Efficacy Evaluation of Tissue Plasminogen Activator with Anti-Vascular Endothelial Growth Factor Drugs for Submacular Hemorrhage Treatment: A Meta-Analysis

Submacular hemorrhage (SMH) is the accumulation of blood in the macular area that can severely damage the macular structure and visual function. Recently, the intraocular administration of tissue plasminogen activator (TPA) with anti-vascular endothelial growth factor (anti-VEGF) drugs was reported to have a positive effect on SMH. This meta-analysis aimed to explore the efficacy and safety of the drug combination. We systematically searched the Web of Science, MEDLINE, EMBASE, and Cochrane Library databases and screened relevant full-length literature reports. The quality of the reports was assessed by two independent reviewers. The best-corrected visual acuity (BCVA) and foveal thickness (FT) were considered the main indicators of efficacy. RevMan 5.4 software was used for this meta-analysis. Twelve studies were analyzed, and the results showed that BCVA at 1 month (p < 0.001), 3 months (p < 0.001), 6 months (p < 0.001), and the last follow-up (p < 0.001) was improved relative to the preoperative value. The postoperative FT was lower than the preoperative FT (p < 0.001). No significant difference in efficacy was observed between subretinal and intravitreal TPA injections (p = 0.37). TPA with anti-VEGF drugs is safe for SMH treatment and can significantly improve BCVA and reduce FT.


Introduction
Submacular hemorrhage (SMH) is characterized by the presence of blood between the retinal pigment epithelium (RPE) and the neurosensory retina in the macular area. It is caused by choroidal and retinal vascular abnormalities [1]. SMH damages photoreceptors in several ways, including iron toxicity, fibrin meshwork contraction, outer retinal shear forces, and reduced nutrient supply, eventually resulting in macular scarring [2]. SMH caused by various ocular diseases such as neovascular age-related macular degeneration (nAMD), polypoid choroidal vasculopathy (PCV), pathological myopia, and retinal aneurysm has a significantly negative impact on the patient's visual ability, with a poor prognosis [3]. A population-based study in 2014 estimated that the annual incidence of new and large SMH complicated with wet AMD was approximately 25 per million per year, which is detrimental to global eye health [4]. The cellular mechanism of SMH remains to be explored, as recent studies have revealed the importance of oxidative stress and inflammation response in arteriovenous pathologies [5,6]. SMH can result in sudden or progressive vision loss depending on the extent and thickness of the bleeding, and the reception of visual information by photoreceptors can be blocked, with subsequent damage. A retrospective review of eyes with massive SMH confirmed that visual acuity correlated inversely with the thickness rather than the diameter of SMH [7].
Several methods have been used for the treatment of SMH secondary to AMD. Surgical progress mainly focuses on vitrectomy along with multiple procedures such as the TPA were also excluded. Articles that met the requirements were listed and evaluated to ensure the inclusion of all eligible studies. Disagreements between the researchers were resolved through consultation with a third author (N.Z.). This meta-analysis was performed in accordance with the PRISMA guidelines (registration number CRD42022358037).

Literature Quality Evaluation
Because all the included studies were retrospective in nature, the Newcastle-Ottawa Scale (NOS) [21] was used by the two authors to assess the methodological quality and risk of bias of the studies. NOS is based on a star system for assessing the quality of case-control or cohort studies for seven items categorized into three broad groups: (1) selection (S, four stars), (2) comparability (C, two stars), and (3) exposure or outcome (O, three stars). Thus, the maximum number of stars that a study can receive is nine. Two authors (X.H. and W.C.) independently assessed the quality of the included studies. Disagreements between the researchers were resolved through consultation with a third author (N.Z.).

Outcome Indicator
The primary outcome measures were BCVA and the foveal thickness (FT) after treatment. The secondary outcomes were hemorrhage displacement and postoperative complications.

Data Extraction
Data from the included studies were independently extracted by two authors (X.H., W.C.). Studies with unclear or missing data records were excluded, and disagreements between the researchers were resolved through consultation with a third author (N.Z.). Data were collected and recorded as follows: (1) literature information for the included studies (first author, publication time, country, or region), (2) basic information about the study participants (sample size, age, sex, duration of disease course, baseline BCVA, baseline FT, and intervention measures), and (3) outcomes (BCVA after treatment, FT after treatment, bleeding displacement, and postoperative complications).

Statistical Methods
Data analysis was performed using Review Manager (RevMan 5.4, The Cochrane Collaboration, Oxford, UK) software. Continuous variables were statistically analyzed using the mean difference and are reported as the weighted mean deviation (WMD) with a 95% confidence interval (CI). Forest plots were used to describe and represent the statistical results.
When follow-up data were available at several time points in the study, the data at each follow-up and the final reported data were extracted. A paired-samples test was used to compare data before and after treatment.
The I 2 statistic was used to evaluate the heterogeneity of the results. I 2 ≤ 50% indicated low heterogeneity, and a fixed effects model was used. When I 2 was >50%, the heterogeneity was considered high, and a random effects model was used. Funnel plots were used for the visual assessment of publication bias.

Search Results
The study selection flowchart is shown in Figure 1. In total, 171 articles (PubMed (n = 70), Embase (n = 58), Cochrane Library (n = 6), and Web of Science (n = 37)) were identified. No Chinese database was included in this study. Endnote literature management software was used to remove 68 duplicates. Based on the literature type and language, 47 articles were excluded, including 13 conference papers, 11 reviews, one letter, 13 case reports, and nine non-English articles. After reading the title and abstract, 30 articles were eliminated, including three clinical trials with unreported results, four articles on animal experiments, and 23 without the combined use of TPA and anti-VEGF drugs. Subsequently, two independent reviewers screened the full text of 26 possible relevant studies, 14 of which were excluded because of missing data, unclear documentation, or loss of follow-up. Thus, 12 retrospective studies [20,[22][23][24][25][26][27][28][29][30][31][32] including a total of 269 eyes from 269 participants were included in this meta-analysis. software was used to remove 68 duplicates. Based on the literature type and language, 47 articles were excluded, including 13 conference papers, 11 reviews, one letter, 13 case reports, and nine non-English articles. After reading the title and abstract, 30 articles were eliminated, including three clinical trials with unreported results, four articles on animal experiments, and 23 without the combined use of TPA and anti-VEGF drugs. Subsequently, two independent reviewers screened the full text of 26 possible relevant studies, 14 of which were excluded because of missing data, unclear documentation, or loss of follow-up. Thus, 12 retrospective studies [20,[22][23][24][25][26][27][28][29][30][31][32] including a total of 269 eyes from 269 participants were included in this meta-analysis.

Description of the Included Studies
The 12 articles included in this meta-analysis were retrospective studies. Table 1 summarizes the main characteristics of the included studies, including the first author, publication time, sample size, number of eyes, country, etiology of SMH, patient age, disease duration, bleeding area, and NOS star level. Based on the conversion relationship between the bleeding area and the optic disc diameter in the study by Arias et al. [22], the bleeding areas in the studies by Avci et al. [20] and Kitagawa et al. [26] were converted into a representation of the optic disc diameter. Abbreviations: No., number; SD, standard deviation; DD, disc diameter; NOS, Newcastle-Ottawa Scale; G1, Group 1; G2, Group 2; G3, Group 3; AMD, age-related macular degeneration. Note: " $" represents the score of the methodological quality of the included studies according to the modified version (nine-star scoring system) of the NOS. Table 2 presents the treatment modalities, complications, and hemorrhage displacement procedures received by the study participants. Grohmann et al. [31] evaluated the effect of three surgical modalities for SMH treatment and termed them Grohmann G1, G2, or G3 according to the different treatment regimens received by each group.
The BCVA and FT data before and after SMH treatment are summarized in Tables 3  and 4, respectively. Based on the conversion scheme of different visual acuity recording methods published by Ferris et al. [33], some of the original values in the studies by Erdogan et al. [30], Kitagawa et al. [26], and Guthoff et al. [24] were converted to logarithms of the minimum angle of resolution units to facilitate subsequent statistical analysis.

Analysis of BCVA
The final BCVA values in the 12 included studies were analyzed, and the comparative results are shown in the first forest plot (Figure 2). The final BCVA significantly improved relative to the initial BCVA (MD = −0.52, 95% CI= (−0.68, −0.37), I 2 = 62%, p < 0.001). Heterogeneity analysis showed that I 2 (62%) was more than 50%; therefore, a more conservative random effects model was used. The findings suggested that TPA combined with anti-VEGF therapy is effective in improving the final visual acuity of patients with SMH.

Analysis of BCVA
The final BCVA values in the 12 included studies were analyzed, and the comparative results are shown in the first forest plot (Figure 2). The final BCVA significantly improved relative to the initial BCVA (MD = −0.52, 95% CI= (−0.68, −0.37), I 2 = 62%, p < 0.001). Heterogeneity analysis showed that I 2 (62%) was more than 50%; therefore, a more conservative random effects model was used. The findings suggested that TPA combined with anti-VEGF therapy is effective in improving the final visual acuity of patients with SMH. Among the included studies, six, five, and seven documented BCVA at 1, 3, and 6 months, respectively. Grohmann et al. [31] studied the influence of different surgical    ticipants were not grouped according to the site of injection; therefore, this subgroup analysis was not included in the meta-analysis. The subgroup analysis showed that the SRI or IVI of TPA could increase BCVA (SRI group: MD = −0.63, 95% CI = (−0.92, −0.34), I 2 = 71%, p < 0.001; IVI group: MD = −0.46, 95% CI = (−0.69, −0.23), I 2 = 59%, p < 0.001; overall: MD = −0.54, 95% CI = (−0.72, −0.37), I 2 = 64%, p = 0.37). A random effects model was used because I 2 was >50%. Overall, the site of TPA administration did not affect the trend in BCVA improvement (p = 0.37 > 0.05 for the difference between groups).  [20,23,27,[29][30][31][32] of TPA versus (B) BCVA after the IVI of TPA [24][25][26]28,31]. Both subgroups show improved postoperative BCVA relative to the preoperative BCVA, with no significant difference between the two subgroups. This indicates that the site of TPA injection does not affect BCVA. BCVA, best-corrected visual acuity; SRI, subretinal injection; TPA, tissue plasminogen activator; IVI, intravitreal injection. Figure 5 shows the results of comparisons between the final FT and preoperative FT in cases subjected to combined TPA and anti-VEGF treatment for SMH. FT decreased postoperatively, and the difference was statistically significant (MD = −384.54, 95% CI = (−513.66, −255.42), I 2 = 84%, p < 0.001). I 2 was >50%, and the random effects model was used. The findings indicated that combined treatment with TPA and anti-VEGF drugs can promote a decrease in FT, which is beneficial for the structural recovery of the fovea in patients with SMH.  [24][25][26]28,31]. Both subgroups show improved postoperative BCVA relative to the preoperative BCVA, with no significant difference between the two subgroups. This indicates that the site of TPA injection does not affect BCVA. BCVA, best-corrected visual acuity; SRI, subretinal injection; TPA, tissue plasminogen activator; IVI, intravitreal injection. Figure 5 shows the results of comparisons between the final FT and preoperative FT in cases subjected to combined TPA and anti-VEGF treatment for SMH. FT decreased postoperatively, and the difference was statistically significant (MD = −384.54, 95% CI = (−513.66, −255.42), I 2 = 84%, p < 0.001). I 2 was >50%, and the random effects model was used. The findings indicated that combined treatment with TPA and anti-VEGF drugs can promote a decrease in FT, which is beneficial for the structural recovery of the fovea in patients with SMH.  [25,26,28,31,32]. The foveal thickness is expressed in microns (μm), and it has decreased after treatment. This indicates that the combination of TPA and anti-VEGF drugs is beneficial for the structural restoration of the fovea. TPA, tissue plasminogen activator; anti-VEGF, anti-vascular endothelial growth factor.

Discussion
The accumulation of blood in the macula can cause irreversible damage to photoreceptors within 24 h, and damage to visual function can be particularly serious [34]. The etiology of SMH can generally be divided into two categories: CNV (such as nAMD, PCV, and pathologic myopia) and non-CNV (such as retinal aneurysm, Terson syndrome, and trauma). CNV-associated SMH is more common. The choice of treatment for SMH is closely related to the cause of the hemorrhage. In non-CNV SMH, visual acuity usually improves to varying degrees after the blood is absorbed or cleared [35]. In CNV SMH, retinal neovasculari-  [25,26,28,31,32]. The foveal thickness is expressed in microns (µm), and it has decreased after treatment. This indicates that the combination of TPA and anti-VEGF drugs is beneficial for the structural restoration of the fovea. TPA, tissue plasminogen activator; anti-VEGF, anti-vascular endothelial growth factor.

Discussion
The accumulation of blood in the macula can cause irreversible damage to photoreceptors within 24 h, and damage to visual function can be particularly serious [34]. The etiology of SMH can generally be divided into two categories: CNV (such as nAMD, PCV, and pathologic myopia) and non-CNV (such as retinal aneurysm, Terson syndrome, and trauma). CNV-associated SMH is more common. The choice of treatment for SMH is closely related to the cause of the hemorrhage. In non-CNV SMH, visual acuity usually improves to varying degrees after the blood is absorbed or cleared [35]. In CNV SMH, retinal neovascularization hinders physiological metabolic processes and acts as a basic factor for hemorrhage. Therefore, a reduction in the damage from hemorrhage in the macula and the inhibition of the persistent impact of neovascularization should be simultaneously achieved.
TPA is widely found in the aqueous humor, vitreous humor, and retina of the eye and plays an important role in eye development [36]. TPA can activate plasminogen into plasmin, which hydrolyzes fibrin and promotes blood clot dissolution and absorption [37]. The retinal expression of VEGF increases under hypoxia and induces neovascularization in the short term [38]. Based on the results of existing clinical trials, this article discusses the influence of the TPA injection site on the efficacy of combination therapy. Dr. Hilel Lewis showed in animals that labeled intravitreally injected TPA was present on the vitreous surface and failed to reach the neural retina or subretinal clots. This indicated that TPA does not diffuse through intact ILM in animals, and there is no scientific basis for the pure IVI of TPA in the treatment of SMH without vitreous hemorrhage, which may be caused by the rupture of the overlying retina [14]. This prompts doctors to select therapies according to the condition in clinical practice. Although we analyzed and concluded that the site of TPA administration does not affect the trend in BCVA improvement, rational selection of the TPA injection site according to the presence or absence of concomitant vitreous hemorrhage may provide greater benefits to patients. Anti-VEGF drugs can effectively inhibit neovascularization and reduce the associated damage to the eye [39]. A large number of studies have shown that the intraocular injection of TPA and anti-VEGF drugs can effectively and safely treat subretinal hemorrhage of different etiologies. However, the efficacy of combining the two drugs for the treatment of CNV SMH has not been systematically analyzed.
This meta-analysis included 269 eyes of 269 participants from 12 articles. Only a few of the reported complication rates in the included studies exceeded 30%, with most ranging from 2.4% to 20%; this indicates that TPA combined with anti-VEGF drugs is relatively safe. BCVA and FT were used as the main indicators of the efficacy of TPA with anti-VEGF drugs for the treatment of SMH. The results showed that TPA with anti-VEGF drug therapy significantly improved the BCVA of patients. At 1, 3, and 6 months after treatment, BCVA improved relative to that before treatment. In addition, five of these studies indicated that the combination of the two drugs reduced FT and promoted the structural recovery of the fovea. Furthermore, no significant differences in BCVA improvement were observed between subretinal and intravitreal TPA injections; this finding was not consistent with those of some other studies. For example, Wilkins et al. [40] and Ohayon et al. [41] found that the SRI of TPA can effectively promote the recovery of vision and facilitate the displacement and absorption of blood. In contrast, Tranos et al. [42] and Bell et al. [43] found that the IVI of TPA is beneficial for reducing the incidence of complications and has the same effect as SRI, consistent with our results. Notably, efficacy was limited by underlying diseases regardless of the type of treatment. Therefore, more research is needed to evaluate the efficacy of TPA and anti-VEGF therapy for SMH complicated by multiple eye diseases besides AMD.
This meta-analysis had some limitations. Several outcomes showed high heterogeneity in this research, probably because of the study population, duration of disease, and measurement methods. Random effects models were used for each of the primary outcomes to make the conclusions more reasonable, and subgroup analyses were performed according to the site of TPA injection. The 12 included articles were all retrospective studies; therefore, randomized controlled studies were lacking. Moreover, after the onset of SMH, patients are expected to receive the best individualized treatment; thus, setting a control group would be morally difficult. Therefore, the NOS scores of the included stud-ies were generally five or seven stars (Table 1). After receiving the combined TPA and anti-VEGF treatment, some patients received additional anti-VEGF treatment, which was not included in this meta-analysis. During SMH treatment, in addition to TPA and anti-VEGF combination therapy, patients may have undergone other treatments such as surgery, gas replacement, and follow-up care, for a full recovery. These treatments are affected by various factors, such as the surgeon's proficiency, medical conditions, and patient cooperation, and these factors may have introduced bias in the results of the clinical studies and secondary analyses. In addition, the sample size of some studies was slightly small, and multicenter, large-sample randomized controlled trials are needed to overcome the limitations of this study.
With the in-depth study of molecular mechanisms and the application of various experimental techniques, the treatment of SMH will predictably show standardization and precision in the future. Among vascular abnormality-related diseases, cerebral cavernous malformations have gained a lot of attention in recent years, and some studies have more deeply explored the dysregulated pathways such as oxidative stress and inflammation response in the pathogenesis by applying next-generation sequencing technology [5,6]. In ophthalmology, causative genes for abnormal angiogenic pathways are also being investigated [44]. These pioneering results based on transcriptome analysis point to genes that may be involved in pathogenesis and serve as potential therapeutic targets to diversify the diagnosis and treatment of diseases. Therefore, exploring and targeting pathogenesis at the cellular level may also become an important and insightful component of future SMH therapy.

Conclusions
In summary, this meta-analysis suggests that TPA with anti-VEGF drugs for SMH treatment is safe and can significantly improve BCVA and reduce FT, with no statistically significant difference in the treatment effect between subretinal and intravitreal TPA injections. Further studies should assess the optimal therapeutic doses of various anti-VEGF drugs and focus on monitoring complications.