 A Gallo

D Rosenbaum

C Kanagasabapathy

X Girerd

Effects of carotid baroreceptor stimulation on retinal arteriole remodeling evaluated with adaptive optics camera in resistant hypertensive patients. Effets de la stimulation du barorécepteur carotidien sur le remodelage artériolaire rétinien évalué par l'Optique Adaptative chez l'hypertendu résistant

Keywords: adaptive optics camera, resistant hypertension, retina, arteriolar remodeling, microcirculation Résumé Optique adaptative, microcirculation, hypertension résistante, rétine, remodelage artériolaire

Background and Aim. Baroreceptor activation therapy (BAT) leads to a decrease in blood pressure (BP) in patients affected by resistant hypertension (RH) by reducing sympathetic outflow.

This study aimed at evaluating the effects of BAT on RH patients' retinal arteriolar microvasculature, a territory devoid of adrenergic innervation.

Patients and Methods.

Five patients defined as affected by RH after excluding secondary causes of hypertension and based on number of anti-hypertensive treatments, underwent the implantation of Barostim TM neo TM . Systolic Blood Pressure (SBP) and Diastolic Blood Pressure (DBP) were assessed by office and 24-hours ambulatory BP monitoring (ABPM). Adaptive Optics Camera RTX1® (ImagineEye, Orsay, France) was used to measure wall thickness (WT), internal diameter (ID), wall cross sectional area (WCSA) and wall-to-lumen ratio (WLR). A cohort of 21 notcontrolled hypertensive patients matched for age, gender and follow-up time, undergoing standardantihypertensive therapy changes, was selected as a control group. SBP and DBP were assessed by office and home BP monitoring (HBPM). Evaluations were performed at baseline and after 6 months mean follow-up.

Results. Office SBP decreased by 9.7±12.3% and 29.7±12.4% in standard-therapy and BAT group respectively, while office DBP decreased by 7.6±17.4% and 14.8±15.7%. Concerning ABPM/HBPM, a mean reduction of both SBP and DBP of 7.9±11% was observed for the standardtherapy while a reduction of 15.8±10.5% and 15.8%±5.3% was observed for SBP and DBP respectively in BAT group. While in the standard therapy group a significant reduction in WLR (-5.9%) due to both ID increase (+2.3%) and WT reduction (-5.7%) was observed, without changes in WCSA (-0.3%), RH patients had a significant reduction in WCSA (-12.1%), due to a trend in both WT and ID reduction (-6.5% and -1.7% respectively), without significant changes in WLR (-2%) Conclusion. While a reverse eutrophic remodeling was observed in patients undergoing a standardantihypertensive treatment, hypotrophic changes were found in RH patients undergoing BAT. Despite the lack of adrenergic receptors on retinal vessels, chronic baroreflex stimulation may exert an effect on retinal microvasculature in RH patients by more systemic than local mechanisms.

Introduction

Novel strategies for a better management of Resistant Hypertension (RH) are being sought. Apart from pharmacologic treatment, which by definition should already be at adequate dose, novel invasive therapeutic strategies have been introduced lately, among which the Barorefex Activation Therapy (BAT).

RH is characterized by an increased sympathetic activity that regulates vascular smooth muscle tone by increased circulating levels of noradrenaline [START_REF] Mäki-Petäjä | The Role of the Autonomic Nervous System in the Regulation of Aortic Stiffness[END_REF]. Simultaneously, a decrease in parasympathetic activity is found. By stimulating the afferent nerve fibers of carotid sinus baroreceptors, BAT determines a central reduction in sympathetic outflow with an increased parasympathetic activity [START_REF] Heusser | Carotid baroreceptor stimulation, sympathetic activity, baroreflex function, and blood pressure in hypertensive patients[END_REF].

Apart from the renovated interest in Sympathetic Nervous System (SNS) activity in arterial hypertension, solid evidences exist about micro-and macrovascular remodeling taking place in arterial hypertension. Effects on large vessels like an increase in wall thickness and a reduction in lumen diameter, as well as an increased stiffness have been shown early in hypertension, independently from aging effects [START_REF] Schiffrin | Vascular remodeling in hypertension: mechanisms and treatment[END_REF].

Retinal microvasculature is the only microvascular territory directly approachable in a non-invasive fashion. Retinal arteriolar remodeling has been found to have the same characteristics of large vessel remodeling, with an increase in wall-to-lumen ratio (WLR) observed in hypertensive patients, representing an inward eutrophic remodeling. A reverse remodeling process has also been shown after the start of standard antihypertensive therapy in newly diagnosed or non-controlled non-resistant hypertensive patients [START_REF] Rosenbaum | Effects of age, blood pressure and antihypertensive treatments on retinal arterioles remodeling assessed by adaptive optics[END_REF][START_REF] De Ciuceis | Effect of antihypertensive treatment on microvascular structure, central blood pressure and oxidative stress in patients with mild essential hypertension[END_REF]. As retinal vessels are typically devoid of adrenergic receptors and are not directly influenced by SNS, no direct effects of acute BAT-induced BP changes are expected on retinal microcirculation. Long-term BAT effects on retinal arterioles in RH have never been analyzed.

Hence, the main objectives of our study were 1/ to study the effects of BAT on retinal microcirculation in patients with RH and 2/ to compare them with a group of not-controlled hypertensive patients undergoing an adaptation of antihypertensive therapy.

Materials and Methods

Study population

Between June 2015 and December 2015 in the Cardiovascular Prevention Unit of Pitié-Salpêtrière University Hospital, Paris, we consecutively enrolled 5 patients affected by RH (as defined by 2013 ESC guidelines), aged from 20 to 60 years old, without contraindication for the implantation of the device, with no recent history of cardiovascular disease. In the same time period we selected 21 not-controlled not-RH patients without recent history of cardiovascular disease. They were matched for age, gender and follow-up time. Exclusion criteria were refusal to give consent and technical obstacles to AOC assessment. The study was carried out according to the principles outlined in the Declaration of Helsinki. Approval of the local Ethics Committee was obtained and informed consent was given by all participants.

BP Measurements

Office brachial BP was assessed in standard conditions in seated position during the outpatient visit using an oscillometric device simultaneously to AOC assessment. In RH patients, BP was confirmed with 24-h ABPM using an oscillometric device (Spacelabs) applied to the non-dominant arm. Home BP measurements (HBPM) were performed in standard-therapy group. HBPM were taken using validated electronic arm devices. Values were recorded as the mean systolic BP and diastolic BP of three consecutive measurements made every 2 minutes in the morning and in the evening, after 5 minutes in sitting position, for three consecutive days. Both ABPM and HBPM were obtained at baseline and follow-up.

Therapeutic device

The stimulation system used was the Barostim TM neo TM (CVRx., Minneapolis, MN, USA). A detailed description of the device has been already provided [START_REF] Hoppe | Minimally invasive system for baroreflex activation therapy chronically lowers blood pressure with pacemaker-like safety profile: results from the Barostim neo trial[END_REF]. All 5 patients had been implanted on the right side. After successful implantation the intensity, frequency and pulse duration were progressively adapted to patients' response in terms of BP control and local symptoms during the first month, being the pulse generator programmable by radio-frequency control through an external software. Stimulation was constant during the whole circadian cycle.

Small arteries: wall to lumen ratio estimation by adaptive optics

Adaptive Optics Camera (AOC) fundus images were obtained using a commercially available flood-illumination AOC retinal camera (rtx1™; Imagine Eyes, Orsay, France).The detailed procedure of images acquisition and treatment has been previously reported [START_REF] Koch | Morphometric analysis of small arteries in the human retina using adaptive optics imaging: relationship with blood pressure and focal vascular changes[END_REF]. Retinal imaging was performed by an operator blinded to clinical data at the Cardiovascular Prevention Unit. The ratio of total parietal thickness over the lumen diameter averaged along 250μm length defined the WLR. Wall thickness (WT) was defined as WT = (Outer Diameter (OD) -Internal Diameter (ID)) and total wall cross sectional area (WCSA) as π * ((OD/2)²-(ID/2)²). Intra observer reproducibility of microvasculature anatomical indices were already published [START_REF] Koch | Morphometric analysis of small arteries in the human retina using adaptive optics imaging: relationship with blood pressure and focal vascular changes[END_REF][START_REF] Gallo | Retinal arteriolar remodeling evaluated with adaptive optics camera: Relationship with blood pressure levels[END_REF].

Definition of Resistant Hypertension

Patients were considered as affected by RH if their office and home BP had been respectively >140/90 and > 135/85 mmHg for at least six months despite triple anti-hypertensive therapy at adequate dose, including a diuretic.

Patients follow-up

Baseline evaluation was made during the outpatients visit before the implantation of the device for BAT group and at the first consulting for standard-therapy group. Antihypertensive therapy for the standard-therapy group was chosen according to recent guidelines [START_REF] Mancia | ESH/ESC Guidelines for the management of arterial hypertension: the Task Force for the management of arterial hypertension of the European Society of Hypertension (ESH) and of the European Society of Cardiology (ESC)[END_REF]. Patients from both treatment groups were re-evaluated at one and six months after either device implantation or antihypertensive therapeutic adaptation.

Statistical analysis

All continuous variables are expressed as mean (SD), unless otherwise stated. Differences between groups were evaluated by variance analysis and Student's t test. Distribution of categorical variables between groups was evaluated using the χ 2 test. Before-and-after observations were made by paired t-test. Normality of continuous variables distribution was tested using Kolmogorov-Smirnov test. For parametric tests non parametric variables were log-transformed. Analyses were performed using SPSS v23 (Chicago, Illinois). A probability value <0.05 was considered statistically significant.

Results

Key baseline characteristics were comparable in the two groups (Table 1). Interestingly, a trend toward a higher ID and WCSA was observed in RH patients, although not statistically significant. As compared to standard-therapy group, BAT group had a higher number of medications and higher office and home SBP, as expected. The median (IQR) follow-up time was 182 (122-196) days for BAT group and 192 (74-417) days for standard-therapy group (p=0.8). Interestingly, patient in the BAT group had had a failed attempt of renal denervation. Both therapeutic interventions were efficient, as shown in Table 2: office SBP decreased by 9.7±12.3% and 29.7±12.4% in standardtherapy and BAT group respectively, while office DBP decreased by 7.6±17.4% and 14.8±15.7%. Concerning ABPM/HBPM, a mean reduction of 7.9±11% for both SBP and DBP was observed for the standard-therapy while a reduction of 15.8±10.5% and 15.8%±5.3% was observed for SBP and DBP respectively in BAT group. As shown in Figure 1, while in the standard therapy group a significant reduction in WLR (-5.9%) due to both ID increase (+2.3%) and WT reduction (-5.7%) was observed, without changes in WCSA (-0.3%), RH patients had a significant reduction in WCSA (-12.1%), due to a trend in both WT and ID reduction (-6.5% and -1.7% respectively), without significant changes in WLR (-2%). Differences in retinal remodeling indexes changes were not statistically significant between the two treatment groups.

Discussion

In this study we analyzed retinal microcirculatory adaptation to BP changes in patients with RH. While a reverse eutrophic remodeling was observed in patients undergoing a standardantihypertensive treatment adaptation, hypotrophic changes were found in RH patients undergoing BAT.

Findings in the standard therapy group are in line previously published results. In a cohort of 45 hypertensive patients undergoing antihypertensive treatment improvement, we found a significant reduction in WLR (-6%) together with an increase of ID (+4.4%), thus confirming the reverse eutrophic remodeling after BP control in hypertensive patients [START_REF] Rosenbaum | Effects of age, blood pressure and antihypertensive treatments on retinal arterioles remodeling assessed by adaptive optics[END_REF].

A difference is the arteriolar reverse remodeling observed in the two groups could be related to the different observed BP drop independently from BAT therapy. This mechanism should be therefore related to a different baseline condition (e.g. hypertrophic remodeling in RH, as observed in secondary hypertension), but this was not the case of our cohort.

In RH patients we only observed a reduction in WCSA, without changes in WLR despite a reduction trend in both WT and ID. To our knowledge, this is the first time that a hypotrophic pattern is observed in vivo on retinal small arteries. To explain this observation, a first assumption could be that these patients may have a damaged myogenic tone response as reflected by the fact that they are not responders to vasoactive antihypertensive drugs [START_REF] Schiffrin | Effect of crossing over hypertensive patients from a β-blocker to an angiotensin receptor antagonist on resistance artery structure and on endothelial function[END_REF][START_REF] Ghiadoni | Effect of the angiotensin II type 1 receptor blocker candesartan on endothelial function in patients with essential hypertension[END_REF][START_REF] Mulvany | Vascular remodeling[END_REF]. A typical myogenic tone response was observed in the standard therapy group after the introduction of calcium-channel blockers, beta-blockers or ACE-Inhibitors/Angiotensin receptor blockers where changes in WLR were due to a combined vasodilatation and wall thinning. Another reason may be that while eutrophic remodeling is the earliest event taking place in the crosstalk between micro and macrocirculation, anticipating the development of prognostically important features (such as left ventricular hypertrophy), a decrease in SNS activity could be responsible of the hypotrophic pattern changes. Renal denervation, as well as BAT, have already been shown to determine a regression of left ventricular hypertrophy, mainly due to myocyte hypertrophy reversal [START_REF] Georgakopoulos | Chronic Baroreflex Activation: A Potential Therapeutic Approach to Heart Failure With Preserved Ejection Fraction[END_REF][START_REF] Mahfoud | Effect of renal denervation on left ventricular mass and function in patients with resistant hypertension: data from a multicenter cardiovascular magnetic resonance imaging trial[END_REF]. Finally, the role of baroreflex stimulation may explain the different long term retinal response in our cohorts. As recently hypothesized [START_REF] Lohmeier | The Baroreflex as a long-term controller of arterial pressure[END_REF], common anti-hypertensive drugs may exert an effect of decreasing baroreceptors stimuli, which may determine a compensatory activation of the SNS. In the long-term this may lead to an inefficient anti-hypertensive effect leading to RH. BAT would counteract this compensatory effect, determining the significant decrease in BP that we as others observed [START_REF] Scheffers | Novel Baroreflex Activation Therapy in Resistant Hypertension Results of a European Multi-Center Feasibility Study[END_REF].

Contrary to choroidal vasculature, retinal microcirculation is characterized by the absence of any sympathetic stimuli influence [START_REF] Kur | Cellular and physiological mechanisms underlying blood flow regulation in the retina choroid in health disease[END_REF]. While baroreflex activation decreases peripheral resistance in sympathetic-stimuli-responder territories, it has no effect on retinal vascular resistance. An explanation for the long-term effect of BAT on retinal microvasculature is that chronic baroreflex stimulation may exert an effect on renal-controlled fluid balance. Interestingly, all 5 patients had undergone renal denervation therapy, without success. Behind the success of BAT compared to renal denervation there may be an insufficient natriuretic effect of renal nerve fibers, which may determine a redundant natriuretic response, mediated by an increase in atrial natriuretic peptide secretion, increased renal interstitial fluid pressure and reduced plasma protein concentration [START_REF] Lohmeier | The Baroreflex as a long-term controller of arterial pressure[END_REF]. This is the first study to evaluate in vivo retinal microvascular long-term effects of baroreflex stimulation in RH, expressed by hypotrophic changes. The direct visualization of retinal microcirculation is a unique opportunity to examine body's microvasculature safely and noninvasively in vivo.

The main limitation of this study is that BAT group consisted of only 5 patients, which makes of this a pilot study without the possibility to draw conclusions. However, even with this small sample, we were able to observe a statistically significant hypotrophic effect of BAT on retinal microvasculature. Adaptive Optics represents a reliable methodology, characterized by small intraobserver variability and allows a high-definition study of arteriolar structure [START_REF] Rosenbaum | Imaging of retinal arteries with adaptative optics, feasibility and reproducibility[END_REF].

Conclusion

A hypotrophic pattern is observed in the retinal vasculature subjected to long-term BAT. Despite the lack of adrenergic receptors on retinal vessels, chronic baroreflex stimulation may exert an effect on retinal microvasculature in RH patients by more systemic than local mechanisms.

Figure 1 .

 1 Figure 1. Retinal microvascular indexes changes in study population stratified by treatment group.

Table 1 .

 1 Characteristics of the patients at baseline

	BAT	Standard antihypertensive therapy	p value

Table 2 .

 2 Blood pressure changes in study population according to treatment groups, at baseline and follow-up

Acknowledgements:

We thank Mme Caroline Kanagasabapathy for AOC images acquisition and analysis.

The authors report no conflict of interest.