemic pulse pressure (difference between systolic blood pressure and diastolic blood pressure), indicating that brain measures of arterial compliance have added predictive utility of WMSA volume over systemic measures of vascular health. Mediation analyses revealed that the relationship between greater age and poorer fluid intelligence (IQ) was mediated sequentially by a reduction in arterial compliance and greater WMSA volume. Additional mediation analyses involving switching the temporal sequence of arterial compliance and WMSA was not statistically significant. Further, substituting WMSA for DTI measures of FA and MD in the mediation analysis also did not reach statistical significance. These results suggest that the cerebrovascular pathway involved in age-related cognitive decline in fluid IQ are mediated primarily through arterial compliance and WMSA, but not changes in white matter microstructure measured by DTI. Tentative findings suggest that vascular damage manifested as poorer arterial compliance and WMSA volume, may converge with degradation to white matter microstructure in the fornix

Using physiological MRI to estimate dynamic cerebral autoregulation metrics: functional MRI feasibility study

Cerebral autoregulation is the homeostatic mechanism that maintains sufficient cerebral circulation despite changes in the perfusion pressure. Dynamic CA refers to the changes that occur in CBF within the first few seconds after an acute MAP change. Assessment of the CA impairment plays important role in the prognosis of many cerebrovascular diseases such as stroke, sub-arachnoid haemorrhage, as well as traumatic brain injury and neurodegenerative disorders. This thesis investigates the feasibility of using physiological MRI to estimate dynamic cerebral autoregulation (dCA) metrics. In particular, this thesis has an emphasis on measuring beat-to-beat arterial blood pressure inside the scanner to provide better understanding of the physiological aspects of dCA. Further, continuous blood pressure (BP) measures in response to different non invasive BP fluctuating methods are acquired to evaluate the reliability of these methods to induce response changes. Blood Oxygen Level Dependent (BOLD) fMRI method was used to estimate the expected variations of tissue oxygenation during induced dCA changes in healthy volunteers. The non invasive arterial blood pressure measurements were acquired using MR compatible arterial blood pressure monitoring device (NIBP-MRI/Caretaker; Biopac®). Further, sudden release of inflated thigh-cuffs (TCR) and inspiratory breath-hold (iBH) methods were used in the scanner to induce dynamic autoregulatory changes. These two methods were investigated in a pilot study, to evaluate the reliability prior to the MR study by comparing BP measurements obtained outside the scanner using non invasive methods. This pilot study included monitoring BP changes in response to four types of non invasive BP fluctuating methods. The reliability of NIBP/MRI Caretaker device was examined by comparing the BP response changes with the simultaneously acquired BP data from Finometer plethysmographic device. The cerebral autoregulation metrics were estimated by calculating the rate of regulation (RoR) following dynamic BP fluctuating events. Rate of regulation defines the rate at which the BOLD signal changes depending on MAP changes at a particular time. Further, the tissue specific regulation parameters were obtained for grey matter (GM), white matter (WM) and water shed areas (WS). The effect of iBH method on cerebral blood flow (CBF) and velocity (CBFV) was explored in a preliminary study by quantitative measures using time resolved 4D PC MRI angiography in two subjects. The mean arterial blood pressure (MAP) changes in response to TCR and iBH method were comparable. The fMRI data demonstrated BOLD signal amplitude change in response to the induced fast MAP changes. The GM and WS areas showed similar rates of regulation, and these were nominally higher than WM RoR in both TCR and iBH methods. Further, the 4D PC MRI data suggested 29% CBF-increase in response to 33% iBH in four minutes acquisition time. The acquired non invasive arterial BP measures concurrent with the BOLD signal amplitude response, allowed deriving the rate of regulation as a metric of dCA. It is not known whether this information is clinically relevant to gauge the haemodynamic risk association to cerebrovascular disease. However, BOLD signal change and CBF changes after iBH are confounded by the extent to which the CO2 gradually accumulate in response to iBH and causes an overshoot in the CBF response-change. In conclusion, the presented study indicates the feasibility of using physiological MRI to measure dCA in response to non-invasively induced MAP changes. Estimation of the dCA metrics could be improved by using advanced data fitting methods as well as controlling for physiological parameters such as PECO2

Using physiological MRI to estimate dynamic cerebral autoregulation metrics: functional MRI feasibility study

Cerebral autoregulation is the homeostatic mechanism that maintains sufficient cerebral circulation despite changes in the perfusion pressure. Dynamic CA refers to the changes that occur in CBF within the first few seconds after an acute MAP change. Assessment of the CA impairment plays important role in the prognosis of many cerebrovascular diseases such as stroke, sub-arachnoid haemorrhage, as well as traumatic brain injury and neurodegenerative disorders. This thesis investigates the feasibility of using physiological MRI to estimate dynamic cerebral autoregulation (dCA) metrics. In particular, this thesis has an emphasis on measuring beat-to-beat arterial blood pressure inside the scanner to provide better understanding of the physiological aspects of dCA. Further, continuous blood pressure (BP) measures in response to different non invasive BP fluctuating methods are acquired to evaluate the reliability of these methods to induce response changes. Blood Oxygen Level Dependent (BOLD) fMRI method was used to estimate the expected variations of tissue oxygenation during induced dCA changes in healthy volunteers. The non invasive arterial blood pressure measurements were acquired using MR compatible arterial blood pressure monitoring device (NIBP-MRI/Caretaker; Biopac®). Further, sudden release of inflated thigh-cuffs (TCR) and inspiratory breath-hold (iBH) methods were used in the scanner to induce dynamic autoregulatory changes. These two methods were investigated in a pilot study, to evaluate the reliability prior to the MR study by comparing BP measurements obtained outside the scanner using non invasive methods. This pilot study included monitoring BP changes in response to four types of non invasive BP fluctuating methods. The reliability of NIBP/MRI Caretaker device was examined by comparing the BP response changes with the simultaneously acquired BP data from Finometer plethysmographic device. The cerebral autoregulation metrics were estimated by calculating the rate of regulation (RoR) following dynamic BP fluctuating events. Rate of regulation defines the rate at which the BOLD signal changes depending on MAP changes at a particular time. Further, the tissue specific regulation parameters were obtained for grey matter (GM), white matter (WM) and water shed areas (WS). The effect of iBH method on cerebral blood flow (CBF) and velocity (CBFV) was explored in a preliminary study by quantitative measures using time resolved 4D PC MRI angiography in two subjects. The mean arterial blood pressure (MAP) changes in response to TCR and iBH method were comparable. The fMRI data demonstrated BOLD signal amplitude change in response to the induced fast MAP changes. The GM and WS areas showed similar rates of regulation, and these were nominally higher than WM RoR in both TCR and iBH methods. Further, the 4D PC MRI data suggested 29% CBF-increase in response to 33% iBH in four minutes acquisition time. The acquired non invasive arterial BP measures concurrent with the BOLD signal amplitude response, allowed deriving the rate of regulation as a metric of dCA. It is not known whether this information is clinically relevant to gauge the haemodynamic risk association to cerebrovascular disease. However, BOLD signal change and CBF changes after iBH are confounded by the extent to which the CO2 gradually accumulate in response to iBH and causes an overshoot in the CBF response-change. In conclusion, the presented study indicates the feasibility of using physiological MRI to measure dCA in response to non-invasively induced MAP changes. Estimation of the dCA metrics could be improved by using advanced data fitting methods as well as controlling for physiological parameters such as PECO2