Advances of Intraocular Fluid for the Application and Treatment of Uveitis

Abstract

Uveitis is a group of inflammatory eye diseases, which may affect the iris, ciliary body, choroid and other structures, and in severe cases may lead to vision loss. Due to the variety of causes and complex pathogenesis of uveitis, the empirical use of drugs in the absence of clear etiology is likely to be counterproductive and delay the patient’s condition. In recent years, the analysis of intraocular fluid (including aqueous humor and vitreous body) has made significant progress in the diagnosis, etiology research and treatment of uveitis. Therefore, this review summarized the latest research progress of intraocular fluid in uveitis, especially its application in etiological diagnosis, biomarkers and treatment strategies, hoping to provide new insights for the clinical diagnosis and treatment of uveitis.

Introduction

Uveitis is a common eye disease that occurs mostly in young adults. There are many kinds of uveitis, and the causes are quite complex.^1,2 Globally, uveitis accounts for about 5–10% of cases of visual impairment and a quarter of cases leading to legal blindness in developing countries.^3–5 The pathogenesis and mechanism of uveitis are quite complex, involving trauma, infection, autoimmunity and other factors.⁶ According to the etiology, the International Uveitis Study Group divides uveitis into four categories: infectious uveitis, non-infectious uveitis, masquerading syndrome and other idiopathic uveitis.⁷ Since the treatment of infectious and non-infectious uveitis is very different, the empirical use of glucocorticoids and immunosuppressants is likely to be counterproductive and delay the patient’s condition when the etiology is not clear.^8,9 Therefore, for some patients with atypical clinical manifestations, unclear diagnosis, highly suspected infectious causes or malignant lesions, conventional clinical diagnostic methods are limited.

The biological rationale for intraocular fluid analysis in uveitis is closely related to the disruption of blood-ocular barriers during intraocular inflammation. Under physiological conditions, the blood–aqueous barrier, mainly formed by tight junctions in the non-pigmented ciliary epithelium and iris vascular endothelium, restricts the entry of plasma proteins and inflammatory cells into the aqueous humor. Similarly, the blood–retinal barrier, consisting of retinal vascular endothelial cells and retinal pigment epithelial cells, maintains the immune-privileged microenvironment of the posterior segment.^10,11 During uveitis, inflammatory mediators such as TNF-α, IL-1β, IL-6, IL-8, IL-17 and CCL2 can alter endothelial tight junctions, increase vascular permeability, and promote leukocyte migration, resulting in the leakage of proteins, cytokines, chemokines, pathogen-derived nucleic acids, and tumor- associated molecular signals into the aqueous humor or vitreous body.^12,13 Therefore, changes in intraocular fluid composition are not only consequences of blood–ocular barrier breakdown, but also provide a molecular window for identifying inflammatory activity, infectious causes, autoimmune responses, and masquerade syndromes in uveitis.

Intraocular fluid is a general term for the fluid in the eye, including aqueous humor and vitreous body, which plays an indispensable role in maintaining the ocular microenvironment.¹⁴ They provide essential nutrients to intraocular structures, regulate intraocular pressure, and facilitate the clearance of metabolic byproducts.^15,16 Numerous studies have demonstrated that a variety of biomolecules in intraocular fluid are involved in the pathogenesis of uveitis and participate in multiple biological processes. Obtaining intraocular fluid (aqueous humor or vitreous humor) and performing laboratory tests are new auxiliary detection methods developed recently.¹⁷ For uveitis in which the etiology and type cannot be determined, intraocular fluid testing plays an important role in determining the cause and guiding treatment.

Because the anatomical location of inflammation determines both the disruption of the blood–ocular barriers and the composition of intraocular fluids, understanding the anatomy and physiological functions of the uvea is essential for interpreting intraocular fluid findings. Alterations in aqueous humor and vitreous composition are closely associated with the site and severity of ocular inflammation, thereby providing important clues for disease classification, etiological diagnosis, and therapeutic decision-making. Therefore, before discussing the clinical applications of intraocular fluid analysis, it is necessary to first review the anatomical characteristics of the uvea and the biological properties of intraocular fluids.

Hence, this review provides a comprehensive overview of the advances of intraocular fluid for the application and treatment of uveitis. Firstly, we introduced the anatomy and function of uvea, as well as the composition of intraocular fluids. Then, we concluded the etiology, classification, clinical manifestations and treatment of uveitis, highlighting the latest advances of intraocular fluid in uveitis. Finally, we emphasize the challenges and future directions of treating uveitis, aiming to provide new ideas for the clinical diagnosis and treatment of uveitis.

The Anatomy and Function of Uvea

The uvea, also known as the pigment membrane, is the second layer of the eye wall. It can be divided into three parts: iris, ciliary body and choroid. The anterior uvea includes the iris and ciliary body, and the posterior uvea is the choroid.^18,19 These three tissues are closely connected anatomically, and interact with each other during lesions. The choroid, located between the sclera and retina, is the largest part of the uveal area. The normal choroid is a soft, smooth and elastic brown film with a thickness of 0.1 ~0.2 mm, which is rich in blood vessels and pigments.^20,21 The uvea has the following four functions: (1) providing nutrition for the eye. (2) producing aqueous humor and regulate intraocular pressure. (3) blocking the action of light and (4) excreting waste and toxic substances. Because the blood flow of the uveal membrane is large and slow, the pathogenic antigen substances in the blood will precipitate here.^22,23 Therefore, it is closely related to systemic diseases and it is one of the sites that are easy to be inflamed.

The Composition of Intraocular Fluids

Intraocular fluid is a general term for the fluid in the eye, and changes in its composition can reflect the physiological and pathological conditions in the eye. Aqueous humor and vitreous are commonly used test samples in uveitis.

Aqueous Humor

Aqueous humor is a colorless transparent fluid filled in the anterior and posterior chambers, which is produced by the ciliary body, and excreted into the blood through the posterior chamber-pupil-anterior chamber angle.^24,25 The total amount of aqueous humor is 0.15–0.3 mL. The main component was water, accounting for 98.75% of the total. Aqueous humor is derived from plasma, so the chemical composition of aqueous humor and plasma is basically similar, but not identical. The main difference between the two is the total solids content, which is 1.08g per 100mL of aqueous humor and 9.5g per 100mL of plasma. In addition, the amount of protein in the plasma is high, while the amount of protein in the aqueous humor is very little, which is one of the greatest characteristics of aqueous humor composition.^26–29 Most importantly, aqueous humor not only provides nutrition for the iris, cornea and lens, but also maintains intraocular pressure, as well as having a certain refractive function, which together with the cornea, lens and vitreous body constitute the refractive system of the eyeball.^30,31

Vitreous Body

Vitreous body, as a kind of eyeball content, is colorless transparent colloid body and its main component is water. The vitreous body is filled between the lens and the retina and is covered by the vitreous membrane, which occupies about 4/5 of the inner cavity of the eye.^32,33 The vitreous body, aqueous humor of the lens and the cornea constitute the refractive stroma of the eye. Furthermore, vitreous body also supports the retina and the eyeball wall and maintains the shape of the eyeball. In addition, with the increase of age, or due to high myopia and other reasons, the semi-solid gel-like vitreous body will gradually become liquid, which is called vitreous liquefaction.^34–36

The Classification, Clinical Manifestations and Treatment of Uveitis

Uveitis is a collective term describing a group of diseases characterized by intraocular inflammation consisting of the iris, ciliary body, and choroid.³⁷ Based on the anatomical site of the eye affected by the disease, uveitis is classified as anterior (inflammation of the anterior chamber), intermediate (inflammation of the vitreous body), or posterior (inflammation of the retina or choroid), with more widespread inflammation across the anatomical area referred to as panuveitis. This protocol was endorsed by the Standardization of Nomenclature in Uveitis Working Group and is now the standard required for publication of uveitis studies in the peer-reviewed literature.^7,38,39 According to the character of inflammation, uveitis can be divided into granulomatous and non-granulomatous uveitis. The granulomatous uveitis has a slow onset, mild irritation symptoms and ciliary hyperemia, large corneal precipitates, mutonic fat, and visible iris nodules. Besides, the course of disease is longer, the recurrence rate is higher, and the prognosis is poor. The non-granulomatous uveitis has an acute onset, obvious irritation symptoms and ciliary hyperemia, small keratic precipitates, short course of disease, and good prognosis.^40,41 Pathologically, the granulomatous uveitis was mainly infiltrated by macrophages and epithelial cells in addition to lymphocytes. The non-granulomatous uveitis was mainly infiltrated by lymphocytes and plasma cells, and mainly invaded the anterior uvea. Based on the etiology, they can be divided into extrinsic, secondary and intrinsic uveitis.^42–44 What’s more, Uveitis is further classified according to onset (sudden or insipid), duration (limited or persistent, with symptoms lasting 3 months or more, respectively), and clinical course (acute, chronic, or recurrent). Acute uveitis represents a brief burst of inflammation, whereas chronic uveitis is characterized by persistent inflammation that recurs within less than 3 months after discontinuation of treatment. Recurrent uveitis refers to disease that recurs after a period of inactivity lasting 3 months or more after discontinuation of treatment.^45–47

When it comes to the clinical assessment of uveitis, a detailed medical history is essential, including ocular, systemic, family, and social history. This will provide substantial information to examine in detail and guide subsequent studies.^1,48

Although uveitis encompasses multiple etiologies and disease phenotypes, its treatment relies on a number of key factors: correct anatomical classification and etiological diagnosis, identification of complications, assessment of disease activity, and the presence of any systemic disease association. The current treatment of uveitis includes: (1) control and prevention of ocular inflammation; (2) treatment of vision-threatening complications (such as cataract, cystoid macular edema, glaucoma); (3) protect and restore vision; (4) improve quality of life.^49,50

Recent Advances of Intraocular Fluid for the Application and Treatment of Uveitis

In recent years, significant research progress has been made in intraocular fluid in the diagnosis and treatment of uveitis, mainly focusing on the innovation of detection technology, the discovery of biomarkers, and the optimization of individualized treatment strategies. The following is the latest research progress of intraocular fluid in the field of uveitis. The major clinical applications of intraocular fluid analysis in uveitis are summarized in Table 1.

Table 1.

Clinical Applications of Intraocular Fluid Analysis in Uveitis

Application	Sample and Clinical Scenario	Main Tests or Biomarkers	Clinical Value and References
Pathogen identification in infectious uveitis	Aqueous humor or vitreous; atypical, recurrent, severe, immunocompromised, or refractory suspected infection	PCR, multiplex PCR, NGS; HSV, VZV, CMV, Toxoplasma gondii, Mycobacterium tuberculosis	Identifies infectious causes and guides antimicrobial or antiviral therapy51–63
Immune and inflammatory profiling in non-infectious uveitis	Mainly aqueous humor; vitreous for posterior-segment disease or unclear inflammatory status	IL-6, IL-10, IL-17, IL-23, TNF-α, CXCL10, CCL2, anti-retinal antibodies, miRNAs	Distinguishes infectious from non-infectious inflammation, reflects disease activity, and supports immunomodulatory therapy64–71
Differential diagnosis of masquerade syndromes	Mainly vitreous; aqueous humor may provide supportive information in suspected VRL or refractory uveitis	IL-10/IL-6 ratio, MYD88 L265P mutation, cytology, flow cytometry, gene rearrangement analysis	Differentiates inflammatory uveitis from vitreoretinal lymphoma or other neoplastic masquerade syndromes56,72,73
Treatment response monitoring	Aqueous humor or vitreous according to disease location and treatment target	Viral load, inflammatory cytokines, TNF-α, IL-6, VEGF, NLRP3 inflammasome-related markers	Evaluates therapeutic response, monitors pathogen clearance or inflammatory activity, and helps optimize treatment74–80
High-throughput and AI-assisted analysis	Aqueous humor or vitreous combined with clinical and imaging data in complex or unclear uveitis	Multiplex PCR, metagenomic NGS, proteomics, metabolomics, OCT features, cytokine panels, machine-learning models	Enables biomarker discovery, improves etiological classification, and may support prognosis prediction and precision medicine17,57–59,81–83

Abbreviations: PCR, polymerase chain reaction; NGS, next-generation sequencing; HSV, herpes simplex virus; VZV, varicella-zoster virus; CMV, cytomegalovirus; VRL, vitreoretinal lymphoma; IL, interleukin; TNF-α, tumor necrosis factor-α; VEGF, vascular endothelial growth factor; OCT, optical coherence tomography; AI, artificial intelligence.

Application of Intraocular Fluid in the Diagnosis of Uveitis

Intraocular fluid analysis has become an important method for the diagnosis of uveitis, especially in cases with unknown etiology. Pathogens such as viruses, bacteria, fungi, parasites, or autoimmune responses can be identified by molecular biological and immunological testing of aqueous humor or vitreous samples.^51,52 Clinically, aqueous humor sampling is generally preferred as a less invasive initial test for anterior-segment inflammation or suspected viral/toxoplasmic infection, whereas vitreous sampling is more suitable for posterior-segment-dominant disease, dense vitritis, negative aqueous results, or suspected endophthalmitis and masquerade syndromes requiring cytology, culture, flow cytometry, or molecular testing.^53–56 At present, intraocular fluid detection can be divided into two categories. One is the detection of pathogens to determine whether the uveitis is caused by viruses, bacteria or fungi. Polymerase chain reaction (PCR) and next generation sequencing (NGS) technologies have been widely used to detect pathogens in intraocular fluids.^57–59 For example, the detection of pathogens such as herpes viruses (HSV, VZV, CMV), Toxoplasma gondii, and Mycobacterium tuberculosis has significantly improved the accuracy of diagnosis.^60,61 Detection of Mycobacterium tuberculosis DNA or specific cytokines (such as IFN-γ) in intraocular fluid can help to confirm the diagnosis of tuberculous uveitis.^62,63 The other category is the detection of immune indicators, which can help distinguish between infectious and non-infectious uveitis by detecting cytokines (such as IL-6, IL-17, TNF-α) and autoantibodies (such as anti-retinal antibodies) in the intraocular fluid.^64–66 Beyond distinguishing infectious from non-infectious uveitis, intraocular fluid analysis also plays an indispensable role in differentiating inflammatory diseases from neoplastic conditions. A clinically important subset of refractory uveitis represents uveitic masquerade syndromes, most notably primary vitreoretinal (intraocular) B-cell lymphoma (PVRL/VRL), whose diagnosis is frequently delayed when it relies solely on vitreous cytology with limited cellularity. Intraocular fluid cytokine profiling shows that PVRL-derived malignant B cells secrete excess IL-10, whereas inflammatory uveitis is dominated by IL-6; an IL-10/IL-6 ratio >1.0 in vitreous fluid (with optimal cutoffs further refined for vitreous vs aqueous sampling) serves as a well-validated ancillary signal favoring lymphoproliferative origin over uveitis.⁷² Beyond cytokines, targeted detection of the MYD88 L265P somatic mutation from vitreous aspirates by allele-specific PCR or NGS substantially improves diagnostic yield even when cytology is equivocal or cellularity is poor, providing a molecular anchor that complements cytokine screening in routine practice.⁷³

Application of Intraocular Fluid in the Biomarkers of Uveitis

Biomarkers in intraocular fluid are of great value in the etiological diagnosis, disease activity assessment and treatment response monitoring of uveitis. It has been found that the levels of certain cytokines (such as IL-6, IL-10, TNF-α) and chemokines (such as CXCL10, CCL2) in intraocular fluid are associated with the activity and etiology of uveitis.^67–69 For example, elevated IL-6 levels are commonly seen in infectious uveitis, whereas IL-17 and IL-23 are significantly elevated in autoimmune uveitis.⁷⁰ In addition, the expression profile of miRNA in uveitis has become a research hotspot, which provides new clues for the diagnosis and classification of uveitis. For example, certain mirnas (such as miR-155 and miR-146a) are abnormally expressed in the intraocular fluid of patients with uveitis, which may serve as potential diagnostic and prognostic markers.⁷¹

Application of Intraocular Fluid in the Treatment Strategy

Analysis of intraocular fluid is not only helpful for diagnosis, but also provides a basis for individualized treatment. The first is targeted therapy, which can guide the selection of targeted therapy drugs by detecting cytokines and inflammatory mediators in the intraocular fluid. For example, anti-TNF-α agents such as adalimumab have shown promising efficacy in autoimmune uveitis.^74,75 It has been found that the activation of NLRP3 inflammasome in intraocular fluid is related to the pathogenesis of uveitis, which may become a new therapeutic target.^76,77 In addition, intraocular fluid analysis can also evaluate the effect of topical treatments, such as intraocular glucocorticoids or anti-VEGF agents, and optimize treatment regimens. Second, gene editing of specific genes, such as IL-10 or IFN-γ, is being explored for the treatment of uveitis.^78,79

More importantly, intraocular fluid can also provide treatment response monitoring and individualized treatment. For example, the efficacy of anti-TNF-α drugs, such as adalimumab, is evaluated by monitoring changes in the levels of cytokines (such as TNF-α, IL-6) in the intraocular fluid.⁸⁰ In viral uveitis, the dynamic monitoring of intraocular fluid viral load is helpful to adjust the antiviral treatment plan. All these results reveal the great advantages and potential of intraocular fluids in the treatment of uveitis.

Application of High-Throughput Detection Techniques and Artificial Intelligence Analysis

Multiplex PCR technology can simultaneously detect a variety of pathogens (such as viruses, bacteria, fungi and parasites), which improves the diagnostic efficiency of infectious uveitis.^57,58 Metagenomic sequencing can comprehensively detect potential pathogens by high-throughput sequencing of intraocular fluid samples, especially for cases with unknown etiology.⁵⁹ Proteomics and metabolomics can explore biomarkers related to uveitis by analyzing proteins and metabolites in intraocular fluid. At the same time, imaging combined with molecular detection, such as optical coherence tomography (OCT) and intraocular fluid analysis, can improve the accuracy and comprehensiveness of diagnosis.^81,82 In addition, with the development of science and technology, machine-learning models derived from multicenter studies are increasingly being used to construct prediction models based on intraocular fluid data combined with clinical features to assist the etiological diagnosis and prognosis evaluation of uveitis. Machine-learning frameworks are increasingly applied to multiplex intraocular fluid readouts (cytokine/chemokine panels, and increasingly proteomic or NGS-derived features) to move beyond single static cut-offs and improve discrimination among infectious uveitis, non-infectious autoimmune uveitis, and masquerade syndromes such as vitreoretinal lymphoma.⁸³ Prospective, multicentre validation with standardized sampling and assay protocols, combined with multimodal clinical/OCT data, will be required before such models transition to bedside decision-support tools. In the future, artificial intelligence may facilitate precision medicine by integrating intraocular fluid biomarkers with multimodal imaging and clinical data, enabling individualized diagnosis and treatment recommendations.

Challenges and Future Perspectives

Despite remarkable progress in the use of intraocular fluid analysis in uveitis, several challenges remain.^84–88

The Etiology Is Complex and Diverse

The causes of uveitis may include infections (eg, viruses, bacteria, fungi, parasites), autoimmune diseases, neoplasms, or idiopathic causes. The analysis of intraocular fluid needs to identify the cause, but the detection methods and technical requirements of different causes are different, which increases the complexity of diagnosis. For example, infectious uveitis requires the detection of pathogens (eg, viruses or bacteria by PCR), whereas autoimmune uveitis may require the detection of specific antibodies or cytokines.

Sampling Difficulties

The acquisition of intraocular fluid (such as anterior chamber fluid or vitreous fluid) requires invasive procedures (such as anterior chamber puncture or vitreous puncture) with some risks, such as infection, hemorrhage, or retinal detachment. Sample sizes are often small, particularly in the anterior chamber fluid, limiting the sensitivity and reproducibility of testing.

Limitations of Detection Techniques

At present, the commonly used detection techniques include PCR, cytokine analysis, antibody detection, etc, but these techniques require high requirements for equipment, reagents and operators, and may not cover all potential pathogens or immune markers.

Detection of certain pathogens, such as fungi or parasites, has low sensitivity and is prone to missed diagnosis.

Complexity of Interpretation

The results of intraocular fluid testing may be affected by a variety of factors, such as sample handling, the specificity of the test method, and individual patient differences.

For example, the elevation of some cytokines or antibodies in the intraocular fluid may suggest inflammation, but the specific cause cannot be directly determined, and comprehensive analysis combined with clinical manifestations and other examination results is needed.

Monitoring of Response to Treatment

Intraocular fluid analysis can be used to monitor inflammatory activity or pathogen clearance during treatment, but repeated sampling is unrealistic and limits its application for dynamic monitoring. In some cases, the results of intraocular fluid testing are inconsistent with the clinical response, adding to the difficulty of treatment decisions.

Cost and Accessibility

Intraocular fluid testing involves equipment and technology with high cost, which may not be universally available in some regions or medical institutions. The financial burden on the patient is also an issue to consider.

The Need for Individualized Treatment

The etiology and manifestations of uveitis are highly heterogeneous. Intraocular fluid analysis needs to provide a basis for individualized treatment, but there is still a lack of unified detection and treatment standards.

Based on these challenges, the development of non-invasive detection methods, such as tear or serum markers, is a future research direction. In addition, Future studies should integrate multi-omics data such as genome, transcriptome, proteome and metabolome to fully reveal the molecular mechanisms of uveitis and develop new diagnostic and therapeutic strategies.

Conclusion

In summary, intraocular fluid analysis plays an important role in the diagnosis, etiology study, biomarker discovery, and treatment strategy of uveitis. The research progress of intraocular fluid in uveitis provides a new direction for accurate diagnosis and individualized treatment of the disease. In the future, with the continuous innovation of detection technology and the in-depth exploration of biomarkers, intraocular fluid analysis will play a more important role in the management of uveitis, and further promote the progress of clinical diagnosis and treatment of uveitis and the development of cutting-edge technologies such as non-invasive detection and gene therapy.

Funding Statement

Funded by Tianjin Key Medical Discipline Construction Project (TJYXZDXK-3-004A-2).

Data Sharing Statement

No new datasets were generated or analyzed in this narrative review. Further information is available from the corresponding author, Yibo Gong (yibogong1988@163.com), upon reasonable request.

Author Contributions

All authors made a significant contribution to the work reported, whether in the conception, study design, execution, acquisition of literature, analysis and interpretation, or in all these areas; took part in drafting, revising, or critically reviewing the manuscript; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work. Specifically, WL contributed to the conception of the review, literature screening, and drafting of the manuscript; WH and HZ contributed to literature retrieval, data extraction, and manuscript revision; YG contributed to supervision, conceptualization, and critical revision of the manuscript. All authors read and approved the final version.

Disclosure

The authors declare no competing interests in this work.