Correlation between lymphotoxin-α concentrations and conjunctival goblet cell density in dry eye patients

doi:10.21203/rs.3.rs-2839120/v1

Purpose

To explore the relationship between the tear lymphotoxin-α (LT-α) concentration, conjunctival goblet cell density (GCD) and ophthalmic parameters in dry eye patients and the value of the LT-α concentration and conjunctival GCD in diagnosing dry eye.

Methods

Prospective case‒control study. Seventy-eight dry eye patients and twenty normal subjects were recruited. After completing the China Dry Eye Questionnaire, all subjects were examined in sequence with slit-lamp microscopy, tear LT-α concentration analysis, tear meniscus height measurement, the Schirmer I test and the conjunctival GCD examination using confocal microscopy.

Results

The conjunctival GCD (54.70 (24.38–126.50) cells/mm²) in the dry eye group was lower than that in the control group (125.80 (72.75–180.00) cells/mm²) (P = 0.011). The LT-α concentration(0.17(0.05–1.18) ng/ml) in the dry eye group was lower than that in the control group(0.30(0.13–1.32) ng/ml) without statistical significance (P = 0.366). And the LT-α concentrations were positively correlated with the tear film breakup time (FBUT) ( r = 0.262, p < 0.01). When LT-α < 1.47 ng/ml (81.6% subjects fell in this range), conjunctival GCD was positively correlated with the LT-α concentration (β = 63.92, P = 0.004). The area under the ROC curve (AUC) of LT-α concentration in the diagnosis of dry eye was 0.5657, the cutoff value was 0.11 ng/ml, the sensitivity was 41.03%, and the specificity was 80.00%. The AUC of conjunctival GCD in the diagnosis of dry eye was 0.6993, the cutoff value was 67.90 cells/mm², the sensitivity was 59.15%, and the specificity was 82.35%.

Conclusion

The LT-α concentration is valuable in evaluating conjunctival GCD and tear film stability. The value of evaluating the conjunctival GCD using confocal microscopy in dry eye diagnosis is worthy of attention.

Health sciences/Diseases

Health sciences/Medical research

Dry eye is a chronic ocular surface disease caused by multiple factors. It can be caused by tear film instability or ocular surface microenvironment imbalance, which can be accompanied by ocular surface inflammatory response, tissue damage and neurological abnormalities, resulting in a variety of ocular symptoms and/or visual dysfunction. The ocular surface is a unified system, and its homeostasis is maintained by a variety of factors. If one or more factors change, the ocular surface microenvironment will be unbalanced, resulting in dry eye[1].

The role of inflammation in maintaining the homeostasis of the ocular surface microenvironment should not be understated. Inflammation in dry eye is related to the activation and infiltration of helper T cells (Th cells)[2]. During development, naive Th cells can be stimulated by different signaling factors in the ocular surface microenvironment and differentiate into Th cell subsets such as Th1, Th2, Th17, and regulatory T cells (Treg cells). Th1 and Th17 cells are two important inflammatory Th cell subsets in dry eye. IFN-γ secreted by Th1 cells and IL-17 secreted by Th17 cells can destroy tear film and ocular surface tissue, inducing epithelial cell apoptosis, goblet cell loss and squamous metaplasia [2–5]. IFN-γ is a well-recognized inflammatory factor that can cause goblet cell secretion dysfunction and induce the unfolded protein response and goblet cell death[6–8]. Moreover, Treg cells, also known as suppressor T cells, can suppress the immune response of Th1 and Th17 cells and maintain the balance between immune activation and tolerance. The quantity and activity of Treg cells are key to maintaining ocular immune homeostasis[9]. The functional loss of Treg cells and the imbalance of ocular surface immune homeostasis are important pathological mechanisms in dry eye disease[3, 10–11]. In a mouse model of aging-related dry eye disease, it was observed that dysfunctional Treg cells could not suppress the activity of Th1 and Th17 cells, leading to the production of IFN-γ and IL-17[10–11].

Lymphotoxin-alpha (LT-α) is a member of the tumor necrosis factor (TNF) superfamily that is expressed by a variety of cells, including T cells, B cells, and natural killer cells [12], and binds to TNF receptors. Most receptors of the TNF superfamily can be divided into one of two categories: TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2). LT-α has a stronger affinity for TNFR2 and mainly acts through it under normal physiological conditions in vivo[13]. Studies have shown that LT-α can selectively bind to the highly expressed TNFR2 receptor in Treg cells, thereby activating the proliferation and function of Treg cells and contributing to tissue repair[14]. A previous study showed that LT-α concentrations in tears of patients with chronic ocular graft-versus-host disease (oGVHD) were significantly lower than those in controls, and were negatively correlated with ocular surface disease index scores and ocular surface staining scores and positively correlated with tear film breakup time, suggesting that LT-α may have a protective role in regulating inflammatory responses on the ocular surface[15].

Based on the regulation of immune homeostasis on the number and function of conjunctival goblet cells, as well as the role of LT-α in the ocular surface, we speculated that LT-α expression in tears may be related to the health of the ocular surface and conjunctival goblet cells. Our study intends to explore the relationship between LT-α concentrations, conjunctival goblet cell density (GCD) and ophthalmic parameters in patients with different degrees of dry eye, investigate the value of LT-α concentrations and conjunctival GCD in tears in the diagnosis of dry eye, and provide broader ideas for the diagnosis and classification of dry eye.

2.1. Patients

This study was approved by the Ethics Committee of the Hankou Aier Eye Hospital (HKAIER2021IRB-008-01) and conducted in accordance with the Declaration of Helsinki. Our trial recruited 78 dry eye patients from January 2021 to January 2022 as the dry eye group, including 51 cases in the mild dry eye group, 27 cases in the moderate and severe dry eye group, and 20 normal subjects as the control group. All subjects were randomly selected. One eye from each patient was evaluated in this study. The "Chinese Expert Consensus on Dry Eye: Definition and Classification (2020)" was referred to for the diagnosis and grading standards of dry eye[16]. Informed consent was obtained from all subjects prior to data collection for this study. Exclusion criteria: (1) Patients who were allergic to fluorescein sodium instillation on the ocular surface; (2) Those who received ocular surgery or trauma within the previous 3 months; (3) Those who underwent punctal plug insertion within 1 month; (4) Those who had ocular surface infection within the previous 1 month; (5) Those who have used antibiotics, glucocorticoids, nonsteroidal anti-inflammatory drugs or immunosuppressants within 1 month; (6) Those who have used diquafosol sodium eye drops; (7) Those with thyroid associated ophthalmopathy or Sjogren's syndrome; (8) Those with severe meibomian gland dysfunction (MGD) (including any one of the following): 1) Lid margin abnormality (vascular engorgement, plugged meibomian gland orifices and irregular lid margins); 2) Inability to express meibum, even with heavy pressure; 3) > 2/3 loss of the total meibomian gland area; and 4) Damaged corneal epithelium and superficial stroma.

2.2. Methods

2.2.1 Clinical examination

All subjects first completed the Chinese Dry Eye Questionnaire (as described in Appendix 1 below)[17] and then underwent tear collection for further LT-α concentration analysis (as described in 2.2.2 below). Corneal staining (Jingming, Tianjin) and fluorescein breakup time (FBUT) were examined with a slit lamp (Shanghai Expo Optoelectronics Instrument Co., Ltd.). An Ocular Keratograph (Oculus, Wezlar, Germany) was used to measure tear meniscus height, a tear strip (Jingming, Tianjin) was used to perform the Schirmer I test, and confocal microscopy (Heidelberg, Germany) was used to determine the density of conjunctival goblet cells in the nasal conjunctiva (as described in 2.2.3 below). All clinical examinations were performed by the same clinician.

2.2.2 Tear collection and LT-α concentration analysis

A tear sample of approximately 2.2 µl was collected with a tear collector (Seinda Biomedical Corporation, Guangzhou, China) from each patient, then added to well A of the LT-α detection reagent (Seinda Biomedical Corporation, Guangzhou, China). Then, the detection solution (sodium chloride solution) was added to well B. After waiting for 15 minutes for color development, the LT-α concentration in the fresh tears was determined using colloidal gold immunochromatography analyzer (Seinda Biomedical Corporation, Guangzhou, China).

2.2.3 Conjunctival GCD analysis

The conjunctival GCD on the nasal side was evaluated under anesthesia by using a laser confocal microscope (Heidelberg, Germany) (the detection head was approximately 2–4 mm away from the limbus). The imaging area was 400 µm*400 µm. After the inspection and image acquisition were completed, we selected 10 clear images in different fields of view, calculated the conjunctival GCD by using the cell counting tool integrated in the laser confocal microscope software, and averaged the values to obtain the result.

2.3 Statistics

All statistical analyses of data were performed using SPSS statistics (22.0) and R software (4.0). Normally distributed variables are expressed as the mean ± standard deviation and were compared by using one-way analysis of variance, and nonnormally distributed variables are expressed as the median (first quartile, third quartile) and were compared by using the Kruskal‒Wallis test. If the count variable had a theoretical number < 10, Fisher's exact probability test was used. Subsequently, Spearman correlation analysis was used to explore the correlation between variables, and one-way analysis of variance was used to explore the relationship between LT-α concentration and other clinical indicators. Then, after adjusting for potential confounders, a generalized additive model (GAM) was used for curve fitting to explore the relationship between LT-α concentration and conjunctival GCD, and a multivariate piecewise linear regression model was further used to find the best inflection point of the curve. Finally, using dry eye as the outcome variable, ROC analysis was performed with LT-α concentration and conjunctival GCD as the test result variables to explore the value of LT-α and conjunctival GCD in diagnosing dry eye. In addition, we also grouped by the optimal diagnostic threshold of GCD, and the Mann‒Whitney U test was used to analyze the differences in LT-α levels. A p value (two-tailed) < 0.05 was considered statistically significant.

3.1 General Information Description

A total of 98 cases and 98 eyes samples were collected. Among them, there were 20 people in the control group, with an average age of 26.25 ± 4.73 years old, and the proportions of males and females were 25.00% and 75.00%, respectively. There were 78 people in the dry eye group, with an average age of 30.27 ± 5.99 years old, and the proportions of males and females were 24.36% and 75.64%, respectively. The basic data of the two groups are shown in Table 1. The dry eye group was divided into subgroups according to severity, including 51 people in the mild dry eye group (29.73 ± 5.87 years old) and 27 people in the moderate to severe dry eye group (31.30 ± 6.19 years old). The Chinese dry eye questionnaire scores, FBUT, and corneal staining scores were significantly different among the control group, mild dry eye group, and moderate to severe dry eye group (P < 0.05) (Table 1). The conjunctival GCD in the mild dry eye group was significantly lower than that in the control group (P = 0.027), and there was no significant difference among the other groups (Fig. 1). There was no significant difference in LT-α concentrations among the 3 groups (P > 0.05) (Fig. 2).

Table 1

Basic description of control group, mild dry eye group, moderate to severe dry eye group and dry eye group
	Control group	Mild dry eye group	Moderate and severe dry eye group	Dry eye group	P value(2 group)	P value(3 group)
Number of people	20	51	27	78
Age, years	26.25 ± 4.73	29.73 ± 5.87	31.30 ± 6.19	30.27 ± 5.99	0.007	0.013
Sex, male/female	5/15	15/36	4/23	19/59	0.953	0.361
Chinese Dry Eye Questionnaire score	3.00 (2.00-5.25)	13.00 (10.00–16.00)	13.00 (11.25-15.00)	13.00 (10.00–15.00)	< 0.001	< 0.001
FBUT (s)	6.00 (5.00-6.75)	4.00 (3.00–5.00)	2.00 (2.00-3.50)	3.00 (2.00–5.00)	< 0.001	< 0.001
Corneal staining score	0.00 (0.00–0.00)	1.00 (0.00–1.00)	4.00 (4.00–5.00)	1.00 (0.00–4.00)	< 0.001	< 0.001
Tear meniscus height (mm)	0.24 (0.21–0.30)	0.22 (0.18–0.28)	0.24 (0.19–0.29)	0.22 (0.18–0.28)	0.111	0.186
Schirmer Ⅰ test (mm/5 min)	15.00(13.00–25.00)	12.00(7.00–25.00)	18.00(10.75-30.00)	12.00(8.00–27.00)	0.179	0.131
Conjunctival GCD (cells/mm²)	125.80(72.75–180.00)	51.15(24.31-100.18)	76.67(25.00-135.90)	54.70(24.38–126.50)	0.011	0.027
LT-α concentration (ng/ml)	0.30(0.13–1.32)	0.19(0.07–1.18)	0.14(0.04–1.12)	0.17(0.05–1.18)	0.366	0.550
P value (2 group) represents the control group and dry eye group, P value (3 group) represents the control group, mild dry group and moderate and severe dry eye group.

3.2 Correlation analysis among LT-α, conjunctival GCD and clinical parameters

Spearman correlation analysis showed that the LT-α concentration was positively correlated with the conjunctival GCD (r = 0.254, p < 0.05) and FBUT (r = 0.262, p < 0.01). The results of the Schirmer Ⅰ test were positively correlated with conjunctival GCD (r = 0.262, p < 0.05) and tear meniscus height (r = 0.328, p < 0.01). Age was negatively correlated with the Schirmer Ⅰ test result (r= -0.301, p < 0.01) and corneal staining score (r= -0.221, p < 0.05). The FBUT was negatively correlated with the corneal staining score (r= -0.448, p < 0.01) and the Chinese Dry Eye Questionnaire score (r=-0.367, p < 0.01). The corneal staining score was positively correlated with the Chinese Dry Eye Questionnaire score (r = 0.324, p < 0.01).

After adjusting for age and sex, a univariate analysis was performed on the relationship between clinical parameters and LT-α concentrations. The results showed that the FBUT was significantly positively correlated with the LT-α concentration (P = 0.001). That is, as the LT-α concentration increased, tear film stability improved. No other clinical indicators were significantly correlated with the LT-α concentration.

3.3 Correlation analysis between tear LT-α concentrations and conjunctival GCD

To further explore the relationship between conjunctival GCD and LT-α concentrations, a GAM was used to perform curve fitting after adjusting for variables such as age and sex. The relationship between conjunctival GCD and LT-α concentration was not a simple linear relationship but an S-shaped curvilinear one (Fig. 3). Multivariate piecewise linear regression based on the log likelihood ratio was used to find the inflection point and verify whether the relationship was significant. We divided the LT-α concentration data into 2 segments according to the inflection point. Table 2 presents the results. When the LT-α concentration < 1.47 (81.6% subjects fell in this range) in the first segment, the conjunctival GCD was positively correlated with the LT-α concentration (β = 63.92, P = 0.004). When the LT-α concentration > 1.47, the conjunctival GCD and LT-α concentration were negatively correlated, but the relationship tended to fluctuate nonlinearly in the second segment (β=-53.93, P = 0.047).

Table 2 Multiple segmented linear regression analysis of LT-α concentration versus conjunctival GCD

3.4 LT-α concentrations versus conjunctival GCD in dry eye diagnostic tests

The ROC curves of LT-α concentrations and conjunctival GCD are shown in Fig. 4. The AUC of LT-α concentrations was 0.5657 (95% CI 0.7067 − 0.4247). The cutoff diagnostic value was 0.11 ng/ml, the sensitivity was 41.03%, and the specificity was 80.00%. The AUC of conjunctival GCD was 0.6993 (95% CI is 0.8272 − 0.5713). The cutoff diagnostic value was 67.90 cells/mm², the sensitivity was 59.15%, and the specificity was 82.35%.

3.5 Comparison of LT-α concentration in different conjunctival GCD groups

The LT-α concentration was divided into group 1 (conjunctival GCD < 67.9/mm2) and group 2 (conjunctival GCD ≥ 67.9/mm2) according to the diagnostic threshold for conjunctival GCD. The LT-α concentrations in group 1 (0.097 (0.040–0.685)) were significantly lower than those in group 2 (0.440 (0.135, 1.633); P = 0.013, Mann–Whitney U test).

Initial studies on LT-α have shown that it plays a role in the occurrence and development of inflammation[12, 18–21], but the specific effects it induces appear to be inconsistent. Studies have indicated that this inconsistency may be due to the fact that LT-α can bind to two different TNF receptors (TNFR1 and TNFR2) to activate specific signal transduction pathways, thereby exerting different physiological effects[18]. In the onset stage of herpes virus keratitis in mice, the expression of LT-α was found to be upregulated[20]. Moreover, LT-α levels in the serum and synovial tissue of patients with rheumatoid arthritis were significantly increased[12]. However, TNF-α and LT-α levels were found to be negatively correlated with fatigue levels in a serum study on patients with primary Sjögren's syndrome, casting doubt on the previous belief that proinflammatory cytokines directly mediate fatigue symptoms in chronic immune diseases[21].

LT-α has been poorly studied in ophthalmic diseases. A prospective cross-sectional study showed that TNF a, IL-10, IL-1b, IL-1ra, IL-17a, and IL-12/23p40 levels were significantly higher in the high LT-α dry eye group (LT-α > 0.7 ng/ml) than those in the low LT-α dry eye group (LT-α < 0.7 ng/ml). Compared with the control group, the levels of IL-10, EGF, IL-17a and IL-12/23p40 were increased in the high LT-α dry eye group, but no difference was found in the low LT-α dry eye group, suggesting that the pathogenesis of dry eye in patients with high and low LT-α is not completely the same. It has also been suggested that LT-α may have different pathophysiological mechanisms in different types of dry eyes[22]. Another study found that the LT-α concentrations in the tears of patients with chronic oGVHD were significantly lower than those in the control group, and the area under the curve for LT-α diagnosis of chronic oGVHD was 0.847. The cutoff value is 0.203 ng/mL, which suggests LT-α concentration could be used a marker for the diagnosis of chronic oGVHD[15]. In addition, the investigators noted that the majority of oGVHD patients had low tear LT-α levels, whereas a minority of oGVHD patients had much higher tear LT-α levels[15]. Based on these findings, it is suggested that LT-α may play a protective role in regulating inflammatory responses in ocular surface immune homeostasis in most cases, but there are indeed different effects, which may be related to the variable pathogenesis and severity of dry eye.

In our study, we explored for the first time the relationship between LT-α concentrations and conjunctival GCD. Spearman correlation analysis showed that the LT-α concentration was positively correlated with conjunctival GCD (r = 0.254, p < 0.05), and the two present with an S-shaped, curvilinear relationship. When the LT-α concentration < 1.47 ng/ml (81.6% subjects fell in this range), it was positively correlated with conjunctival GCD. The results also showed that most dry eye patients have decreased LT-α concentrations and that the LT-α concentrations of moderate to severe dry eye patients are lower than those of mild dry eye patients, which suggests that LT-α concentrations may have a protective effect on the immune regulation of the ocular surface within a certain concentration range. The underlying mechanism of this result may be that the decrease in LT-α levels leads to a decline in the function of Treg cells, which makes ocular surface immune homeostasis unbalanced, while Th1 and Th17-induced inflammatory factors, such as IFN-γ, mediate enhanced inflammation, which can lead to conjunctival goblet cell damage. Moreover, the LT-α concentrations of the low conjunctival GCD group (< 67.90 cells/mm2) were significantly lower than those of the high conjunctival GCD group (≥ 67.90 cells/mm2), verifying the aforementioned underlying mechanism. However, when the LT-α concentration > 1.47 ng/ml, the conjunctival GCD and LT-α concentration demonstrated a fluctuating, negatively correlated relationship, but the exact nature of the relationship is difficult to determine. In the study mentioned above[22], the tear inflammatory factors of patients in the high-level LT-α dry eye group were significantly higher than those in the low-level LT-α dry eye group and the control group, which suggested that inflammatory cytokines were more involved in the high-level LT-α group and that different inflammatory mechanisms may be involved in the pathogenesis of dry eye with high levels of LT-α. This may explain why the change in conjunctival GCD is not completely consistent with the level of LT-α concentration in our research results when LT-α is greater than 1.47 ng/ml. Nonetheless, due to the small number of samples in the two studies, the specific threshold needs to be further explored by expanding the sample size, and the pathophysiological mechanism is also worthy of further investigation.

We also explored the value of LT-α concentration in the diagnosis of dry eye in this study. The area under the ROC curve of LT-α was 0.5657, the cutoff diagnostic threshold was 0.11 ng/ml, the sensitivity was 41.03%, and the specificity was 80.00%. These results suggest that LT-α concentration combined with other clinical examinations may help diagnose dry eye, especially mucin-deficient dry eye. In addition, we found that the FBUT was positively correlated with the LT-α concentration (r = 0.262, p < 0.01; β = 0.27, p = 0.001), which is consistent with a previous study of ocular oGVHD[15]. They also found that tear LT-α concentrations in oGVHD patients and controls were significantly correlated with FBUT (r = 0.608, p < 0.0001), and the correlation strength was higher than that in our study. The slight difference between the two studies may be related to the sample size and examination procedure. LT-α concentrations may help to evaluate tear film stability, but more experimental evidence is still needed.

Many studies have found that dry eye can lead to goblet cell reduction, tear deficiency and inflammatory factor infiltration, which can affect the proliferation and secretion of goblet cells[7, 23–24]. Our research shows that the conjunctival GCD in the dry eye group was significantly lower than that in the control group; this was obvious for the mild dry eye group, but in the moderate to severe dry eye group, the conjunctival GCD was higher than that in the mild dry eye group. Although there was no significant difference, this observation may be related to the more extreme inflammation involved in moderate to severe dry eye. Some inflammatory factors (such as IL13) may stimulate the conjunctiva to induce a compensatory mechanism and stimulate the proliferation of goblet cells. Although the number of compensatory hyperplasia goblet cells increases, they may not have secretory function[25]. We used a confocal microscope to evaluate conjunctival GCD. The diagnostic efficiency of conjunctival GCD is higher than that of LT-α concentration, indicating that the value of GCD in the diagnosis of dry eye should be considered. A study by Pflugfelder[24] confirmed that there was no significant difference in the density of goblet cells in patients with MGD compared with that of normal controls. Shimazaki-Den S et al. [26] reported that dry eye patients, excluding MGD patients, with a shortened FBUT have significantly reduced mucin MUC5AC and MUC16 levels regardless of whether they exhibit tear deficiency. The GCD in the dry eye group was also lower than that in the control group, but the difference was not statistically significant[26]. It may be related to the uneven distribution of goblet cells and the heterogeneity caused by imprint cytology detection. Strict inclusion criteria and accurate examination procedures were adopted to ensure that our results were as representative as possible. Our findings together with those of previous studies indicate that goblet cell loss can exist independently of MGD-related dry eye and aqueous-deficient dry eye, and a quick and noninvasive examination of conjunctival GCD by confocal microscopy can be an important method for the diagnosis of mucin-deficient dry eye. However, the relationship between the morphology and function of goblet cells, the relationship between the density of goblet cells and the levels of various mucins in tears, and the diagnostic threshold for mucin-deficient dry eye need to be further studied.

The number of conjunctival goblet cells has been found to be reduced in aqueous-deficient dry eye and certain ocular surface inflammatory conditions, such as oGVHD [24, 27]. Pflugfelder confirmed that the conjunctival GCD of patients with Sjögren's syndrome was significantly lower than that of the normal control group, and the expression of MUC5AC mucin in Sjogren's syndrome and non-Sjögren's syndrome dry eye groups was also lower than that of MGD patients[24]. Scholars have also confirmed that tear deficiency can lead to higher expression of IFN-γ in the conjunctiva[7], and loss of goblet cells was found to be associated with high expression of IFN-γ in both human and mouse dry eye models[28]. Since our study needed to collect 0.22 µl tear fluid for LT-α concentration analysis, it was difficult to collect tears from patients with aqueous-deficient dry eye. We were biased to the nonaqueous-deficient dry eye population at the time of study, but the results still showed that the density of conjunctival goblet cells was positively correlated with the Schirmer Ⅰ test without topical anesthesia (r = 0.262, p < 0.05). The above studies prove that there is a close relationship between tear secretion in the lacrimal gland and the density of conjunctival goblet cells. Adequate tear secretion plays a protective role in conjunctival goblet cells, and a mild decrease in tear secretion may lead to the loss of conjunctival goblet cells through an increase in inflammatory factors and other pathways.

Of course, our study also has certain limitations. First, the age of the control group was slightly younger, which is related to the high incidence of dry eye in young people, but we adjusted for the influence of age and sex on the observation indicators during statistical analysis. In addition, lissamine green staining of the conjunctiva was not performed in this study. Ocular surface epithelial damage and mucin coverage assessments may help explain the relationship between conjunctival GCD and LT-α levels. Therefore, more in-depth research with larger samples and a wider age range is needed to better clarify the relationship between tear LT-α levels and conjunctival goblet cells in the future.

In conclusion, we found a strong positive correlation between LT-α concentration in tears and conjunctival GCD, and both have a certain value in the diagnosis of dry eye diseases. Apart from this, LT-α concentration is also valuable for evaluating the stability of tear film, but more research evidence is still needed to support this relationship. Moreover, the value of conjunctival GCD evaluation by confocal microscopy in the diagnosis of dry eye is worthy of attention.

Data availability statement: The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Conflicts of Interest

All authors indicate no potential competing of interest.

Disclosure

This study was supported by the Science and Technology Project of Hunan Province (2021SK50101) and the Health Commission of Hubei Province Scientifific Research Project (WJ2019H395), the Health and Family Planning Committee Science Foundation of Wuhan (WX19Q48).

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No competing interests reported.

Appendix1.Dryeyequestionnaire.pdf

Correlation between lymphotoxin-α concentrations and conjunctival goblet cell density in dry eye patients

Status:

Version 1

Abstract

Purpose

Methods

Results

Conclusion

Figures

1. Introduction

2. Materials and Methods

2.1. Patients

2.2. Methods

2.2.1 Clinical examination

2.2.2 Tear collection and LT-α concentration analysis

2.2.3 Conjunctival GCD analysis

2.3 Statistics

3. Results

3.1 General Information Description

3.2 Correlation analysis among LT-α, conjunctival GCD and clinical parameters

3.3 Correlation analysis between tear LT-α concentrations and conjunctival GCD

3.4 LT-α concentrations versus conjunctival GCD in dry eye diagnostic tests

3.5 Comparison of LT-α concentration in different conjunctival GCD groups

4. Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1