Aqueous Humor Research Articles

Phenotypic Biomarkers of Aqueous Extracellular Vesicles from Retinoblastoma Eyes

Recent advancements in aqueous humor (AH) cell-free DNA (cfDNA) genomics have opened new avenues for ex vivo molecular profiling of retinoblastoma (RB), the most common pediatric intraocular malignancy, where biopsy is typically prohibited. While these insights offer a genetic blueprint of the tumor, they lack multi-omic molecular phenotyping, which is essential for understanding the functional state. Extracellular vesicles (EVs), naturally present in AH, are promising by offering time-resolved phenotypic information. We employed multiplex bead-based flow cytometry and Single Extracellular Vesicle Nanoscopy (SEVEN) to analyze EV phenotypes in AH from a cohort of five RB, with three uveal melanoma (UM) and two age-matched glaucoma (GLC) samples serving as controls. The studies identified CD133-enriched EVs uniquely in RB AH, absent in both GLC and UM AH. This was corroborated by further analysis of five RB cell lines, including two commercial (Y79, Weri) and three in-house developed lines, confirming CD133 enrichment and supporting its role as an RB-specific EV marker. Single-vesicle analysis demonstrated a strong association of CD133 with CD81 and CD63, with minimal CD9 presence. These results, validated through complementary techniques, position CD133 as a critical marker in RB-derived EVs, paving the way for enhanced multi-omic RB characterization and potential advancements in clinical diagnostics.

Long‐Term Monitoring of ApoE4 within Aqueous Humor Using Intraocular Lenses Containing Target‐Responsive Inverse Opal Hydrogel

Neurodegenerative diseases, including Alzheimer's disease (AD), pose significant challenges to global healthcare because of their irreversible postsymptomatic progression. Conventional radiographic techniques have limitations in early detection, owing to the time lapse between symptom recognition by individuals and precise inspection using medical instruments. Current research explores ophthalmic approaches to address this gap, investigating the physiological link between ocular health and brain diseases. Intraocular lenses (IOLs), widely used in cataract and refractive surgeries, provide a safe and minimally invasive platform for the continuous monitoring of neurodegenerative disease biomarkers, including those associated with AD. By integrating biomolecule‐responsive sensors with IOLs, it becomes feasible to achieve early diagnosis and long‐term, noninvasive monitoring postimplantation. Herein, a fluorescently labeled inverse opal hydrogel (FIOH) is merged with IOLs for ApoE4 detection, a key risk factor for AD. The permeability and inherent photonic properties of FIOH facilitate the cumulative enhancement of the fluorescence signal. In a simulation of the aqueous humor circulation, the detection limit for ApoE4 is determined to be 0.1 nM. Therefore, FIOH‐integrated IOLs hold promise not only for early detection but also for providing a reliable platform for the continuous, noninvasive monitoring of neurodegenerative diseases after the initial, minimally invasive implantation.

High-fat and high-sucrose diets induce an experimental rabbit model for age-related macular degeneration (AMD).

Age-related macular degeneration (AMD) is a leading cause of blindness. Metabolic disorders and diets are risk factors. We compared lipid profiles and retinal phenotypes with long-term feeding of four diets in male Chinchilla rabbits. Animals were fed with a normal diet (ND), high-fat (HFD), high-sucrose (HSD), or high-fat and high-sucrose diet (HFSD) for six months. The eyes were examined using multimodal imaging modalities and electroretinogram (ERG). Retinal sections were analyzed using H&E staining, toluidine blue staining, immunostaining, and transmission electron microscopy. Lipids and complement C3 in serum or aqueous humour were measured. RNA sequencing was performed to evaluate the retinal transcriptomes. HFD and HSD had minor effects on lipid profiles but synergistically induced severe dyslipidemia. All diets did not cause obesity. HFSD feeding induced retinal lesions like reticular pseudo-drusen (RPD) and pigmentary abnormalities. The RPD-like lesions were mainly lipid droplets around RPE cells. HFSD induced elevated ocular C3 levels and reduced retinal vessel density. In conclusion, HFD and HSD can synergistically induce normal-weight dyslipidemia and RPD-like retinal lesions. HFSD-fed male Chinchilla rabbits are a good model of early AMD.

Targeting the VEGFR2 signaling pathway for angiogenesis and fibrosis regulation in neovascular age-related macular degeneration

Neovascular age-related macular degeneration (nAMD) is characterized by abnormal blood vessel growth from the choroid, leading to complications and eventual blindness. Despite anti-VEGF therapy, subretinal fibrosis remains a major concern, as VEGF/VEGF receptor-2 (VEGFR2) signaling can contribute to both angiogenesis and fibrosis. For the identification of the aqueous humor proteome, we performed liquid chromatography with tandem mass spectrometry analysis. To investigate the potential therapeutic effects of targeting the VEGF signaling pathway using apatinib, a highly selective VEGFR2 tyrosine kinase inhibitor, this study employed in vitro (THP-1 conditioned media-treated ARPE-19 cells) and in vivo (laser-induced choroidal neovascularization mouse) models of nAMD. This study revealed elevated VEGFR2 protein levels in the aqueous humor of nAMD patients, suggesting a potential target to mitigate neovascularization and fibrosis in nAMD. Apatinib effectively reduced VEGFA and αSMA levels in both in vitro and in vivo models. Moreover, apatinib showed improvement in laser-induced subretinal hyper-reflective lesions. The action mechanism was linked to the inhibition of VEGFR2 activation, leading to the suppression of both angiogenesis and fibrosis through the downregulation of STAT3 phosphorylation. Therefore, the VEGFR2 signaling pathway appears to play a central role in the development of nAMD by regulating both angiogenesis and fibrosis.

Short-term changes in the trabecular iris angle and anterior chamber during wear of scleral lenses with different diameters

ABSTRACT Clinical relevance Scleral lens (SL) compression on the conjunctiva/episclera during wear may alter adjacent structures related to aqueous humour drainage. Background The aim of this work was to assess short-term changes in the four quadrants of trabecular iris angle (TIA) and anterior chamber parameters during SL wear, using anterior segment optical coherence tomography (AS-OCT). Methods Prospective study was conducted involving 16 healthy subjects with a mean age of 28 ± 5 years. Two SL of diameter 15.80 mm (L1) and 16.80 mm (L2) with the same parameters were used for 2 hours in different days. The central fluid reservoir (FR), the TIA in superior, inferior, nasal, temporal quadrants, and anterior chamber parameters; anterior chamber depth (ACD), anterior chamber width (ACW), anterior chamber area (ACAr) and anterior chamber volume (ACV) were measured with AS-OCT. These measurements were performed immediately after SL application (0 h), one hour (1 h) and two hours (2 h) of SL wear. Results TIA decreases significantly in the superior quadrant at 2 hours of L2 wear (−5.19 ± 7.79º) (p = 0.04). In this sector, differences were also observed between the changes induced by both lenses during the 2 hours of wear (p = 0.04) and between first and second hours of wear (p = 0.04). ACD was significantly lower with L1 (−0.09 ± 0.14 mm) relative to L2 (0.00 ± 0.06 mm) between immediately after the application and 2 hours of wear (p = 0.02). A significant reduction in ACAr at 1 hour (−0.65 ± 0.75 mm2) (p = 0.01) and 2 hours of wear (−0.81 ± 1.15 mm2) (p = 0.04), as well as in ACW at 2 hours (−0.11 ± 0.14 mm) (p = 0.02) compared to immediately after application of L1 was found. Conclusion Small changes in TIA and anterior chamber occur during two hours of SL wear, but these changes are of limited clinical relevance in healthy subjects.

Gut and intraocular fluid dysbiosis in people with type 2 diabetes-related retinopathy in India: A case for further research.

To explore the relationship between gut microbiome, gut mycobiome, and intraocular (aqueous humor) microbiome dysbiosis in people with type 2 diabetes (T2DM) and diabetic retinopathy (DR). Multiple case-control studies. We evaluated three groups of people: healthy controls (HC), people with T2DM without retinopathy, and those with DR. The study samples included fecal matter (30-50 g) and aqueous humor (0.05-0.1 mL). After amplicon sequencing, we analyzed microbiome profiles (V3-V4 region of bacterial 16S rRNA gene) and mycobiome (ITS2 region of fungal rRNA gene). The main outcome measures were relative abundance, α and β diversity, and dysbiotic bacteria and fungi, analyzed based on the inferred functions of the taxa. We recruited 82 people for gut microbiome (30 HC, 24 DM, and 28 DR); 75 people for gut mycobiome (30 HC, 21 DM, and 24 DR); and 12 people for aqueous humor microbiome (4 each HC, DM, and DR) studies. Generally, there was an increased abundance of pro-inflammatory and pathogenic microorganisms and a decreased abundance of anti-inflammatory and probiotic microorganisms. The differences were higher between HC and DM/DR than between DM and DR. In aqueous humor, there was a wider separation in microbiome profiles of people with DR than their gut microbiome. The gut and aqueous humor microbiota of people with diabetes and DR may differ from those without diabetes. Given these unique observations in individuals living in one region of India, further research involving people from different regions is required to identify indices for possible regional or global use.

Omiganan-Based Synthetic Antimicrobial Peptides for the Healthcare of Infectious Endophthalmitis.

Bacterial endophthalmitis is a severe infection of the aqueous or vitreous humor of the eye that can lead to permanent vision loss. Due to the rapid emergence of antibiotic resistance and dose-limiting toxicities, the standard treatment of bacterial endophthalmitis via the intravitreal injection of broad-spectrum antibiotics remains inadequate. Membrane active cationic antimicrobial peptides (AMPs) have emerged as a promising class of effective and broad-spectrum antimicrobial agents with potential to overcome antibiotic resistance. In this work, we investigate, for the first time, the use of omiganan (IK-12), a 12-amino acid indolicidin derivative for the treatment of bacterial endophthalmitis. Additionally, IK-12 was used as a template to perform amino acid rearrangements, without altering the length or type of amino acids, to yield a series of new derivative AMPs with varying extents of secondary structure formation under membrane mimicking conditions. IK-12 and its derivatives demonstrated strong and broad-spectrum antibacterial activities against a panel of clinically isolated Gram-positive and Gram-negative bacteria, including methicillin-resistant Staphylococcus aureus commonly implicated in bacterial endophthalmitis. Interestingly, two of the new IK-12 derivatives, IP-12 and WP-12, showed lower geometric mean minimum inhibitory concentration and higher 50% hemolysis concentration values, which effectively translated into 2- to 3.4-fold higher bacterial selectivity than the parent IK-12. Furthermore, the intravitreal injection of IK-12, IP-12, and WP-12 in a rabbit model of MRSA-induced endophthalmitis led to considerably improved clinical presentation and reduced recruitment of inflammatory cells. In all, these results demonstrate the potential of IK-12 and its derivatives, IP-12 and WP-12, as promising candidates for the treatment of bacterial endophthalmitis.

Clinical Spectrum and Perspective in Bilateral Acute Retinal Necrosis: Systematic Review

Bilateral acute retinal necrosis (BARN) represents a broad ophthalmological field of severe retinal pathologies associated with poor visual prognosis and blindness. The purpose of this review is to examine the clinical spectrum in detail over the past few years, exploring laboratory and instrumental diagnosis, and providing useful and up-to-date guidance in this field. A systematic review of this field has been performed through the PRISMA guidelines, searching in the PUBMED database. Serological laboratory tests on blood or polymerase chain reaction (PCR) on aqueous humor or vitreous samples are crucial to identifying the underlying cause and choosing the timeliest strategic treatments. Often, the main cause remains herpesviruses, with Varicella zoster (VZV) predominating over the others. There are also other causes that one needs to be carefully aware of. Anatomical and functional recovery is unfavorable if they are not individuated in a quick time. Early diagnosis and timely treatment offer a better chance of visual improvement and the avoidance of complications. Complications worsen the visual prognosis over months and may require a surgical approach.

The effect of mucoadhesive polymers on ocular permeation of thermoresponsive in situ gel containing dexamethasone–cyclodextrin complex

Dexamethasone (DXM) is a commonly used corticosteroid in the treatment of ocular inflammatory conditions that affect more and more people. The aim of this study was to evaluate the effect of the combination of hydroxypropyl-β-cyclodextrin (HPBCD), in situ gelling formulations, and other mucoadhesive polymers, i.e., hydroxypropyl methylcellulose (HPMC) and zinc-hyaluronate (ZnHA), on permeation by applying in vitro and ex vivo ophthalmic permeation models. Additionally, gelling properties, in vitro drug release, and mucoadhesion were measured to determine the impact of these factors on permeation and ultimately on bioavailability. The results showed that GEL1 and GEL2 had an optimal gelling temperature, 36.3 ℃ and 34.6 ℃, respectively. Moreover, the combination of Poloxamer 407 (P407) with other polymers improved the mucoadhesion (GEL1: 1333.7 mN) compared with formulations containing only P407 (P12: 721.8 mN). Both HPBCD and the gel matrix had a considerable influence on the drug release and permeability of DXM, and the combination could facilitate the permeation into the aqueous humor. After 30 min of treatment, the DXM concentration in the aqueous humor was 1.16–1.37 µg∕mL in case of the gels, whereas DXM could not be detected when treated with the DXM suspension. The results of the experiments using an in vitro cell line indicated that the formulations could be considered safe for topical treatment of the eye. In conclusion, with application of a small amount of HPMC (0.2 % w∕w), the concentration of P407 could be reduced to 12 % w/w while maintaining the ideal gelling properties and gel structure without negatively affecting permeability compared with the formulation containing a higher amount of P407. Furthermore, the gel matrix may also provide programmed and elongated drug release.

Aqueous Humor Angiography.

Aqueous humor angiography, or aqueous angiography, is an anterior segment imaging technique capable of visualizing the conventional/trabecular aqueous humor outflow pathways. As a translational technique, it is applicable for in vivo imaging in living subjects and ex vivo imaging using whole-globe preparations or anterior segment perfusion setups. Excellent spatial and temporal resolution has enabled insights into the segmental distribution of aqueous humor outflow in physiological conditions as well as after trabecular bypass surgery. In this chapter, we thoroughly describe aqueous humor angiography in various experimental setups. The necessary materials for different settings and their application for in vivo and ex vivo experiments as well as notes on dye choice, dye sequences, and special considerations on performing aqueous humor angiography during surgery are presented.

Effectiveness and biocompatibility of a novel Schlemm’s canal microstent for glaucoma management

To evaluate the effectiveness and biocompatibility of Wistend, a novel Schlemm’s canal (SC) microstent made of Nitinol designed to improve aqueous humor outflow. New Zealand white (NZW) rabbits were divided into blank, sham-operated and Wistend groups. ICare® Tonovet Plus®, swept-source optical coherence tomography (SS-OCT), slit lamp biomicroscopy, retinal camera and scanning electron microscopy (SEM) were used for preoperative and postoperative observations. Hematoxylin and Eosin (H&E) tissue staining was adopted for biocompatibility. A significant difference in intraocular pressure (IOP) between the Wistend group and the control groups was observed during the six-month follow-up. SS-OCT identified arc line internal reflections within the SC in the anterior chamber angle. Conjunctival congestion and edema gradually diminished in the early stages. No corneal vascularization, no anterior chamber inflammatory response and no significant tissue reactions were noted in any groups. SEM showed the Wistend’s windows and orifices remained clear, encircled by minimal incidental ocular tissue and free from blockage. Histopathological examination revealed no discernible differences between the Wistend-implanted and sham-operated eyes. These in vivo studies demonstrate the effectiveness and biocompatibility of the microstent. Our findings suggest a promising potential for Wistend in significantly reducing IOP and effectively facilitating the outflow of aqueous humor.

Time-Lapse Live-Cell Imaging Using Fluorescent Protein Sensors in Outflow Pathway Cells Under Fluid Flow Conditions.

The role of shear stress in regulating aqueous humor (AH) outflow and intraocular pressure (IOP) in the trabecular meshwork (TM) and Schlemm's canal (SC) of the eye is an emerging field. Shear stress has been shown to activate mechanosensitive ion channels in TM cells and induce nitric oxide production in SC cells, which can affect outflow resistance and lower IOP. Live-cell imaging using fluorescent protein sensors has provided real-time data to investigate the physiological relationship between fluid flow and shear stress in the outflow pathway cells. The successful application of time-lapse live-cell imaging in primary cultured cells has led to the identification of key cellular and molecular mechanisms involved in regulating AH outflow and IOP, including the role of autophagy and primary cilia as mechanosensors. This chapter presents a detailed protocol for conducting time-lapse live-cell imaging under fluid flow conditions in the outflow pathway cells.

Protocol for the Extraction and Characterization of Trabecular Meshwork Cell Cytoskeleton Fraction.

Among various cellular attributes modulating aqueous humor (AH) outflow through the trabecular pathway and eventually intraocular pressure, the involvement of actomyosin regulated cellular contraction and relaxation, cell-extracellular matrix adhesion and cell-cell junctions, and mechanotransduction are well recognized. Although various biological and pharmacological agent-modulated AH outflows were associated with altered actin cytoskeletal organization and cell adhesive interactions of trabecular meshwork (TM) cells, these changes were analyzed largely with a biased approach to examine the specific proteins, but there were very few efforts in examining them with hypothesis-free unbiased approach in their native state. Therefore, in this chapter, we describe a protocol tailored to characterize the cytoskeleton of TM cells. This simple protocol can be applied to identifying the differentially regulated TM cell cytoskeleton proteins under any given treatment condition in conjunction with quantitative proteomic analysis.

Measurements of Phagocytosis in Trabecular Meshwork Cells.

The restriction in the movement of aqueous humor through the trabecular meshwork is known to play an important role in the pathogenesis of glaucomas. Among the functions of the trabecular meshwork that help to prevent this restriction and maintain the movement of aqueous humor is phagocytosis. Cells in the trabecular meshwork actively phagocytose pigment, cellular debris, and other materials in order to help keep the drainage pathway clear. A decrease in phagocytosis has thus been implicated in the development of glaucomas. In this chapter, we outline two methods using fluorescent markers that can be used to measure the phagocytic properties of trabecular meshwork cells in culture.

Autophagy activation ameliorates the fibrosis of trabecular meshwork cells induced by TGFβ2 through the promotion of fibrotic proteins degradation.

The level of transforming growth factor-beta2 (TGFβ2) is elevated in aqueous humor of partial glaucoma patients, and induced trabecular meshwork (TM) fibrosis, which could cause TM cells dysfunction and lead to intraocular pressure (IOP) elevation. Autophagy is a dynamic process of bulk degradation of organelles and proteins under stress condition, while its functions in fibrotic development remain controversial. Meanwhile, it is still unclear if activation of autophagy could ameliorate TGFβ2-induced fibrosis in TM cells. In this study, we demonstrated that autophagy activation with Rapamycin or Everolimus could ameliorate TM fibrosis induced by TGFβ2. We also proved that activation of autophagy may decrease TM cells fibrosis and reduce elevated IOP induced by TGFβ2 in vivo, while Rapamycin or Everolimus has no effect on TGFβ/Smad3 pathway activity and fibrotic genes expression. However, when Chloroquine phosphate blocks autophagy-lysosome pathway, the protective effect of Rapamycin or Everolimus on fibrosis was weakened. We established that autophagy activation ameliorates TM fibrosis through promoting fibrotic proteins degradation.

Evaluation of Cross-Linked Actin Networks (CLANs) in Human Trabecular Meshwork Cells and Tissues.

Elevated intraocular pressure (IOP) is a major risk factor for the development and progression of glaucoma, the leading cause of irreversible vision loss and blindness. An overall increase in resistance to aqueous humor outflow causes sustained elevation in IOP. Glaucomatous insults in the aqueous humor outflow pathway, including the trabecular meshwork (TM), precede such chronic physiological changes in IOP. These insults include ultrastructural changes with excessive extracellular matrix deposition and actin cytoskeletal reorganization that leads to pathological stiffening of the ocular tissues. One of the most common cytoskeletal changes associated with TM tissue stiffness in glaucoma is the increased prevalence of cross-linked actin networks (CLANs) in cells of the trabecular meshwork (TM) and lamina cribrosa (LC). In glaucomatous cells, rearrangement of linear actin stress fibers leads to formation of polygonal arrays within the cytoplasm, resembling a geodesic dome-like structure, that we identified as CLANs. In addition to increased amounts of CLANs in POAG TM cells and tissues, we also discovered that glucocorticoid (GC) and TGFβ2 signaling pathways associated with the development of ocular hypertension (OHT) and glaucoma also induced CLANs in the TM. Despite a clear association, we are yet to completely understand the mechanisms involved in CLAN formation and their direct relevance to disease pathology. In this chapter, we will describe methods to identify and characterize CLANs using fluorescent microscopy in primary TM cell cultures, ex vivo perfusion cultured human anterior segments, and in situ in human donor eyes.

Human Anterior Segment Perfusion Organ Culture.

The human anterior segment perfusion organ culture is an ex vivo model system for studying the human conventional outflow pathway with reference to pressure regulation. In this model, anterior segments dissected from human donor eyes can be fixed to a modified petri dish and perfused with media along with various study agents at the physiological flow rate of 2.5μL/min. The model mimics the one-way flow of aqueous humor in human eyes and can be used to evaluate the effects of various drugs on eye pressure in real time. Using this model, cells and tissues of the anterior segment can be maintained for up to 28days, enabling histological and molecular evaluations.

Nanotubules and Cellular Communication in Trabecular Meshwork Cells.

Glaucoma causes dysfunction to tissues located in the anterior and posterior eye. In the anterior eye, the trabecular meshwork (TM) is the site of pathogenesis, where decreased TM cell numbers and alterations to the amount and composition of extracellular matrix hinder outflow of aqueous humor fluid from the anterior chamber. This causes intraocular pressure (IOP) elevation. Elevated IOP, a main risk factor for primary open-angle glaucoma, damages the axons of retinal ganglion cells in the posterior eye, which ultimately leads to blindness. Thus, clinical treatment paradigms for glaucoma are focused on reducing IOP. Normotensive IOPs are established by balancing the production of aqueous fluid from the ciliary body with drainage through the TM to Schlemm's canal. When IOP becomes elevated, TM cells coordinate a homeostatic response to lower IOP, which requires effective and efficient cellular communication. Tunneling nanotubes (TNTs) are transient specialized structures that allow cells to communicate with one another. Actin-rich tubes allow direct transmission of signals and cargoes between cells. This is important to overcome limitations of diffusion-based signaling in aqueous environments such as the anterior eye. Here, we describe a live-cell imaging method for monitoring TNTs in primary TM cells.

Glutathione Modulates Hydrogen Sulfide Release and the Ocular Hypotensive Action of Diallyl Polysulfide Compounds.

Hydrogen sulfide (H2S) is an endogenous transmitter with the potential to regulate aqueous humor dynamics and protect retinal neurons from degeneration. The aim of the present study was two-fold: (a) to evaluate the release of H2S from two polysulfides, diallyl disulfide (DADS), and diallyl trisulfide (DATS); and (b) to investigate their ocular hypotensive actions in normotensive male and female rabbits in the presence and absence of GSH. H2S was quantified hourly for up to 6 h using a H2S-Biosensor (World Precision Instruments, Sarasota, Fl). Intraocular pressure (IOP) was assessed in normotensive New Zealand Albino rabbits using a pneumotonometer (model 30 classic; Reichert Ophthalmic Instruments, Depew, NY, USA). In the presence of GSH, there was an increase in the in vitro release of H2S produced by DADS and DATS. Both DADS and DATS also caused a dose-dependent reduction in IOP in male and female rabbits, in both treated and untreated eyes. For instance, in male animals, the presence of GSH (3% and 5%) significantly (p < 0.05, n = 5) enhanced the ocular hypotensive action of DADS (2%) and DATS (2%) from 14.02 ± 2.89% to 18.67 ± 5.6% and from 16.22 ± 3.48 to 23.62 ± 5.79%, respectively. GSH enhanced both H2S release and ocular hypotensive action of the polysulfides in a manner that was dependent on the number of sulfur atoms present in each polysulfide. Furthermore, female animals were less sensitive to the IOP-lowering action of the polysulfides, when compared to their male counterparts.

Vascular endothelial growth factor/connective tissue growth factor and proteomic analysis of aqueous humor after intravitreal conbercept for proliferative diabetes retinopathy.

To investigate the role of connective tissue growth factor (CTGF) and vascular endothelial growth factor (VEGF) in the protein profile of the aqueous humor in patients with proliferative diabetic retinopathy (PDR) following intravitreal injection of conbercept. This study included 72 PDR patients and 8 cataract patients as controls. PDR patients were divided into 3 groups according to the intervals of 3, 5, and 7d between intravitreal conbercept (IVC, 0.5 mg/0.05 mL) injection and pars plana vitrectomy (PPV) performed. Aqueous humor samples were collected before and after IVC and PPV for VEGF and CTGF levels detected with enzyme-linked immunosorbent assay (ELISA). The differential proteomics of 10 patients who underwent PPV surgery 5d after IVC and 8 normal controls was studied, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed on the data, and the protein interaction network of 23 differential proteins was studied. Post-IVC, VEGF levels decreased and CTGF levels increased significantly in aqueous humor, with the CTGF/VEGF ratio rising significantly at all intervals. Liquid chromatography tandem mass spectrometry (LC-MS/MS) identified differentially expressed proteins between pre- and post-IVC samples. GO and KEGG analyses revealed involvement in immune response, stress response, complement and coagulation cascades, ferroptosis, and PPAR signaling pathways. PPI analysis highlighted key proteins like APOA1, C3, and transferrin (TF). ELISA assay confirmed the differential expression of proteins such as HBA1, SERPINA1, COL1A1, and ACTB, with significant changes in the IVC groups. The study demonstrates that IVC effectively reduces VEGF levels while increasing CTGF levels, thereby modifying the CTGF/VEGF ratio, and IVC significantly alters the protein profile in the aqueous humor of patients with PDR. Proteomic analysis reveals that these changes are associated with critical biological pathways and protein interactions involved in immune response, stress response, and cellular metabolism.