Tear fluid and Complement activation products in tears after ocular surgery.

doi:10.21203/rs.3.rs-2506004/v1

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Tear fluid and Complement activation products in tears after ocular surgery.

https://doi.org/10.21203/rs.3.rs-2506004/v1

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published 18 Jul, 2023

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Purpose

To investigate changes in the tear film complement activation products (CAPs) and ocular surface after vitrectomy combined with cataract surgery.

Methods

Forty-three patients (23 women; median age, 69 years) were enrolled in this prospective study and underwent phacoemulsification and vitrectomy. We measured Schirmer's test (ST) and CAPs in the tears at baseline, 4 days and 1 month after the surgery. Tears were collected in microtubes. The CAPs in the tear fluid were analyzed by cytometric bead array.

Results

A negative correlation was found between the ST and C3a (r² = -0.37, P = 0.0015) at baseline. The median ST (8.5 mm) at baseline increased to 16 mm at 4days ( P < 0.001) and 10 mm at 1 month (P = 0.44). The C3a levels (1202 pg/ml) at baseline increased to 2753 pg/ml at 4 days (P < 0.001), and 1763 pg/ml at 1month (P = 0.049). The C4a levels (476 pg/ml) at baseline increased to 880 pg/ml at 4days (P < 0.001), and 657 pg/ml at 1 month (P = 0.013). The C5a levels (22.6 pg/ml) at baseline increased to 470.9 pg/ml at 4 days (P < 0.001), and 38.3 pg/ml (P = 0.0048). The surgical eyes were divided into the short ST group ( ≦ 10 mm, n = 22) and long ST group (> 10 mm, n = 21) based on the preoperative ST values. At 1 month postoperatively, the C3a levels was 2194 pg/ml in the preoperative short ST group and 1391 pg/ml in the long ST group, with significantly higher C3a concentrations in the short ST group (P < 0.001).

Conclusions

The CAPs levels in tears increased after vitrectomy combined with cataract surgery. A preoperative deficit in tear secretion might induce prolonged complement activation and delayed recovery of ocular surface parameters postoperatively.

Tears

Schirmer’s test

Complement activation products

Ocular surface

Tear

Inflammation

In developed countries, the number of patients who develop cataracts is increasing with the increasing elderly population. Recent reports have suggested that the prevalence of dry eye also is increasing in older populations [1–3]. Ophthalmologists frequently likely perform cataract surgery on eyes with dry eye [4–7]. Recent advancements in technology and instrumentation have reduced the surgical invasiveness. However, other harmful factors, such as light projection into the eyes during cataract surgery, the toxicity of disinfectants with povidone iodine, and increasing evaporation from the ocular surface while using a speculum, may induce postoperative dry eye disease [4–8]. Postoperative exacerbation of dry eye symptoms may be problematic for cataract surgeons and patients; however, there is no established treatment.

The innate immune system may be involved in postoperative inflammation in which complement may play an important role in protecting against all foreign pathogens, homeostasis, and wound healing [9–11]. There are three pathways of complement activation, i.e., the classical, lectin, and alternative pathways. Complement is associated with many acute inflammatory processes and host defenses [9–11]. In ophthalmology, the complement system is involved in age-related macular degeneration [12–16], bacterial keratitis [17], and allergic conjunctivitis [18]. In Sjögren's syndrome and dry eye, tear fluid secretion is decreased, causing inflammation of the ocular surface, which has been reported to be associated with complement activation products (CAPs) [19, 20].

In wound healing, hemostasis, debris clearance, and tissue reconstruction are necessary. At the site of wound healing, platelet aggregate at the wound to establish hemostasis. Degranulation of platelets then induces C3 [21], which generates C3a and C5a that cause migration, activation, and aggregation of mast cells, polymorphonuclear cells, and macrophages [22, 23], all involved in tissue clearance. This cellular response after complement activation may be associated with the wound-healing process in intraocular surgery. Investigating the relationship between the complement activated proteins and ocular surface parameters postoperatively may provide clues to inflammation.

We expected postoperative inflammation to increase in association with the combined surgery. In this study, we investigated tear secretion and postoperative changes in C3a, C4a, and C5a levels in tears in the patients who underwent vitrectomy and cataract surgery.

The Institutional Review Board of Fukushima Medical University, Fukushima, Japan, approved the study protocol (ID: 30046). The research was conducted in accordance with the tenets of the Declaration of Helsinki, and all patients provided written informed consent after they received a detailed explanation of the study protocols and possible consequences associated with participation. The study included consecutive patients who underwent vitrectomy combined with cataract surgery for epiretinal membranes and macular holes at Fukushima Medical University Hospital. No patients received the treatments for dry eye. Two experienced surgeons (YK, AK) performed all surgeries. No patients used dry eye treatments preoperatively.

The exclusion criteria included a history of ophthalmic surgery; the presence of severe conjunctival chalasis, superior limbic keratoconjunctivitis, lid-wiper epitheliopathy, pterygium. Other exclusion criteria included systemic diseases that could affect the ocular surface, such as type 2 diabetes, rheumatoid arthritis, and Sjögren's syndrome.

Vitrectomy combined with cataract surgery

Levofloxacin 1.5% eye drops (Pfizer, New York, NY, USA) were applied four times daily for 3 days preoperatively in all cases. Immediately before surgery, 0.25% polyvinyl alcohol iodine (Rohto Nitten, Nagoya, Japan) was applied, and the surgical field was disinfected according to the package insert.

Vitrectomy combined with cataract surgery was performed using the Constellation Vision System (Alcon Japan Pharma, Tokyo, Japan). Cataract surgery was performed through a 2.4-mm corneal incision, and an intraocular lens was inserted. A 25-gauge pars plana vitrectomy was performed under a non-contact wide-viewing system (Resight Fundus Imaging System; Carl Zeiss Meditec Japan Co., Ltd, Tokyo, Japan). Saline was applied to the surgical field to hydrate the ocular surface intraoperatively. Postoperatively, the puls planner wound was sutured with 8 − 0 Vicryl (Mani, Tochigi, Japan) in all cases.

Postoperative eye drops were instilled, i.e., levofloxacin 1.5% eye drop for 2 weeks, 0.1% fluorometholone (Rohto Nitten) for 4 weeks, and bromfenac ophthalmic solution 0.1% (Nisshin, Yamagata, Japan) for 12 weeks postoperatively in all cases.

The Schirmer's test and tear fluid collection and measurements

The Schirmer's test (ST) on the day before surgery (baseline) and 4 days and 1 month postoperatively to investigate lacrimal fluid secretion. The tear fluid was collected non-invasively during the ocular surface evaluation as reported previously [24]. The same investigator (HM) collected the tear fluid in all cases. To avoid the effect of eye drops on the ocular surface measurements, tear collection was performed at least 1 hour after the eye drops were instilled. The patient lay on his or her back on the bed and the clinician used a micropipette (Drummond Scientific, Broomall, PA, USA) to collect two µl of tear fluid from the external eyelid margins. The collected tear fluid was immediately stored at -80 ° C until analysis. The collected tears were measured using the BD CBA Human Anaphylatoxin Kit (BD Biosciences, San Jose, CA, USA) for CAPs (C3a, C4a, and C5a), according to the manufacturer's instructions [15–17].

Statistical analysis

The Steel-Dwass tests were used to compare preoperative measurements to those during the follow-up period. Correlation coefficients were performed to identify the association of changes in ST. P < 0.05 was considered statistically significant. Statistical analyses were performed using JMP16 software (SAS Institute, Cary, NC, USA).

Vitrectomy combined with cataract surgery was performed in 43 eyes of 43 patients (23 women, 20 men; median age, 69 years; interquartile range [IQR], 64–73 years). All patients underwent surgery on one eye. Thirty eyes had an epiretinal membrane, and the remaining 13 eyes had a macular hole. The median surgical time was 60.0 minutes (IQR, 49.8–79 minutes).

Table 1 summarizes the clinical data during the follow-up period. All parameters were measured at baseline, 4 days, and 1 month postoperatively, in the surgical eyes.

The ST (mm) values at baseline, 4 days, and 1 month postoperatively were 8.5 (IQR, 4–15), 16 (IQR, 7.5–20), and 10 (IQR, 5–15), respectively, in the surgical eyes. The ST values increased 4 days after the surgery compared to baseline (P < 0.001) and decreased 1 month postoperatively compared to baseline (P = 0.44).

The C3a levels at baseline, 4 days, and 1 month postoperatively were 1202 pg/ml (IQR, 860–2304), 2753 pg/ml (IQR, 1710–4453), 1763 pg/ml (IQR, 1234–2464), respectively, in the surgical eyes. The C3a levels increased 4 days postoperatively and decreased at 1 month. The differences in the C3a levels between baseline and 4 days and between 4 days and 1 month postoperatively was significant (P < 0.001, P = 0.049, respectively) (Table 1).

The C4a levels at baseline, 4 days, and 1 month postoperatively were, respectively, 476 pg/ml (IQR, 196–742), 880 pg/ml (IQR, 479-1,464), and 657 pg/ml (IQR, 295–974) in the surgical eyes. The C4a levels increased significantly at 4 days (P < 0.001) and 1 month postoperatively (P = 0.013) compared to baseline (Table 1).

The C5a level at baseline, 4 days, and 1 month postoperatively were, respectively, 22.6 pg/ml (IQR, 15.6–36.0), 470.9 pg/ml (IQR, 98.4-1,061.4), and 38.3 pg/ml (IQR, 18.0-66.8) in the surgical eyes. The C5a levels increased at 4 days (P < 0.001) and 1 month (P = 0.0048) postoperatively (Table 1).

The correlation between ST and CAPs in tear fluid was investigated (Fig. 1). This figure showed that the C3a levels was correlated negatively with the ST values at base line (P = 0.015).

A further analysis was performed to clarify the relationship between the C3a and ST. The surgical eyes were divided into the short ST group ( ≦ 10 mm, n = 22) and long ST group (> 10 mm, n = 21) based on the preoperative ST values. A significant difference was seen in the C3a level between the short and long ST groups (short ST, long ST, 1602 pg/ml [IQR, 960–3193], 882 pg/ml [IQR, 624–1677]) (P = 0.041). In the short ST group, the C3a level increased (2210 pg/ml; [IQR, 1506–4328]) at 4 days and remained high (2194 pg/ml; [IQR, 1348–3192]) at 1 month, while in the long ST group, the elevated C3a level 2063 pg/ml; [IQR, 1986–5775] at 4 days decreased to 1391 pg/ml

[IQR, 881–1872] at 1 month (P = 0.032) (Table 2). The C4a and C5a levels in the short and long ST groups increased at 4 days and decreased to the preoperative levels 1 month postoperatively (Table.2).

Table 2. Relationship between complement activation products and ST during follow-up period.

Median(IQR)		Short ST group	P value vs baseline	P value vs 4 days	Long ST group	P value vs baseline	P value vs 4 days	P value of short ST vs Long ST
Median(IQR)		Short ST group	P value vs baseline	P value vs 4 days	Long ST group	P value vs baseline	P value vs 4 days	P value of short ST vs Long ST
C3a (pg/ml)	base line	1602 (960-3193)	-	-	887 (624-1677)	-	-	< 0.001
	4 days	2210 (1506-4328)	0.012	-	3063 (1986-5775)	< 0.001	-	0.29
	1 month	2194 (1348-3192)	0.059	0.076	1391 (881-1872)	0.11	< 0.001	< 0.001
C4a (pg/ml)	base line	386 (171-831)	-	-	519 (241-723)	-	-	0.97
	4 days	762 (451-2061)	< 0.001	-	911 (534-1360)	< 0.001	-	0.95
	1 month	453 (275-876)	0.46	0.0025	620 (295-1030)	0.12	< 0.001	0.60
C5a (pg/ml)	base line	23.4 (16.4-40.4)	-	-	21.2 (11.7-27.6)	-	-	0.42
	4 days	470.9 (93.8-1153.3)	< 0.001	-	565.7 (155.3-670.9)	< 0.001	-	0.70
	1 month	37.0 (16.8-55.3)	0.043	< 0.001	45.6 (22.2-128.1)	0.056	< 0.001	0.53
ST; schirmer's test, IQR; interquartile range

We measured the CAPs in tears and ST after vitrectomy with cataract surgery. Four days postoperatively ST increased significantly compared to baseline. The C3a, C4a, and C5a levels increased 4 days postoperatively compared to baseline and remained high at 1 month. Since the ST was correlated negatively with the C3a level at baseline, we analyzed the data in the short and long ST groups. The C3a level in the long ST group decreased to the baseline level 1 month postoperatively, while in the short ST group the C3a level remained high 1 month postoperatively.

Development of inflammation postoperatively is one of the greatest concerns for cataract surgeons. Specialists in this field have discussed this issue; however, proper management for postoperative care of patients with inflammation has not been established. The innate immune system also plays a pivotal role in wound healing [25]. However, hyperactivity of the complement system may induce unfavorable responses [26, 27]. Experimental research has suggested that complement activity can be associated with an unfavorable postoperative response [27, 28]. Considering this, we expected that the ocular surface parameters might be associated with complement activity in the postoperative period.

The elevated C3a, C4a, and C5a levels 4 days postoperatively were considered a common tissue response to surgical injury [29]. The preoperative C3a level in the tear fluid was correlated negatively with the ST value. The C3a and C5a elevation immediately postoperatively continued for 1 month in the short ST group, suggesting that postoperative complement activation in the alternative pathway may be prolonged in eyes with low basal secretion of tear fluid. In addition, the recent proteome analyses [30, 31] showed the involvement of the complement system in dry eye. This may also be related to decreased CAPs and tear production. In the short ST group in this study, C3a in tear fluid had not returned to preoperative values even one month after surgery. This suggests that short ST may be a factor contributing to postoperative inflammation. Instillation of artificial tears in the eyes with a short ST time might reduce the persistent complement activation and ocular symptoms related to tear film instability. The ST should be measured in predicting postoperative inflammation, and if short ST is present, adequate anti-inflammatory and tear fluid replacement may be beneficial.

Limitations

The sample size was too small to determine a relation between dry eye and a surgical effect postoperatively. In addition, because we used absorbable sutures to close the vitrectomy wound to prevent postoperative infection, biodegraded products (glycol ether) can be toxic to the cornea and conjunctive and induce inflammation. Because the suture was absorbed by 2 weeks in all cases, the effect of the biodegraded materials might be minimal at 1 month. Finally, we did not investigate the relation between the complement system and inflammatory cytokines and cellular immunity. Further studies are needed to determine the role of innate immunity in dry eye.

We found that CAPs increased postoperatively. A preoperative decrease in tear secretion can induce prolonged complement activation and delay recovery of ocular surface parameters after vitrectomy combined with cataract surgery.

complement activation products (CAPs) Schirmer's test (ST)

Ethics approval and consent to participate

Consent for publication

Written informed consent for the publication was obtained from all patients in this study.

Consent for publication- Not applicable

Availability of date and materials

Date are available from Hiroki Maehara ([email protected]) for researchers who meet the criteria for accessto confidential data.

Competing interests

The authors declare that they have no competing interests.

Fundings

We have no conflicts of interest in this study and have received no financial support.

Author’s contributions:

TS has been involebed indrafting the manuscript and revising it critically for important intellectual content.

HM has collected data and helped for data analysis and drafting the manuscript.

KN has collected data.

KT has collected data.

YK has collected data.

AK has collected data.

TO has collected data.

TM has collected data.

HS has collected data.

All the authors read and approved the final manuscript.

Acknowledgement

Not applicable.

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No competing interests reported.

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You are reading this latest preprint version

Tear fluid and Complement activation products in tears after ocular surgery.

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Abbreviations

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