Differential Changes in Retinal Cyst Characteristics in inner versus outer retina in Diabetic Macular Edema Treated With Ang-2/VEGF-A Dual Inhibitor

Differential Changes in Retinal Cyst Characteristics in inner versus outer retina in Diabetic Macular Edema Treated With Ang-2/VEGF-A Dual Inhibitor | 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 Research Article Differential Changes in Retinal Cyst Characteristics in inner versus outer retina in Diabetic Macular Edema Treated With Ang-2/VEGF-A Dual Inhibitor Camilla Alovisi, Vanda Bucceri, Gabriele Piccoli, Celeste Limoli, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8563078/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose: Location of retinal cysts in diabetic macular edema (DME) can have clinical implications and a layer-specific response to treatment may aid in predicting therapeutic response. This study aimed to compare quantitative and qualitative changes in inner versus outer retinal cysts after loading dose of faricimab in center-involved cystoid DME using spectral-domain OCT (SD-OCT). Methods: 435 retinal cysts (194 inner, 241 outer) from eighteen eyes of 12 patients were analyzed on 612 SD-OCT B-scans within the 1 central mm. Cysts were classified as inner (IC) or outer (OC) based on the location relative to the outer plexiform layer (OPL). Features of the cysts were quantified using ImageJ (1.53), including reflectivity as optical density (ODN), ODN ratio (ODR), area, and circularity index (CI). Non-parametric analyses were performed, with significance set at p < 0.05. Results: Faricimab significantly reduced central retinal thickness (CRT),(p<.01), inner, outer, total cyst area and cyst count (p≤.03, for all). IC showed significant reductions in number, ODN, and CI (p≤.01, for all). OC exhibited a reduction in number (p=.08) and a significant increase in ODN (p=.04). Higher baseline CRT and larger total cyst area were associated with greater %CRT reduction (p=.008 and p=.02), whereas higher baseline OC reflectivity was associated with smaller %CRT reductions (p=.04). Conclusions: Faricimab induces a compartment-specific response of intraretinal cysts in DME. Inner and outer retinal fluid show distinct qualitative behaviour, reflecting different blood–retinal barrier dynamics. OCT-based assessment of cyst location and reflectivity may complement CRT in evaluating treatment response. Diabetic Macular Edema Optical Coherence Tomography Retinal Cyst Characteristics Ang-2/VEGF-A Dual Inhibition Retinal Imaging Biomarkers Figures Figure 1 Figure 2 Key Message Anti-VEGF/Ang-2 therapy improves diabetic macular edema (DME) by reducing intraretinal fluid and retinal thickness, as assessed by OCT. This study shows that inner and outer retinal cysts behave differently under treatment , with distinct temporal and spatial changes, highlighting compartment-specific fluid dynamics in DME. Changes in cyst characteristics by location provide additional OCT biomarkers beyond central retinal thickness, offering insights into treatment response and retinal recovery. INTRODUCTION Diabetic macular edema (DME) results from the accumulation of extracellular fluid within the retinal layers due to disruption of both the inner (iBRB) and the outer (oBRB) blood–retinal barriers [ 1 ]. Fluid may accumulate in the inner retina, outer retina, or subretinal space, with each compartment carrying distinct clinical implications [ 2 , 3 ]. Inner retinal fluid volume correlates with best-corrected visual acuity (BCVA) [ 2 ] while large cysts—particularly within the outer nuclear layer (ONL)—and the presence of subretinal fluid (SRF) have been related to reduced retinal sensitivity [ 3 ]. Outer retinal fluid is typically associated with more advanced diabetic retinopathy (DR) and worse visual outcomes [ 3 – 6 ]. Moreover, the reflectivity of cystoid spaces on OCT and OCT angiography (OCTA) has recently emerged as a noninvasive biomarker of BRB integrity and therapeutic responsiveness in DME [ 7 – 10 ]. Among molecular mediators involved in BRB destabilization, angiopoietin-2 (Ang-2) plays a central role [ 11 – 13 ]. At the iBRB, Ang-2 potentiates VEGF-A-induced endothelial permeability and disrupts endothelial–pericyte interactions [ 11 , 12 ]. At the oBRB, Ang-2 compromises tight-junction integrity in the retinal pigment epithelium (RPE), increases paracellular permeability, alters angiogenic signalling, and reduces metabolic support to photoreceptors [ 13 ]. Faricimab, a bispecific antibody targeting both VEGF-A and Ang-2, provides a pathophysiologically targeted treatment that can stabilize both BRB components [ 14 ]. However, its differential impact on inner versus outer retina in treatment- naïve DME has not been fully characterized. The purpose of this study was to evaluate early morphological and reflectivity changes in inner (IC) and outer retinal cysts (OC) following the loading dose of faricimab in previously treatment-naïve, center-involved cystoid DME. METHODS This retrospective observational study was conducted at the Medical Retina & Imaging Unit at the Eye Clinic IRCCS MultiMedica, Milan, Italy, from October 2023 to April 2024. The study adhered to the tenets of Declaration of Helsinki with collected written informed consent from all study participants from DR institutional registry and approval by the Institutional Ethics Committee of IRCCS MultiMedica (CE 55.2022). Patients with type 1 or 2 diabetes mellitus (DM), aged 30 or older, diagnosed with treatment-naive cystoid center-involved DME and with available full retina imaging, were enrolled. Based on observed effect sizes in key OCT biomarkers, approximately 30–40 eyes would be required to confirm these findings with adequate statistical power (80%) in future prospective studies; the present sample is suitable for exploratory analysis. All patients underwent 4 monthly loading doses of faricimab (Vabysmo 6mg; Roche, Switzerland) with evaluations at baseline and 2 months after the fourth injection. Exclusion criteria included previous intravitreal treatments, active PDR characterized by leaking neovascularization of the optic disc or elsewhere within the last 6 months, significant media opacities hindering image quality, subfoveal neuroretinal detachment (SND), which can compromise the identification of IC and OC due to retina distortion from SRF, history of macular laser photocoagulation or macular surgery, or other macular comorbidities. Both eyes were enrolled if eligible. All patients had a full ophthalmic examination, including best-corrected visual acuity (BCVA), ultra-widefield color fundus photography (UWF-CFP), ultra-widefield fluorescein angiography (UWF-FA) and OCT. VA Assessment The BCVA was determined using the standard Early Treatment Diabetic Retinopathy Study (ETDRS) protocol with a modified ETDRS distance charts trans-illuminated with a chart illuminator (180 candela/m 2 Precision Vision, Bloomington, IL, USA) at 4 m. Visual acuity was scored as the total number of letters read correctly, calculated according to the ETDRS score method. Color fundus photos and fluorescein angiography Ultra-widefield color fundus photography (UWF-CFP) and fluorescein angiography (UWF-FA) (Optos, California; Optos plc, Dunfermline, UK) were used to assess the severity of DR based on the International Clinical Diabetic Retinopathy Severity Scale [ 15 ]. UWF-FA additionally provided a qualitative evaluation of leakage in the late phases of the exam. All patients had multimodal imaging at baseline and at the end of the follow-up period. Image Analysis B-scans were acquired using the Spectralis HRA-OCT system (Heidelberg Engineering, Heidelberg, Germany). The OCT volume scan encompassed a 20°x20 ° area, consisting of 97 raster lines scanned at 60 µm intervals, with high resolution (HR) and automatic real-time (ART) 25. For each patient, 17 spectral-domain OCT (SD-OCT) B-scans within the central 1 mm of the OCT-ETDRS grid were analyzed at baseline and after the loading dose, totalling 612 SD-OCT scans. Only images with signal strength above 20 decibels were included, and the signal strength remained consistent across all examinations. Using ImageJ (version 1.53 t, freely available at https://imagej.net/ij/,provided in the public domain by the National Institutes of Health, Bethesda, MD, USA), 435 individual cysts were manually segmented and classified by retinal location (Fig. 1 ,a). IC—those situated above the outer plexiform layer (OPL)—accounted for 194 lesions, whereas 241 cysts located below the OPL were classified as OC. From each patient's 17 B-scans, we quantified mean cyst-specific parameters, including the number of cysts, total cyst area, circularity index, optical density (ODN) measured in pixels, and optical density ratio (ODR), divided into inner and outer layers using the OPL as the boundary. The ODR was defined as the mean ODN of the cyst divided by the mean ODN of the vitreous, as previously described [ 16 ]. STATISTICAL ANALYSIS Continuous variables are reported as mean ± standard deviation (SD), and categorical variables as frequencies and percentages. The distribution of continuous variables was assessed using the Shapiro–Wilk test. Given the limited sample size and the non-normal distribution of most variables, non-parametric statistical methods were applied throughout the analysis. Baseline comparisons between IC and OC were performed using unpaired analyses (Mann–Whitney U test). Longitudinal changes in OCT and cyst-related parameters between baseline and post-loading follow-up were evaluated using paired analyses within the same eyes (Wilcoxon signed-rank test). To avoid clustering bias due to multiple cysts and B-scans originating from the same eye, cyst-related parameters were averaged per eye, and all correlation and regression analyses were conducted at the eye level. In addition to p-values, effect sizes were calculated where appropriate to quantify the magnitude of observed differences. Effect sizes were reported to aid interpretation in this exploratory study and should not be considered confirmatory. Effect sizes for non-parametric tests were expressed as r values, and standardized beta coefficients were reported for regression analyses. The percentage change in central retinal thickness (ΔCRT%) after the faricimab loading dose was used as the primary anatomical outcome measure to quantify treatment response. Specifically, ΔCRT% was calculated as: $$\:{\Delta\:}\text{C}\text{R}\text{T}\text{\%}=\frac{\text{C}\text{R}{\text{T}}_{\text{post-loading}}-\text{C}\text{R}{\text{T}}_{\text{baseline}}}{\text{C}\text{R}{\text{T}}_{\text{baseline}}}\times\:100.$$ where CRT post−loading refers to the measurement obtained after four consecutive monthly faricimab injections. A negative ΔCRT% represents a reduction in retinal thickness, i.e., anatomical improvement. Univariable relationships between baseline imaging parameters and ΔCRT% were explored using both Spearman correlation coefficients and linear regression models. Statistical analyses were performed using SPSS v.29 (IBM Corp., Armonk, NY), and p < 0.05 was considered statistically significant. Multivariate models were adjusted for age and sex. RESULTS In this study, we evaluated 194 IC and 241 OC, from 18 eyes of 12 patients (7 males and 5 females) with a mean age of 64.8 ± 8.7 years. Eleven patients had type 2 DM and one had type 1 DM, with a mean disease duration of 11.55 ± 5.8 years and an average glycosylated haemoglobin (HbA1c) of 6.7 ± 1.3%. Eleven eyes had moderate non-proliferative diabetic retinopathy (NPDR), one had severe NPDR, and six had stable proliferative diabetic retinopathy (PDR). All eyes were treatment-naïve and presented with cystoid center-involved DME. Baseline demographic and clinical characteristics are summarized in Table 1 . Table 1 Baseline Demographic and Ocular Characteristics Demographic characteristics Patients (n = 12) Age, mean (SD), y 64.8 (8.7) Sex, Female, No. (%) 5 (41.6) Type 1 DM (%) 1 (8.3) Type 2 DM (%) 11(91.6) Duration of DM, mean (SD), y 11.55 (5.8) HbA1c, (%) mean (SD) 6.7 (1.3) Bilateral eyes (%) 6 (50) Eye characteristics Eye (n = 18) BCVA, mean (SD) (ETDRS letters) 64 (15.2) Pseudophakic (%) 11(61.1) DR Grading (%) Moderate NPDR 11(61.1) Severe NPDR 1(5.5) PDR 6 (33.3) Baseline OCT Characteristics At baseline, the mean number of cysts within the central 1-mm area did not differ significantly between IC and OC (8.77 ± 5.9 vs 8.03 ± 5.2; p = .41). However, the total cyst area was larger in OC compared to IC (0.19 ± 0.2 vs 0.03 ± 0.03, respectively, p = 0.10). Mean ODN and optical ODR were comparable between compartments (ODN: 53.486 ± 12.321 vs 50.535 ± 10.361; p = .22; ODR: 6.86 ± 3.3 vs 7.15 ± 4.6; p = .09). Circularity was higher in OC than in IC (0.65 ± 0.07 vs 0.57 ± 0.09; p = .27), indicating a more regular cyst morphology in the outer retina. The percentage of IC fluid showed no correlation with either CRT (r = 0.20, p = .43) or BCVA (r = 0.03, p = .91). Anatomical and Functional Outcomes after Faricimab Treatment From baseline to final follow-up, faricimab treatment induced significant anatomical improvements, with distinct responses between retinal compartments. CRT decreased significantly from 454 ± 136.2 µm to 306 ± 89.6 µm (p < .01). BCVA showed a slight, non-significant increase from 64 ± 15.2 to 65 ± 14.6 ETDRS letters (p = .79). The total number of cysts declined significantly following the loading dose with a large effect size (from 16.49 ± 10.4 to 7.32 ± 6.1; p < .01, r = 0.81). This reduction was primarily driven by IC, in which cyst count decreased significantly, with a large effect size, from 8.77 ± 5.9 to 2.03 ± 3.1 (p < .01, r = 0.86). OC demonstrated a moderate effect size reduction in cyst number, with a statistical trend that did not reach significance (8.03 ± 5.2 to 5.32 ± 3.5; p = .08, r = 0.61) (Table 2 ). In UWF-FA imaging, a visibly reduced leakage was observed at the posterior pole during the late phases of the examination (Fig. 1 c-d). Table 2 Cysts Parameters Cysts Parameters (Mean) Baseline End of the loading dose P value Total cysts No. (SD) 16.49 (10.4) 7.32 (6.1) < .01* IC No. (SD) 8.77 (5.9) 2.03 (3.1) < .01* OC No. (SD) 8.03 (5.2) 5.32 (3.5) .08 ODN IC Px (SD) 53.486 (12.321) 33.410 (28.834) .01* ODN OC Px (SD) 50.535 (10.361) 58.582 (12.659) .04* ODR IC Px (SD) 6.86 (3.3) 4.39 (4.6) .07 ODR OC Px (SD) 7.15 (4.6) 9.10 (4.5) .21 Total cysts area Px 2 (SD) 0.40 (0.2) 0.20 (16.9) .03* Area IC Px 2 (SD) 0.03 (0.03) 0.01 (0.03) .03* Area OC Px 2 (SD) 0.19 (0.2) 0.06 (0.04) .03* Circularity IC (SD) 0.57 (0.09) 0.33 (0.3) < .01* Circularity OC (SD) 0.65 (0.07) 0.64 (0.08) .68 Abbreviations: IC: Inner Cysts; OC: Outer Cysts; ODN: Optical Density; Px: Pixel; ODR: Optical Density Ratio. SD: Standard Deviation; p* significative. Changes in Optical Density and Reflectivity A divergent behaviour between IC and OC compartments was observed in optical density metrics. The mean ODN of IC cysts decreased significantly with a large effect size over follow-up (from 53.486 ± 12.321 to 33.410 ± 28.834 pixels; p = .01, r = 0.87). In contrast, OC exhibited a significant increase in ODN with a moderate effect size (from 50.535 ± 10.361 to 58.582 ± 12.659 pixels; p = .04, r = 0.52). Similarly, the mean ODR of IC showed a decreasing trend with a small-to-moderate effect size (from 6.86 ± 3.3 to 4.39 ± 4.6 pixels; p = .07, r = 0.30), consistent with reduced relative reflectivity. OC, however, demonstrated a tendency toward higher ODR values, with a small-to-moderate effect size that did not reach statistical significance (from 7.15 ± 4.6 to 9.10 ± 4.5 pixels; p = .21, r = 0.35) (Table 2 ). Cyst Area and Morphological Changes Both inner and outer retinal compartments showed significant reductions in total cyst areas after treatment. IC area decreased with a moderate effect size from 0.03 ± 0.03 to 0.01 ± 0.03 pixels 2 (p = .03, r = 0.63), and OC area with a small-to-moderate effect size from 0.19 ± 0.2 to 0.06 ± 0.04 pixels 2 (p = .03, r = 0.30). Morphologically, IC became significantly less circular over time, with a moderate effect size (from 0.57 ± 0.09 to 0.33 ± 0.30; p < .01, r = 0.63). In contrast, OC circularity remained stable, showing a negligible effect size and no significant change (0.65 ± 0.07 to 0.64 ± 0.08; p = .68, r = 0.05) (Table 2 ). Association of Baseline OCT Biomarkers With Anatomical Response Several baseline OCT parameters were significantly associated with the percentage change in CRT (ΔCRT%) after the loading dose with faricimab. Baseline CRT showed a strong negative association with ΔCRT% (ρ = − 0.607, p < .01; p = .008; B = − 0.064 ± 0.024, β=−0.552, p = .01), indicating greater proportional thickness reduction in eyes with greater retinal thickening at presentation. Similarly, total intraretinal cyst area was also negatively associated with ΔCRT% (ρ = − 0.525, p = .02) and was a significant predictor in univariable linear regression (B = − 47.866 ± 18.159, β=−0.550, p = .01), suggesting that a higher baseline intraretinal fluid burden was associated with a greater anatomical response (Fig. 2 ). In contrast, the mean reflectivity ratio of cysts below the OPL (bODR) was positively associated with ΔCRT% (ρ = 0.476, p = .04) and emerged as a significant positive predictor in univariable regression (B = + 1.918 ± 0.695, β = 0.568, p = .01), indicating a smaller CRT reduction in eyes with more reflective outer cystic fluid. These associations remained significant after adjustment for age and sex. Other parameters, including cyst circularity and the number of cysts located above or below the OPL, were not significantly associated with ΔCRT% (all p > .05). DISCUSSION This study documents compartment-specific anatomical responses to faricimab treatment in DME, highlighting differences between IC and OC responses and characteristics. Analysis of OCT images was essential for layer-specific characterization of intraretinal cysts, enabling differentiation of inner and outer retinal fluid behaviour and revealing treatment-related changes that cannot be captured by global thickness measures alone. IC exhibited a significant reduction in number, size, and reflectivity, a pattern consistent with the pharmacological effects of dual Ang-2/VEGF-A inhibition on the iBRB. The rapid restoration of endothelial–pericyte signalling likely underlies the more prompt and pronounced regression of inner retinal fluid. In contrast, OC showed a different anatomical response. The slower resolution may reflect the chronic and multifactorial nature of oBRB dysfunction, which is characterized by disruption of RPE tight junctions, impaired metabolic support to photoreceptors, and reduced efficiency of fluid transport [ 1 , 14 ]. Notably, an increase in OC optical density was observed during treatment, suggesting persistent alterations in exudate composition. This finding may indicate exudation or biochemical remodelling within outer retinal fluid compartments, consistent with a slower clearance of protein-rich material [ 5 ]. Moreover, the area and circularity index of OC remained relatively stable over time. Such morphological characteristics suggest that OC may represent a more established and less dynamic form of edema [ 17 ]. Finally, baseline OCT biomarkers were associated with treatment response: greater intraretinal fluid and a larger total cystic area were linked to greater anatomical improvement, whereas higher OC reflectivity was associated with a smaller reduction in CRT, suggesting a more treatment-resistant edema phenotype. This finding suggests that retinal compartment and qualitative fluid features may respond differently to dual Ang-2/VEGF-A inhibition and can reveal aspects of disease that are not indicated by fluid volume alone [ 14 ]. The relationship between intraretinal cyst location and macular function has been widely explored, although findings remain partly heterogeneous [ 1 , 5 ]. Previous studies have suggested that fluid involving the outer retinal layers may be more frequently observed in eyes with more advanced DR and may be associated with poorer visual outcomes [ 1 , 5 ]. Conversely, recent analyses using automated and deep learning–based OCT quantification have reported that inner retinal fluid volume shows a stronger association with BCVA, whereas outer retinal fluid may be less directly related to visual function [ 2 ]. Studies integrating microperimetry (MP) with detailed structural analysis using artificial intelligence (AI) have further indicated that cyst size and retinal layer localization may influence retinal sensitivity [ 3 , 18 ]. In particular, larger cysts within the ONL and the presence of SRF have been associated with greater reductions in retinal sensitivity, while smaller cysts confined to the inner nuclear layer appear to have a more limited functional impact [ 3 ]. These structural features have been described more frequently in later disease stages and may reflect long-standing BRB dysfunction and altered retinal microenvironment. Importantly, symptom duration alone did not correlate with functional impairment, suggesting that the extent and distribution of structural alterations may be more relevant than disease chronicity per se [ 3 ]. In the present study, faricimab treatment resulted in a significant reduction in CRT and intraretinal fluid, with distinct compartment-specific responses. BCVA showed a minimal, non-significant change over the short follow-up period. The lack of significant improvement in BCVA likely reflects the short follow-up period and the limited sensitivity of BCVA to compartment-specific structural changes. Additionally, we evaluated cyst reflectivity utilizing structural OCT. Prior research on cyst reflectivity, including the suspended scattering particles in motion, mainly employed OCT angiography [ 7 – 10 ]. However, this approach may limit precise anatomical localization. Therefore, our results cannot be directly compared to previous studies. The increased OC reflectivity could reflect an optical artifact due to reabsorption of overlying inner retinal fluid and improved signal penetration. However, reflectivity was quantified using a normalized ODR, and although the ODN of OC increased, the corresponding ODR change was not significant, arguing against a pure transmission effect [ 16 ]. In addition, baseline OC reflectivity independently predicted a smaller CRT reduction, supporting its role as a true biomarker of chronic, treatment-resistant edema rather than an imaging artifact. Our findings align with the literature showing that outer retinal pathology—whether represented by the number of cysts, reflectivity, or area changes—reflects more advanced disease and reduced therapeutic responsiveness [ 9 , 10 ]. In contrast, the rapid reduction in IC size and reflectivity after faricimab treatment suggests a faster recovery of the iBRB and more efficient removal of extracellular fluid in the inner retina. This is further supported by the decreased leakage observed in UWF-FA, as previously detailed in a post hoc analysis of YOSEMITE/RHINE [ 19 ]. Limitations of the study include its small sample size and retrospective design, which may limit generalizability. The short follow-up, restricted to the loading dose, precludes evaluation of long-term structural and functional outcomes, particularly for OC. Finally, functional assessment was limited to BCVA, and more sensitive measures of visual function were not included. Larger studies with advanced imaging, AI quantification, functional assessments and longer follow-up are necessary to validate these findings and evaluate the prognostic significance of cyst characteristics and retinal fluid compartmentalization as an OCT biomarker in DME. CONCLUSION This study provides clinical evidence that intraretinal cysts in DME are heterogeneous and exhibit compartment-specific responses to dual Ang-2/VEGF-A inhibition with faricimab, consistent with DME pathogenesis and the distinct mechanisms by which Ang-2 affects permeability in the iBRB and oBRB. From a clinical perspective, the differential behaviour of IC and OC may help explain the variability in anatomical outcomes observed in treatment-naïve DME patients despite standardized anti-VEGF therapy. These observations highlight the significance of retinal fluid compartmentalization as a key biomarker for treatment response, which could also assist in the early detection of patients needing closer monitoring, prolonged loading doses, or different therapeutic approaches to personalize DME treatment. Abbreviations SD Standard Deviation DM Diabetes Mellitus BCVA Best Corrected Visual Acuity DR Diabetic Retinopathy NPDR Non-Proliferative Diabetic Retinopathy PDR Non-active Proliferative Diabetic Retinopathy Declarations Clinical trial number : not applicable. Statements and Declarations Ethics: The study was conducted in accordance with the Declaration of Helsinki and approval was granted by the Institutional Ethics Committee of IRCCS MultiMedica (CE 55.2022). The authors confirm that the ethical guidelines of the journal were adhered to and that approval was obtained from the appropriate ethics committee.Written consent for publication of identifying information and accompanying images was obtained. Ethics committee approval: The retrospective study was conducted in accordance with the Declaration of Helsinki and approved by Institutional Ethics Committee of IRCCS MultiMedica (CE 55.2022). Consent to publish: The authors affirm that human research partici­pants provided informed consent for publication of the images. Conflict of Interest C.A., V.B., C.L., J.C., S.D.S., S.B.: no financial disclosures relevant to this paper G.P.: EssilorLuxottica Group – Espansione Group S.p.A. Employee, Nidek Co LT. honorarium for symposium. P.N.: Thea, Hoya, Essilor Luxottica e Fondazione One-sight, Sifi, Bausch& Lomb, Alfa Intes, Alcon, Doc, Santen, Eyerising SV: Consultant to Abbvie, Adverum, Apellis, Bayer, Boehringer & Ingelheim, Novartis, Hoffman La Roche, RetinAI, Zeiss; Commercial Relationships Disclosure: No conflicting relationship exists for the other authors in relation to this study. All authors have completed and submitted the ICMJE disclosures form. Financial Support: None References Cunha-Vaz J (2017) Mechanisms of Retinal Fluid Accumulation and Blood-Retinal Barrier Breakdown. 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Can J Ophthalmol 60(5):297–305. 10.1016/j.jcjo.2025.02.013 Goldberg RA, Mar FA, Csaky K, Amador M, Khanani AM, Gibson K et al (2025) Resolution of Angiographic Macular Leakage with Faricimab versus Aflibercept in Patients with Diabetic Macular Edema in YOSEMITE/RHINE. Ophthalmol Retina 9(6):515–526. 10.1016/j.oret.2024.11.015 Additional Declarations Competing interest reported. C.A., V.B., C.L., J.C., S.D.S., S.B.: no financial disclosures relevant to this paper G.P.: EssilorLuxottica Group – Espansione Group S.p.A. Employee, Nidek Co LT. honorarium for symposium. P.N.: Thea, Hoya, Essilor Luxottica e Fondazione One-sight, Sifi, Bausch& Lomb, Alfa Intes, Alcon, Doc, Santen, Eyerising SV: Consultant to Abbvie, Adverum, Apellis, Bayer, Boehringer & Ingelheim, Novartis, Hoffman La Roche, RetinAI, Zeiss; Supplementary Files Table1.docx Table2.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-8563078","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":573857510,"identity":"9be60969-1ff3-48fc-909e-9d80782e2a09","order_by":0,"name":"Camilla Alovisi","email":"","orcid":"","institution":"Eye Clinic, Ospedale Oftalmico","correspondingAuthor":false,"prefix":"","firstName":"Camilla","middleName":"","lastName":"Alovisi","suffix":""},{"id":573857512,"identity":"73d081b1-a5d2-498a-ac96-6ddef7f411fe","order_by":1,"name":"Vanda Bucceri","email":"","orcid":"","institution":"Eye Clinic, IRCCS MultiMedica","correspondingAuthor":false,"prefix":"","firstName":"Vanda","middleName":"","lastName":"Bucceri","suffix":""},{"id":573857515,"identity":"72cc8212-d048-4a56-a7cb-bbed96c1887f","order_by":2,"name":"Gabriele Piccoli","email":"","orcid":"","institution":"Eye Clinic, IRCCS MultiMedica","correspondingAuthor":false,"prefix":"","firstName":"Gabriele","middleName":"","lastName":"Piccoli","suffix":""},{"id":573857516,"identity":"2159f540-96b3-4e05-bba2-9c0a82b9675d","order_by":3,"name":"Celeste Limoli","email":"","orcid":"","institution":"Eye Clinic, IRCCS MultiMedica","correspondingAuthor":false,"prefix":"","firstName":"Celeste","middleName":"","lastName":"Limoli","suffix":""},{"id":573857518,"identity":"295dedeb-d161-4a35-abeb-fa956c3f4b1b","order_by":4,"name":"Jelena Cuk","email":"","orcid":"","institution":"University Clinical Centre of Serbia","correspondingAuthor":false,"prefix":"","firstName":"Jelena","middleName":"","lastName":"Cuk","suffix":""},{"id":573857519,"identity":"1f9fa9f4-3b6b-47f9-b3ee-080f5d264167","order_by":5,"name":"Samuele Di Stefano","email":"","orcid":"","institution":"Eye Clinic, IRCCS MultiMedica","correspondingAuthor":false,"prefix":"","firstName":"Samuele","middleName":"Di","lastName":"Stefano","suffix":""},{"id":573857520,"identity":"2e1c7627-2142-4936-a274-fc5dc95f2e74","order_by":6,"name":"Sofia Bugakova","email":"","orcid":"","institution":"Eye Clinic, IRCCS MultiMedica","correspondingAuthor":false,"prefix":"","firstName":"Sofia","middleName":"","lastName":"Bugakova","suffix":""},{"id":573857521,"identity":"60ba8b39-f060-4f7d-a1e2-f502077eb112","order_by":7,"name":"Paolo Nucci","email":"","orcid":"","institution":"University of Milan","correspondingAuthor":false,"prefix":"","firstName":"Paolo","middleName":"","lastName":"Nucci","suffix":""},{"id":573857524,"identity":"4dc4678f-fc44-4a6a-b320-03fc641cb68f","order_by":8,"name":"Stela Vujosevic","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA90lEQVRIiWNgGAWjYHACxgNgsgFEGjDIgZkJDBK4NbAxMKBoMYZpwa0HpgUGEhugDJxa5Oc3Hzjwg+FwYnN7+8PHPAV26RtuN7dueMBgUYdLi8ExtoSDPUAtjT1njI15DJJzN9w52HYDn8MM2HgMDvAwpCU2zshhk5xhcCB3w41E/Frk2/g/HPwD1pL+/CdQS7oBIS0Mx3gYDvMw2AC1JJgxfDA4kEBQi8GxNIPDMgY2xiC/SHwwSDacCdZiICHZgMthzYcfPnxTISG7ERhiHxL+2Mnz3Uh/dvNHRR0/TodB7GJgMGxAFyEI5IlQMwpGwSgYBSMUAAC/jlvfXv1LLgAAAABJRU5ErkJggg==","orcid":"","institution":"Eye Clinic, IRCCS MultiMedica","correspondingAuthor":true,"prefix":"","firstName":"Stela","middleName":"","lastName":"Vujosevic","suffix":""}],"badges":[],"createdAt":"2026-01-09 16:53:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8563078/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8563078/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":100857643,"identity":"3cd7d233-1f62-4890-8507-e8e9cc37edfc","added_by":"auto","created_at":"2026-01-22 07:15:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1082303,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e: example of SD-OCT B-scan segmentation and analysis of the cysts using ImageJ; \u003cstrong\u003ec-d\u003c/strong\u003e: transformation of cyst number, characteristics and leakage on OCT and ultra-widefield fluorescein angiography (UWF-FA) from baseline (c) to the end of the loading dose with faricimab (d). LD: loading dose\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-8563078/v1/e32a502da66ebecd4dc0bb69.png"},{"id":100857640,"identity":"8f36767f-1ebc-4e7a-8865-1f1ea503b838","added_by":"auto","created_at":"2026-01-22 07:15:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":121408,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScatterplots of significant associations with ΔCRT%:\u003c/strong\u003e\u003cem\u003e \u003c/em\u003eAssociations between baseline OCT biomarkers and percentage change in central retinal thickness (ΔCRT%) after loading dose with faricimab.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-8563078/v1/94a85e0ff500adb7d1d77b81.png"},{"id":104973212,"identity":"6c1e0214-de89-45c4-af50-dfa9b3685c5f","added_by":"auto","created_at":"2026-03-19 11:27:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1998728,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8563078/v1/ffe46f41-0413-483a-a9f2-a9cb63627c56.pdf"},{"id":100949266,"identity":"1dd6fdbf-6df1-4085-a9bd-395bae7363d2","added_by":"auto","created_at":"2026-01-23 06:56:41","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":18679,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8563078/v1/6ed0a0700ed36048bb5ee40b.docx"},{"id":101202389,"identity":"5caebc37-010e-46f6-a60f-65ca8d8c71b4","added_by":"auto","created_at":"2026-01-27 09:29:45","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":20011,"visible":true,"origin":"","legend":"","description":"","filename":"Table2.docx","url":"https://assets-eu.researchsquare.com/files/rs-8563078/v1/210b5e3c6c24a8dbe4b702f7.docx"}],"financialInterests":"Competing interest reported. C.A., V.B., C.L., J.C., S.D.S., S.B.: no financial disclosures relevant to this paper\nG.P.: EssilorLuxottica Group – Espansione Group S.p.A. Employee, Nidek Co LT. honorarium for symposium. \nP.N.: Thea, Hoya, Essilor Luxottica e Fondazione One-sight, Sifi, Bausch\u0026amp; Lomb, Alfa Intes, Alcon, Doc, Santen, Eyerising\nSV: Consultant to Abbvie, Adverum, Apellis, Bayer, Boehringer \u0026 Ingelheim, Novartis, Hoffman La Roche, RetinAI, Zeiss;","formattedTitle":"Differential Changes in Retinal Cyst Characteristics in inner versus outer retina in Diabetic Macular Edema Treated With Ang-2/VEGF-A Dual Inhibitor","fulltext":[{"header":"Key Message","content":"\u003cul\u003e\n \u003cli\u003eAnti-VEGF/Ang-2 therapy improves diabetic macular edema (DME) by reducing intraretinal fluid and retinal thickness, as assessed by OCT.\u003c/li\u003e\n \u003cli\u003eThis study shows that \u003cstrong\u003einner and outer retinal cysts behave differently under treatment\u003c/strong\u003e, with distinct temporal and spatial changes, highlighting compartment-specific fluid dynamics in DME.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eChanges in cyst characteristics by location provide additional OCT biomarkers beyond central retinal thickness, offering insights into treatment response and retinal recovery.\u003c/strong\u003e\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"INTRODUCTION","content":"\u003cp\u003eDiabetic macular edema (DME) results from the accumulation of extracellular fluid within the retinal layers due to disruption of both the inner (iBRB) and the outer (oBRB) blood\u0026ndash;retinal barriers [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Fluid may accumulate in the inner retina, outer retina, or subretinal space, with each compartment carrying distinct clinical implications [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Inner retinal fluid volume correlates with best-corrected visual acuity (BCVA) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] while large cysts\u0026mdash;particularly within the outer nuclear layer (ONL)\u0026mdash;and the presence of subretinal fluid (SRF) have been related to reduced retinal sensitivity [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Outer retinal fluid is typically associated with more advanced diabetic retinopathy (DR) and worse visual outcomes [\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Moreover, the reflectivity of cystoid spaces on OCT and OCT angiography (OCTA) has recently emerged as a noninvasive biomarker of BRB integrity and therapeutic responsiveness in DME [\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Among molecular mediators involved in BRB destabilization, angiopoietin-2 (Ang-2) plays a central role [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. At the iBRB, Ang-2 potentiates VEGF-A-induced endothelial permeability and disrupts endothelial\u0026ndash;pericyte interactions [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. At the oBRB, Ang-2 compromises tight-junction integrity in the retinal pigment epithelium (RPE), increases paracellular permeability, alters angiogenic signalling, and reduces metabolic support to photoreceptors [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Faricimab, a bispecific antibody targeting both VEGF-A and Ang-2, provides a pathophysiologically targeted treatment that can stabilize both BRB components [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, its differential impact on inner versus outer retina in treatment- na\u0026iuml;ve DME has not been fully characterized.\u003c/p\u003e \u003cp\u003eThe purpose of this study was to evaluate early morphological and reflectivity changes in inner (IC) and outer retinal cysts (OC) following the loading dose of faricimab in previously treatment-na\u0026iuml;ve, center-involved cystoid DME.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003eThis retrospective observational study was conducted at the Medical Retina \u0026amp; Imaging Unit at the Eye Clinic IRCCS MultiMedica, Milan, Italy, from October 2023 to April 2024. The study adhered to the tenets of Declaration of Helsinki with collected written informed consent from all study participants from DR institutional registry and approval by the Institutional Ethics Committee of IRCCS MultiMedica (CE 55.2022).\u003c/p\u003e \u003cp\u003ePatients with type 1 or 2 diabetes mellitus (DM), aged 30 or older, diagnosed with treatment-naive cystoid center-involved DME and with available full retina imaging, were enrolled. Based on observed effect sizes in key OCT biomarkers, approximately 30\u0026ndash;40 eyes would be required to confirm these findings with adequate statistical power (80%) in future prospective studies; the present sample is suitable for exploratory analysis. All patients underwent 4 monthly loading doses of faricimab (Vabysmo 6mg; Roche, Switzerland) with evaluations at baseline and 2 months after the fourth injection. Exclusion criteria included previous intravitreal treatments, active PDR characterized by leaking neovascularization of the optic disc or elsewhere within the last 6 months, significant media opacities hindering image quality, subfoveal neuroretinal detachment (SND), which can compromise the identification of IC and OC due to retina distortion from SRF, history of macular laser photocoagulation or macular surgery, or other macular comorbidities. Both eyes were enrolled if eligible. All patients had a full ophthalmic examination, including best-corrected visual acuity (BCVA), ultra-widefield color fundus photography (UWF-CFP), ultra-widefield fluorescein angiography (UWF-FA) and OCT.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eVA Assessment\u003c/h2\u003e \u003cp\u003eThe BCVA was determined using the standard Early Treatment Diabetic Retinopathy Study (ETDRS) protocol with a modified ETDRS distance charts trans-illuminated with a chart illuminator (180 candela/m\u003csup\u003e2\u003c/sup\u003e Precision Vision, Bloomington, IL, USA) at 4 m. Visual acuity was scored as the total number of letters read correctly, calculated according to the ETDRS score method.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eColor fundus photos and fluorescein angiography\u003c/h3\u003e\n\u003cp\u003eUltra-widefield color fundus photography (UWF-CFP) and fluorescein angiography (UWF-FA) (Optos, California; Optos plc, Dunfermline, UK) were used to assess the severity of DR based on the International Clinical Diabetic Retinopathy Severity Scale [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. UWF-FA additionally provided a qualitative evaluation of leakage in the late phases of the exam. All patients had multimodal imaging at baseline and at the end of the follow-up period.\u003c/p\u003e\n\u003ch3\u003eImage Analysis\u003c/h3\u003e\n\u003cp\u003eB-scans were acquired using the Spectralis HRA-OCT system (Heidelberg Engineering, Heidelberg, Germany). The OCT volume scan encompassed a 20\u0026deg;x20 \u0026deg; area, consisting of 97 raster lines scanned at 60 \u0026micro;m intervals, with high resolution (HR) and automatic real-time (ART) 25. For each patient, 17 spectral-domain OCT (SD-OCT) B-scans within the central 1 mm of the OCT-ETDRS grid were analyzed at baseline and after the loading dose, totalling 612 SD-OCT scans. Only images with signal strength above 20 decibels were included, and the signal strength remained consistent across all examinations. Using ImageJ (version 1.53 t, freely available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://imagej.net/ij/,provided\u003c/span\u003e\u003cspan address=\"https://imagej.net/ij/,provided\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e in the public domain by the National Institutes of Health, Bethesda, MD, USA), 435 individual cysts were manually segmented and classified by retinal location (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e,a). IC\u0026mdash;those situated above the outer plexiform layer (OPL)\u0026mdash;accounted for 194 lesions, whereas 241 cysts located below the OPL were classified as OC. From each patient's 17 B-scans, we quantified mean cyst-specific parameters, including the number of cysts, total cyst area, circularity index, optical density (ODN) measured in pixels, and optical density ratio (ODR), divided into inner and outer layers using the OPL as the boundary. The ODR was defined as the mean ODN of the cyst divided by the mean ODN of the vitreous, as previously described [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eSTATISTICAL ANALYSIS\u003c/h2\u003e \u003cp\u003eContinuous variables are reported as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD), and categorical variables as frequencies and percentages. The distribution of continuous variables was assessed using the Shapiro\u0026ndash;Wilk test. Given the limited sample size and the non-normal distribution of most variables, non-parametric statistical methods were applied throughout the analysis. Baseline comparisons between IC and OC were performed using unpaired analyses (Mann\u0026ndash;Whitney U test). Longitudinal changes in OCT and cyst-related parameters between baseline and post-loading follow-up were evaluated using paired analyses within the same eyes (Wilcoxon signed-rank test). To avoid clustering bias due to multiple cysts and B-scans originating from the same eye, cyst-related parameters were averaged per eye, and all correlation and regression analyses were conducted at the eye level. In addition to p-values, effect sizes were calculated where appropriate to quantify the magnitude of observed differences. Effect sizes were reported to aid interpretation in this exploratory study and should not be considered confirmatory. Effect sizes for non-parametric tests were expressed as r values, and standardized beta coefficients were reported for regression analyses.\u003c/p\u003e \u003cp\u003eThe percentage change in central retinal thickness (ΔCRT%) after the faricimab loading dose was used as the primary anatomical outcome measure to quantify treatment response. Specifically, ΔCRT% was calculated as:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:{\\Delta\\:}\\text{C}\\text{R}\\text{T}\\text{\\%}=\\frac{\\text{C}\\text{R}{\\text{T}}_{\\text{post-loading}}-\\text{C}\\text{R}{\\text{T}}_{\\text{baseline}}}{\\text{C}\\text{R}{\\text{T}}_{\\text{baseline}}}\\times\\:100.$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere CRT\u003csub\u003epost\u0026minus;loading\u003c/sub\u003e refers to the measurement obtained after four consecutive monthly faricimab injections. A negative ΔCRT% represents a reduction in retinal thickness, i.e., anatomical improvement.\u003c/p\u003e \u003cp\u003eUnivariable relationships between baseline imaging parameters and ΔCRT% were explored using both Spearman correlation coefficients and linear regression models. Statistical analyses were performed using SPSS v.29 (IBM Corp., Armonk, NY), and p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. Multivariate models were adjusted for age and sex.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003eIn this study, we evaluated 194 IC and 241 OC, from 18 eyes of 12 patients (7 males and 5 females) with a mean age of 64.8\u0026thinsp;\u0026plusmn;\u0026thinsp;8.7 years. Eleven patients had type 2 DM and one had type 1 DM, with a mean disease duration of 11.55\u0026thinsp;\u0026plusmn;\u0026thinsp;5.8 years and an average glycosylated haemoglobin (HbA1c) of 6.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3%. Eleven eyes had moderate non-proliferative diabetic retinopathy (NPDR), one had severe NPDR, and six had stable proliferative diabetic retinopathy (PDR). All eyes were treatment-na\u0026iuml;ve and presented with cystoid center-involved DME. Baseline demographic and clinical characteristics are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBaseline Demographic and Ocular Characteristics\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDemographic characteristics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePatients (n\u0026thinsp;=\u0026thinsp;12)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge, mean (SD), y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e64.8 (8.7)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSex, Female, No. (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5 (41.6)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eType 1 DM (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 (8.3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eType 2 DM (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11(91.6)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDuration of DM, mean (SD), y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.55 (5.8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHbA1c, (%) mean (SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.7 (1.3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBilateral eyes (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6 (50)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEye characteristics\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eEye (n\u0026thinsp;=\u0026thinsp;18)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBCVA, mean (SD) (ETDRS letters)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e64 (15.2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePseudophakic (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11(61.1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eDR Grading (%)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eModerate NPDR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11(61.1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSevere NPDR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1(5.5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePDR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6 (33.3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBaseline OCT Characteristics\u003c/h2\u003e \u003cp\u003eAt baseline, the mean number of cysts within the central 1-mm area did not differ significantly between IC and OC (8.77\u0026thinsp;\u0026plusmn;\u0026thinsp;5.9 vs 8.03\u0026thinsp;\u0026plusmn;\u0026thinsp;5.2; p\u0026thinsp;=\u0026thinsp;.41). However, the total cyst area was larger in OC compared to IC (0.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 vs 0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03, respectively, p\u0026thinsp;=\u0026thinsp;0.10). Mean ODN and optical ODR were comparable between compartments (ODN: 53.486\u0026thinsp;\u0026plusmn;\u0026thinsp;12.321 vs 50.535\u0026thinsp;\u0026plusmn;\u0026thinsp;10.361; p\u0026thinsp;=\u0026thinsp;.22; ODR: 6.86\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3 vs 7.15\u0026thinsp;\u0026plusmn;\u0026thinsp;4.6; p\u0026thinsp;=\u0026thinsp;.09). Circularity was higher in OC than in IC (0.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 vs 0.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09; p\u0026thinsp;=\u0026thinsp;.27), indicating a more regular cyst morphology in the outer retina. The percentage of IC fluid showed no correlation with either CRT (r\u0026thinsp;=\u0026thinsp;0.20, p\u0026thinsp;=\u0026thinsp;.43) or BCVA (r\u0026thinsp;=\u0026thinsp;0.03, p\u0026thinsp;=\u0026thinsp;.91).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnatomical and Functional Outcomes after Faricimab Treatment\u003c/h3\u003e\n\u003cp\u003eFrom baseline to final follow-up, faricimab treatment induced significant anatomical improvements, with distinct responses between retinal compartments. CRT decreased significantly from 454\u0026thinsp;\u0026plusmn;\u0026thinsp;136.2 \u0026micro;m to 306\u0026thinsp;\u0026plusmn;\u0026thinsp;89.6 \u0026micro;m (p\u0026thinsp;\u0026lt;\u0026thinsp;.01). BCVA showed a slight, non-significant increase from 64\u0026thinsp;\u0026plusmn;\u0026thinsp;15.2 to 65\u0026thinsp;\u0026plusmn;\u0026thinsp;14.6 ETDRS letters (p\u0026thinsp;=\u0026thinsp;.79).\u003c/p\u003e \u003cp\u003eThe total number of cysts declined significantly following the loading dose with a large effect size (from 16.49\u0026thinsp;\u0026plusmn;\u0026thinsp;10.4 to 7.32\u0026thinsp;\u0026plusmn;\u0026thinsp;6.1; p\u0026thinsp;\u0026lt;\u0026thinsp;.01, r\u0026thinsp;=\u0026thinsp;0.81). This reduction was primarily driven by IC, in which cyst count decreased significantly, with a large effect size, from 8.77\u0026thinsp;\u0026plusmn;\u0026thinsp;5.9 to 2.03\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1 (p\u0026thinsp;\u0026lt;\u0026thinsp;.01, r\u0026thinsp;=\u0026thinsp;0.86). OC demonstrated a moderate effect size reduction in cyst number, with a statistical trend that did not reach significance (8.03\u0026thinsp;\u0026plusmn;\u0026thinsp;5.2 to 5.32\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5; p\u0026thinsp;=\u0026thinsp;.08, r\u0026thinsp;=\u0026thinsp;0.61) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In UWF-FA imaging, a visibly reduced leakage was observed at the posterior pole during the late phases of the examination (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec-d).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCysts Parameters\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eCysts Parameters (Mean)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBaseline\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEnd of the loading dose\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eTotal cysts No. (SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16.49 (10.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.32 (6.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;.01*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eIC No. (SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.77 (5.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.03 (3.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;.01*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eOC No. (SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.03 (5.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.32 (3.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eODN IC Px (SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e53.486 (12.321)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e33.410 (28.834)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e.01*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eODN OC Px (SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50.535 (10.361)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e58.582 (12.659)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e.04*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eODR IC Px (SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.86 (3.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.39 (4.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eODR OC Px (SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.15 (4.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.10 (4.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e.21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eTotal cysts area Px\u003csup\u003e2\u003c/sup\u003e (SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.40 (0.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.20 (16.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e.03*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eArea IC Px\u003csup\u003e2\u003c/sup\u003e (SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.03 (0.03)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.01 (0.03)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e.03*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eArea OC Px\u003csup\u003e2\u003c/sup\u003e (SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.19 (0.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.06 (0.04)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e.03*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eCircularity IC (SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.57 (0.09)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.33 (0.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;.01*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eCircularity OC (SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.65 (0.07)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.64 (0.08)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e.68\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eAbbreviations: IC: Inner Cysts; OC: Outer Cysts; ODN: Optical Density; Px: Pixel; ODR: Optical Density Ratio. SD: Standard Deviation; p* significative.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eChanges in Optical Density and Reflectivity\u003c/h3\u003e\n\u003cp\u003eA divergent behaviour between IC and OC compartments was observed in optical density metrics. The mean ODN of IC cysts decreased significantly with a large effect size over follow-up (from 53.486\u0026thinsp;\u0026plusmn;\u0026thinsp;12.321 to 33.410\u0026thinsp;\u0026plusmn;\u0026thinsp;28.834 pixels; p\u0026thinsp;=\u0026thinsp;.01, r\u0026thinsp;=\u0026thinsp;0.87). In contrast, OC exhibited a significant increase in ODN with a moderate effect size (from 50.535\u0026thinsp;\u0026plusmn;\u0026thinsp;10.361 to 58.582\u0026thinsp;\u0026plusmn;\u0026thinsp;12.659 pixels; p\u0026thinsp;=\u0026thinsp;.04, r\u0026thinsp;=\u0026thinsp;0.52). Similarly, the mean ODR of IC showed a decreasing trend with a small-to-moderate effect size (from 6.86\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3 to 4.39\u0026thinsp;\u0026plusmn;\u0026thinsp;4.6 pixels; p\u0026thinsp;=\u0026thinsp;.07, r\u0026thinsp;=\u0026thinsp;0.30), consistent with reduced relative reflectivity. OC, however, demonstrated a tendency toward higher ODR values, with a small-to-moderate effect size that did not reach statistical significance (from 7.15\u0026thinsp;\u0026plusmn;\u0026thinsp;4.6 to 9.10\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5 pixels; p\u0026thinsp;=\u0026thinsp;.21, r\u0026thinsp;=\u0026thinsp;0.35) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCyst Area and Morphological Changes\u003c/h2\u003e \u003cp\u003eBoth inner and outer retinal compartments showed significant reductions in total cyst areas after treatment. IC area decreased with a moderate effect size from 0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 to 0.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 pixels\u003csup\u003e2\u003c/sup\u003e (p\u0026thinsp;=\u0026thinsp;.03, r\u0026thinsp;=\u0026thinsp;0.63), and OC area with a small-to-moderate effect size from 0.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 to 0.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 pixels\u003csup\u003e2\u003c/sup\u003e (p\u0026thinsp;=\u0026thinsp;.03, r\u0026thinsp;=\u0026thinsp;0.30). Morphologically, IC became significantly less circular over time, with a moderate effect size (from 0.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 to 0.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30; p\u0026thinsp;\u0026lt;\u0026thinsp;.01, r\u0026thinsp;=\u0026thinsp;0.63). In contrast, OC circularity remained stable, showing a negligible effect size and no significant change (0.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 to 0.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08; p\u0026thinsp;=\u0026thinsp;.68, r\u0026thinsp;=\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAssociation of Baseline OCT Biomarkers With Anatomical Response\u003c/h2\u003e \u003cp\u003eSeveral baseline OCT parameters were significantly associated with the percentage change in CRT (ΔCRT%) after the loading dose with faricimab. Baseline CRT showed a strong negative association with ΔCRT% (ρ = \u0026minus;\u0026thinsp;0.607, p\u0026thinsp;\u0026lt;\u0026thinsp;.01; p\u0026thinsp;=\u0026thinsp;.008; B\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;0.064\u0026thinsp;\u0026plusmn;\u0026thinsp;0.024, β=\u0026minus;0.552, p\u0026thinsp;=\u0026thinsp;.01), indicating greater proportional thickness reduction in eyes with greater retinal thickening at presentation. Similarly, total intraretinal cyst area was also negatively associated with ΔCRT% (ρ = \u0026minus;\u0026thinsp;0.525, p\u0026thinsp;=\u0026thinsp;.02) and was a significant predictor in univariable linear regression (B\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;47.866\u0026thinsp;\u0026plusmn;\u0026thinsp;18.159, β=\u0026minus;0.550, p\u0026thinsp;=\u0026thinsp;.01), suggesting that a higher baseline intraretinal fluid burden was associated with a greater anatomical response (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn contrast, the mean reflectivity ratio of cysts below the OPL (bODR) was positively associated with ΔCRT% (ρ\u0026thinsp;=\u0026thinsp;0.476, p\u0026thinsp;=\u0026thinsp;.04) and emerged as a significant positive predictor in univariable regression (B\u0026thinsp;=\u0026thinsp;+\u0026thinsp;1.918\u0026thinsp;\u0026plusmn;\u0026thinsp;0.695, β\u0026thinsp;=\u0026thinsp;0.568, p\u0026thinsp;=\u0026thinsp;.01), indicating a smaller CRT reduction in eyes with more reflective outer cystic fluid. These associations remained significant after adjustment for age and sex.\u003c/p\u003e \u003cp\u003eOther parameters, including cyst circularity and the number of cysts located above or below the OPL, were not significantly associated with ΔCRT% (all p\u0026thinsp;\u0026gt;\u0026thinsp;.05).\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis study documents compartment-specific anatomical responses to faricimab treatment in DME, highlighting differences between IC and OC responses and characteristics. Analysis of OCT images was essential for layer-specific characterization of intraretinal cysts, enabling differentiation of inner and outer retinal fluid behaviour and revealing treatment-related changes that cannot be captured by global thickness measures alone. IC exhibited a significant reduction in number, size, and reflectivity, a pattern consistent with the pharmacological effects of dual Ang-2/VEGF-A inhibition on the iBRB. The rapid restoration of endothelial\u0026ndash;pericyte signalling likely underlies the more prompt and pronounced regression of inner retinal fluid. In contrast, OC showed a different anatomical response. The slower resolution may reflect the chronic and multifactorial nature of oBRB dysfunction, which is characterized by disruption of RPE tight junctions, impaired metabolic support to photoreceptors, and reduced efficiency of fluid transport [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Notably, an increase in OC optical density was observed during treatment, suggesting persistent alterations in exudate composition. This finding may indicate exudation or biochemical remodelling within outer retinal fluid compartments, consistent with a slower clearance of protein-rich material [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Moreover, the area and circularity index of OC remained relatively stable over time. Such morphological characteristics suggest that OC may represent a more established and less dynamic form of edema [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Finally, baseline OCT biomarkers were associated with treatment response: greater intraretinal fluid and a larger total cystic area were linked to greater anatomical improvement, whereas higher OC reflectivity was associated with a smaller reduction in CRT, suggesting a more treatment-resistant edema phenotype. This finding suggests that retinal compartment and qualitative fluid features may respond differently to dual Ang-2/VEGF-A inhibition and can reveal aspects of disease that are not indicated by fluid volume alone [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe relationship between intraretinal cyst location and macular function has been widely explored, although findings remain partly heterogeneous [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Previous studies have suggested that fluid involving the outer retinal layers may be more frequently observed in eyes with more advanced DR and may be associated with poorer visual outcomes [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Conversely, recent analyses using automated and deep learning\u0026ndash;based OCT quantification have reported that inner retinal fluid volume shows a stronger association with BCVA, whereas outer retinal fluid may be less directly related to visual function [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Studies integrating microperimetry (MP) with detailed structural analysis using artificial intelligence (AI) have further indicated that cyst size and retinal layer localization may influence retinal sensitivity [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In particular, larger cysts within the ONL and the presence of SRF have been associated with greater reductions in retinal sensitivity, while smaller cysts confined to the inner nuclear layer appear to have a more limited functional impact [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. These structural features have been described more frequently in later disease stages and may reflect long-standing BRB dysfunction and altered retinal microenvironment. Importantly, symptom duration alone did not correlate with functional impairment, suggesting that the extent and distribution of structural alterations may be more relevant than disease chronicity \u003cem\u003eper se\u003c/em\u003e [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In the present study, faricimab treatment resulted in a significant reduction in CRT and intraretinal fluid, with distinct compartment-specific responses. BCVA showed a minimal, non-significant change over the short follow-up period. The lack of significant improvement in BCVA likely reflects the short follow-up period and the limited sensitivity of BCVA to compartment-specific structural changes.\u003c/p\u003e \u003cp\u003eAdditionally, we evaluated cyst reflectivity utilizing structural OCT. Prior research on cyst reflectivity, including the suspended scattering particles in motion, mainly employed OCT angiography [\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, this approach may limit precise anatomical localization. Therefore, our results cannot be directly compared to previous studies. The increased OC reflectivity could reflect an optical artifact due to reabsorption of overlying inner retinal fluid and improved signal penetration. However, reflectivity was quantified using a normalized ODR, and although the ODN of OC increased, the corresponding ODR change was not significant, arguing against a pure transmission effect [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In addition, baseline OC reflectivity independently predicted a smaller CRT reduction, supporting its role as a true biomarker of chronic, treatment-resistant edema rather than an imaging artifact.\u003c/p\u003e \u003cp\u003eOur findings align with the literature showing that outer retinal pathology\u0026mdash;whether represented by the number of cysts, reflectivity, or area changes\u0026mdash;reflects more advanced disease and reduced therapeutic responsiveness [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In contrast, the rapid reduction in IC size and reflectivity after faricimab treatment suggests a faster recovery of the iBRB and more efficient removal of extracellular fluid in the inner retina. This is further supported by the decreased leakage observed in UWF-FA, as previously detailed in a post hoc analysis of YOSEMITE/RHINE [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLimitations of the study include its small sample size and retrospective design, which may limit generalizability. The short follow-up, restricted to the loading dose, precludes evaluation of long-term structural and functional outcomes, particularly for OC. Finally, functional assessment was limited to BCVA, and more sensitive measures of visual function were not included. Larger studies with advanced imaging, AI quantification, functional assessments and longer follow-up are necessary to validate these findings and evaluate the prognostic significance of cyst characteristics and retinal fluid compartmentalization as an OCT biomarker in DME.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThis study provides clinical evidence that intraretinal cysts in DME are heterogeneous and exhibit compartment-specific responses to dual Ang-2/VEGF-A inhibition with faricimab, consistent with DME pathogenesis and the distinct mechanisms by which Ang-2 affects permeability in the iBRB and oBRB. From a clinical perspective, the differential behaviour of IC and OC may help explain the variability in anatomical outcomes observed in treatment-na\u0026iuml;ve DME patients despite standardized anti-VEGF therapy. These observations highlight the significance of retinal fluid compartmentalization as a key biomarker for treatment response, which could also assist in the early detection of patients needing closer monitoring, prolonged loading doses, or different therapeutic approaches to personalize DME treatment.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eStandard Deviation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDiabetes Mellitus\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBCVA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBest Corrected Visual Acuity\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDiabetic Retinopathy\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNPDR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNon-Proliferative Diabetic Retinopathy\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePDR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNon-active Proliferative Diabetic Retinopathy\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e: not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatements and Declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics:\u0026nbsp;\u003c/strong\u003eThe study was conducted in accordance with the Declaration of Helsinki and approval was granted by the Institutional Ethics Committee of IRCCS MultiMedica (CE 55.2022). The authors confirm that the ethical guidelines of the journal were adhered to and that approval was obtained from the appropriate ethics committee.Written consent for publication of identifying information and accompanying images was obtained.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics committee approval:\u0026nbsp;\u003c/strong\u003eThe retrospective study was conducted in accordance with the Declaration of Helsinki and approved by Institutional Ethics Committee of IRCCS MultiMedica (CE 55.2022).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish:\u0026nbsp;\u003c/strong\u003eThe authors affirm that human research partici­pants provided informed consent for publication of the images.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC.A., V.B., C.L., J.C., S.D.S., S.B.: no financial disclosures relevant to this paper\u003c/p\u003e\n\u003cp\u003eG.P.: EssilorLuxottica Group – Espansione Group S.p.A. Employee, Nidek Co LT. honorarium for symposium.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eP.N.: Thea, Hoya, Essilor Luxottica e Fondazione One-sight, Sifi, Bausch\u0026amp;amp; Lomb, Alfa Intes, Alcon, Doc, Santen, Eyerising\u003c/p\u003e\n\u003cp\u003eSV: Consultant to Abbvie, Adverum, Apellis, Bayer, Boehringer \u0026amp; Ingelheim, Novartis, Hoffman La Roche, RetinAI, Zeiss;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCommercial Relationships Disclosure:\u0026nbsp;\u003c/strong\u003eNo conflicting relationship exists for the other authors in relation to this study. All authors have completed and submitted the ICMJE disclosures form.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFinancial Support:\u003c/strong\u003e None\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCunha-Vaz J (2017) Mechanisms of Retinal Fluid Accumulation and Blood-Retinal Barrier Breakdown. 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Can J Ophthalmol 60(5):297\u0026ndash;305. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jcjo.2025.02.013\u003c/span\u003e\u003cspan address=\"10.1016/j.jcjo.2025.02.013\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoldberg RA, Mar FA, Csaky K, Amador M, Khanani AM, Gibson K et al (2025) Resolution of Angiographic Macular Leakage with Faricimab versus Aflibercept in Patients with Diabetic Macular Edema in YOSEMITE/RHINE. Ophthalmol Retina 9(6):515\u0026ndash;526. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.oret.2024.11.015\u003c/span\u003e\u003cspan address=\"10.1016/j.oret.2024.11.015\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Diabetic Macular Edema, Optical Coherence Tomography, Retinal Cyst Characteristics, Ang-2/VEGF-A Dual Inhibition, Retinal Imaging Biomarkers","lastPublishedDoi":"10.21203/rs.3.rs-8563078/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8563078/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003ePurpose:\u003c/strong\u003e Location of retinal cysts in diabetic macular edema (DME) can have clinical implications and a layer-specific response to treatment may aid in predicting therapeutic response. This study aimed to compare quantitative and qualitative changes in inner versus outer retinal cysts after loading dose of faricimab in center-involved cystoid DME using spectral-domain OCT (SD-OCT).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e 435 retinal cysts (194 inner, 241 outer) from eighteen eyes of 12 patients were analyzed on 612 SD-OCT B-scans within the 1 central mm. Cysts were classified as inner (IC) or outer (OC) based on the location relative to the outer plexiform layer (OPL). Features of the cysts were quantified using ImageJ (1.53), including reflectivity as optical density (ODN), ODN ratio (ODR), area, and circularity index (CI). Non-parametric analyses were performed, with significance set at p \u0026lt; 0.05.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Faricimab significantly reduced central retinal thickness (CRT),(p\u0026lt;.01), inner, outer, total cyst area and cyst count (p≤.03, for all). IC showed significant reductions in number, ODN, and CI (p≤.01, for all). OC exhibited a reduction in number (p=.08) and a significant increase in ODN (p=.04). Higher baseline CRT and larger total cyst area were associated with greater %CRT reduction (p=.008 and p=.02), whereas higher baseline OC reflectivity was associated with smaller %CRT reductions (p=.04).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e Faricimab induces a compartment-specific response of intraretinal cysts in DME. Inner and outer retinal fluid show distinct qualitative behaviour, reflecting different blood–retinal barrier dynamics. OCT-based assessment of cyst location and reflectivity may complement CRT in evaluating treatment response.\u003c/p\u003e","manuscriptTitle":"Differential Changes in Retinal Cyst Characteristics in inner versus outer retina in Diabetic Macular Edema Treated With Ang-2/VEGF-A Dual Inhibitor","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-22 07:15:51","doi":"10.21203/rs.3.rs-8563078/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ea24dfc5-f247-4b38-bde8-d42a8dffc390","owner":[],"postedDate":"January 22nd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-19T11:26:27+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-22 07:15:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8563078","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8563078","identity":"rs-8563078","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}