Tear fluid and complement activation products in tears after ocular surgery

In developed countries, the number of cataract patients is increasing due to the growing elderly population. There is also a rise in the prevalence of dry eye among older individuals[1‐3]. Ophthalmologists often perform cataract surgery on patients with dry eye. While advancements in technology have made surgery less invasive, certain factors such as light exposure during surgery, disinfectant toxicity, and evaporation from the ocular surface can contribute to postoperative dry eye [4‐8]. This can be problematic for both surgeons and patients, but there is no established treatment for it.

The innate immune system may play a role in postoperative inflammation, and complement, an important component of this system, is involved in protecting against foreign pathogens, maintaining homeostasis, and promoting wound healing [9‐11]. Complement activation occurs through three pathways: the classical, lectin, and alternative pathways. Complement is associated with various acute inflammatory processes and helps in defending the host against infections [9‐11]. The classical pathway of complement activation is initiated by IgM and IgG immunoglobulins. This leads to the cleavage of C4 and C2, resulting in the activation of C3 and C5. Additionally, the classical pathway can also be activated through the Lectin pathway without the involvement of immunoglobulins. The alternative pathway, on the other hand, operates independently of antibodies and can rapidly activate complement even on the ocular surface. When tissue is damaged, it contains proteases that promote the activation of C3 and C5 [10]. C3 is then cleaved to generate C3a and C3b, while C5 is activated to form C5a and C5b. Ultimately, the complement pathway forms a membrane attack complex (MAC), which leads to cell lysis and death [11]. C3a is known to act as a mediator in inflammatory reactions, affecting polymorphonuclear leukocytes, vasoconstriction, and causing leakage. It is responsible for increased vascular permeability [12]. C5a has been found to mobilize neutrophils and activate basophils, monocytes, and neutrophils, resulting in increased vascular permeability [12, 13]. In ophthalmology, the complement system is involved in age-related macular degeneration [14‐18], bacterial keratitis [19], and allergic conjunctivitis [20]. 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) [21, 22].

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 [12], which generates C3a and C5a that cause migration, activation, and aggregation of mast cells, polymorphonuclear cells, and macrophages [23, 24], 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 hypothesized that postoperative inflammation would increase in patients who underwent combined vitrectomy and cataract surgery. Therefore, in this study, we examined tear secretion and analyzed the changes in levels of C3a, C4a, and C5a in tears of these patients following the surgical procedures.

A total of 43 patients (23 women, 20 men) with a median age of 69 years (interquartile range [IQR]: 64–73 years) underwent combined vitrectomy and cataract surgery in 43 eyes. The surgical procedure was performed on 1 eye per patient. Among the eyes, 30 had an epiretinal membrane, while the remaining 13 had a macular hole. The median duration of the surgical procedure was 60.0 min (IQR: 49.8–79 min). 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 examined (Fig. 1). This figure demonstrated a negative correlation between C3a levels and baseline ST values (P = 0.015).

To further investigate the relationship between C3a and ST, we conducted an additional analysis. 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 levels of C4a and C5a in both the short and long ST groups showed an increase at 4 days after surgery and then decreased to the preoperative levels after 1 month (Table 2).

In our study, we measured the levels of CAPs in tears and the ST after performing vitrectomy with cataract surgery. Four days after the surgery, we observed a significant increase in ST compared to the baseline measurement. Additionally, the levels of C3a, C4a, and C5a also showed an increase at the 4-day postoperative mark compared to the baseline levels. These elevated levels of CAPs remained high even at the 1-month follow-up.The development of inflammation after cataract surgery is a significant concern for surgeons in this field. Experts have discussed this issue extensively, but there is currently no established protocol for effectively managing postoperative inflammation in patients. Additionally, the innate immune system plays a crucial role in the process of wound healing [26]. However, excessive activation of the complement system can lead to undesirable reactions [27, 28]. Previous experimental studies have indicated a potential association between complement activity and unfavorable postoperative responses [28, 29]. Based on this, we hypothesized that ocular surface parameters might be linked to complement activity during the postoperative period.

The increase in C3a, C4a, and C5a levels 4 days after surgery were considered a common tissue response to surgical injury [30]. The preoperative C3a level in tear fluid showed a negative correlation with the ST value. The levels of C3a and C5a remained elevated immediately after surgery and persisted for 1 month in the short ST group. This suggests that postoperative complement activation through the alternative pathway may be prolonged in eyes with low basal tear fluid secretion. Recent proteome analyses have also indicated the involvement of the complement system in dry eye [31, 32].

This could be associated with reduced CAPs and tear production. In the short ST group examined in this study, the levels of C3a in tear fluid had not returned to their preoperative values even after 1 month of surgery. This indicates that a short ST may contribute to postoperative inflammation. Applying artificial tears to eyes with a short ST could potentially decrease persistent complement activation and alleviate ocular symptoms associated with tear film instability. Measuring ST can help predict postoperative inflammation, and if a short ST is detected, appropriate anti-inflammatory measures and tear fluid replacement may be beneficial. This study is also the first to measure complement activation products in tear fluid. We believe that this research will serve as a foundation for future investigations into inflammation in tear fluid.

Our findings indicate that there was an increase in CAPs after surgery. A reduction in tear secretion before the surgery can lead to prolonged complement activation and a slower recovery of ocular surface parameters following combined vitrectomy and cataract surgery.