Effect Of Smoking on Retinal Vessel Morphology and Functionality: a case- control, Clinical Trial

Effect Of Smoking on Retinal Vessel Morphology and Functionality: a case- control, Clinical Trial | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Effect Of Smoking on Retinal Vessel Morphology and Functionality: a case- control, Clinical Trial Riccardo Sacconi, Seraphine Saladino, Paolo Forte, Federico Beretta, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8916335/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract There is a lack of knowledge about the effect of smoking activity on the anatomy and functionality of retinal vessels. The aim of this low-risk, single-center, case-control, interventional clinical trial (ClinicalTrials.gov ID NCT07261176) was to evaluate the effect of smoking activity on the functionality and anatomy of the retinal vessels. Forty healthy subjects were enrolled: 20 healthy volunteers with regular daily tobacco activity, and 20 healthy volunteers without tobacco activity (negative control). Each subject underwent a comprehensive ophthalmologic examination, including optical coherence tomography angiography(OCT-A), evaluation of vascular functionality by the dynamic vessel analyzer(DVA), and retinal vessel analyzer(RVA) at baseline and repeated for the "smoking" group 3 and 30 minutes after the smoking activity (8 and 35 minutes after baseline in controls). By means of RVA, the static analysis disclosed a significant decrease of central retinal artery equivalent 3 minutes after cigarette smoking, which is maintained even after 30 minutes after the smoking activity(p = 0.014). By means of DVA, a significant difference was disclosed in arterial dilation percentage post-flicker stimulation 3 minutes after smoking activity, comparing smokers and controls(mean arterial dilation 3.880 ± 2.284% and 2.130 ± 1.751%, respectively, p = 0.010). Interestingly, no significant differences in RVA and DVA analyses were disclosed at the baseline between smoker group and controls. Finally, using OCT-A, no significant change was found. We disclosed an “acute” side effect of smoking activity on ocular vascular system, showing an immediate vasoconstriction after smoking activity. However, no chronic effects from smoking activity were disclosed in our population of young people with low smoking activity. Health sciences/Diseases Health sciences/Health care Health sciences/Medical research Health sciences/Risk factors Introduction Tobacco smoking is a popular addictive habit and a growing health problem. Smoking activity causes cardiovascular pathology, influencing several pathways: dysfunction of vascular endothelial cells, instabilities of systemic hemostatic and coagulation, and lipid abnormalities. 1 , 2 The influence of tobacco smoking on endothelial dysfunction is due to diminished production of nitric oxide (NO). Smokers showed a reduction of nitrate and nitrite in the serum, and a reduction of metabolic end-products of NO in comparison with non-smokers. 1 , 3 Smoker population showed an increase rate and mortality from ischemic stroke and subarachnoid hemorrhage. 4 – 8 An estimated overall relative risk of stroke, from a 32-study meta-analysis, was found to be 1.5 in smokers versus non-smokers. 7 The retina is characterized by the great advantage of direct visualization of vessels and capillaries (directly or using noninvasively imaged). This allows us to visualize vessel damage that could reflect the systemic vascular status 9 . The oxidative stress of smoking activity was related to several ocular pathologies. 10 – 13 However, the effects of smoking on retinal microcirculation remain incompletely understood. Alterations of retinal blood flow were associated by smoking activity. 14 Both morphological (i.e. vessel wall damage and capillary loss) and functional alterations (i.e. sequestration of blood in the microcirculation and reactive hyperemia) were linked to smoking activity. 15 Several processes are involved in the complex mechanism of the autoregulation of circulation in the human body in order to maintain constant blood flow perfusion, such as the local myogenic response, endothelium-derived substances, local metabolic factors, and the autonomic nerves. 16 During smoking activity, the level of norepinephrine and epinephrine in the blood increases significantly, resulting into a blood pressure increase, and thus activation of autoregulation process. 17 , 18 The impact of smoking on retinal autoregulation, vascular reactivity, and other ocular retinal hemodynamic parameters in otherwise healthy individuals is controversial. 19 Recently, thanks to the development of optical coherence tomography angiography (OCT-A) in the clinical practice and research, we are able to analyze in vivo changes of retinal vessels and choriocapillaris in different situations. 20 – 23 Even if smoking activity was reported as a risk factor for chronic reduced retinal capillaries, 24 a couple of studies reported no acute effects of smoking activity on retinal vessels using OCT-A 14,25 . However, no previous studies reported the effect of smoking activity using an anatomic-functional test as dynamic vessel analyzer (DVA). Indeed, DVA, thanks to the principle of neurovascular coupling, studies in a non-invasive way the endothelial function. 26 , 27 The aim of this clinical trial is to evaluate the effect of smoking activity on the functionality and anatomy of the retinal vessels using OCT-A and DVA. Methods Study participants This is a low-risk, single-center, case-control, interventional study. Forty healthy subjects were enrolled at the Medical Retina & Imaging Unit of the Department of Ophthalmology, University Vita-Salute, San Raffaele Hospital in Milan from April 2021 to December 2024. In detail, 20 healthy volunteers with regular daily tobacco activity (about 10 cigarettes per day, who have been smoking for 5 to 10 years) as a normal life habit were enrolled. In addition, 20 healthy volunteers who do not engage in tobacco activity as a normal life habit were enrolled as negative controls. The study was conducted in accordance with the Declaration of Helsinki for research involving human participants and was approved by the San Raffaele Ethics Committee (approval number 179/INT/2020). All participants provided written informed consent prior to enrollment in the study. The trial was registered on ClinicalTrials.gov (ID NCT07261176, registration date: 21/11/2025). Inclusion criteria for the study group were: 1) adults of both sexes (aged 20 to 30 years old); 2) healthy volunteers who can understand and sign informed consent. Exclusion criteria included: 1) inability to follow planned procedures; 2) opacity of the dioptric means that do not allow the execution of the exams; 3) known allergies that do not allow pupillary dilation; 4) ocular pathologies and systemic hypertension or diabetes. Study Protocol Study Protocol Based on the normal lifestyle habits of the included healthy volunteers, they were enrolled in the "smoking" or "non-smoking" group (negative control group). Only healthy volunteers using cigarettes with a nicotine amount of 0.8 grams were included. In order to avoid the recent smoking activity being a confounding factor, each healthy volunteer was asked not to engage in tobacco activity in the previous 24 hours, and it was also recommended the exclusion of any other vasoconstrictor substance (e.g., coffee and alcohol). At baseline, all healthy volunteers underwent the planned clinical examinations including comprehensive ophthalmologic examination, best-corrected visual acuity (BCVA), structural spectral domain-OCT (SD-OCT, Spectralis, Heidelberg Engineering, Heidelberg, Germany), OCT-A (PlexElite 9000, SS-OCTA, Carl Zeiss Meditec, Inc., Dublin, USA), evaluation of retinal vascular functionality by DVA (Imedos, Jena, Germany), and evaluation of retinal vessel diameter using retinal vessel analyzer (RVA, Imedos GmbH, Jena, Germany). These examinations were repeated for the "smoking" group 3 minutes and 30 minutes after the healthy volunteers performed their usual smoking activity. Only one eye per healthy volunteer was included. For the "non-smoking" group, examinations were repeated at 8 and 35 minutes after baseline (with no tobacco activity). The time was studied as 5 minutes of the usual time of smoking activity in the "smoking" group in addition to the intervals already defined in the "smoking" group. The examinations were also repeated in the non-smoking group to assess the influence of the learning effect of the examination and to evaluate the intervariability of the examination. OCT-A image acquisition and analysis In all patients, a scanning area of 6 x 6mm was adopted, centered on the foveal area. FAZ area was manually outlined using the polygon selection tool in the whole retinal plexus, and its dimension was expressed as square millimeters (mm 2 ). Specifically, obtained images were imported into ImageJ 1.50 software (National Institutes of Health, Bethesda, Maryland, USA) and the mean’s thresholding was used to binarize each image of superficial and deep capillary plexus (SCP and DPC), whereas choriocapillaris images were binarized through Phansalkar’s thresholding (radius, 15 pixels). Perfusion density (PD) was calculated for SCP, DCP, and choriocapillaris (CC) as the ratio between the white pixels and the total pixels after FAZ exclusion. Dynamic Vessel Analysis Dynamic Vessel Analyzer assesses the degree of arterial and venous dilation upon stimulation with flicker light. During the examination, 3 cycles of flicker/non-flicker light were registered. The DVA creates flicker with an optoelectronic shutter that interrupts the light source with a bright-to-dark ratio of 25:1 at a frequency of 12.5 Hz, to maximize vasodilation and blood flow during flicker 28 – 30 . The flicker stimulation had a duration of 20s (to allow vessel dilation). Vessel diameters were expressed in measurement units (MU); vessel dilation was measured by calculating the percentage increase in vessel diameter relative to baseline after 20s of flicker stimulation and averaging the 3 measurement cycles. Static Vessel Analysis The static vessel analysis was performed on fundus images acquired by a fundus camera (FF450; Carl Zeiss GmbH, Jena, Germany) and analyzed by VISUALIS and VesselMap Software (Imedos Systems, Ltd, Jena, Germany). This analysis gives us the central retinal artery equivalent (CRAE), which is strictly related to the central retinal artery diameter, and the central retinal vein equivalent (CRVE), which is strictly related to the central retinal vein diameter. Statistical Analysis Statistical analysis was performed using SPSS Statistics software version 22.0 (SPSS Inc.,cIBM, Chicago, IL, USA). Catergorical variable were reported as counts and percentages, whereas quantitative variables as means ± standard deviation. Chi-squared test was performed to compare qualitative variables. The intraclass correlation coefficient (ICC; 95% CI) was used to estimate the agreement between individual measurements from both readers. Normal distribution of continuous variables was tested using the Kolmogorov-Smirnov test. Comparisons of arterial diameter, arterial dilation, arterial constriction, venous diameter, venous dilation, CRAE, CRVE, FAZ, PD of SCP, PD of DCP, and PD of CC between different timepoints (before and after tobacco smoking, and 30 minutes after tobacco smoking) were performed using repeated measures analysis of variance (ANOVA) with Bonferroni post hoc analysis 20 . Comparisons of arterial diameter, arterial dilation, arterial constriction, venous diameter, venous dilation, CRAE, CRVE, FAZ, PD of SCP, PD of DCP, and PD of CC between smokers and controls at the same timepoint were performed using independent sample t-test 20 . In all analyses, p-values < 0.05 were considered statistically significant. Results Characteristics of subjects included in the analysis Forty eyes of 40 healthy subjects were enrolled. Twenty-one subjects (52.5%) were females, and 19 subjects were males (47.5%). All subjects were Caucasian, and the mean age was 25 ± 2 years (median 25, range 22–30). BCVA was 20/20 Snellen equivalent in all included eyes. All patients were phakic, and the mean refractive error was − 1.1 ± 2.0 diopters (median, -0.37, range − 5.0/+4.0). By means of structural OCT, the mean CMT was 273 ± 21 µm (median 275, range 230–330). Subfoveal ChT was 329 ± 92 µm (median 322, range 186–574), whereas mean ChT (obtained as the mean between subfoveal ChT, ChT measured 1000 µm nasally to the fovea, and ChT measured 1000 µm temporally to the fovea 31 ) was 336 ± 91 µm (median 330, range 199–585). Among 40 eyes, 20 eyes of 20 subjects were enrolled in the smoker group, whereas 20 eyes of 20 subjects were enrolled in the control group. No statistical differences were disclosed between the two groups analysing age (p = 0.713) and sex (p = 0.342). All the demographics of the study population were reported in Table 1 . Table 1 Demographics and main clinical features at the baseline of the study population Age, years (Mean ± SD) Study population (n = 40) Smoker group (n = 20) Control group (n = 20) P value 25 ± 2 24.7 ± 1.7 24.9 ± 1.7 0.713 Sex, n (%) - Females 21 (52.5) 9 (45%) 12 (60%) - Males 19 (47.5) 11 (55%) 8 (40%) 0.342 BCVA, LogMAR (Mean ± SD) 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 1 Refractive Error, D (Mean ± SD) -1.1 ± 2.0 -0.4 ± 1.6 -1.9 ± 2.2 0.018 CMT, µm (mean ± SD) 273 ± 21 280 ± 23 267 ± 15 0.054 Subfoveal ChT, µm (mean ± SD) 329 ± 92 350 ± 68 308 ± 109 0.156 Mean ChT, µm (mean ± SD) 336 ± 91 356 ± 69 317 ± 107 0.179 n: number; SD: standard deviation; BCVA: best-corrected visual acuity; D: diopters; CMT: central macular thickness; ChT: choroidal thickness. Static Vessel Analysis By means of RVA, in the smoker group, the static analysis disclosed a significant decrease of CRAE after the smoking activity (p = 0.014). In detail, CRAE was 263.9 ± 54.5 MU at the baseline, 223.7 ± 36.3 MU 3 minutes after the smoker activity (p = 0.009), and 227.5 ± 49.2 MU 30 minutes after the smoker activity (p = 0.044). On the other hand, no significant differences were disclosed in the controls analyzing CRAE in the 3 time-points (p = 0.561). Analyzing CRVE, in the smoker group, the mean CRVE showed a trend of decrease after smoking activity, although not statistically significant (p = 0.095). Of note, no significant differences were disclosed in the controls analyzing CRVE in the 3 time-points (p = 0.618). All the analyses and p-values were reported in Table 2 . Table 2 Dynamic and static vessel analysis, and OCT-A analysis of the smoker population (n = 20) ANOVA Baseline 3-minute post smoking 30-minute post smoking P value* Mean ± SD Mean ± SD P value + Mean ± SD P value + P value § Arterial Dilation, % 0.217 2.850 ± 2.393 3.880 ± 2.284 0.332 3.425 ± 3.448 1 1 Arterial Constriction, % 0.490 -0.580 ± 1.624 -0.230 ± 1.216 1 -0.665 ± 1.766 1 0.739 Venous Dilation, % 0.109 4.335 ± 3.262 3.485 ± 2.344 0.303 3.050 ± 1.717 0.103 1 CRAE, MU 0.014 263.9 ± 54.5 223.7 ± 36.3 0.009 227.5 ± 49.2 0.044 1 CRVE, MU 0.095 292.0 ± 58.6 263.9 ± 31.1 0.109 265.7 ± 42.9 0.124 1 FAZ, mm 2 0.587 0.190 ± 0.135 0.190 ± 0.137 1 0.193 ± 0.141 1 0.926 PD SCP, % 0.159 41.7 ± 2.7 40.9 ± 2.5 0.706 42.1 ± 2.8 1 0.156 PD DCP, % 0.200 40.2 ± 2.0 40.7 ± 2.2 1 41.0 ± 2.1 0.267 0.950 PD CC, % 0.930 73.6 ± 2.6 73.4 ± 1.9 1 73.5 ± 1.7 1 1 ANOVA: Analysis of variance; SD: standard deviation; CRAE: central retinal artery equivalent; CRVE central retinal vein equivalent; MU: measurement units; FAZ: foveal avascular zone; PD: perfusion density; SCP: superficial capillary plexus; DCP: deep capillary plexus. Statistical Analysis: Analysis of variance (ANOVA) for repeated measures with Bonferroni post-hoc analysis. *: ANOVA for repeated measures +: comparison with baseline using Bonferroni post-hoc analysis §: comparison with 3-minute value using Bonferroni post-hoc analysis Dynamic Vessel Analysis By means of DVA, the diameter of the artery and of the vein selected for the analyses was not different between the two groups at the baseline (p = 0.591 and p = 0.379 for the arterial diameter and vein diameter analysis, respectively). Analyzing the response of the retinal vessels to the flicker light, we disclosed no significant difference in the arterial dilation percentage after the smoking activity (p = 0.217), although there was a trend with increased arterial dilation response (mean arterial dilation of 2.850 ± 2.393% at the baseline, 3.880 ± 2.284% 3 minutes after smoking activity, 3.425 ± 3.448% 30 minutes after smoking activity). On the other hand, no significant difference or trend was disclosed in the control group comparing examinations of the 3 timepoints (p = 0.710). Analyzing the arterial constriction after the dilation due to the flicker light and the vein dilation percentage, no significant difference was disclosed in both groups. Comparing the arterial dilation percentage, arterial constriction percentage, and vein dilation percentage between smoker group and controls in the respective time-points, a significant difference was disclosed in arterial dilation percentage at 8 minutes (mean arterial dilation 3.880 ± 2.284% and 2.130 ± 1.751%, respectively, p = 0.010). This difference was reduced considering the 35-minute examination (mean arterial dilation 3.425 ± 3.448% and 1.875 ± 1.687%, respectively, p = 0.079). No other differences were disclosed. All the analyses about the DVA parameters were reported in Tables 2 and 3 . Table 3 Dynamic and static vessel analysis, and OCT-A analysis of the control population (n = 20) ANOVA Baseline 8-minute later 35-minute later P value* Mean ± SD Mean ± SD P value + Mean ± SD P value + P value § Arterial Dilation, % 0.710 2.265 ± 1.965 2.130 ± 1.751 1 1.875 ± 1.687 1 1 Arterial Constriction, % 0.910 -0.525 ± 1.028 -0.370 ± 1.484 1 -0.460 ± 1.773 1 1 Venous Dilation, % 0.628 3.130 ± 2.657 2.670 ± 1.817 1 2.745 ± 2.286 1 1 CRAE, MU 0.561 225.4 ± 28.6 219.2 ± 36.9 1 217.9 ± 40.1 0.900 1 CRVE, MU 0.618 252.0 ± 31.1 245.8 ± 42.3 1 253.7 ± 42.8 1 0.994 FAZ, mm 2 0.063 0.224 ± 0.084 0.223 ± 0.079 0.279 0.225 ± 0.081 0.060 1 PD SCP, % 0.980 42.6 ± 3.3 42.6 ± 3.2 1 42.5 ± 2.8 1 1 PD DCP, % 0.679 40.3 ± 2.9 40.1 ± 3.1 1 40.3 ± 2.7 1 1 PD CC, % 0.919 73.2 ± 2.1 73.2 ± 2.0 1 73.1 ± 2.3 1 1 ANOVA: Analysis of variance; SD: standard deviation; CRAE: central retinal artery equivalent; CRVE central retinal vein equivalent; MU: measurement units; FAZ: foveal avascular zone; PD: perfusion density; SCP: superficial capillary plexus; DCP: deep capillary plexus. Statistical Analysis: Analysis of variance (ANOVA) for repeated measures with Bonferroni post-hoc analysis. *: ANOVA for repeated measures +: comparison with baseline using Bonferroni post-hoc analysis §: comparison with 8-minute value using Bonferroni post-hoc analysis OCT-Angiography Analysis Examinations of FAZ did not show any significant microvascular change after the smoking activity (Table 2 ). Interobserver variability between readers was excellent for FAZ measurements (ICC = 0.991 [0.978–0.996]). Perfusion density among SCP, DCP, and CC did not show any significant change before and after smoking activity in smoking group (p = 0.159, p = 0.200, and p = 0.930 for SCP, DCP, and CC analysis, respectively) and by comparing examinations of 3 timepoints in controls (p = 0.980, p = 0.679, and p = 0.919 for SCP, DCP, and CC analysis, respectively). No significant differences were disclosed comparing FAZ, PD of SCP, DCP, and CC between smoking group and controls in the respective time-points (p > 0.1 in all analyses). The signal strength index (SSI) of examinations did not show any significant difference among the different examinations (p > 0.2 in all analyses). Discussion In this study, we used a multimodal imaging evaluation including DVA and OCT-A to analyze vascular retinal changes induced by cigarette smoking. We disclosed a significant arterial constriction of retinal vessels 3 minutes after cigarette smoking, which is maintained even after 30 minutes. The innovation of our study is to possibly clarify which are the smoking effects on retinal vessel morphology and function by adding to the classical retinal imaging techniques the DVA analysis. Cigarette smoking increases the risk of cardiovascular pathology through several pathways 1 . Effects of smoking cigarettes on the microvessels involve diminished endothelial-dependent vasodilation, endothelial cell dysfunction, and accelerated platelet aggregation. These modifications lead to vascular blood flow changes due to vasodilatation or vasoconstriction. 32 Furthermore, smoking increases the chances of atherosclerotic plaque rupture, leading to acute myocardial infarction, stroke, or acute peripheral vascular diseases 33 . In our trial, the static analysis of the retinal vessels confirmed a significant decrease of CRAE in the smoking group 3 minutes after smoking (p = 0.009) and a maintenance of vasoconstriction 30 minutes after smoking (p = 0.044). Moreover, in the smoking group, mean CRVE showed a trend of decrease after smoking activity. Since CRAE and CRVE are related to the diameter of the central retinal artery and vein, respectively, we disclosed a significant constriction of the central retinal artery and a trend of constriction of the central retinal vein after smoking. This was not disclosed in the control group. These data agree with the systemic effects of smoking. As discussed before, smoking causes vasoconstriction in other cardiovascular systems, as in coronary blood flow. 2 Thanks to DVA, we have confirmed and reported the direct vasoconstriction effect of smoking activity on the retinal vessels. Interestingly, analyzing the CRAE and CRVE at baseline, there was no difference between the group of smokers and controls. This means that smokers showed no chronic vasoconstriction in comparison to controls. There are several reasons for this: the volunteers enrolled in the study are young people with low smoking activity (maximum 10 cigarettes/day) who have been smoking for up to 10 years. Furthermore, the volunteers were asked to do a cigarette smoke wash-out 24 hours before being subjected to the study. For all these reasons, the smokers included in our analysis may not yet have developed chronic vascular effects. However, we cannot exclude that greater smokers could also show chronic vasoconstriction as suggested by Zhu et al 24 . They showed that cigarette smoking is an independent risk factor for reduced vessel density or retinal capillaries, and this is strictly related to increased smoking pack-years. This data suggested a time-dependent and dose-dependent effect of chronic vascular retinal damage induced by smoking activity. Analyzing the vessel dilation (arterial and venous) in response to the flicker light, we disclosed a trend with increased arterial dilation response after the smoking activity in smokers. On the other hand, no significant difference or trend was disclosed in the control group comparing examinations of the 3 timepoints. Comparing the arterial dilation percentage between the smoking group and controls in the respective time-points, a significant difference was disclosed at 8 minutes, comparing smokers and controls. This difference was reduced considering the 35-minute examination. These results could be explained because after smoking activity, the artery responds with vasoconstriction; as the post-flicker dilation is a percentage of the pre-stimulation diameter, the result is influenced by the last one. We have seen that the pre-stimulation diameter is smaller after cigarette smoking, so the percentage expressing arterial dilation will be increased. However, DVA analyses the vasodilation response to the flicker stimulus on large retinal vessels and not on small vessels. On the other hand, the major effect of smoking cigarettes on vessel response was previously reported involving the micro-vessels and endothelial-dependent vasorelaxation, which were not investigated using DVA. 32 Regarding the analysis of retinal plexuses using OCT-A, our results confirm the literature. Ayhan et al. 14 have reported that smoking cigarettes does not cause significant changes in the size of the FAZ area and the macular vessel density in healthy habitual smokers. Ciesielski et al. 25 disclosed the lack of immediate effects of cigarette smoking on the vascular density of the central retina in healthy habitual smokers and no significant changes in the FAZ. All these results were confirmed in our analysis. Analyzing the PD of the retinal plexuses (SCP and DCP) and of the CC, we did not disclose any significant changes immediately after and 30 minutes after the smoking activity. Limitations of our study should be kept in mind. First of all, the present study is mainly limited by its small sample size. Moreover, the sample does not reflect all smokers, because we selected young people who have not smoked for more than 10 years and who smoke about 10 cigarettes a day. But this is also a strength because it makes our population homogeneous, and thus, we were able to study chronic and acute effects of smoking activity on retinal vessels in this kind of smoker. As further strength, our study is the first to add DVA and RVA analysis as an objective parameter to the evaluation of possible smoking effects on visual function. In conclusion, our study demonstrates that there is a significant acute arterial constriction response after cigarette smoking, which is maintained even after 30 minutes (although slightly reduced). Furthermore, we reported no chronic vasoconstriction of retinal vessels in comparison to controls, suggesting that low-dose and low-time of smoking activity, as in our population (about 10 cigarettes/day for 5 to 10 years), is not enough to induce chronic vasoconstriction at the level of the retinal vessels. Stopping smoking at this stage could result in beneficial effects for smoking-induced vascular diseases. However, further studies are warranted to better understand the physiology behind this mechanism and to confirm the same effects on other vascular systems outside the eye. Declarations Financial support none. Disclosures : Riccardo Sacconi is a consultant for: AbbVie (North Chicago, Illinois, USA), Bayer Shering-Pharma (Berlin, Germany), Boehringer Ingelheim (Ingelheim, Germany), Carl Zeiss Meditec (Dublin, USA), Hoffmann-La-Roche (Basel, Switzerland), Novartis (Basel, Switzerland). Seraphine Saladino, Paolo Forte, Federico Beretta: none. Francesco Bandello is a consultant for Alcon (Fort Worth,Texas,USA), Alimera Sciences (Alpharetta, Georgia, USA), AbbVie (North Chicago, Illinois, USA), Farmila-Thea (Clermont-Ferrand, France), Bayer Shering-Pharma (Berlin, Germany), Bausch And Lomb (Rochester, New York, USA), Genentech (San Francisco, California, USA), Hoffmann-La-Roche (Basel, Switzerland), NovagaliPharma (Évry, France), Novartis (Basel, Switzerland), Sanofi-Aventis (Paris, France), Thrombogenics (Heverlee,Belgium), Zeiss (Dublin, USA). Giuseppe Querques is a consultant for Alimera Sciences (Alpharetta, Georgia, USA), AbbVie (North Chicago, Illinois, USA), Amgen (Thousand Oaks, USA), Heidelberg (Germany), KBH (Chengdu, China), LEH Pharma (London, UK), Lumithera (Poulsbo, USA), Novartis (Basel, Switzerland), Bayer Shering-Pharma (Berlin, Germany), Sandoz (Berlin, Germany), Sifi (Catania, Italy), Soof-Fidia (Albano, Italy), Zeiss (Dublin, USA). Author Contribution RS, GQ: research design, data acquisition and analysis, interpretation of data, drafting the manuscript and critical revision of the manuscript. SS, PF, FB: data acquisition and analysis, critical revision of the manuscript. FB: interpretation of data and critical revision of the manuscript. Acknowledgement Acknowledgments: none.Disclosures: Riccardo Sacconi is a consultant for: AbbVie (North Chicago, Illinois, USA), Bayer Shering-Pharma (Berlin, Germany), Boehringer Ingelheim (Ingelheim, Germany), Carl Zeiss Meditec (Dublin, USA), Hoffmann-La-Roche (Basel, Switzerland), Novartis (Basel, Switzerland). Seraphine Saladino, Paolo Forte, Federico Beretta: none. Francesco Bandello is a consultant for Alcon (Fort Worth,Texas,USA), Alimera Sciences (Alpharetta, Georgia, USA), AbbVie (North Chicago, Illinois, USA), Farmila-Thea (Clermont-Ferrand, France), Bayer Shering-Pharma (Berlin, Germany), Bausch And Lomb (Rochester, New York, USA), Genentech (San Francisco, California, USA), Hoffmann-La-Roche (Basel, Switzerland), NovagaliPharma (Évry, France), Novartis (Basel, Switzerland), Sanofi-Aventis (Paris, France), Thrombogenics (Heverlee,Belgium), Zeiss (Dublin, USA). Giuseppe Querques is a consultant for Alimera Sciences (Alpharetta, Georgia, USA), AbbVie (North Chicago, Illinois, USA), Amgen (Thousand Oaks, USA), Heidelberg (Germany), KBH (Chengdu, China), LEH Pharma (London, UK), Lumithera (Poulsbo, USA), Novartis (Basel, Switzerland), Bayer Shering-Pharma (Berlin, Germany), Sandoz (Berlin, Germany), Sifi (Catania, Italy), Soof-Fidia (Albano, Italy), Zeiss (Dublin, USA). 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Eye 4 , 319–325 (1990). Cryer, P. E., Haymond, M. W., Santiago, J. V. & Shah, S. D. Norepinephrine and Epinephrine Release and Adrenergic Mediation of Smoking-Associated Hemodynamic and Metabolic Events. N Engl. J. Med. 295 , 573–577 (1976). Robinson, F., Riva, C. E., Grunwald, J. E., Petrig, B. L. & Sinclair, S. H. Retinal blood flow regulation in response to an acute increase in blood pressure. Investig. Ophthalmol. Vis. Sci. 27 , 722–726 (1986). Gilmore, E. D., Hudson, C., Venkataraman, S. T., Preiss, D. & Fisher, J. Comparison of Different Hyperoxic Paradigms to Induce Vasoconstriction: Implications for the Investigation of Retinal Vascular Reactivity. ; 45 :3207–3212. (2004). Sacconi, R. et al. Benefits of dark chocolate intake on retinal vessels functionality: a randomized, blind, crossover clinical trial. Sci. Rep. 14 , 20203 (2024). Sacconi, R. et al. Optical coherence tomography angiography in pseudophakic cystoid macular oedema compared to diabetic macular oedema: qualitative and quantitative evaluation of retinal vasculature. Br. J. Ophthalmol. 102 , 1684–1690 (2018). Querques, L. et al. Anatomical and functional changes in neovascular AMD in remission: comparison of fibrocellular and fibrovascular phenotypes. Br. J. Ophthalmol. 104 , 47–52 (2020). Borrelli, E. et al. Peripapillary hyperreflective ovoid mass-like structures (PHOMS): OCTA may reveal new findings. Eye (Lond) . 35 , 528–531 (2021). Zhu, X. et al. Association of cigarette smoking with retinal capillary plexus: an optical coherence tomography angiography study. Acta Ophthalmol. 100 , e1479–e1488 (2022). Ciesielski, M., Rakowicz, P. & Stopa, M. Immediate effects of smoking on optic nerve and macular perfusion measured by optical coherence tomography angiography. Sci. Rep. 9 , 1–8 (2019). Sacconi, R. et al. Multimodal Imaging Assessment of Vascular and Neurodegenerative Retinal Alterations in Type 1 Diabetic Patients without Fundoscopic Signs of Diabetic Retinopathy. J. Clin. Med. 8 , 1409 (2019). Newman, E. A. Functional hyperemia and mechanisms of neurovascular coupling in the retinal vasculature. J. Cereb. Blood Flow. Metab. 33 , 1685–1695 (2013). Polak, K., Schmetterer, L. & Riva, C. E. Influence of flicker frequency on flicker-induced changes of retinal vessel diameter. Investig Ophthalmol. Vis. Sci. 43 , 2721–2726 (2002). Riva, C. E., Falsini, B. & Logean, E. Flicker-evoked responses of human optic nerve head blood flow: Luminance versus chromatic modulation. Investig Ophthalmol. Vis. Sci. 42 , 756–762 (2001). Falsini, B., Riva, C. E. & Logean, E. Flicker-evoked changes in human optic nerve blood flow: Relationship with retinal neural activity. Investig Ophthalmol. Vis. Sci. 43 , 2309–2316 (2002). Sacconi, R. et al. SD-OCT Choroidal Thickness in Advanced Primary Open-Angle Glaucoma. J. Glaucoma . 26 , 523–527 (2017). Barua, R. S. et al. Dysfunctional endothelial nitric oxide biosynthesis in healthy smokers with impaired endothelium-dependent vasodilatation. Circulation 104 , 1905–1910 (2001). Ishida, M., Sakai, C., Kobayashi, Y. & Ishida, T. Cigarette Smoking and Atherosclerotic Cardiovascular Disease. J. Atheroscler Thromb. 31 , 189–200 (2024). Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8916335","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":622725526,"identity":"ffdb22c6-878c-4d01-b166-116f174f4049","order_by":0,"name":"Riccardo Sacconi","email":"","orcid":"","institution":"Vita-Salute San Raffaele University","correspondingAuthor":false,"prefix":"","firstName":"Riccardo","middleName":"","lastName":"Sacconi","suffix":""},{"id":622725527,"identity":"e35fd2ed-e7a0-480e-bf5a-7b43b6dcdffc","order_by":1,"name":"Seraphine Saladino","email":"","orcid":"","institution":"Vita-Salute San Raffaele University","correspondingAuthor":false,"prefix":"","firstName":"Seraphine","middleName":"","lastName":"Saladino","suffix":""},{"id":622725528,"identity":"2186a3ca-77dd-463e-936f-39ccb6261d24","order_by":2,"name":"Paolo Forte","email":"","orcid":"","institution":"Fondazione Italiana Macula ETS","correspondingAuthor":false,"prefix":"","firstName":"Paolo","middleName":"","lastName":"Forte","suffix":""},{"id":622725529,"identity":"c2016d44-7d2a-416b-85b9-489f34561887","order_by":3,"name":"Federico Beretta","email":"","orcid":"","institution":"Vita-Salute San Raffaele University","correspondingAuthor":false,"prefix":"","firstName":"Federico","middleName":"","lastName":"Beretta","suffix":""},{"id":622725530,"identity":"d7b1d0c1-e97c-4907-81b1-2a8e49e2109a","order_by":4,"name":"Francesco Bandello","email":"","orcid":"","institution":"Vita-Salute San Raffaele University","correspondingAuthor":false,"prefix":"","firstName":"Francesco","middleName":"","lastName":"Bandello","suffix":""},{"id":622725531,"identity":"87b67c10-c5fe-4e6b-8080-c63b4d6cf4c5","order_by":5,"name":"Giuseppe Querques","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABKUlEQVRIiWNgGAWjYDACZgwR9h4wxcPHwNhApBaeMwwMB4AUGwNjI3Y9GEAiB6yFgY0BuzXy7jzGHz7uuCPHwJ5j+Lii7LCc/My3Bx9/zLGTYWNgbn+ARYvhYR4zyZlnnhkz8LwxNjxz7rCxwe28ZIOD25JxOsywmceMmbftcGKDRI6ZZCOQsUE6x0zi4DZmfFqMP/9tO1wP1GL+E6ilfv7MMyAt9Ti1yDPzGEgzth1OAPrajLERxLjBA9JyGKcWA2a2MsneM88M23ieFUs2nEs33HAmx9jg7LbjPGzMjI0zsNnSf3jzh5877sjzsydv/NhQZi0v337G8EHltmp7fvb2Bx+w2XKAART8B4CxkMAAjgsEwIxliC0NUC0MmFpGwSgYBaNgFEAAAOCbZHo4AGW8AAAAAElFTkSuQmCC","orcid":"","institution":"University of Modena and Reggio Emilia","correspondingAuthor":true,"prefix":"","firstName":"Giuseppe","middleName":"","lastName":"Querques","suffix":""}],"badges":[],"createdAt":"2026-02-19 10:09:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8916335/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8916335/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107078886,"identity":"8813a29c-3b75-4207-b438-4d6c9469ea9d","added_by":"auto","created_at":"2026-04-16 13:52:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":958409,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8916335/v1/b6e78085-89ed-4513-b4ec-c71ecba1f8b1.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effect Of Smoking on Retinal Vessel Morphology and Functionality: a case- control, Clinical Trial","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTobacco smoking is a popular addictive habit and a growing health problem. Smoking activity causes cardiovascular pathology, influencing several pathways: dysfunction of vascular endothelial cells, instabilities of systemic hemostatic and coagulation, and lipid abnormalities.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e The influence of tobacco smoking on endothelial dysfunction is due to diminished production of nitric oxide (NO). Smokers showed a reduction of nitrate and nitrite in the serum, and a reduction of metabolic end-products of NO in comparison with non-smokers.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e Smoker population showed an increase rate and mortality from ischemic stroke and subarachnoid hemorrhage.\u003csup\u003e\u003cspan additionalcitationids=\"CR5 CR6 CR7\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e An estimated overall relative risk of stroke, from a 32-study meta-analysis, was found to be 1.5 in smokers versus non-smokers.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe retina is characterized by the great advantage of direct visualization of vessels and capillaries (directly or using noninvasively imaged). This allows us to visualize vessel damage that could reflect the systemic vascular status\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. The oxidative stress of smoking activity was related to several ocular pathologies.\u003csup\u003e\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e However, the effects of smoking on retinal microcirculation remain incompletely understood. Alterations of retinal blood flow were associated by smoking activity.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e Both morphological (i.e. vessel wall damage and capillary loss) and functional alterations (i.e. sequestration of blood in the microcirculation and reactive hyperemia) were linked to smoking activity. \u003csup\u003e15\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eSeveral processes are involved in the complex mechanism of the autoregulation of circulation in the human body in order to maintain constant blood flow perfusion, such as the local myogenic response, endothelium-derived substances, local metabolic factors, and the autonomic nerves.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e During smoking activity, the level of norepinephrine and epinephrine in the blood increases significantly, resulting into a blood pressure increase, and thus activation of autoregulation process.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e The impact of smoking on retinal autoregulation, vascular reactivity, and other ocular retinal hemodynamic parameters in otherwise healthy individuals is controversial.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eRecently, thanks to the development of optical coherence tomography angiography (OCT-A) in the clinical practice and research, we are able to analyze in vivo changes of retinal vessels and choriocapillaris in different situations.\u003csup\u003e\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e Even if smoking activity was reported as a risk factor for chronic reduced retinal capillaries,\u003csup\u003e24\u003c/sup\u003e a couple of studies reported no acute effects of smoking activity on retinal vessels using OCT-A\u003csup\u003e14,25\u003c/sup\u003e. However, no previous studies reported the effect of smoking activity using an anatomic-functional test as dynamic vessel analyzer (DVA). Indeed, DVA, thanks to the principle of neurovascular coupling, studies in a non-invasive way the endothelial function.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe aim of this clinical trial is to evaluate the effect of smoking activity on the functionality and anatomy of the retinal vessels using OCT-A and DVA.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy participants\u003c/h2\u003e \u003cp\u003eThis is a low-risk, single-center, case-control, interventional study. Forty healthy subjects were enrolled at the Medical Retina \u0026amp; Imaging Unit of the Department of Ophthalmology, University Vita-Salute, San Raffaele Hospital in Milan from April 2021 to December 2024. In detail, 20 healthy volunteers with regular daily tobacco activity (about 10 cigarettes per day, who have been smoking for 5 to 10 years) as a normal life habit were enrolled. In addition, 20 healthy volunteers who do not engage in tobacco activity as a normal life habit were enrolled as negative controls.\u003c/p\u003e \u003cp\u003e The study was conducted in accordance with the Declaration of Helsinki for research involving human participants and was approved by the San Raffaele Ethics Committee (approval number 179/INT/2020). All participants provided written informed consent prior to enrollment in the study. The trial was registered on ClinicalTrials.gov (ID NCT07261176, registration date: 21/11/2025).\u003c/p\u003e \u003cp\u003e Inclusion criteria for the study group were: 1) adults of both sexes (aged 20 to 30 years old); 2) healthy volunteers who can understand and sign informed consent.\u003c/p\u003e \u003cp\u003eExclusion criteria included: 1) inability to follow planned procedures; 2) opacity of the dioptric means that do not allow the execution of the exams; 3) known allergies that do not allow pupillary dilation; 4) ocular pathologies and systemic hypertension or diabetes.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStudy Protocol\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eStudy Protocol\u003c/div\u003e \u003cp\u003eBased on the normal lifestyle habits of the included healthy volunteers, they were enrolled in the \"smoking\" or \"non-smoking\" group (negative control group). Only healthy volunteers using cigarettes with a nicotine amount of 0.8 grams were included. In order to avoid the recent smoking activity being a confounding factor, each healthy volunteer was asked not to engage in tobacco activity in the previous 24 hours, and it was also recommended the exclusion of any other vasoconstrictor substance (e.g., coffee and alcohol).\u003c/p\u003e \u003cp\u003eAt baseline, all healthy volunteers underwent the planned clinical examinations including comprehensive ophthalmologic examination, best-corrected visual acuity (BCVA), structural spectral domain-OCT (SD-OCT, Spectralis, Heidelberg Engineering, Heidelberg, Germany), OCT-A (PlexElite 9000, SS-OCTA, Carl Zeiss Meditec, Inc., Dublin, USA), evaluation of retinal vascular functionality by DVA (Imedos, Jena, Germany), and evaluation of retinal vessel diameter using retinal vessel analyzer (RVA, Imedos GmbH, Jena, Germany). These examinations were repeated for the \"smoking\" group 3 minutes and 30 minutes after the healthy volunteers performed their usual smoking activity. Only one eye per healthy volunteer was included.\u003c/p\u003e \u003cp\u003eFor the \"non-smoking\" group, examinations were repeated at 8 and 35 minutes after baseline (with no tobacco activity). The time was studied as 5 minutes of the usual time of smoking activity in the \"smoking\" group in addition to the intervals already defined in the \"smoking\" group. The examinations were also repeated in the non-smoking group to assess the influence of the learning effect of the examination and to evaluate the intervariability of the examination.\u003c/p\u003e\n\u003ch3\u003eOCT-A image acquisition and analysis\u003c/h3\u003e\n\u003cp\u003eIn all patients, a scanning area of 6 x 6mm was adopted, centered on the foveal area.\u003c/p\u003e \u003cp\u003eFAZ area was manually outlined using the polygon selection tool in the whole retinal plexus, and its dimension was expressed as square millimeters (mm\u003csup\u003e2\u003c/sup\u003e). Specifically, obtained images were imported into ImageJ 1.50 software (National Institutes of Health, Bethesda, Maryland, USA) and the mean\u0026rsquo;s thresholding was used to binarize each image of superficial and deep capillary plexus (SCP and DPC), whereas choriocapillaris images were binarized through Phansalkar\u0026rsquo;s thresholding (radius, 15 pixels). Perfusion density (PD) was calculated for SCP, DCP, and choriocapillaris (CC) as the ratio between the white pixels and the total pixels after FAZ exclusion.\u003c/p\u003e\n\u003ch3\u003eDynamic Vessel Analysis\u003c/h3\u003e\n\u003cp\u003eDynamic Vessel Analyzer assesses the degree of arterial and venous dilation upon stimulation with flicker light. During the examination, 3 cycles of flicker/non-flicker light were registered. The DVA creates flicker with an optoelectronic shutter that interrupts the light source with a bright-to-dark ratio of 25:1 at a frequency of 12.5 Hz, to maximize vasodilation and blood flow during flicker\u003csup\u003e\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. The flicker stimulation had a duration of 20s (to allow vessel dilation). Vessel diameters were expressed in measurement units (MU); vessel dilation was measured by calculating the percentage increase in vessel diameter relative to baseline after 20s of flicker stimulation and averaging the 3 measurement cycles.\u003c/p\u003e\n\u003ch3\u003eStatic Vessel Analysis\u003c/h3\u003e\n\u003cp\u003eThe static vessel analysis was performed on fundus images acquired by a fundus camera (FF450; Carl Zeiss GmbH, Jena, Germany) and analyzed by VISUALIS and VesselMap Software (Imedos Systems, Ltd, Jena, Germany). This analysis gives us the central retinal artery equivalent (CRAE), which is strictly related to the central retinal artery diameter, and the central retinal vein equivalent (CRVE), which is strictly related to the central retinal vein diameter.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using SPSS Statistics software version 22.0 (SPSS Inc.,cIBM, Chicago, IL, USA). Catergorical variable were reported as counts and percentages, whereas quantitative variables as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Chi-squared test was performed to compare qualitative variables. The intraclass correlation coefficient (ICC; 95% CI) was used to estimate the agreement between individual measurements from both readers. Normal distribution of continuous variables was tested using the Kolmogorov-Smirnov test. Comparisons of arterial diameter, arterial dilation, arterial constriction, venous diameter, venous dilation, CRAE, CRVE, FAZ, PD of SCP, PD of DCP, and PD of CC between different timepoints (before and after tobacco smoking, and 30 minutes after tobacco smoking) were performed using repeated measures analysis of variance (ANOVA) with Bonferroni post hoc analysis\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Comparisons of arterial diameter, arterial dilation, arterial constriction, venous diameter, venous dilation, CRAE, CRVE, FAZ, PD of SCP, PD of DCP, and PD of CC between smokers and controls at the same timepoint were performed using independent sample t-test\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. In all analyses, p-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003eCharacteristics of subjects included in the analysis\u003c/h2\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003eForty eyes of 40 healthy subjects were enrolled. Twenty-one subjects (52.5%) were females, and 19 subjects were males (47.5%). All subjects were Caucasian, and the mean age was 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2 years (median 25, range 22\u0026ndash;30). BCVA was 20/20 Snellen equivalent in all included eyes. All patients were phakic, and the mean refractive error was \u0026minus;\u0026thinsp;1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0 diopters (median, -0.37, range\u0026thinsp;\u0026minus;\u0026thinsp;5.0/+4.0). By means of structural OCT, the mean CMT was 273\u0026thinsp;\u0026plusmn;\u0026thinsp;21 \u0026micro;m (median 275, range 230\u0026ndash;330). Subfoveal ChT was 329\u0026thinsp;\u0026plusmn;\u0026thinsp;92 \u0026micro;m (median 322, range 186\u0026ndash;574), whereas mean ChT (obtained as the mean between subfoveal ChT, ChT measured 1000 \u0026micro;m nasally to the fovea, and ChT measured 1000 \u0026micro;m temporally to the fovea\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e) was 336\u0026thinsp;\u0026plusmn;\u0026thinsp;91 \u0026micro;m (median 330, range 199\u0026ndash;585).\u003c/div\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\n \u003cp\u003eAmong 40 eyes, 20 eyes of 20 subjects were enrolled in the smoker group, whereas 20 eyes of 20 subjects were enrolled in the control group. No statistical differences were disclosed between the two groups analysing age (p\u0026thinsp;=\u0026thinsp;0.713) and sex (p\u0026thinsp;=\u0026thinsp;0.342). \u0026nbsp;\u003c/p\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n \u003cp\u003eAll the demographics of the study population were reported in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eDemographics and main clinical features at the baseline of the study population\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth rowspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge, years (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eStudy population (n\u0026thinsp;=\u0026thinsp;40)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSmoker group (n\u0026thinsp;=\u0026thinsp;20)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eControl group (n\u0026thinsp;=\u0026thinsp;20)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eP value\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e24.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e24.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e0.713\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eSex, n (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e- \u003cstrong\u003eFemales\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21 (52.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9 (45%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12 (60%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e- \u003cstrong\u003eMales\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19 (47.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11 (55%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8 (40%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.342\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eBCVA, LogMAR (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eRefractive Error, D (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.018\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCMT, \u0026micro;m (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e273\u0026thinsp;\u0026plusmn;\u0026thinsp;21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e280\u0026thinsp;\u0026plusmn;\u0026thinsp;23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e267\u0026thinsp;\u0026plusmn;\u0026thinsp;15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.054\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eSubfoveal ChT, \u0026micro;m (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e329\u0026thinsp;\u0026plusmn;\u0026thinsp;92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e350\u0026thinsp;\u0026plusmn;\u0026thinsp;68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e308\u0026thinsp;\u0026plusmn;\u0026thinsp;109\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.156\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean ChT, \u0026micro;m (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e336\u0026thinsp;\u0026plusmn;\u0026thinsp;91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e356\u0026thinsp;\u0026plusmn;\u0026thinsp;69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e317\u0026thinsp;\u0026plusmn;\u0026thinsp;107\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.179\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003en: number; SD: standard deviation; BCVA: best-corrected visual acuity; D: diopters; CMT: central macular thickness; ChT: choroidal thickness.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eStatic Vessel Analysis\u003c/h2\u003e\n \u003cp\u003eBy means of RVA, in the smoker group, the static analysis disclosed a significant decrease of CRAE after the smoking activity (p\u0026thinsp;=\u0026thinsp;0.014). In detail, CRAE was 263.9\u0026thinsp;\u0026plusmn;\u0026thinsp;54.5 MU at the baseline, 223.7\u0026thinsp;\u0026plusmn;\u0026thinsp;36.3 MU 3 minutes after the smoker activity (p\u0026thinsp;=\u0026thinsp;0.009), and 227.5\u0026thinsp;\u0026plusmn;\u0026thinsp;49.2 MU 30 minutes after the smoker activity (p\u0026thinsp;=\u0026thinsp;0.044). On the other hand, no significant differences were disclosed in the controls analyzing CRAE in the 3 time-points (p\u0026thinsp;=\u0026thinsp;0.561).\u003c/p\u003e\n \u003cp\u003eAnalyzing CRVE, in the smoker group, the mean CRVE showed a trend of decrease after smoking activity, although not statistically significant (p\u0026thinsp;=\u0026thinsp;0.095). Of note, no significant differences were disclosed in the controls analyzing CRVE in the 3 time-points (p\u0026thinsp;=\u0026thinsp;0.618).\u003c/p\u003e\n \u003cp\u003eAll the analyses and p-values were reported in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eDynamic and static vessel analysis, and OCT-A analysis of the smoker population (n\u0026thinsp;=\u0026thinsp;20)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth rowspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eANOVA\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eBaseline\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e3-minute post smoking\u003c/p\u003e\n \u003c/th\u003e\n \u003cth colspan=\"3\" align=\"left\"\u003e\n \u003cp\u003e30-minute post smoking\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP value*\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP value\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP value\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP value\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026sect;\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eArterial Dilation, %\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e0.217\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e2.850\u0026thinsp;\u0026plusmn;\u0026thinsp;2.393\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e3.880\u0026thinsp;\u0026plusmn;\u0026thinsp;2.284\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.332\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e3.425\u0026thinsp;\u0026plusmn;\u0026thinsp;3.448\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eArterial Constriction, %\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e0.490\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e-0.580\u0026thinsp;\u0026plusmn;\u0026thinsp;1.624\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e-0.230\u0026thinsp;\u0026plusmn;\u0026thinsp;1.216\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e-0.665\u0026thinsp;\u0026plusmn;\u0026thinsp;1.766\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.739\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eVenous Dilation, %\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e0.109\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e4.335\u0026thinsp;\u0026plusmn;\u0026thinsp;3.262\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e3.485\u0026thinsp;\u0026plusmn;\u0026thinsp;2.344\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.303\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e3.050\u0026thinsp;\u0026plusmn;\u0026thinsp;1.717\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.103\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCRAE, MU\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e0.014\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e263.9\u0026thinsp;\u0026plusmn;\u0026thinsp;54.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e223.7\u0026thinsp;\u0026plusmn;\u0026thinsp;36.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.009\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e227.5\u0026thinsp;\u0026plusmn;\u0026thinsp;49.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.044\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCRVE, MU\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e0.095\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e292.0\u0026thinsp;\u0026plusmn;\u0026thinsp;58.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e263.9\u0026thinsp;\u0026plusmn;\u0026thinsp;31.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.109\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e265.7\u0026thinsp;\u0026plusmn;\u0026thinsp;42.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.124\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eFAZ, mm\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e0.587\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e0.190\u0026thinsp;\u0026plusmn;\u0026thinsp;0.135\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e0.190\u0026thinsp;\u0026plusmn;\u0026thinsp;0.137\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e0.193\u0026thinsp;\u0026plusmn;\u0026thinsp;0.141\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.926\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePD SCP, %\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e0.159\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e41.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e40.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.706\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e42.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.156\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePD DCP, %\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e0.200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e40.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e40.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e41.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.267\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.950\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePD CC, %\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e0.930\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e73.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e73.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e73.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eANOVA: Analysis of variance; SD: standard deviation; CRAE: central retinal artery equivalent; CRVE central retinal vein equivalent; MU: measurement units; FAZ: foveal avascular zone; PD: perfusion density; SCP: superficial capillary plexus; DCP: deep capillary plexus.\u003c/p\u003e\n \u003cp\u003eStatistical Analysis: Analysis of variance (ANOVA) for repeated measures with Bonferroni post-hoc analysis.\u003c/p\u003e\n \u003cp\u003e*: ANOVA for repeated measures\u003c/p\u003e\n \u003cp\u003e+: comparison with baseline using Bonferroni post-hoc analysis\u003c/p\u003e\n \u003cp\u003e\u0026sect;: comparison with 3-minute value using Bonferroni post-hoc analysis\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eDynamic Vessel Analysis\u003c/h2\u003e\n \u003cp\u003eBy means of DVA, the diameter of the artery and of the vein selected for the analyses was not different between the two groups at the baseline (p\u0026thinsp;=\u0026thinsp;0.591 and p\u0026thinsp;=\u0026thinsp;0.379 for the arterial diameter and vein diameter analysis, respectively).\u003c/p\u003e\n \u003cp\u003eAnalyzing the response of the retinal vessels to the flicker light, we disclosed no significant difference in the arterial dilation percentage after the smoking activity (p\u0026thinsp;=\u0026thinsp;0.217), although there was a trend with increased arterial dilation response (mean arterial dilation of 2.850\u0026thinsp;\u0026plusmn;\u0026thinsp;2.393% at the baseline, 3.880\u0026thinsp;\u0026plusmn;\u0026thinsp;2.284% 3 minutes after smoking activity, 3.425\u0026thinsp;\u0026plusmn;\u0026thinsp;3.448% 30 minutes after smoking activity). On the other hand, no significant difference or trend was disclosed in the control group comparing examinations of the 3 timepoints (p\u0026thinsp;=\u0026thinsp;0.710). Analyzing the arterial constriction after the dilation due to the flicker light and the vein dilation percentage, no significant difference was disclosed in both groups.\u003c/p\u003e\n \u003cp\u003eComparing the arterial dilation percentage, arterial constriction percentage, and vein dilation percentage between smoker group and controls in the respective time-points, a significant difference was disclosed in arterial dilation percentage at 8 minutes (mean arterial dilation 3.880\u0026thinsp;\u0026plusmn;\u0026thinsp;2.284% and 2.130\u0026thinsp;\u0026plusmn;\u0026thinsp;1.751%, respectively, p\u0026thinsp;=\u0026thinsp;0.010). This difference was reduced considering the 35-minute examination (mean arterial dilation 3.425\u0026thinsp;\u0026plusmn;\u0026thinsp;3.448% and 1.875\u0026thinsp;\u0026plusmn;\u0026thinsp;1.687%, respectively, p\u0026thinsp;=\u0026thinsp;0.079). No other differences were disclosed.\u003c/p\u003e\n \u003cp\u003eAll the analyses about the DVA parameters were reported in Tables\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eDynamic and static vessel analysis, and OCT-A analysis of the control population (n\u0026thinsp;=\u0026thinsp;20)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth rowspan=\"2\" align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eANOVA\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eBaseline\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth colspan=\"2\" align=\"left\"\u003e\n \u003cp\u003e8-minute later\u003c/p\u003e\n \u003c/th\u003e\n \u003cth colspan=\"3\" align=\"left\"\u003e\n \u003cp\u003e35-minute later\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP value*\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP value\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP value\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP value\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026sect;\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eArterial Dilation, %\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e0.710\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e2.265\u0026thinsp;\u0026plusmn;\u0026thinsp;1.965\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e2.130\u0026thinsp;\u0026plusmn;\u0026thinsp;1.751\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e1.875\u0026thinsp;\u0026plusmn;\u0026thinsp;1.687\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eArterial Constriction, %\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e0.910\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e-0.525\u0026thinsp;\u0026plusmn;\u0026thinsp;1.028\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e-0.370\u0026thinsp;\u0026plusmn;\u0026thinsp;1.484\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e-0.460\u0026thinsp;\u0026plusmn;\u0026thinsp;1.773\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eVenous Dilation, %\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e0.628\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e3.130\u0026thinsp;\u0026plusmn;\u0026thinsp;2.657\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e2.670\u0026thinsp;\u0026plusmn;\u0026thinsp;1.817\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e2.745\u0026thinsp;\u0026plusmn;\u0026thinsp;2.286\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCRAE, MU\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e0.561\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e225.4\u0026thinsp;\u0026plusmn;\u0026thinsp;28.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e219.2\u0026thinsp;\u0026plusmn;\u0026thinsp;36.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e217.9\u0026thinsp;\u0026plusmn;\u0026thinsp;40.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.900\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCRVE, MU\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e0.618\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e252.0\u0026thinsp;\u0026plusmn;\u0026thinsp;31.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e245.8\u0026thinsp;\u0026plusmn;\u0026thinsp;42.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e253.7\u0026thinsp;\u0026plusmn;\u0026thinsp;42.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.994\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eFAZ, mm\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e0.063\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e0.224\u0026thinsp;\u0026plusmn;\u0026thinsp;0.084\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e0.223\u0026thinsp;\u0026plusmn;\u0026thinsp;0.079\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.279\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e0.225\u0026thinsp;\u0026plusmn;\u0026thinsp;0.081\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.060\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePD SCP, %\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e0.980\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e42.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e42.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e42.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePD DCP, %\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e0.679\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e40.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e40.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e40.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePD CC, %\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\"\u003e\n \u003cp\u003e0.919\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e73.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e73.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\"±\"\u003e\n \u003cp\u003e73.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eANOVA: Analysis of variance; SD: standard deviation; CRAE: central retinal artery equivalent; CRVE central retinal vein equivalent; MU: measurement units; FAZ: foveal avascular zone; PD: perfusion density; SCP: superficial capillary plexus; DCP: deep capillary plexus.\u003c/p\u003e\n \u003cp\u003eStatistical Analysis: Analysis of variance (ANOVA) for repeated measures with Bonferroni post-hoc analysis.\u003c/p\u003e\n \u003cp\u003e*: ANOVA for repeated measures\u003c/p\u003e\n \u003cp\u003e+: comparison with baseline using Bonferroni post-hoc analysis\u003c/p\u003e\n \u003cp\u003e\u0026sect;: comparison with 8-minute value using Bonferroni post-hoc analysis\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eOCT-Angiography Analysis\u003c/h2\u003e\n \u003cp\u003eExaminations of FAZ did not show any significant microvascular change after the smoking activity (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Interobserver variability between readers was excellent for FAZ measurements (ICC\u0026thinsp;=\u0026thinsp;0.991 [0.978\u0026ndash;0.996]). Perfusion density among SCP, DCP, and CC did not show any significant change before and after smoking activity in smoking group (p\u0026thinsp;=\u0026thinsp;0.159, p\u0026thinsp;=\u0026thinsp;0.200, and p\u0026thinsp;=\u0026thinsp;0.930 for SCP, DCP, and CC analysis, respectively) and by comparing examinations of 3 timepoints in controls (p\u0026thinsp;=\u0026thinsp;0.980, p\u0026thinsp;=\u0026thinsp;0.679, and p\u0026thinsp;=\u0026thinsp;0.919 for SCP, DCP, and CC analysis, respectively).\u003c/p\u003e\n \u003cp\u003eNo significant differences were disclosed comparing FAZ, PD of SCP, DCP, and CC between smoking group and controls in the respective time-points (p\u0026thinsp;\u0026gt;\u0026thinsp;0.1 in all analyses).\u003c/p\u003e\n \u003cp\u003eThe signal strength index (SSI) of examinations did not show any significant difference among the different examinations (p\u0026thinsp;\u0026gt;\u0026thinsp;0.2 in all analyses).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we used a multimodal imaging evaluation including DVA and OCT-A to analyze vascular retinal changes induced by cigarette smoking. We disclosed a significant arterial constriction of retinal vessels 3 minutes after cigarette smoking, which is maintained even after 30 minutes. The innovation of our study is to possibly clarify which are the smoking effects on retinal vessel morphology and function by adding to the classical retinal imaging techniques the DVA analysis.\u003c/p\u003e \u003cp\u003eCigarette smoking increases the risk of cardiovascular pathology through several pathways\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Effects of smoking cigarettes on the microvessels involve diminished endothelial-dependent vasodilation, endothelial cell dysfunction, and accelerated platelet aggregation. These modifications lead to vascular blood flow changes due to vasodilatation or vasoconstriction.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e Furthermore, smoking increases the chances of atherosclerotic plaque rupture, leading to acute myocardial infarction, stroke, or acute peripheral vascular diseases\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn our trial, the static analysis of the retinal vessels confirmed a significant decrease of CRAE in the smoking group 3 minutes after smoking (p\u0026thinsp;=\u0026thinsp;0.009) and a maintenance of vasoconstriction 30 minutes after smoking (p\u0026thinsp;=\u0026thinsp;0.044). Moreover, in the smoking group, mean CRVE showed a trend of decrease after smoking activity. Since CRAE and CRVE are related to the diameter of the central retinal artery and vein, respectively, we disclosed a significant constriction of the central retinal artery and a trend of constriction of the central retinal vein after smoking. This was not disclosed in the control group. These data agree with the systemic effects of smoking. As discussed before, smoking causes vasoconstriction in other cardiovascular systems, as in coronary blood flow.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e Thanks to DVA, we have confirmed and reported the direct vasoconstriction effect of smoking activity on the retinal vessels.\u003c/p\u003e \u003cp\u003eInterestingly, analyzing the CRAE and CRVE at baseline, there was no difference between the group of smokers and controls. This means that smokers showed no chronic vasoconstriction in comparison to controls. There are several reasons for this: the volunteers enrolled in the study are young people with low smoking activity (maximum 10 cigarettes/day) who have been smoking for up to 10 years. Furthermore, the volunteers were asked to do a cigarette smoke wash-out 24 hours before being subjected to the study. For all these reasons, the smokers included in our analysis may not yet have developed chronic vascular effects. However, we cannot exclude that greater smokers could also show chronic vasoconstriction as suggested by Zhu et al\u003csup\u003e24\u003c/sup\u003e. They showed that cigarette smoking is an independent risk factor for reduced vessel density or retinal capillaries, and this is strictly related to increased smoking pack-years. This data suggested a time-dependent and dose-dependent effect of chronic vascular retinal damage induced by smoking activity.\u003c/p\u003e \u003cp\u003eAnalyzing the vessel dilation (arterial and venous) in response to the flicker light, we disclosed a trend with increased arterial dilation response after the smoking activity in smokers. On the other hand, no significant difference or trend was disclosed in the control group comparing examinations of the 3 timepoints. Comparing the arterial dilation percentage between the smoking group and controls in the respective time-points, a significant difference was disclosed at 8 minutes, comparing smokers and controls. This difference was reduced considering the 35-minute examination. These results could be explained because after smoking activity, the artery responds with vasoconstriction; as the post-flicker dilation is a percentage of the pre-stimulation diameter, the result is influenced by the last one. We have seen that the pre-stimulation diameter is smaller after cigarette smoking, so the percentage expressing arterial dilation will be increased. However, DVA analyses the vasodilation response to the flicker stimulus on large retinal vessels and not on small vessels. On the other hand, the major effect of smoking cigarettes on vessel response was previously reported involving the micro-vessels and endothelial-dependent vasorelaxation, which were not investigated using DVA.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eRegarding the analysis of retinal plexuses using OCT-A, our results confirm the literature. Ayhan et al.\u003csup\u003e14\u003c/sup\u003e have reported that smoking cigarettes does not cause significant changes in the size of the FAZ area and the macular vessel density in healthy habitual smokers. Ciesielski et al.\u003csup\u003e25\u003c/sup\u003e disclosed the lack of immediate effects of cigarette smoking on the vascular density of the central retina in healthy habitual smokers and no significant changes in the FAZ. All these results were confirmed in our analysis. Analyzing the PD of the retinal plexuses (SCP and DCP) and of the CC, we did not disclose any significant changes immediately after and 30 minutes after the smoking activity.\u003c/p\u003e \u003cp\u003eLimitations of our study should be kept in mind. First of all, the present study is mainly limited by its small sample size. Moreover, the sample does not reflect all smokers, because we selected young people who have not smoked for more than 10 years and who smoke about 10 cigarettes a day. But this is also a strength because it makes our population homogeneous, and thus, we were able to study chronic and acute effects of smoking activity on retinal vessels in this kind of smoker. As further strength, our study is the first to add DVA and RVA analysis as an objective parameter to the evaluation of possible smoking effects on visual function.\u003c/p\u003e \u003cp\u003eIn conclusion, our study demonstrates that there is a significant acute arterial constriction response after cigarette smoking, which is maintained even after 30 minutes (although slightly reduced). Furthermore, we reported no chronic vasoconstriction of retinal vessels in comparison to controls, suggesting that low-dose and low-time of smoking activity, as in our population (about 10 cigarettes/day for 5 to 10 years), is not enough to induce chronic vasoconstriction at the level of the retinal vessels. Stopping smoking at this stage could result in beneficial effects for smoking-induced vascular diseases. However, further studies are warranted to better understand the physiology behind this mechanism and to confirm the same effects on other vascular systems outside the eye.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eFinancial support\u003c/h2\u003e \u003cp\u003enone.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003e \u003cb\u003eDisclosures\u003c/b\u003e:\u003c/strong\u003e \u003cp\u003eRiccardo Sacconi is a consultant for: AbbVie (North Chicago, Illinois, USA), Bayer Shering-Pharma (Berlin, Germany), Boehringer Ingelheim (Ingelheim, Germany), Carl Zeiss Meditec (Dublin, USA), Hoffmann-La-Roche (Basel, Switzerland), Novartis (Basel, Switzerland). Seraphine Saladino, Paolo Forte, Federico Beretta: none. Francesco Bandello is a consultant for Alcon (Fort Worth,Texas,USA), Alimera Sciences (Alpharetta, Georgia, USA), AbbVie (North Chicago, Illinois, USA), Farmila-Thea (Clermont-Ferrand, France), Bayer Shering-Pharma (Berlin, Germany), Bausch And Lomb (Rochester, New York, USA), Genentech (San Francisco, California, USA), Hoffmann-La-Roche (Basel, Switzerland), NovagaliPharma (\u0026Eacute;vry, France), Novartis (Basel, Switzerland), Sanofi-Aventis (Paris, France), Thrombogenics (Heverlee,Belgium), Zeiss (Dublin, USA). Giuseppe Querques is a consultant for Alimera Sciences (Alpharetta, Georgia, USA), AbbVie (North Chicago, Illinois, USA), Amgen (Thousand Oaks, USA), Heidelberg (Germany), KBH (Chengdu, China), LEH Pharma (London, UK), Lumithera (Poulsbo, USA), Novartis (Basel, Switzerland), Bayer Shering-Pharma (Berlin, Germany), Sandoz (Berlin, Germany), Sifi (Catania, Italy), Soof-Fidia (Albano, Italy), Zeiss (Dublin, USA).\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eRS, GQ: research design, data acquisition and analysis, interpretation of data, drafting the manuscript and critical revision of the manuscript. SS, PF, FB: data acquisition and analysis, critical revision of the manuscript. FB: interpretation of data and critical revision of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eAcknowledgments: none.Disclosures: Riccardo Sacconi is a consultant for: AbbVie (North Chicago, Illinois, USA), Bayer Shering-Pharma (Berlin, Germany), Boehringer Ingelheim (Ingelheim, Germany), Carl Zeiss Meditec (Dublin, USA), Hoffmann-La-Roche (Basel, Switzerland), Novartis (Basel, Switzerland). Seraphine Saladino, Paolo Forte, Federico Beretta: none. Francesco Bandello is a consultant for Alcon (Fort Worth,Texas,USA), Alimera Sciences (Alpharetta, Georgia, USA), AbbVie (North Chicago, Illinois, USA), Farmila-Thea (Clermont-Ferrand, France), Bayer Shering-Pharma (Berlin, Germany), Bausch And Lomb (Rochester, New York, USA), Genentech (San Francisco, California, USA), Hoffmann-La-Roche (Basel, Switzerland), NovagaliPharma (\u0026Eacute;vry, France), Novartis (Basel, Switzerland), Sanofi-Aventis (Paris, France), Thrombogenics (Heverlee,Belgium), Zeiss (Dublin, USA). Giuseppe Querques is a consultant for Alimera Sciences (Alpharetta, Georgia, USA), AbbVie (North Chicago, Illinois, USA), Amgen (Thousand Oaks, USA), Heidelberg (Germany), KBH (Chengdu, China), LEH Pharma (London, UK), Lumithera (Poulsbo, USA), Novartis (Basel, Switzerland), Bayer Shering-Pharma (Berlin, Germany), Sandoz (Berlin, Germany), Sifi (Catania, Italy), Soof-Fidia (Albano, Italy), Zeiss (Dublin, USA).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data used to support the findings of this study are available from the corresponding author upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eYanbaeva, D. G., Dentener, M. A., Creutzberg, E. C., Wesseling, G. \u0026amp; Wouters, E. F. M. Systemic effects of smoking. \u003cem\u003eChest\u003c/em\u003e \u003cb\u003e131\u003c/b\u003e, 1557\u0026ndash;1566 (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBenowitz, N. L. 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Atheroscler Thromb.\u003c/em\u003e \u003cb\u003e31\u003c/b\u003e, 189\u0026ndash;200 (2024).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8916335/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8916335/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThere is a lack of knowledge about the effect of smoking activity on the anatomy and functionality of retinal vessels. The aim of this low-risk, single-center, case-control, interventional clinical trial (ClinicalTrials.gov ID NCT07261176) was to evaluate the effect of smoking activity on the functionality and anatomy of the retinal vessels.\u003c/p\u003e \u003cp\u003eForty healthy subjects were enrolled: 20 healthy volunteers with regular daily tobacco activity, and 20 healthy volunteers without tobacco activity (negative control). Each subject underwent a comprehensive ophthalmologic examination, including optical coherence tomography angiography(OCT-A), evaluation of vascular functionality by the dynamic vessel analyzer(DVA), and retinal vessel analyzer(RVA) at baseline and repeated for the \"smoking\" group 3 and 30 minutes after the smoking activity (8 and 35 minutes after baseline in controls).\u003c/p\u003e \u003cp\u003eBy means of RVA, the static analysis disclosed a significant decrease of central retinal artery equivalent 3 minutes after cigarette smoking, which is maintained even after 30 minutes after the smoking activity(p\u0026thinsp;=\u0026thinsp;0.014). By means of DVA, a significant difference was disclosed in arterial dilation percentage post-flicker stimulation 3 minutes after smoking activity, comparing smokers and controls(mean arterial dilation 3.880\u0026thinsp;\u0026plusmn;\u0026thinsp;2.284% and 2.130\u0026thinsp;\u0026plusmn;\u0026thinsp;1.751%, respectively, p\u0026thinsp;=\u0026thinsp;0.010). Interestingly, no significant differences in RVA and DVA analyses were disclosed at the baseline between smoker group and controls. Finally, using OCT-A, no significant change was found.\u003c/p\u003e \u003cp\u003eWe disclosed an \u0026ldquo;acute\u0026rdquo; side effect of smoking activity on ocular vascular system, showing an immediate vasoconstriction after smoking activity. However, no chronic effects from smoking activity were disclosed in our population of young people with low smoking activity.\u003c/p\u003e","manuscriptTitle":"Effect Of Smoking on Retinal Vessel Morphology and Functionality: a case- control, Clinical Trial","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-16 13:51:39","doi":"10.21203/rs.3.rs-8916335/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-14T15:34:11+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-03T20:11:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"296663811915178553534005263883896133309","date":"2026-04-10T08:37:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"175568563562476314785543843559517626418","date":"2026-04-09T08:21:07+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-08T13:44:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-03T13:17:44+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-03-11T12:58:41+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-10T08:00:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-03-10T07:01:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b887db3d-724e-4820-a890-1e36acb41677","owner":[],"postedDate":"April 16th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-14T15:34:11+00:00","index":95,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-03T20:11:04+00:00","index":94,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":66264802,"name":"Health sciences/Diseases"},{"id":66264803,"name":"Health sciences/Health care"},{"id":66264804,"name":"Health sciences/Medical research"},{"id":66264805,"name":"Health sciences/Risk factors"}],"tags":[],"updatedAt":"2026-04-16T13:51:39+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-16 13:51:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8916335","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8916335","identity":"rs-8916335","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}