Cholestasis in the newborn: experience of a level III Neonatal Intensive Care Unit

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(1)2015/2016. João Pedro Brandão Madureira da Silva. Assessment of cerebral vasoreactivity with transcranial. doppler in healthy portuguese adults. março, 2016.

(2) João Pedro Brandão Madureira da Silva. Assessment of cerebral vasoreactivity with transcranial doppler in healthy portuguese adults. Mestrado Integrado em Medicina. Área: Neurologia Tipologia: Dissertação. Trabalho efetuado sob a Orientação de: Doutora Elsa Irene Peixoto Azevedo Silva E sob a Coorientação de: Dr. Pedro Miguel Araújo Campos de Castro. Trabalho organizado de acordo com as normas da revista: Journal of the Neurological Sciences março, 2016.

(3) Projeto de Opção do 6º ano - DECLARAÇÃO DE INTEGRIDADE. Eu, João Pedro Brandão Madureira da Silva, abaixo assinado, nº mecanográfico 201006158, estudante do 6º ano do Ciclo de Estudos Integrado em Medicina, na Faculdade de Medicina da Universidade do Porto, declaro ter atuado com absoluta integridade na elaboração deste projeto de opção. Neste sentido, confirmo que NÃO incorri em plágio (ato pelo qual um indivíduo, mesmo por omissão, assume a autoria de um determinado trabalho intelectual, ou partes dele). Mais declaro que todas as frases que retirei de trabalhos anteriores pertencentes a outros autores, foram referenciadas, ou redigidas com novas palavras, tendo colocado, neste caso, a citação da fonte bibliográfica.. Faculdade de Medicina da Universidade do Porto, 23/03/2015. Assinatura conforme cartão de identificação:. _________________________.

(4) Projecto de Opção do 6º ano – DECLARAÇÃO DE REPRODUÇÃO. NOME. João Pedro Brandão Madureira da Silva NÚMERO DE ESTUDANTE. DATA DE CONCLUSÃO. 201006158 DESIGNAÇÃO DA ÁREA DO PROJECTO. Neurologia TÍTULO DISSERTAÇÃO/MONOGRAFIA (riscar o que não interessa). Assessment of cerebral vasoreactivity with transcranial doppler in healthy portuguese adults ORIENTADOR. Doutora Elsa Irene Peixoto Azevedo Silva. COORIENTADOR (se aplicável). Dr. Pedro Miguel Araújo Campos de Castro. ASSINALE APENAS UMA DAS OPÇÕES: É AUTORIZADA A REPRODUÇÃO INTEGRAL DESTE TRABALHO APENAS PARA EFEITOS DE INVESTIGAÇÃO, MEDIANTE DECLARAÇÃO ESCRITA DO INTERESSADO, QUE A TAL SE COMPROMETE. É AUTORIZADA A REPRODUÇÃO PARCIAL DESTE TRABALHO (INDICAR, CASO TAL SEJA NECESSÁRIO, Nº MÁXIMO DE PÁGINAS, ILUSTRAÇÕES, GRÁFICOS, ETC.) APENAS PARA EFEITOS DE INVESTIGAÇÃO, MEDIANTE DECLARAÇÃO ESCRITA DO INTERESSADO, QUE A TAL SE COMPROMETE. DE ACORDO COM A LEGISLAÇÃO EM VIGOR, (INDICAR, CASO TAL SEJA NECESSÁRIO, Nº MÁXIMO DE PÁGINAS, ILUSTRAÇÕES, GRÁFICOS, ETC.) NÃO É PERMITIDA A REPRODUÇÃO DE QUALQUER PARTE DESTE TRABALHO.. X. Faculdade de Medicina da Universidade do Porto, 23 / 03 / 2016. Assinatura conforme cartão de identificação: ______________________________________________.

(5) À minha família, em particular aos meus pais e irmã, aos meus amigos e a todos aqueles que me apoiaram durante o meu percurso académico… e fora dele..

(6) Clinical Research Paper. Assessment of cerebral vasoreactivity with transcranial doppler in healthy portuguese adults. Authors: João Brandão Madureira a, Pedro Miguel Castro b, Elsa Azevedo c. Faculty of Medicine, University of Porto, Alameda Professor Hernâni Monteiro, 4200-319 Porto, Portugal; [email protected] a Neurology Department, São João Hospital Center, Faculty of Medicine of University of Porto, Alameda Professor Hernâni Monteiro, 4200-319 Porto, Portugal; [email protected] b Neurology Department, São João Hospital Center, Faculty of Medicine of University of Porto, Alameda Professor Hernâni Monteiro, 4200-319 Porto, Portugal; [email protected] c. Corresponding author: João Brandão Madureira Faculty of Medicine of University of Porto, Alameda Professor Hernâni Monteiro, 4200-319 Porto, Portugal E-mail: [email protected] 1.

(7) Abbreviations. ABP – Arterial blood pressure CA – Cerebral autoregulation CBF – Cerebral blood flow CBFV – Cerebral blood flow velocity CBFVIDX – CBFV during Identify X CBFVNB – CBFV during the N-Back CVR – Cerebrovascular reactivity CVRi – Cerebrovascular resistance index EtCO2 – End-tidal CO2 HF – High frequency HR – Heart rate LF – Low frequency MoCA – Montreal Cognitive Assessment NVC – Neurovascular coupling TCD – Transcranial Doppler TFA – Transfer function analysis VLF – Very-low frequency VMR – Vasomotor reactivity. 2.

(8) Abstract Background: Cerebrovascular reactivity (CVR) maintains an adequate cerebral blood flow by three major regulatory mechanisms: cerebral autoregulation (CA), vasomotor reactivity (VMR) to CO2 variations and neurovascular coupling (NVC). However, most studies generalize their results based in the response to only one of these tests. Using a neurovascular stress tests battery, our study aims to evaluate if there is any relationship between CVR mechanisms and how they might be influenced by demographic and systemic hemodynamic factors. Methods: Fifty-eight healthy adults from 20 to 80 years-old were included. Arterial blood pressure (Finometer), cerebral blood flow velocity in both middle cerebral arteries (transcranial Doppler), electrocardiogram and end-tidal-CO2 were monitored. We assessed CA by transfer function analysis, VMR at hypo and hypercapnia (carbogen 5%) and NVC response during N-Back Task. Montreal Cognitive Assessment score was recorded. Results: Neurovascular stress tests were not affected by age or gender and no correlation was found between their outputs (p>0.05). Baseline and variations of hemodynamic parameters had no correlation with their outputs (p>0.05). Cognitive score was uncorrelated with NVC (p>0.05). Conclusions: Neurovascular stress tests measure different aspects of CVR control and a full battery might be more useful for future studies on neurovascular control. Being independent of age and cognitive status, CVR tests seem promising for studying several cerebrovascular conditions affecting the aging brain.. Keywords Cerebrovascular reactivity; cerebral blood flow; cerebral autoregulation; vasomotor reactivity; neurovascular coupling; transcranial Doppler.. 3.

(9) 1.. Introduction. Cerebrovascular reactivity (CVR) is crucial for the maintenance of an adequate cerebral blood perfusion in response to a myriad of vasoactive stimuli [1-3]. In order to compensate for high metabolic rate and limited energy stores of brain tissue, a complex interplay of metabolic [4, 5], neuronal [6, 7], pressure- and shear-dependent [4-6, 8] myogenic mechanisms modulate cerebral resistance, allowing the cerebrovascular bed to constrict or dilate in response to hemodynamic and neurophysiological stimuli [9].. Transcranial Doppler (TCD) is a powerful and versatile tool for non-invasive assessment of intracranial vessels. With the advent of this technique, which has a remarkable temporal resolution, it became possible to easily assess the health of the cerebrovascular bed using neurovascular stress tests [10, 11]. These procedures allow the evaluation of three major physiological processes that underlie cerebral blood flow preservation: (i) cerebral autoregulation (CA), which is regarded as the capacity of the cerebral blood vessels to maintain constant cerebral blood flow (CBF) regardless of changes in cerebral perfusion pressure (CBP) [12]; (ii) vasomotor reactivity (VMR), which allows flow control in response to changes in carbon dioxide (CO2) and oxygen (O2) levels [13]; and (iii) neurovascular coupling (NVC), or functional hyperaemia, which adapts local flow in response to neuronal activity and metabolic demand [14].. Impairment of CVR has been linked to several disorders such as Alzheimer’s and Parkinson’s diseases [15-18], head trauma [19], subarachnoid haemorrhage [20], carotid artery disease [21], stroke [22], metabolic diseases [23] and autonomic. 4.

(10) failure [24]. However, in most reports only one of the CVR components is studied. Therefore, there is a lack of evidence on the interplay between CA, VMR and NVC in both healthy and pathological states, and how age, gender and other demographic factors influence them per se, regardless of disease. It is also essential to test the reproducibility of TCD results across several populations, so that confounding factors are identified.. Using a neurovascular stress tests battery, this study seeks to evaluate if there is any relationship between CA, VMR and NVC and how they might be individually influenced by age or gender. It also aims to standardize a reference set of values for neurovascular stress tests in a healthy population.. 2.. Methods. This study was conducted in São João Hospital Center, a university hospital in Porto, Portugal. It was approved by the appropriate local institutional ethical committee and performed in accordance with the Declaration of Helsinki ethical standards. All participants gave written and signed informed consent.. 2.1.. Population studied. Healthy subjects were selected from hospital or faculty staff and by advertising within university facilities. We predefined to include 10 participants with the same number of males and females in each decade of age strata, ranging from 20 to 80 years. All participants fulfilled a comprehensive medical questionnaire to exclude if they had common cardiovascular risk factors (hypertension, diabetes,. 5.

(11) smoking) or history of a cardiovascular or neurological disease affecting the central nervous system. Systolic and diastolic blood pressure was averaged from three measurements in the sitting position with an oscillometric cuff (Omron M6, Japan). Body mass index was calculated for each participant. All participants above 40 years underwent cervical and transcranial ultrasound studies (Vivid e; GE) before evaluation to exclude hemodynamically significant extra- or intracranial stenosis. All subjects were refrained from caffeinated beverages, alcohol and exercise for at least 12 hours before the study.. 2.2.. Cognitive assessment. The subjects performed a formal cognitive assessment using Montreal Cognitive Assessment (MoCA) test [25].. 2.3.. Monitoring protocol. Evaluations were carried out in a dim lighted room, with temperature around 20ºC. All participants were monitored in supine position in a bed with the head at 0º. Arterial blood pressure (ABP) was continuously monitored with a finger cuff in non-dominant side using Finometer MIDI (FMS, Amsterdam, Netherlands). Heart rate (HR) was assessed from lead II of a standard 3-lead electrocardiogram (ECG). Cerebral blood flow velocity (CBFV) was recorded bilaterally from M1 segment of the middle cerebral artery (MCA), at a depth of 50-55 mm, with 2MHz monitoring probes secured with a standard headband (Doppler BoxX, DWL, Singen, Germany). End-tidal carbon dioxide (EtCO2) was continuously recorded with nasal cannula attached to Respsense capnograph (Nonin, Amsterdam, Netherlands). All data were synchronized and digitized at 400 Hz with Powerlab. 6.

(12) (AD Instruments, Oxford, UK) and stored for offline analysis. Data was recorded in resting state for 10 minutes to posteriorly assess CA. Afterwards CO2 and neurovascular coupling protocols were performed, as explained below.. 2.3.1. CO2 vasoreactivity protocol After a 2-minute resting period, subjects inspired a gas mixture of 5% CO2, 21% O2 and balance nitrogen for 2 minutes, then rested for 2 minutes and finally hyperventilated to an end-tidal CO2 of approximately 20 mmHg for another 2 minutes. VMR was determined as the slope of relationship between ETCO2 plotted against averaged mean CBFV at the last 30 seconds of hypo or hypercapnia and expressed as change in cerebral blood flow per mmHg change in ETCO2. VMR was calculated separately for hypercapnia and hypocapnia.. 2.3.2. Neurovascular coupling protocol N-Back Task was performed and analysed as described by Sorond F. et al [26]. While in supine position, a sequence of single letters was displayed onto the ceiling. While watching the sequence of letters, subjects were instructed to press the button of a computer mouse each time a letter was repeated (1-Back) or each time a letter was repeated every other letter (2-Back). A control task was performed before each task – Identify the X; measured parameters were baseline CBFV and its difference during the N-Back Tasks. The sequence of testing was as follows: 2 minutes of control task; 2 minutes of 1-Back; 2 minutes of control task; and finally 2 minutes of 2-Back. The mean percent change for each MCA was calculated as a ratio of the percent difference between the CBFV during the N-Back (CBFVNB) and its corresponding “Identify the letter X” control period. 7.

(13) (CBFVIDX) divided by CBFV during Identify X (CBFVIDX) and multiplied by 100 using the following formula: [(CBFVNB -CBFVIDX)/ (CBFVIDX)] *100.. 2.4.. Data analysis and CA calculations. All signals were inspected and artifacts removed by linear interpolation. Systolic, mean and diastolic values of ABP and CBFV were calculated. For each heartbeat, cerebrovascular resistance index (CVRi) was calculated by ABP/CBFV reflecting vasomotor function [27]. Autoregulation was assessed in conformity with a recent set of recommendations from the Cerebral Autoregulation Research Network (CARNet) [28]. Transfer function analysis (TFA) was used to assess dynamic CA by calculating coherence, gain and phase parameters from beat-to-beat spontaneous oscillations in CBFV and ABP as previously reported [29, 30]. In resume, 10 minutes of normalized data were interpolated at 100 Hz into uniform time basis; averaged periodogram was calculated by Welch [29] method with Hanning window of 100 seconds, with 50% overlap. Coherence was calculated between input auto-spectra of ABP over cross-spectra of CBFV/ABP and transfer functions of phase and gain were determined by dividing the cross-spectrum by the input auto-spectrum [29]. Coherence, gain and phase are reported in three bands: very-low (VLF: 0.020.07 Hz), low (LF: 0.07–0.20 Hz) and high (HF: 0.20-0.50 Hz) frequencies. CA is usually believed to operate at VLF and LF bands [29-31]. In short, coherence is the coefficient of correlation between the signals; higher coherence between the oscillations is reflective of less effective CA. Gain quantifies the damping effect of CA on the magnitude of ABP oscillations. Phase shift represents the time delay. 8.

(14) between ABP and CBFV oscillations. Lower gain and higher phase represent tighter, more effective autoregulatory response [29, 32].. 2.5.. Statistics. Normality of the variables was determined by Shapiro-Wilk test. Right and left differences in MCA measurements were examined using paired t-tests and Wilcoxon signed-rank test. As there were no significant differences between sides, right and left mean CBFV was computed for all subsequent analysis. Student’s t-test and one-way between subjects ANOVA test were used to compare subjects baseline characteristics between genders and age groups. MoCA scores were compared between age groups using Kruskal-Wallis H test. Significant differences of hemodynamic parameters, CA, VMR and NVC results between age strata and genders were evaluated by multiple linear regression. Pearson or Spearman correlation coefficients were used as appropriate to assess the influence of ABP, HR and EtCO2 on CA, VMR, NVC results as well as in the relationship between these tests. Paired t-tests and Wilcoxon signed-rank tests were used to assess the significance of the variation of ABP, HR and EtCO2 between the rest and activation phases of VMR and NVC protocols.. 3.. Results. Table 1 summarizes demographic and baseline measurements of all subjects (n=58). Older participants had higher MAP (MAP F(53,1)=30.34, p<0.00001). No other. statistical. significant. differences. were. found. between. baseline. characteristics. A multiple regression studied the prediction of CBFV according. 9.

(15) to gender and age (F(2, 50)=10.840, p=0.00012, R2=0.302) (table 2). Age was analysed as a continuous variable. Mean CBFV decreased approximately 3.4% by decade (β=-0.342, p=0.0002), and women had a mean CBFV 7,5 cm/s higher than men (β=7.529, p=0.0134). The same analysis was performed with CVRi as the dependent variable. This model was statistical significant (F(2, 50)=5.765, p=0.00559, R2=0.187). However only age was a significant predictor of CVR (β=0.008, p=0.0016). Increased age was also associated with lower MoCA scores (χ2(5)=28.826, p=0.00003). This was particularly significant in the visuospatial/executive (χ2(5)=12.949, p=0.02386), attention (χ2(5)=18.456, p=0.00243), language (χ2(5)=14.118, p=0.01488), abstraction (χ2(5)=11.259, p=0.04648) and delayed recall (χ2(5)=11.421, p=0.04364) domains (table A.1). No statistical significant differences were detected between right and left MCA hemodynamic measurements (table A.2). Thus, mean CBFV of right and left MCA was calculated and used as parameter for subsequent analysis. We found no effect of age and gender concerning CA, VMR and NVC tests.. Figure 1 summarizes transfer function gain, phase and coherence in the frequency domains of VLF (0.02–0.07 Hz), LF (0.07–0.20 Hz) and HF (0.20-0.50 Hz) for spontaneous oscillations during the CA protocol. Coherence and gain were positively correlated between themselves in the VLF and LF domains (VLF: r(58)=0.518, p=0.00003; LF: r(58)=0.57122, p<0.000001) while phase was largely independent (p>0.05) (table 3).. Figures 2 depicts the percentage of change in CBFV, MAP, HR and EtCO2 during hypercapnia and hypocapnia. Mean values of MAP, HR and EtCO2 during the. 10.

(16) resting and activation phases, as well as the value of variation during these periods, are depicted in table 4. During the hypercapnic challenge there was an increase in EtCO2 of approximately 9 ± 3 mmHg, while it decreased by 17 ± 3 mmHg during hyperventilation. Hypocapnia gain was correlated with EtCO2 observed during the procedure (r(55)=0.331, p=0.01365). While MAP increased modestly during hypercapnia, HR was the main systemic hemodynamic parameter changing during hyperventilation. These changes did not affect VMR results (p>0.05). VMR global gain was positively correlated with hypercapnic gains (r(56)=0.413, p=0.00155), but not with the hypocapnic (r(56)= 0.222; p=0.09938). Curiously, hypercapnic and hypocapnic gains were not correlated (r(56)=0.214, p=0.11311) (table 3).. The average CBFV increase during 2-back task was 6.27 ± 4.64 % (figure 3). There was a statistical significant but mild change in MAP, HR and EtCO2 (p<0.01) during the task, as reported on table 4. Systemic hemodynamic parameters did not influence test outcomes. NVC results did not correlate with MoCA global and subtotal scores (p>0.05).. CA, VMR and NVC results did not correlated significantly with each other except for VMR global gain, which was correlated with both LF and HF TFA gain (r(56)=0.326, p=0.01406; r(56)=0.324; p=0.01481; respectively) (table 3).. 11.

(17) 4.. Discussion. This study suggests, for the first time, that there is no clear correlation between the outputs of CA, VMR and NVC protocols. Neurovascular stress tests seemed to be independent of age and gender, making it easier to compare results across different demographic groups. Thus far, no consensus has been reached on the role of aging on CVR. In fact, while some studies point to a negative correlation between age and CVR [33-36], others report no association [37-40]. In the present study, aging was associated with a decrease of CBFV and an increase in CVRi. These findings might result from systemic changes in the vascular tree that are known to accumulate during lifetime. Thus, it would be expected that aging per se would result in an impairment of CVR. However, we demonstrated that aging had no influence on neurovascular stress tests, in spite of CBFV decline with age [41-45]. Past reports on cognitive performance have shown that successful aging is associated with higher brain activation, compensating for agerelated neural changes and achieving an accuracy that equals young subjects [40]. These compensatory mechanisms have been reported in other studies, not only during neuronal activation, but also during metabolic and autoregulatory stimuli [39, 46]. Globally, data seems to suggest that cerebrovascular adaptive mechanisms have a functional reserve, compensating for the age-related deterioration of the cerebrovascular bed and enabling the maintenance of cerebrovascular homeostasis.. Several studies have reported higher CBFV in women, compared to men [47]. Similarly, gender-related differences have been noticed in CA and VMR studies [47, 48]. Aging seems to be related to this findings, since women have higher. 12.

(18) hypercapnic gains than men in younger ages, but lower values after menopause in the absence of hormonal supplements [49]. Thus, hormonal status might play an important role in the maintenance of cerebrovascular homeostasis. Changes in the microcirculation, such as vessel tonus, reactivity and metabolism might also be related to this finding. Our results show that, although there is a decrease of CBFV with age in both genders, women have higher mean velocities, regardless of age. On the other hand, this difference does not seem to transpose to CA, VMR or NVC. However, we did not evaluate the phase of menstrual cycle, if women were pre or postmenopausal, or if they were on hormonal supplements, which might have an effect on cerebrovascular hemodynamics, thus acting as a potential confounding factor.. Cerebral autoregulation might be assessed using a correlation coefficient [50], autoregulatory index [51] or transfer function analysis [52], although no consensus exists on what should be the gold standard. In this study we used TFA, which has been increasing in popularity amongst research groups [28]. Gain, phase shift and coherence were computed using fast-Fourrier transformation. The curves obtained for each of these parameters were similar to those reported in literature [53]. Taking into account that coherence values were lower than 0.5 below 0.05 Hz, any assumption below this frequency should be made with caution, since linearity may not be applicable. However, it steadily increases and surpasses 0.5 after 0.05 Hz. On the other hand, high frequency (HF) domains show high coherence and gain values, suggesting that autoregulatory mechanisms are unable to control these oscillations. Phase shift values are approximately 0 from 0.30 Hz onwards. Therefore, there is no lag. 13.

(19) between systemic and cerebral oscillations in the HF range and ABP is transmitted undamped to CBFV. The opposite occurs towards the LF band, with low gain and high phase shift values. Thus, it is thought that CA exerts its effects in the lower band of frequencies, presumably below 0.30 Hz.. There is still a large heterogeneity between the various methods of TFA in the literature. Phase shift has been shown to be more stable than other CA parameters. Our results show lower dispersion of values for phase shift than for gain or correlation, as can be noted in figure 1. It also shows less variation after 0.30 Hz, which is pointed as the limit for the cerebral autoregulatory capacity. On the other hand, phase was not correlated with gain or coherence, which might favour the use of this parameter as an indicator of autoregulation. However, it has one major drawback: it requires the assumption of stationary conditions, which might be difficult to accomplish even in controlled protocols where multiple sources of non-linearities are possible.. CVR, which is dependent on cerebrovascular endothelial function, can be assessed from the CBFV responses to rapid changes in EtCO2. Vascular tonus reacts to these changes, favouring vasodilation in the onset of hypercapnia and vasoconstriction with hypocapnia. In this study, hypercapnia was induced by CO2 administration and hypocapnia by hyperventilation. VMR global gain, as well as hypercapnic and hypocapnic gains were independent of age and gender and no correlation was found between these gains. However, global gain was correlated with the hypercapnic gain. Thus, it seems that VMR is more linked to vasodilatory than vasoconstrictive responses. A higher dispersion of values for the. 14.

(20) hypercapnic gains than for hypocapnic was also observed, suggesting an increased variability in the response to hypercapnia than to hypocapnia. Since the tone of cerebral vessels seems to be the function of arterial blood gases, pH, cerebrospinal fluid composition and nitric oxide [9], it is possible that these mechanisms might play a differential role during vasodilatory or constrictive responses.. Neuronal activation is related to increased metabolic activity which demands for a higher regional blood supply. We observed that there was a significant increase during 2-Back Task (compared to 1-Back), which was expected since 2-Back is a more demanding task. The lack of statistical significant differences between right and left CBFV measurements during NVC tasks suggests that there is a bilateral activation during this protocol. No correlation between the results of MoCA test score and NVC gains was found. Similarly, no correlation was found between NVC and any of the subtotal scores of the tests. Sorond et al demonstrated that there was a positive association between 2-Back Task performance and Trail B scores, which specifically tests for executive function [54]. However, no correlation was found between 2-Back Task performance and the Mini-mental examination, which assesses global cognitive impairment, similarly to MoCA test. Consequently, it is possible that more refined and specific cognitive tests are better predictors of NVC performance. On the other hand, it is also possible that N-Back Task is not specifically dependent on the cognitive level of the subject, but rather on the activation status, possibly mediated by the sympathetic nervous system, once HR and MAP increase during this task. Thus, it would be interesting to compare our results with outcomes obtained from the. 15.

(21) application of other NVC protocols which elicit neuronal activation independently of cognition.. At last we analysed the association between neurovascular stress tests results. There was a lack of correlation between CVR components, apart from some occasional findings in the autoregulatory HF domain. Since autoregulation is thought to be inexistent above 0.30 Hz, these findings lack significance.. One limitation of the Doppler studies is the use CBFV measurements instead of CBF. However, it is unlikely that the calibre of insonated vessels changed in the limited time of the procedures, as shown by Serrador et al [55]. On the other hand, some participants had mild dyslipidaemia and were on pharmacological control with statins. However, none of them had episodes of vascular disease and carotid atherosclerosis was excluded by cervical and transcranial ultrasound studies. Although it is unlikely that these factors had influence in the results, we cannot exclude the presence of microvascular disease or the presence of CVR impairment caused by the disease or drug. It is also important to state that we had a small sample size and, although we did not find evidence of the role of aging on CVR, data on elderly individuals, especially on those older than 80 years, continues to be sparse.. Overall, our data suggests that neurovascular stress tests measure different aspects of CVR control and that a full battery might be more useful in future studies on neurovascular control. It is possible that autonomic, myogenic or metabolic stimuli might have a differential role in each of CVR components, even. 16.

(22) if some common mechanisms might exist between themselves. We have also demonstrated that age and gender do not have an influence in cerebrovascular regulation or neurovascular stress tests. Being independent of age and cognitive status, CVR tests seem promising for studying several cerebrovascular conditions affecting the aging brain. On the other hand, cerebrovascular regulation did not seem to be affected by MAP, HR or EtCO2. However, even if fluctuations of these physiological functions might not influence CVR, it seems important to include them on monitoring routines as a control parameter. The lack of correlation between protocols’ results supports the need to define gold standards for each component of CVR and clarify the terminology in use. Thus, CA, VMR and NVC should not be used interchangeably to describe individual response to CVR stimuli. Analysis of the mechanisms that underlie CVR, by both laboratory and imaging studies, are needed to understand the intricate interplay of factors that are responsible for CBF regulation and their role on pathology.. Conflicts of interest None.. Acknowledgements The authors thank Joana Carvalho, PhD, from the Faculty of Sport Sciences and Physical Education of the University of Porto, and São João Hospital Center Volunteers Association for their help recruiting participants for the present study. This study is part of Madureira JP MD thesis.. 17.

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(30) Table 1 – Baseline characteristics stratified by gender Total. Male. Female. 58. 28. 30. 23 (22-26) 34 (32-39) 46 (41-50) 55 (51-60) 65 (61-69). 24 (22-26) 35 (32-39) 46 (41-50) 56 (55-60) 63 (61-66). 22 (23-23) 33 (32-34) 47 (44-50) 54 (51-58) 66 (62-69). 75 (71-80). 77 (74-80). 74 (71-78). 24.6 ± 3.6. 25.1 ± 3.0. 24.1 ± 4.0. 26.6 ± 3.1. 27.1 ± 2.6. 26.2 ± 3.4. SBP, mmHg. 125.5 ± 16.3. 126.6 ± 11.6. 124.5 ± 19.5. DBP, mmHg. 77.3 ± 9.1. 78.0 ± 8.0. 76.8 ± 10.0. MAP, mmHg. 93.4 ± 10.7. 94.2 ± 8.5. 92.7 ± 12.4. HR, bpm. 68.8 ± 9.5. 69.1 ± 10.9. 68.5 ± 8.3. PP, mmHg. 61.7 ± 14.6. 58.2 ± 15.7. 64.6 ± 13.3. EtCO2, mmHg. 38.2 ± 2.8. 39.0 ± 2.6. 37.6 ± 2.9. CVRi, mmHg·cm-1.s-2. 1.3 ± 0.3. 1.3 ± 0.4. 1.2 ± 0.3. n Age, y 21-30 31-40 41-50 51-60 61-70 71-80 BMI, kg.m. -2. MoCA test score. All values are given in mean ± SD, except for age which is given in mean with ranges in parentheses. BMI = Body mass index; MoCA test = Montreal Cognitive Assessment; SBP = Systolic blood pressure; DBP = Diastolic blood pressure; MAP = Mean arterial blood pressure; HR = Heart Rate; PP = Pulse pressure; EtCO2 = End-tidal CO2; CVRi = Cerebrovascular resistance index.. 25.

(31) Figure 1 – Transfer function gain (A and B), phase (C and D) and coherence (E and F) between changes in MAP and mean CBFV during a resting period. Group-averaged results are shown. Solid lines and shaded regions represent Mean ± SE. Vertical lines limit the intervals for VLF = Very-low frequencies (0.02– 0.07 Hz); LF = Low-frequencies (0.07–0.20 Hz); HF = High frequencies (0.200.50 Hz). MCA = Middle cerebral artery; MAP = Mean arterial pressure; CBFV = Cerebral blood flow velocity; a.u. = arbitrary units.. 26.

(32) Total. N (M:F ratio). 21-30. 31-40. 41-50. 51-60. 61-70. 70-80. Model. Age. Gender. R. β. p. β. p. 2. 58 (14:15). 10 (1:1). 10 (1:1). 12 (1:1). 10 (1:1). 7 (3:4). 9 (4:5). --------. --------. --------. --------. --------. 65.67 ± 12.51 62.05 ± 13.59 68.91 ± 10.69. 73.68 ± 14.85 70.60 ± 19.08 76.15 ± 12.28. 67.68 ± 8.69 63.29 ± 7.12 74.99 ± 5.87. 68.94 ± 10.49 62.39 ± 11.37 74.39 ± 6.16. 63.13 ± 12.16 60.65 ± 11.71 65.60 ± 13.43. 62.88 ± 11.92 66.56 ± 18.49 60.11 ± 5.71. 55.80 ± 11.58 45.81 ± 10.57 61.79 ± 7.69. 0.302** 0.186* 0.330*. -0.343 -0.352 -0.336. <0.001 0.031 0.001. 7.529 ---------------. 0.013 ---------------. 1.27 ± 0.34 1.30 ± 0.40 1.25 ± 0.30. 1.10 ± 0.34 1.15 ± 0.51 1.06 ± 0.20. 1.13 ± 0.28 1.25 ± 0.30 0.98 ± 0.20. 1.28 ± 0.32 1.30 ± 0.40 1.27 ± 0.28. 1.30 ± 0.45 1.32 ± 0.60 1.29 ± 0.36. 1.36 ± 0.28 1.22 ± 0.26 1.46 ± 0.29. 1.51 ± 0.27 1.64 ± 0.30 1.42 ± 0.25. 0.183* 0.107 0.295*. 0.008 0.008 0.009. 0.002 0.120 0.002. -0.069 ---------------. 0.434 ---------------. 0.47 ± 0.16 0.63 ± 0.16 0.74 ± 0.14. 0.48 ± 0.17 0.62 ± 0.11 0.74 ± 0.11. 0.46 ± 0.17 0.68 ± 0.11 0.80 ± 0.06. 0.42 ± 0.15 0.63 ± 0.20 0.75 ± 0.17. 0.54 ± 0.12 0.69 ± 0.14 0.71 ± 0.15. 0.47 ± 0.23 0.56 ± 0.09 0.67 ± 0.20. 0.49 ± 0.18 0.61 ± 0.22 0.77 ± 0.11. 0.010 0.035 0.019. 0.001 -0.001 -0.001. 0.510 0.406 0.524. -0.015 0.047 -0.029. 0.726 0.254 0.439. 0.68 ± 0.28 1.03 ± 0.29 1.32 ± 0.40. 0.70 ± 0.23 1.05 ± 0.45 1.41 ± 0.55. 0.70 ± 0.36 1.12 ± 0.27 1.48 ± 0.52. 0.68 ± 0.36 1.08 ± 0.27 1.34 ± 0.31. 0.64 ± 0.18 1.02 ± 0.20 1.30 ± 0.29. 0.63 ± 0.19 0.91 ± 0.24 1.15 ± 0.15. 0.71 ± 0.34 0.92 ± 0.28 1.18 ± 0.43. 0.001 0.055 0.071. <0.001 -0.004 -0.006. 0.913 0.105 0.052. 0.013 0.057 0.056. 0.863 0.460 0.591. 1.04 ± 0.45 0.63 ± 0.23 0.17 ± 0.21. 1.05 ± 0.44 0.74 ± 0.20 0.19 ± 0.17. 0.83 ± 0.52 0.60 ± 0.26 0.14 ± 0.18. 1.31 ± 0.27 0.56 ± 0.19 0.13 ± 0.25. 1.20 ± 0.29 0.69 ± 0.24 0.19 ± 0.23. 0.91 ± 0.53 0.49 ± 0.21 0.24 ± 0.26. 0.81 ± 0.51 0.65 ± 0.25 0.15 ± 0.21. 0.025 0.014 0.006. -0.002 -0.001 <0.001. 0.483 0.403 0.788. 0.115 -0.014 -0.029. 0.340 0.820 0.609. Vasoreactivity Hypercapnia, %.mmHg-1 Hypocapnia, %.mmHg-1 Global, %.mmHg-1. 4.87 ± 1.75 1.74 ± 0.46 4.19 ± 1.76. 5.15 ± 2.71 1.64 ± 0.61 4.31 ± 1.29. 4.82 ± 2.35 1.79 ± 0.58 5.16 ± 3.49. 4.29 ± 1.11 1.84 ± 0.31 4.03 ± 1.35. 5.56 ± 1.75 1.69 ± 0.38 4.37 ± 1.1. 4.89 ± 0.96 1.77 ± 0.45 3.54 ± 0.49. 4.58 ± 0.72 1.66 ± 0.47 3.55 ± 1.2. 0.015 0.041 0.047. -0.004 <0.001 -0.022. 0.793 0.924 0.114. -0.396 -0.182 -0.015. 0.402 0.140 0.974. Neurovascular coupling 1-Back gain, %-1 2-Back gain, % -1. 0.22 ± 7.31 6.27 ± 4.64. 4.96 ± 17.12 5.63 ± 4.93. -0.80 ± 2.45 6.10 ± 6.50. -1.51 ± 2.21 7.14 ± 4.80. 0.23 ± 3.46 7.08 ± 3.88. -1.54 ± 2.01 3.41 ± 2.25. 0.07 ± 3.40 7.34 ± 4.00. 0.067 0.014. -0.058 -0.001. 0.298 0.983. -3.088 1.103. 0.115 0.384. Mean CBFV, cm.sec-1 Male Female Mean CVRi, mmHg·cm-1.s-2 Male Female Autorregulation Coherence, a.u. VLF LF HF Gain, cm.sec-1.mmHg-1 VLF LF HF Phase, radius VLF LF HF. 27. Table 2 – Hemodynamic characterisation of age groups: linear regression modelling examining the influence of age and gender on cerebrovascular measurements. All values are given as mean ± SD. CBFV = Cerebral blood flow velocity; CVRi = Cerebrovascular resistance index; a.u. = arbitrary units; VLF = Very-low frequencies (0.02–0.07 Hz); LF = Low-frequencies (0.07–0.20 Hz); HF = High frequencies (0.20–0.50 Hz). * p value < 0.05 ** p value < 0.001. 27.

(33) Figure 2 – Time-course of CBFV (A and D), MAP/HR (B and E) and EtCO2 (C and F) during the hypercapnic challenge (left) and hyperventilation induced hypocapnia (right). Graphs of all subjects were synchronized and averaged. Solid lines and shaded regions represent Mean ± SE. The arrows ( ) in the horizontal axis mark the beginning and end of each challenge. Vasodilatory range, which measures the % of variation between the lowest and highest CBFV values, was 61 ± 16 %. CBFV = Cerebral blood flow velocity; MCA = Middle cerebral artery; MAP = Mean arterial pressure; HR = Heart rate; EtCO2 = Endtidal CO2.. 28.

(34) 29. Table 3 - Correlation between CVR measurements 1 1. Autoregulation. Coherence, a.u.. 2. 3. 4. LF. 0.11. 3. HF. -0.02. 0.25. VLF. 0.52**. -0.13. -0.06. LF. -0.23. 0.57**. 0.16. -0.17. Gain, cm.sec. 6. 7. 8. -1. 5 6. HF. -0.20. 0.30*. 0.10. -0.34**. 0.65**. VLF. -0.04. 0.09. 0.15. -0.09. -0.02. 0.05. 8. LF. -0.14. 0.01. -0.03. -0.24. 0.14. 0.27*. 0.17. 9. HF. 0.18. -0.03. -0.22. -0.11. -0.21. 0.10. -0.04. 0.37**. 7. 10. Phase, radius. 11. 12. -1. 0.24. 0.05. -0.17. 0.16. 0.05. 0.01. -0.07. 0.07. 0.04. Hypocapnia, %.mmHg-1. -0.22. -0.16. -0.04. -0.02. 0.04. -0.02. 0.05. 0.04. 0.04. 0.21. 12. Global, %.mmHg-1. -0.01. 0.14. -0.06. 0.10. 0.33*. 0.32*. 0.02. 0.06. -0.08. 0.41**. 0.22. 1-Back gain, %. 0.16. -0.02. -0.08. -0.04. 0.13. 0.19. -0.04. 0.11. 0.19. 0.20. 0.13. 0.07. 2-Back gain, %. -0.02. 0.13. -0.01. 0.04. 0.05. -0.02. -0.18. -0.18. -0.08. 0.05. -0.03. -0.01. 14. Neurovascular coupling. Hypercapnia, %.mmHg. 10. 11. 13. Vasoreactivity. 9. VLF. 2. 4. 5. CVR = Cerebrovascular reactivity; a.u. = arbitrary units; VLF = Very-low frequencies (0.02–0.07 Hz); LF = Low-frequencies (0.07–0.2 Hz); HF = High frequencies (0.2–0. 5). † Spearman’s correlation coefficient between cerebrovascular measurements is significant at < 0,05 ‡ Spearman’s correlation coefficient between cerebrovascular measurements is significant at < 0,01. 29.

(35) 30. Table 4 – MAP, HR and EtCO2 variation during CVR protocols and their correlation with CVR measurements Resting phase. Activation phase. Difference (95% CI). MAP. HR. EtCO2. MAP,. HR. EtCO2. MAP. HR. EtCO2. mmHg. bpm. mmHg. mmHg. bpm. mmHg. mmHg. bmp. mmHg. 80 ± 13. 69 ± 9. 38 ± 3. --------. --------. --------. --------. --------. --------. Hypercapnia, %.mmHg-1. 80 ± 15. 68 ± 9. 37 ± 4. 91 ± 17. 68 ± 10. 46 ± 4. 11 ± 8 †. 1±7†. 9±3†. Hypocapnia, %.mmHg-1. 81 ± 15. 69 ± 10. 36 ± 4. 83 ± 17. 88 ± 15. 19 ± 3 ʄ. 2±8†. 20 ± 13†. -17 ± 3 †. 1-Back gain, %. 88 ± 18. 70 ± 12. 37 ± 4. 88 ± 18. 72 ± 12. 38 ± 6. 0±3†. 1±2†. 1±4†. 2-Back gain, %. 87 ± 18. 71 ± 12. 38 ± 4. 90 ± 18. 76 ± 14. 37 ± 5. 3± 5†. 5±6†. 0±2†. Autorregulation. Vasoreactivity. Neurovascular coupling. All values are given as mean ± SD. MAP = Mean arterial pressure; HR = Heart rate; EtCO2 = End-tidal CO2; CVR = Cerebrovascular reactivity. † Paired t test p value for comparisons between resting phase and activation phase monitoring parameters is significant at < 0.001 ʄ Spearman’s correlation coefficient P score for correlations between cerebrovascular measurements and monitoring parameters is significant at < 0.05. 30.

(36) Figure 3 – Time-course of CBFV (A), MAP/HR (B) and EtCO2 (C) during the 2-Back Task. Graphs of all subjects were synchronized and averaged. Solid lines and shaded regions represent Mean ± SE. The arrows ( ) in the horizontal axis mark the beginning and end of the respective challenge. CBFV = Cerebral blood flow velocity; MCA = Middle cerebral artery; MAP = Mean arterial pressure; HR = Heart rate; EtCO2 = End-tidal CO2.. 31.

(37) 32. Appendices. Table A.1 – Influence of age on MoCA scores. Scores. Mean. 21-30. 31-40. 41-50. 51-60. 61-70. 70-80. p. MoCA test score. 26.57 (17-30). 29.44 (27-30). 27.3 (23-30). 27.91 (22-30). 26.75 (25-28). 24.00 (18-28). 23.11 (17-29). <0.001. Visuospatial/Executive. 4.56 (2-5). 4.89 (4-5). 5.00 (5-5). 4.64 (3-5). 4.50 (4-5). 4.29 (2-5). 3.89 (2-5). 0.024. Naming. 2.96 (2-3). 3.00 (3-3). 3.00 (3-3). 3.00 (3-3). 3.00 (3-3). 3.00 (3-3). 2.78 (2-3). 0.070. Attention. 5.17 (1-6). 5.78 (5-6). 5.50 (4-6). 5.73 (5-6). 5.25 (4-6). 4.43 (2-6). 4.00 (1-6). 0.002. Language. 2.11 (0-3). 2.89 (2-3). 2.30 (1-3). 2.27 (1-3). 1.63 (1-3). 1.86 (0-3). 1.56 (1-3). 0.015. Abstraction. 1.80 (0-2). 2.00 (2-2). 1.80 (1-2). 1.91 (1-2). 2.00 (2-2). 1.57 (1-2). 1.44 (0-2). 0.046. Delayed recall. 4.15 (0-5). 4.78 (3-5). 4.30 (2-5). 4.45 (3-5). 4.38 (3-5). 3.14 (0-5). 3.56 (1-5). 0.044. Orientation. 5.92 (5-6). 6.00 (6-6). 6.00 (6-6). 5.91 (5-6). 6.00 (6-6). 5.71 (5-6). 5.89 (5-6). 0.234. All values are given as mean with ranges in parentheses. MoCA = Montreal Cognitive Assessment.. 32.

(38) Table A.2 – Comparison of CVR measurements between right and left MCA Right MCA. Left MCA. Mean MCA. p. VLF. 0.48 ± 0.17. 0.47 ± 0.17. 0.47 ± 0.16. 0.530a. LF. 0.64 ± 0.15. 0.62 ± 0.17. 0.63 ± 0.16. 0.051a. 0.75 ± 0.14. 0.74 ± 0.16. 0.74 ± 0.14. 0.297b. VLF. 0.69 ± 0.30. 0.67 ± 0.29. 0.68 ± 0.28. 0.285b. LF. 1.04 ± 0.30. 1.01 ± 0.29. 1.03 ± 0.29. 0.048b. HF. 1.33 ± 0.41. 1.31 ± 0.41. 1.32 ± 0.40. 0.592b. VLF. 1.05 ± 0.50. 1.02 ± 0.50. 1.04 ± 0.45. LF. 0.63 ± 0.24. 0.62 ± 0.26. 0.63 ± 0.23. 0.615a. HF. 0.17 ± 0.22. 0.17 ± 0.22. 0.17 ± 0.21. 0.690a. Hypercapnia, %.mmHg-1. 4.93 ± 1.86. 4.80 ± 1.76. 4.87 ± 1.75. 0.276a. Hypocapnia, %.mmHg-1. 1.81 ± 0.63. 1.79 ± 0.71. 1.74 ± 0.46. 0.495a. 1-Back gain, %. -0.61 ± 2.90. 1.05 ± 13.88. 0.22 ± 7.31. 0.671b. 2-Back gain, %. 5.93 ± 4.86. 6.61 ± 5.02. 6.27 ± 4.64. 0.242b. Autorregulation Coherence, a.u.. HF -1. Gain, cm.sec .mmHg. -1. Phase, radius. Vasoreactivity. Neurovascular coupling. CVR = Cerebrovascular reactivity; MCA = Middle cerebral artery; a.u. = arbitrary units; VLF = Very-low frequencies (0.02–0.07 Hz); LF = Lowfrequencies (0.07–0.20 Hz); HF = High frequencies (0.20-0.50). a Paired t test p value b Wilcoxon signed-rank test. 33.

(39) Highlights . No correlation between cerebrovascular reactivity components was found;. . Cerebrovascular reactivity variation seems to be independent of age and gender;. . MoCA test does not correlate with neurovascular coupling results;. . Results suggest that cerebrovascular reactivity has a functional reserve with age;. . Cerebrovascular reactivity tests seem promising for studying the aging brain.. 34.

(40) Annexes 1. Guide for authors 2. São João Hospital Center Ethical Committee Approval 3. MoCA test. 35.

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