Analysis of Cytokine Levels Based on Optical Coherence Tomography Findings in Acute and Chronic Central Serous Chorioretinopathy

Abstract

Purpose: To investigate the characteristics of optical coherence tomography (OCT) and aqueous humor cytokine differences between acute and chronic central serous chorioretinopathy (CSC) and to evaluate the relevance of these findings.

Methods: This was a cross-sectional, observational study. Patients with CSC were divided into acute and chronic groups based on the symptom duration and were compared with normal controls. Best-corrected visual acuity (BCVA), central macular thickness (CMT), subfoveal choroidal thickness (CT), hyperreflective foci (HF), and cytokines including vascular endothelial growth factor (VEGF), interleukin (IL)-6, IL-8, IL-10, interferon-inducible protein-10 (IP-10), and monocyte chemoattractant protein-1 (MCP-1) were used as comparison metrics.

Results: A total of 62 patients (62 eyes) with CSC (22 with acute CSC and 40 with chronic CSC) and 35 patients as controls were included in this study. The chronic CSC group had significantly older average ages and worse BCVA than the acute CSC group (both p < 0.05). Both CSC groups showed significant increases in CMT and CT (both p < 0.05). In chronic CSC, the CMT was thinner, with more HF in the neuroretina (p = 0.034). VEGF levels were significantly higher in patients with chronic CSC than in those with acute CSC and controls (p < 0.05). The levels of inflammatory cytokines showed no significant difference between the CSC and control groups. Spearman’s correlation analysis showed that the number of HF was positively correlated with disease duration (r = 0.311, p = 0.014), logMAR BCVA (r = 0.487, P < 0.001) and MCP-1 levels (r = 0.256, p = 0.045).

Conclusions: Chronicity of CSC could lead to upregulation of VEGF. HF was associated with a more severe visual impairment in CSC patients and had relations with the levels of MCP-1.

Keywords:

• Central serous chorioretinopathy
• aqueous humor
• cytokine
• hyperreflective foci
• spectral-domain optical coherence tomography

Introduction

Central serous chorioretinopathy (CSC) is characterized by localized retinal detachment at the posterior pole, resulting from the hyperpermeability of the choroidal vessels and dysfunction of the retinal pigment epithelium (RPE).¹ Currently, CSC is proposed to have two distinct subcategories, namely acute and chronic forms, based on the duration of the retinal detachment and characteristic changes on multimodal images.² There is no consensus on the duration threshold for distinguishing acute and chronic forms of CSC in most published papers, generally set within six months.³

Spectral-domain optical coherence tomography (SD-OCT) was necessary to identify and assess CSC owing to the ability to visualize the retinal substructure.⁴ Variable microstructural features, including subretinal fluid (SRF), pigment epithelium detachment (PED), and RPE changes have been well studied in the acute and chronic forms of CSC.⁵ Hyperreflective foci (HF) have also been detected in the detached neuroretina and subretinal space in eyes with CSC. The number of these foci tends to increase with disease progression and is related to worse visual acuity.^6,7 However, the origin and element of HF remain controversial.

In addition to morphological changes, some studies have targeted the discrepancy in cytokines expression between acute and chronic CSC.^8–11 The vascular endothelial growth factor (VEGF) family and inflammatory cytokines, such as interleukin-6 (IL-6), IL-8, interferon-inducible protein-10 (IP-10), and monocyte chemoattractant protein-1 (MCP-1) are discussed in the molecular pathogenesis of CSC. However, it remains controversial whether the inflammatory or angiogenic cytokines are upregulated from the acute to the chronic form of CSC. Additionally, the comparison of cytokines between the different types of CSC and normal controls has not been fully elucidated.

A correlation between HF and aqueous humor inflammatory cytokines has been observed in macular edema.^12,13 However, little is known about the correlation in patients with acute and chronic CSC. In this study, we evaluated the levels of VEGF and inflammatory cytokines in the aqueous humor of patients with acute CSC, chronic CSC, and normal controls. In addition, we analyzed the association between cytokines and morphological changes in OCT to better understand the progression of CSC.

Materials and methods

Patients diagnosed with CSC between May 2019 and May 2021 in the Zhejiang Provincial People’s Hospital and affiliated eye hospital of Wenzhou Medical University were included in this cross-sectional observational study, in accordance with the principles of the Declaration of Helsinki. Patients undergoing cataract surgeries were recruited as controls for comparison. This study was approved by the institutional review board of the Eye Hospital of Wenzhou Medical University. Informed signed consent was obtained from participants.

Patients

All subjects underwent comprehensive ophthalmic evaluation with a slit lamp, intraocular pressure examination, best-corrected visual acuity (BCVA) testing, SD-OCT, optical coherence tomography angiography (OCTA), fundus fluorescein angiography (FFA) and indocyanine green angiography (ICGA). The diagnosis of CSC was based on clinical features and multimodal images.^1–3 Acute CSC (Figure 1) was characterized by dome-shaped serous retinal detachment (SRD) with small focal PED and leakage through the RPE alterations on FFA, with a duration of less than 6 months. Conversely, chronic CSC (Figure 2) was diagnosed based on the diffuse lesions of the RPE with multiple hyperfluorescence leakage points on FFA and symptoms lasting more than 6 months. Patients who were diagnosed with CSC and accepted intravitreal anti-VEGF injection for the first time were included. Patients were excluded if they had macular neovascularization (MNV), age-related macular degeneration, polypoidal choroidal vasculopathy, glaucoma, high myopia, or serious systemic disease. Patients with a history of Diamox, psycho-regulatory and corticosteroid medications use, photodynamic therapy, laser treatment, anti-VEGF therapy, or excessive consumption of caffeine were also excluded from this study.

Figure 1. Multimodal images from the left eye of a 38-year-old male patient with acute CSC. (A) FFA showed focal hyperfluorescence leakage points at macular (white arrow). (B) ICGA showed dilated choroidal vessels at the leakage area (white circle). (C) B-Scan OCT showed serous retinal detachment at macular with slight hyperreflective foci in the outer neuroretina (white arrow).

Figure 2. Multimodal images from the right eye of a 53-year-old male patient with chronic CSC. (A) FFA showed diffuse hyperfluorescence leakage points. (B) ICGA showed dilated choroidal vessels at the leakage area. (C) B-Scan OCT showed serous retinal detachment at macular with moderate hyperreflective foci.

The control group included patients who had undergone cataract surgery. The exclusion criterion for controls was a history of severe oculopathy and systemic disease, such as glaucoma, high myopia, unstable cardiovascular diseases, diabetes, and autoimmune disease.

Optical coherence tomography measurements

The measurement parameters of SD-OCT included central macular thickness (CMT), subfoveal choroidal thickness (CT), and HF. All measurements were manually conducted at the macular central fovea using horizontal SD-OCT B-scan images. CMT was measured from the internal limiting membrane to Bruch’s membrane. CT was measured from Bruch’s membrane to the inner edge of the sclera. The average values of the three times measurements were available for the analysis.

HF was defined as isolated hyperreflective dots with clear demarcation in the neuroretina on OCT, which was 20–50 µm in diameter (Figures 1 and 2).¹⁴ HF were manually counted at a length of 3000 µm centered on the fovea by two experienced masked ophthalmologists. In addition, the patients were classified into two grades based on the number of HF. A slight grade represented a small number (0–5 HF). A moderate grade represented approximately six or more HF.

Collection and analysis of aqueous humor cytokines

Aqueous humor samples were collected before intravitreal injection of anti-VEGF drugs or before cataract surgery. After topical anesthesia, approximately 0.05 ml of aqueous humor was withdrawn using a 30-gauge insulin syringe from the anterior chamber.¹⁵ The sample was immediately kept in a sterile Eppendorf tube and frozen at −84 °C until assayed. Cytokines including VEGF, IL-6, IL- 8, IL-10, IP-10, and MCP-1 were analyzed using Luminex200 (BIO-RAD, Hercules, CA, USA) with the Luminex Performance Assay kit (LXSAHM-06, RnD) and calculated using the standard curve. All the steps were conducted at room temperature under dark illumination to avoid the light-induced degradation of the samples.

Statistical analysis

Statistical analyses were performed using SPSS 20.0 (IBM SPSS Inc., Chicago, IL, USA). A P value less than 0.05 was considered statistically significant. Quantitative data are expressed as means and standard deviations. Categorical variables were compared using the chi-square test. Continuous variables and nonparametric variables between the acute CSC and chronic CSC groups were compared using independent t-tests and Mann–Whitney U-tests. One-way analysis of variance (ANOVA) was used to compare the continuous variables, and the Kruskal–Wallis test was used to compare cytokine levels among the three groups. Correlations between variables were assessed using Spearman’s correlation analysis.

Results

Twenty-two patients (22 eyes) with acute CSC, 40 patients (40 eyes) with chronic CSC, and 35 controls (35 eyes) were included in this study. The demographic data for each group are presented in Table 1. There were significant differences in age (p < 0.001), symptom duration (p < 0.001), and BCVA (p = 0.011) between the acute and chronic CSC groups. There was no significant difference in sex between the CSC and control groups (χ²=0.252, p = 0.882).

Table 1. Demographic data and clinical characteristics of patients with CSC and controls.

	Acute CSC	Chronic CSC	Controls	p value
Number	22	40	35	–
Age, years	43.3 ± 7.1	50.2 ± 7.7	68.6 ± 5.8	<0.001
Sex				0.882
Male	17	29	25
Female	5	11	10
Symptom duration, months	3.82 ± 1.30 (1 ∼ 5)	10.03 ± 6.42 (6 ∼ 35)	–	<0.001
BCVA, logMAR	0.15 ± 0.14	0.30 ± 0.25	–	0.011

CSC: central serous chorioretinopathy; BCVA: best-corrected visual acuity; logMAR: logarithm of minimum angle of resolution.

More hyperreflective foci in patients with chronic CSC

OCT parameters are listed in Table 2. CMT and CT in acute and chronic CSC eyes were significantly higher than those in controls (p < 0.001). Further analysis revealed that CMT in the acute CSC group was higher than that in the chronic CSC group (p = 0.033). However, there was no significant difference in subfoveal CT between the acute and chronic CSC groups (p = 0.917).

Table 2. OCT characteristics of patients with CSC and controls.

Characteristics	Acute CSC	Chronic CSC	Controls	p Value
CMT, µm	391.41 ± 165.47	324.38 ± 133.29	216.37 ± 19.39	<0.001
CT, µm	458.00 ± 92.36	455.88 ± 96.21	230.23 ± 21.93	<0.001
Total number of HF	3.91 ± 2.22	5.58 ± 3.80	–	0.034
Number range of HF				0.008
0 ∼ 5, n%	17 (77.3%)	17 (42.5%)	–
6-13, n%	5(22.7%)	23 (57.5%)	–

OCT: optical coherence tomography; CSC: central serous chorioretinopathy; CMT: central macular thickness; CT: choroidal thickness; HF: hyperreflective foci.

The total number of HF in chronic CSC eyes was 5.58 ± 3.80 (range, 0–13), which was more than that in the acute CSC eyes, 3.91 ± 2.22 (range, 0–8) (p = 0.034). According to the number of HF, patients were then classified into two grades. In the acute CSC group, 17 (77.3%) eyes were graded as slight (range, 0–5) and five (22.7%) as moderate (range, 6–13). In the chronic CSC group, 17 (42.5%) eyes were graded as slight and 23 (57.5%) as moderate. There was a statistically significant difference in the grade of HF number between the acute and chronic CSC groups (χ²=6.93, p = 0.008).

Upregulated levels of VEGF in patients with chronic CSC

As shown in Table 3, six aqueous humor cytokines (VEGF, IL-6, IL-8, IL-10, IP-10, and MCP-1) were analyzed in controls and patients with CSC. We noted significant differences in the concentrations of VEGF (p = 0.004) among the three groups. Aqueous humor levels of VEGF were significantly higher in the chronic CSC group than in the control and acute CSC groups (p < 0.05, Figure 3). However, VEGF levels did not differ significantly between patients with acute CSC and controls (p = 0.749). The other cytokines showed no significant differences among the three groups.

Figure 3. Box-and-whisker plot of vascular endothelial growth factor levels in the aqueous humor in the acute CSC, chronic CSC, and control groups.

Table 3. Cytokines levels of patients with CSC and controls.

Cytokines	Acute CSC	Chronic CSC	Controls	p Value
VEGF	28.64 ± 15.72	37.36 ± 10.21	29.86 ± 12.15	0.004
IL-6	4.59 ± 1.78	6.30 ± 5.89	6.37 ± 8.01	0.212
IL-8	6.99 ± 3.37	9.70 ± 6.34	9.01 ± 6.11	0.150
IL-10	0.65 ± 0.06	0.66 ± 0.09	0.70 ± 0.25	0.750
IP-10	124.97 ± 68.52	212.56 ± 169.13	176.93 ± 105.36	0.143
MCP-1	396.73 ± 111.67	463.20 ± 146.48	435.56 ± 195.16	0.278

CSC: central serous chorioretinopathy; VEGF: vascular endothelial growth factor; IL: interleukin; IP: interferon-inducible protein; MCP: monocyte chemoattractant protein.

Upregulated levels of IL-8 in CSC patients with moderate HF

Cytokine levels in patients with CSC classified by the number of HF are compared in Table 4. IL-8 levels were significantly higher in patients with moderate HF than in those with slight HF (p = 0.047). However, the higher levels of VEGF, IL-6, IP-10, and MCP-1 and lower levels of IL-10 in patients with moderate HF were not statistically different from those in patients with slight HF.

Table 4. Cytokines levels of CSC patients with slight or moderate number of HF.

	HF (slight)	HF (moderate)	p Value
Number	34	28
VEGF	33.02 ± 13.85	35.78 ± 12.00	0.372
IL-6	4.50 ± 1.56	7.14 ± 6.87	0.062
IL-8	7.44 ± 3.40	10.36 ± 7.22	0.047
IL-10	0.67 ± 0.08	0.65 ± 0.09	0.234
IP-10	179.11 ± 160.85	182.07 ± 129.48	0.552
MCP-1	407.99 ± 101.31	478.01 ± 166.38	0.079

CSC: central serous chorioretinopathy; HF: hyperreflective foci; VEGF: vascular endothelial growth factor; IL: interleukin; IP: interferon-c-inducible protein; MCP: monocyte chemoattractant protein.

OCT characteristics associated with disease progression

We evaluated correlations between age, disease duration, BCVA, and OCT characteristics (Table 5). Notably, the number of HF was positively correlated with disease duration (r = 0.311, p = 0.014) and logMAR BCVA (r = 0.487, P < 0.001) in patients with acute and chronic CSC. Subfoveal CT was correlated with age (r=–0.281, p = 0.027). Other OCT characteristics were not correlated with age, disease duration, or BCVA.

Table 5. Correlation between the OCT characteristics and age, duration of the disease, or logMAR BCVA in the patients with CSC.

	Age	Duration of the disease	logMAR BCVA
HF	r = 0.196	r = 0.311	r = 0.487
	p = 0.127	p = 0.014	p < 0.001
CMT	r=-0.135	r=-0.194	r=-0.143
	p = 0.297	p = 0.131	p = 0.268
CT	r=-0.281	r = 0.111	r=-0.013
	p = 0.027	p = 0.389	p = 0.427

OCT: optical coherence tomography; BCVA: best-corrected visual acuity; CSC: central serous chorioretinopathy; HF: hyperreflective foci; CMT: central macular thickness; CT: choroidal thickness.

Cytokines associated with disease progression

For the entire CSC cohort, there were strong positive correlations between IL-6 and IL-8 concentrations (r = 0.686, p < 0.001), IL-6 and IP-10 concentrations (r = 0.671, p < 0.001), IL-6, and MCP-1 concentrations (r = 0.693, p < 0.001), IL-8 and IP-10 concentrations (r = 0.719, P < 0.001), IL-8 and MCP-1 concentrations (r = 0.605, p < 0.001), and IP-10 and MCP-1 concentrations (r = 0.540, p < 0.001).

Disease duration, logMAR BCVA, and the number of HF were correlated with multiple cytokines in patients with CSC (Table 6). Disease duration was positively correlated with IL-6 (r = 0.260, p = 0.041), IL-8 (r = 0.306, p = 0.018) and IP-10 concentrations (r = 0.337, p = 0.008). The logMAR BCVA was positively correlated with MCP-1 concentrations (r = 0.269, p = 0.034). The number of HF was positively correlated with MCP-1 concentrations (r = 0.256, p = 0.045). Moreover, cytokines levels were not correlated with age and other OCT characteristics.

Table 6. Correlation between duration of the disease, logMAR BCVA, HF and six cytokine concentrations in the patients with CSC.

	Duration of the disease	logMAR BCVA	HF
VEGF	r = 0.155	r = 0.216	r = 0.023
	p = 0.228	p = 0.091	p = 0.858
IL-6	r = 0.260	r = 0.109	r = 0.203
	p = 0.041	p = 0.400	p = 0.114
IL-8	r = 0.306	r = 0.063	r = 0.166
	p = 0.018	p = 0.632	p = 0.205
IL-10	r=−0.187	r=−0.230	r=−0.193
	p = 0.229	p = 0.138	p = 0.214
IP-10	r = 0.337	r=−0.023	r = 0.086
	p = 0.008	p = 0.862	p = 0.514
MCP-1	r = 0.211	r = 0.269	r = 0.256
	p = 0.100	p = 0.034	p = 0.045

BCVA, best-corrected visual acuity; HF, hyperreflective foci; CSC, central serous chorioretinopathy; VEGF, vascular endothelial growth factor; IL, interleukin; IP, interferon-c-inducible protein; MCP, monocyte chemoattractant protein. P values calculated by Spearman’s correlation analysis.

Discussion

This study aimed to describe the differences in retinal microstructural and cytokines levels between acute and chronic CSC patients. First, we noted lower central retinal thickness and higher HF with worse visual acuity in chronic CSC patients. In addition, VEGF levels were significantly increased in chronic CSC patients compared with acute CSC patients and controls, whereas inflammatory cytokines, including IL-6, IL-8, IP-10, and MCP-1, were elevated, but the difference was not statistically significant. Moreover, an increased number of HF was associated with some upregulated inflammatory cytokines in CSC.

In CSC, adjacent hyperperfusion and focal choriocapillary ischemia are currently considered the primary underlying pathogenic mechanisms.^16,17 The pathological processes leading to choroidal abnormalities may include autonomic dysregulation, inflammation, abnormal angiogenesis, and the complement system.^2,18 Recently, several studies have reported cytokine levels in patients with CSC. Lim et al.¹⁰ found that VEGF was correlated with symptom duration in a small sample size study. However, they did not detect differences in VEGF levels between patients with chronic CSC and controls. Terao et al.⁸ revealed that inflammatory cytokines were significantly increased along with CSC progression. Angiogenic cytokines such as VEGF almost reached a significant increase in chronic CSC. In contrast, Jung et al.⁹ found no significant difference in cytokine levels between acute and chronic CSC patients. The conflicting findings of aqueous humor cytokines in previous reports indicated that the role of cytokines in CSC remains to be further studied.

On this basis, we analyzed the discrepancy of angiogenic and inflammatory cytokines among controls, acute and chronic CSC patients. In this study, VEGF levels were significantly higher in the chronic CSC group than in the acute CSC and control groups. VEGF, angiogenic cytokines, can theoretically modify choroidal hyperpermeability.¹⁹ Therefore, anti-VEGF injection has been proposed as a treatment for CSC, although it is still off-label.² Further analysis of our results indicated that the distribution of VEGF in chronic CSC showed great differences from the lowest to the highest. Hence, anti-VEGF treatment for CSC should be selectively conducted and considered with some biomarkers, such as flat irregular PED.²⁰

Classic inflammation is generally not involved in the onset of CSC which would be induced by corticosteroids. There was no significant difference in inflammatory cytokine levels between the CSC and control groups in this study. However, IL-6, IL-8, IP-10, and MCP-1 levels tended to be upregulated in chronic CSC patients compared to those in acute CSC patients. These results indicate that persistent SRF in chronic CSC may have an impact on immune regulation of the retina and choroid, which gradually leads to the alteration of these inflammatory cytokines.

In CSC, HF is mainly distributed in the outer neuroretina and more frequently detected in the chronic form and deemed as a sign of poor visual prognosis.^6,21,22 The presence of HF in SD-OCT has been proposed as a biomarker of the inflammatory response in several ocular fundus diseases.^23,24 Some have also considered it to be correlated with the migration of RPE cells, lipoprotein exudation from the choriocapillaris, or degeneration of outer segments.¹⁴ In this study, we found the level of IL-8 was elevated in the patients with a moderate number of HF compared to those with a slight number of HF. IL-8 is produced by retinal endothelial and glial cells and is involved in ischemic angiogenesis.¹⁰ It plays a role in chemotaxis and activation of inflammatory cells such as neutrophils and T lymphocytes.⁹ In addition, the number of HF showed a positive correlation with MCP-1, which is related to leukostasis due to hypoxic retinas.²⁵ The activation and aggregation of inflammatory cells induced by MCP-1 lead to endothelial damage of capillaries, which may further affect the permeability of choroidal vessels.²⁶ This may suggest that inflammatory cytokines participate in the formation of HF in CSC.

This study has several limitations. First, this was a cross-sectional, observational study. Further research with follow-up data is needed to evaluate the prognostic value of HF and cytokines. Second, the difference in the average age among the three groups was caused by the clinical characteristics of acute and chronic CSC. To avoid the influence of age, we assessed the relationship between cytokine levels and age and found no significant correlations. Moreover, cytokines were obtained from the aqueous humor, not vitreous, to represent the intraocular levels.

In conclusion, we assessed the discrepancy between acute and chronic CSC based on cytokines and OCT characteristics. Our results indicated that the higher expression of VEGF in chronic CSC might be evidence for individualized treatment. Additionally, HF as a biomarker was increased in chronic CSC and related to the upregulation of MCP-1. The pathogenesis and clinical value of cytokines in CSC still need further investigation.

Author contributions

C Zhang, J Mao and L Shen: conceived and designed the experiments; J Mao: performed the treatment; S Zhang, Z Zheng, Z Zhang, and Z Xiang: collected the data; C Zhang: analyzed the data and wrote the paper; Y Chen and L Shen: approved the final version of the manuscript.

Ethical statement

All procedures performed in this study involving human participants were in accordance with the ethical standards of the institutional and the national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. This study was approved by the Institutional Review Board of the Affiliated Eye Hospital of Wenzhou Medical University (No.121-K-107-01).

Disclosure statement

No conflict of interest was reported by the author(s).

Data availability statement

The data used to support the findings of this study were supplied by Lijun Shen under license and so cannot be made freely available. Requests for access to these data should be made to Lijun Shen ([email protected]).

Additional information

Funding

This work was supported by the natural Science Foundation of Zhejiang Province (LTGY23H120005) and the Medical Health Science and Technology Project of Zhejiang Provincal Health Commission (23KY490).