Molecular mechanisms and treatments for ocular symblephara

Abstract

There are currently no effective methods to prevent or durably treat ocular symblephara, the adhesions between the palpebral and bulbar conjunctiva. How symblephara form at the molecular level is largely unknown. We present here an overview of current clinical symblephara treatments and describe potential molecular mechanisms behind conjunctival adhesion formation that may inform future symblephara treatment and prevention options. Understanding how symblephara form at the molecular level will facilitate treatment development. Preventative therapies may be possible by targeting symblephara progenitor cells immediately after injuries, while novel therapeutics should be aimed at modulating TGF-β pathways and effector cells in conjunctival scarring to treat symblephara formation more effectively.

1. Introduction

The term symblepharon (plural, symblephara) was first described in Fuchsʼ classic 1892 ophthalmology textbook as the “cicatricial adhesion between the eyelid and eyeball conjunctiva”.¹⁸ Symblephara are characterized by bands that extend from the inner eyelid surface (palpebral conjunctiva) to the ocular surface (bulbar conjunctiva). In 1919, Wilder categorized symblephara into three subtypes: 1. anterior symblepharon, when the eyelid edge or a part of it is attached to the eyeball, 2. posterior symblepharon, when the cul-de-sac or conjunctival fornix is involved, and 3. total symblepharon, when the whole eyelid surface is attached to the globe and the fornix is obliterated.⁸⁹ Conjunctival epithelium, a thin transparent mucous membrane covering the anterior globe and the posterior eyelid, is the tissue that becomes adherent in symblepharon formation. The conjunctival epithelium consists of three regions: the bulbar conjunctiva covering the anterior globe surface, the palpebral conjunctiva covering the posterior eyelids, and the forniceal conjunctiva in the region between the palpebral and bulbar conjunctiva (Figure 1).^60,67

Figure 1:

Symblephara is characterized by band formation between the palpebral and bulbar conjunctiva.

Symblephara is one of the most challenging ocular surface problems and may result in ocular motility restriction, inadequate blinking, entropion, ptosis, and secondary harmful ocular surface effects on the cornea that compromise vision.³⁵ Symblephara may occur from exogenous causes such as chemical/thermal burns and pterygium or endogenous causes such as Stevens-Johnson syndrome (SJS), ocular cicatricial pemphigoid (OCP)/mucous membrane pemphigoid (MMP), and dry eye disease.³⁵ Symblephara may also form as a severe drug reaction side effect or after glaucoma filtering surgery (GFS) as a result of conjunctival epithelial surface denuding.^36,84 Cicatrizing conjunctival disorders may be difficult to diagnose, but account for about 0.8 per million OCP cases, 0.2 per million SJS cases, and 0.2 per million cases from other causes as reported in the UK in 2012.⁵⁹

The conjunctiva from the limbus to the lid margin composes the conjunctival fornix.⁴⁰ An advantage of a deep fornix is to maintain ocular surface health by maintaining the tear reservoir, thereby preventing dry eye disease, allowing contact between the lid margin and globe, and facilitating ocular motility.⁴⁰ One symblepharon hallmark is forniceal shortening from continuous scar tissue accumulation. Forniceal shortening affects ocular motility and may obstruct tear egress from the lacrimal gland ducts, leading to dry eye disease. It may also cause blink-related microtrauma, cicatricial entropion, inadequate blinking and closure, limited Bell phenomenon, and vision defects due to restriction in ocular motility and ptosis.⁴⁰ Once symblephara form in OCP, they usually progress to plica flattening and caruncle keratinization.⁶⁷

Symblepharon assessment in cicatrizing conjunctival disorders has been difficult owing to a lack of a validated measurement system. Multiple grading and scoring methods developed by Foster, Mondino, and Tauber have been used in the past to characterize cicatricial changes in ocular MMP.^45,56 These methods use a qualitative grading system based on inferior fornix shrinkage. While these grading systems were widely used until recently, they do not assess progressive scarring well. To overcome this, a concise tool was developed based on grading of inflammation, scarring, and ocular morbidity using a fornix depth measurer, which allows for quantitative forniceal depth assessment.^56,91 Symblephara severity can be assessed based on fornix shortening in affected eyes compared with normal upper and lower fornix depth and intercanthal distance, depending on age and sex.^33,37,39 Additionally, Kheirkhah and coworkers proposed a symblephara medical management strategy based on severity by using a grading system of length, width and inflammatory activity.⁴⁰ Based on length, defined as the shortest distance between the lid margin to the limbus, Grade I symblephara have a length equal to or more than the normal palpebral conjunctiva. Grade II symblephara are shorter than the normal palpebral conjunctiva, but equal in length to the normal tarsus. Grade III are shorter than the normal tarsus, and grade IV are close to zero in length.⁴⁰ Based on width, defined as the longest horizontal distance compared to the length, the gradation is ʻaʼ if 1/3 eyelid length is covered by the symblephara, ʻbʼ if 2/3 eyelid length is covered, and ʻcʼ if greater than 2/3 is covered. Based on inflammatory activity, symblephara are graded 0 if conjunctival hyperemia is absent, 1+ if mild, 2+ if moderate and 3+ if severe (Figure 2, representative images).⁴⁰

Figure 2:

Representative images showing the different grades of symblephara. A. A representative image of no symblephara present. B. Symblephara grade Ia1+ of the superior fornix caused by alkaline chemical burn. C. Grade Ib0 symblephara following treatment for ocular cicatricial pemphigoid. D. Grade IIb0 medial symblephara following treatment for ocular cicatricial pemphigoid. E. Symblepharon IIb1+ secondary to ocular cicatricial pemphigoid. F. Grade IIb2+ central inferior symblephara of a patient with active ocular cicatricial pemphigoid. G. Symblephara grade IIIc2+ due to Stevens-Johnson Syndrome H. Symblephara grade IVb2+ in a patient with active ocular cicatricial pemphigoid I. Symblephara grade IVc3+ secondary to Stevens-Johnson Syndrome.

While symblephara formation at the molecular level is not fully understood, the known phases include conjunctival inflammation, tissue remodeling and fibrosis. Symblephara prevention or correction can be characterized based on these phases ranging from acute to chronic symblephara formation involving different cellular and biological processes. If left untreated, symblephara may eventually lead to vision loss. We summarize here symblephara treatment options and discuss how their formation at the molecular level will inform future therapy development.

2. Current clinical symblephara treatments

Symblephara formation involves adhesion of the palpebral and bulbar conjunctiva. Therefore, the main preventive technique developed in the 1950s was to create a barrier between the injured conjunctival surfaces to physically stop them from adhering. Tables 1, 2, and 3 summarize the main methods used to treat or prevent symblephara formation. Early symblephara prevention efforts involved using glass rods to manually separate symblephara;³⁶ however, the rods did not keep the raw surfaces apart while the epithelium healed, and the conjunctival tearing may have caused further inflammation. Another approach used a therapeutic soft contact lens and conformers placed into the fornix for 2 weeks to prevent symblephara formation.³⁶ While contact lenses and conformers managed to keep the palpebral and bulbar surfaces apart until re-epithelialization occurred, they were not as effective in preventing symblephara caused by severe chemical burns where subconjunctival fibrosis and scarring caused foreshortened fornices. In these patients, mucous membrane grafts from the inner lip were placed after scar tissue removal. Unfortunately, no treatment method is particularly effective for progressive conjunctival cicatrization such as in MMP where recurrent adhesions frequently occur.³⁶ Some common current therapy limitations include: bilateral or severe symblephara formation, insufficient conjunctival tissue for transplantation., buccal graft shrinkage, and mitomycin C necrosis or ocular perforation.

Table 1:

Physical barrier treatments

Authors	Main Results	Pros	Cons
D.C. Hartman 195123	Conjunctival material for grafting was better cosmetically and, in some instances, functionally, compared to mucous membrane grafts from inside the mouth or cheek	Conjunctival grafts match the cornea better than other materials and heal better	Mucous membrane grafts remain a deep red color after time
H. E. Kaufman et al 197936	Symblephara prevented by separating the injured surfaces for 2 weeks using soft contact lens and conjunctival rings without a corneal center. A device may not be necessary in conditions where only one surface is injured, where adhesions tend not to form	In extensive subconjunctival fibrosis, mucous membrane grafts in addition to scar tissue removal help prevent symblephara	If all scarring is not removed before mucous membrane grafting, cicatrix contraction may re-obliterate the fornix
P. T. Finger 199017	Individually molded 0.03mm clear acrylic sheet, suture-fixed, plaque barrier device prevented conjunctival adhesions after cryotherapy for conjunctival melanomas	Patient tolerated the device without discomfort. Even after bulbar scarring, there was no symblephara formation.	Bulbar scarring was observed 12 months after treatment
M. Farid and N. Lee 201514	Scar removal and keratolimbal allograft sutured as a spacer to prevent scar formation.	Keratolimbal allograft provides a healthy supply of epithelial stem cells	Only few patients tested. More randomized studies required to prove efficacy. Deep fornix not reached to prevent symblephara formation.

Table 2:

AMT treatments

Authors	Main results	Pros	Cons
J. Shimazaki et al 199871	Complete symblephara associated pterygium observed in 3 out of 4 patients treated with AMT along with limbal autograft	Amniotic membrane has antiadhesive properties along with low immunogenicity therefore does not induce rejection. Reconstruction with limbal autograft along with fibrosis suppression is useful in 75% cases	AMT alone results in37.5% recurrence in eyes treated for pterygium. Limbal autografts may only be used to treat monocular injuries and diseases
S. Jain and A. Rastogi 200431	Evaluated AMT as a surgical treatment for symblephara and found 100% reepithelialization rate and 30% cicatrization rate. Preexisting dry eye condition and prior conjunctival surgeries affect success rate	AMT for symblephara prevention is safe and efficient in 100% cases	Severity in subconjunctival fibroblastic response may lead to symblephara recurrence after AMT treatment
G. Singh and H. S. Bhinder 200874	Evaluated DALK with and without AMT as symblephara treatment after severe chemical burns. They found that after treatment, symblephara was found in 2% DALK as compared to 62% in control group that were only given medical therapy and no surgery	1/50 patients showed symblephara after DALK as compared to 31/50 in the control group	38% patients had vascularization in the DALK group compared to 100% in control
A. Kheirkhah et al 200840	Symblephara treatment based on severity. For Grade I and II only cicatrix lysis and AMT showed 92.8% and 100% success rate respectively. Grades III and IV cicatrix lysis, AMT and anchoring sutures were not as effective with 71.4% success. However with either oral mucosal graft or conjunctival graft addition, the success was 100%	Good correlation between symblephara severity grading and surgery type required for 100% success	Completely successful symblephara treatment required intraoperative Mitomycin C
W. Shi, T. Wang et al 200970	Lamellar keratoplasty combined with limbal stem cell treatment along with AMT, autologous conjunctival transplantation and pseudo pterygium for symblephara treatment after burns	Symblephara completely resolved in 19/29 eyes. It remained in 10/29 eyes in strip-like form which was further treated in 7/10 eyes with conjunctival grafts	Multi-step surgeries to treat symblephara
Gregory, D 201122	Acute SJS and TEN treated with cryopreserved AMT applied to lid margins, palpebral conjunctiva and ocular surface together with a symblepharon ring to separate the eyelids from the globe	6/10 patients had completely resolution, while mild symblephara was remained in 4/10 patients	Limited ProKera or AM coverage in adult eyes may lead to symblephara formation in exposed areas
A. Kheirkhah et al 201341	Discusses fornix reconstruction based on symblephara severity using cicatricial lysis, AMT, intraoperative Mitomycin C, anchoring sutures, oral mucosal or conjunctival autograft. They found 84.4% complete success, 9.4% partial success and 6.2% failure with this technique.	Success in this technique is likely due to intraoperative Mitomycin C controlling the inflammation and fibrin glue rather than anchoring sutures.	Complications included entropion in 2 eyes ocular surface keratinization in 1 eye and pyogenic granuloma in 1 eye. Additionally, no control group was used in this study
H. Ghaddar et al 201621	Symblephara caused due to ocular surface Squamous neoplasia after chemical injury treated with human AMT and symblepharon ring	This technique can be used if using ProKera is financially not viable	Patients’ recovery and visual acuity were suboptimal
N. Kara 201835	AMT with modified ocular surface ring made from feeding tube expands the amniotic graft coverage to the entire ocular surface reaching the deeper fornix	AMT with modified ocular surface ring does not require sutures, therefore eliminates problems such as subconjunctival hemorrhage, infection, foreign body rejection and tissue necrosis. It is also inexpensive	Only tested in a single patient so far
S. Shanbagh et al 201968	AMT secured with cyanoacrylate in SJS/TEN patients combined with symblepharon ring prevents symblephara formation.	Sutureless bedside technique shows long term benefit in 4 patients.	Care needs to be taken while using cyanoacrylate as it may be toxic and abrasive to the ocular surface

Table 3:

Mucosal membranes and topical treatments

Authors	Main results	Pros	Cons
A. Tsuji et al 201184	Pterygium prevention for at least 6 months by Tranilast ophthalmic solution instillation after conjunctival free flap transplantation	No symblephara recurrence at 6-month follow up. Preoperative Tranilast instillation may have been effective for symblephara prevention	Tranilast effects over time are not known
A. Sant’Anna, et al 201264	Minor salivary gland and labial mucosal membrane transplantation to treat sever symblephara caused by SJS	Schirmer I test improved in 73.7% patients and greater tear production was observed	Neovascularization increased post operation in 29.4% patients. Approximately 50% improvement in symptoms after surgery
T. Li et al 201846	Mucosal membrane from the lip containing goblet cells to repair the bulbar conjunctiva and a reversed split-thickness skin graft to repair the palpebral conjunctiva and the conjunctival fornix.	Treatment option for recurrent symblephara. No symblephara recurrence at 6-month follow-up	Only tested in a single patient so far.

Abbreviations: AMT-Amniotic Membrane Transplantation; SJS-Stevens-Johnson syndrome; TEN-Toxic Epidermal Necrolysis; DALK-Deep Anterior Lamellar Keratoplasty.

Silicone spacers may give temporary relief, though symblephara may recur when the spacers are removed.¹⁴ Different treatment options have been proposed over the years based on symblephara severity. Tables 1, 2 and 3 summarize symblephara clinical treatment methods in human patients subdivided by technique type. Close analysis yields recent AMT, mucosal membrane, and topical treatments such as AMT with modified ocular surface ring made from feeding tubes, mucosal membrane from lip and tranilast ophthalmic solution may be promising treatments for symblephara; however, more data will be required to determine if they are beneficial over long periods, and they need to be tested in more patients.

3. Molecular mechanisms involved in symblephara formation

To prevent symblephara, one must understand the molecular basis behind their formation, yet currently these molecular mechanisms are largely unknown or poorly understood. Here we propose potential underlying molecular pathways involved in symblephara formation based on its precursor stages: subconjunctival inflammation, fibrosis and scarring. According to Fosterʼs OCP stages, symblephara develops at stage 3 after subconjunctival scarring.⁶⁷ This characterization suggests that one initial symblephara formation sign might be subconjunctival scarring. We describe several pathways involved in ocular diseases that lead to subconjunctival fibrosis. Based on these, we predict the cells (Figure 3) and pathways (Figure 4) that may initiate symblephara formation.

Figure 3:

Diagram showing the different cell types that may be involved in symblephara formation including fibroblasts, myofibroblasts, fibrocytes, cell adhesion molecules and immune cells

Figure 4:

Schematic diagram showing the molecular basis of Symblephara. Symblephara may be caused by subconjunctival fibrosis that can be induced due to multiple pathways including the Hippo pathway that has been implicated in fibrosis upregulation. TGF-β is a master regulator of fibrosis. Symblephara may be governed by TGF-β regulation, which may lead to ECM production, decrease in matrix-degrading metalloproteases, integrins and increase in protease inhibitors. TGF-β can in turn be regulated by BMP signaling, which also effects Smad. Future therapies should be aimed at controlling TGF-β signaling either through direct down regulation or through the modulation of fibrosis-forming factors.

3.1. Cells involved in symblephara formation

3.1.1. Fibroblasts and myofibroblasts

Conjunctival fibroblasts from ocular mucous membrane pemphigoid (ocular MMP) have been implicated in acute and chronic progressive conjunctival fibrosis.⁶⁶ In most fibrotic disorders, inflammation generally precedes fibrosis development; however, cicatrization may progress even when inflammation is visibly absent. Saw and coworkers determined a profibrotic phenotypic change in ocular MMP fibroblasts compared to normal fibroblasts based on increased cell migration, cell division, chemotaxis and matrix metalloproteinase synthesis in vitro, that may cause chronic progressive fibrosis.⁶⁶ Additionally, interleukin-13 (IL-13) was found to be highly expressed in MMP conjunctival stromal cells, suggesting that IL-13 might have a profibrotic, proinflammatory effect on conjunctival fibroblasts, leading to symblephara formation.⁶⁵ Since fibrosis is characterized by TGF-β activation along with collagen deposition, conjunctival fibroblasts in vitro were treated with TGF-β induced heat shock protein 47 (HSP47), which may promote pro-collagen synthesis and fibrosis from collagen type 1 production.^{61, 10}

A critical stage in fibrosis is myofibroblast development and persistence.^50,72 Myofibroblasts get activated in response to tissue injury and their main function is to repair lost or damaged ECM thereby leading to fibrosis.²⁶ Myofibroblasts classically originate from fibroblasts and have contractile features similar to smooth muscle.^26,62 Hypertrophic scarring and fornix contracture due to symblephara formation may be caused by myofibroblast activation. TGF-β plays a major role in myofibroblast trans-differentiation. At the injury site, epithelial cells release TGF-β and platelet-derived growth factors (PDGF) into the tears from the conjunctiva and lacrimal gland and initiate myofibroblast development from precursors. Subconjunctival scarring has also been associated with fibroblast migration in wound healing by TGF-β2 upregulation.^81,8

Myofibroblasts can also originate through epithelial mesenchymal transition (EMT) during fibrosis as an inflammatory response where epithelial cells transition into fibrogenic myofibroblasts.⁷² EMT was found to play an important role in mucosal membrane and exocrine chronic graft-versus-host disease where conjunctival basal epithelia were observed in the subconjunctival cell nuclei beneath the basal lamina.⁵⁴ This suggests that during conjunctival fibrosis epithelial cells may gain the mesenchymal phenotype and migrate to the subepithelial stroma, which may also be responsible in symblephara formation.

Perivascular mesenchymal stem/stromal cells (MSCs)-like cells are an additional source of myofibroblasts in fibrotic diseases in various organs triggered by chronic exposure to pro-inflammatory and pro-fibrotic cytokines.¹ Although MSCs have been shown to have profibrotic properties, they have also been explored as a fibrotic disease treatment because of their immunosuppressive properties.⁸⁵ MSCs are activated pericytes that respond to injury by secreting bioactive agents that have been identified in the conjunctival stroma apart from other sources and may be considered as a topical therapeutic for symblephara.^7,53,5

3.1.2. Fibrocytes

Fibrocytes are bone-marrow derived fibroblast-like cells that circulate in the blood, entering injured tissues to produce connective tissue proteins like vimentin and collagen I and III.^58,55 Wound repair involves cytokine, chemokine, and growth factor secretion that leads to different cell types being recruited to the injured site.⁵⁸ During the inflammatory stage, neutrophils and monocytes are recruited to the wound. In the next stage, re-epithelialization occurs, and fibroblasts secrete collagen to form ECM. In the final healing stage, fibrocytes, recruited to the injury site by TGF-β regulation, differentiate into myofibroblasts that pull the wound edges together.⁵⁸

Fibrocytes contribute towards fibrosis formation by differentiation into myofibroblasts, matrix metalloproteinase production and TGF-β release. They have been identified in many fibrotic diseases such as lung, liver and renal fibrosis, bronchopulmonary dysplasia and skin scarring.⁴⁴ Fibrocytes have been previously implicated in corneal wound healing and fibrosis, where the cornea may modulate and stimulate fibrocytes to mature myofibroblasts that are then involved in fibrosis.^55,44 Fibrocytes are also found in the subconjunctival connective tissue however their involvement in conjunctival scarring is poorly understood.⁸ Understanding fibrocyte activation and involvement in symblephara formation could facilitate novel symblephara therapy development.

3.1.3. T cells

T cells have been implicated in fibrotic diseases as an immune response to scarring leading to cytokine production that stimulate fibroblasts to produce collagen. In chronic diseases and inherited cicatrizing diseases such as epidermolysis bullosa and pemphigus vulgarus the conjunctiva is infiltrated by CD8⁺ T cells.¹² Additionally, mast cells and eosinophils are also found in acute scarring leading to profibrogenic cytokine production.¹⁰ Autoreactive T cells, which may arise as an inflammatory response from tolerance loss to epithelial basement membrane proteins, and fibrinogenic growth factor upregulation in OCP patients has been reported in the subepithelial conjunctival stroma.^10,49,6 T cells may also be involved in symblephara formation in other autoimmune bullous diseases where autoantibodies lead to basement membrane zone disruption.¹⁶

3.1.4. Dendritic cells

Dendritic cells are antigen presenting cells found in skin, mucous, membrane and lymphoid tissue that initiate the primary immune response.^48,9 Classical dendritic cells are CD11c+ myeloid-derived cells that are T cell stimulators.⁹ Dale and coworkers described the emerging role of dendritic cells as the cause for ocular scarring and tissue remodeling in allergic eye diseases such as atopic keratoconjunctivitis.⁹ They suggest that CD11b+ dendritic cells that express pro-inflammatory TNF-α may contribute to inflammation and fibroblast activation in the conjunctiva. CD11b+ dendritic cells may indirectly activate fibroblasts through TH2 stimulation, which produces IL-13 and TGF-β. This leads to eosinophil recruitment and consequent inflammation that promotes tissue damage. Activated fibroblasts in turn lead to inflammation via inflammatory/monocyte-derived dendritic cells, or additional CD11b+ dendritic cells to the injured conjunctiva.⁹ Additionally, fibrosis also results from aldehyde dehydrogenase/retinoic acid (ALDH/RA) mediated dendritic cell paracrine effect that activates a profibrotic phenotype in fibroblasts.^3,10 In an allergic eye disease mouse model with ovalbumin-induced conjunctival inflammation, ALDH/RA autoregulation in ocular MMP fibroblasts was the underlying mechanism for conjunctival scarring, with ALDH inhibition with disulfiram as a potential therapeutic for alleviating fibrosis.² Symblephara formation may share a similar molecular mechanism employing immune-mediated fibrosis pathways. It is not known whether dendritic cells directly or indirectly activate conjunctival fibroblasts. Symblephara formation from allergic eye diseases would be better understood if these critical questions are answered.

3.1.5. Neutrophils

Neutrophils are involved in acute inflammation and may play an important role in adaptive immunity by secreting chemokines that attract other immune cells to the injury site.⁴³ In addition to acute inflammation, conjunctival epithelial neutrophils were also found to be directly correlated with progressive conjunctival scarring and inferior forniceal foreshortening in ocular MMP patients.⁹⁰ In SJS/TEN patients, CD45^INTCD11b⁺CD16⁺CD14⁻ neutrophils were found to infiltrate into even mildly involved or clinically quiescent conjunctival mucosa indicating inflammation persistence.⁹² Conjunctival neutrophils could therefore be considered a biomarker for ocular surface disease progression, including symblephara formation, to assess progressive fornix shrinkage when visible inflammation is absent.⁹⁰

3.1.6. Stem cells

Under normal circumstances, stem cells help regenerate adjacent tissue after injury, such as when limbal stem cells found in the palisades of Vogt regenerate and replenish corneal epithelial cells as a regular process or after injury;²⁵ however, physical or chemical injury to the cornea such as alkali burns may cause limbal stem cell deficiency (LSCD) leading to corneal conjunctivalization and eventual blindness.²⁵ Histological changes include ocular surface epithelial hyperplasia, squamous metaplasia, active stromal fibrosis and severe inflammation suggesting that stem cells loss may contribute to squamous metaplasia.¹⁵ Similarly, while conjunctival stem cells may be important in preventing conjunctival scarring and symblephara formation, their injury or loss after chemical burns may also contribute to symblephara formation.

Currently, there is a debate regarding conjunctival stem cell location. Conjunctival stem cells have been reported in the limbus, forniceal region, and bulbar/palpebral conjunctiva.⁶⁰ The fornix may be a preferred location to maintain conjunctival stem cells based on its ability to provide greater physical protection and because it is rich in goblet cells, intraepithelial mucous crypts, vasculature and immune cells.⁶⁰ To corroborate this, corneal stem cell marker expression like ABCG2 and p63 were found in the medial canthal and inferior forniceal regions of human conjunctivas.⁷⁷ Previous studies have explored culturing conjunctival progenitor cells on amniotic membrane for conjunctival reconstruction, which may be useful as a symblephara treatment.⁵¹ Additionally. in vitro induced pluripotent stem cells (iPSC) have been used to model progressive organ fibrosis to mimic common cellular and molecular pathways involved in fibrosis including chronic injury, inflammation, aberrant repair and tissue remodeling.⁸⁶ The iPSC-derived tissues produced endogenous TGF-β, chemokines and cytokines, activate fibroblasts to myofibroblasts, and lead to progressive collagen deposition similar to human fibrotic disease.⁸⁶ This system could be used to evaluate small molecule therapies for symblephara.

3.2. Pathways involved in symblephara formation

3.2.1. Transforming growth factor-β

As a first response to tissue injury, transforming growth factor (TGF)-β is upregulated leading to fibrosis.¹³ TGF-β is widely known as the master regulator of fibrosis and has been implicated in renal, hepatic, pulmonary, and cardiac fibrosis.^30,52 TGF-β has three main isoforms: TGF-β1, expressed in endothelial, hematopoietic and connective tissue, TGF-β2, expressed in epithelial and neuronal cells, and TGF-β3 that is mainly expressed in mesenchymal cells.³⁰ TGF-β1 regulates fibrosis by extracellular matrix (ECM) secretion in conjunction with an upregulation of matrix proteins, a decrease in matrix-degrading protease production, and an increase in protease inhibitor production. It also modulates integrin expression in a manner that increases cellular adhesion to the matrix.¹³ Conjunctival fibrosis in patients with OCP has been shown to be associated with upregulated mRNA levels of TGF-β1 and TGF-β3 but not TGF-β2 in stromal fibroblasts and macrophages; however, wound healing after GFS and subsequent conjunctival fibrosis formation has been associated with the Hippo pathway where conjunctival fibrosis was caused by TGF-β2 mediated signaling. Verteporfin, a YAP/TAZ inhibitor, is used to suppress the TGF-β2-YAP/TAZ/smad pathway, leading to antifibrotic effects.²⁰ TGF-β1 also stimulates human tenon capsule fibroblast proliferation after GFS, thus further contributing to wound healing and fibrosis.⁸⁰ These studies suggest that TGF-β might play a major role in symblephara formation.

3.2.2. Histone deacetylases

Histone deacetylase (HDAC) is a form of posttranslational modification that removes the acetyl group from histone tails, which regulates transcriptional activity by modulating chromatin compaction.⁹³ HDACs have previously been implicated in fibrogenesis acceleration, while HDAC inhibitors have been shown to regulate fibrosis. HDACs function as pro-inflammatory molecules that release profibrotic cytokines such as IL-1β, IL-6 and TNF-α.⁹³ HDAC1/2 and HDAC3 regulate inflammatory expression in epithelial cells.⁹³

HDAC1 overexpression was found to accelerate conjunctival fibrosis in a rat trabeculectomy model.⁷⁸ Trichostatin A, a HDAC inhibitor, reduced bleb vascularity, leukocyte infiltration, α-SMA and TGF-β1 in rat conjunctiva without any apparent corneal epithelial toxicity.⁷⁸ In another study, the HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA), was reported to prevent postoperative fibrosis in a GFS rabbit model where SAHA treatment reduced bleb vascularity and collagen deposition at the surgery site.^19,69 They found that SAHA suppressed TGF-β2-induced angiogenic cytokines thereby inhibiting myofibroblast differentiation and ECM production. These results suggest that HDAC inhibitors could be considered in potential symblephara therapy.

3.2.3. Bone morphogenic proteins

Part of the TGF-β superfamily, bone morphogenic protein (BMP), while initially known for its involvement in bone morphogenesis, has also been linked to fibrosis.¹¹ BMPs induce Smad-dependent (canonical) or Smad-independent (non-canonical) signaling pathways that affect gene transcription.²⁴ BMP signaling has been studied in the context of liver, kidney, lung and heart fibrosis displaying a complex interplay between TGF-β and BMP signaling resulting in either enhancing or antagonizing fibrosis and regeneration.^11,24

BMP7, one of the 15 different BMP ligands, has been shown to play an antagonistic role in fibrosis. BMP7 can inhibit EMT in renal fibrosis and also plays a protective role in hepatic and cardiac fibrosis.⁸⁷ Several studies have explored BMP7ʼs role in ocular fibrosis including BMP7ʼs potential therapeutic effect in a murine corneal alkali injury model.⁶³ They demonstrated that BMP7 overexpression using adenovirus was effective for ocular burn treatment, mainly via TGF-β suppression and Smad1/5/8 signaling activation.⁶³ Ectopic BMP7 was shown to decrease monocytes/macrophages invasion into corneas after chemical burns, which accelerated healing.⁶³ BMP7 has also been shown to inhibit TGF-β2 ECM production.^87,73 The role of different BMP ligands might differ, however, where one group found BMP6 and activinA mRNA and protein expression significantly increased in scar tissue compared to normal conjunctiva after GFS.⁴ While all BMPs have not been evaluated in the conjunctiva, BMP7 appears to play a therapeutic role in symblephara treatment and prevention.

3.2.4. Cell adhesion molecules

Cell adhesion molecules (CAM) are required for the induction of processes that accompany tissue regeneration after inflammatory damage, such as cell proliferation and differentiation. In allergic diseases such as conjunctivitis, CAMs including E-selectin, ICAM-1, and vascular cell adhesion molecule-1 (VCAM-1) are upregulated in the conjunctiva.^75,88,82 Mast cells also regulate ICAM-1 and VCAM-1 expression. Whitcup and coworkers showed that ICAM-1 expression in the conjunctival vascular endothelium was upregulated after ragweed administration in a mouse conjunctivitis model. Allergic patients also showed conjunctival epithelial ICAM-1 expression upregulation after topical antigen challenge. After injecting the mice with anti-ICAM-1 and anti-LFA-1, they observed inflammatory cell upregulation, predominantly neutrophils and eosinophils infiltrating the conjunctiva. H&E staining demonstrated that lymphocytes and eosinophils infiltrate the injured fornix.⁸⁸ These results indicate that ICAM-1 may also be expressed in symblephara tissue.

Subepithelial mononuclear infiltration was also observed in patients with conjunctivalized corneas due to chronic SJS.³⁸ They observed anti-LFA-1, anti-CD4, anti-CD8, anti-CD68 anti-ICAM-1 and anti-HLA-DR expression. HLA-DR and ICAM-1 immunoreactivities were detected in blood vessels on subepithelial infiltrating cells, epithelial and endothelial cells while LFA-1, CD4, CD8, and CD68 immunoreactivities were detected on subepithelial mononuclear infiltrating cells only. IFN-γ immunoreactivity was detected in subepithelial infiltrating cells, conjunctivalized corneal epithelial basal layer and vascular endothelial cells. Kawasaki and coworkers found cell surface antigen and cytokine immunoreactivity patterns similar in conjunctivalized corneas from three chemically injured eyes and SJS corneas.³⁸ Since symblephara are caused by SJS and chemical injury, symblephara might also show a similar cytokine expression pattern.

4. Current research and future treatment options

4.1. Animal models

Animal models have thus far been the gold standard for understanding disease mechanism and progression and are critical for testing potential anti-adhesion strategies. In 2019, Kang and coworkers demonstrated symblephara formation in a rabbit alkali injury model.³⁴ Their protocol involved placing a 10mm diameter Whatman filter disc soaked in 2N NaOH under the rabbit superior eyelid for 60, 90 and 120 seconds and then rinsing with saline until the pH is approximately 7. Over 4 weeks, they observed symblephara formation in these rabbits and graded the symblephara severity based on two parameters, including fornix depth, defined as the center distance from the lid margin to the deepest fornix, and volume reduction measured via conjunctival sac casting. They found progressive symblephara formation and fornix shortening in the late stages of chemical burns along with fibroblast recruitment in the subconjunctival collagen fibers, which is characteristic in human symblephara formation as well.

The only other animal symblephara model was described by Tajiri and coworkers.⁷⁹ They developed a canine symblephara model using 1N NaOH for 15, 60 and 90 seconds, followed over a 5 week time course. In the 15 second injury group, they observed no symblephara formation after 5 weeks. In the other two groups, symblephara formed within 5 weeks. As in the rabbit model, they also observed vimentin-positive fibroblasts at the injury site, where α-SMA-positive myofibroblasts that produce actin filaments caused stronger adhesion due to tissue shrinkage.

While these animal models are useful in gleaning information about symblephara formation, there are disadvantages to using rabbit and dog injury models, such as their larger size and difficulty implementing high throughput studies. Mouse symblephara models may be most useful in conducting large scale studies to determine effective preventative therapies.

4.2. Anti-adhesion strategies

Anti-adhesion strategies have been described in other organs such as the intestines and abdomen. Isotonic solutions containing macromolecular dehydrating agents may have therapeutic value when applied topically for epithelial discontinuities.²⁸ Using a rat cecum adhesion model, Horii and coworkers²⁹ found that a thermally cross-linked gelatin film had better antiadhesion properties, physical strength, and ductility, without being cytotoxic, in comparison to conventional hyaluronic and carboxymethylcellulose film. In a rat laparotomy adhesion model, Khorshidi and coworkers⁴² found sodium hyaluronate and sesame oil to be effective at preventing adhesions due to their anti-inflammatory and anti-oxidative properties. Antiadhesion agents have been studied in abdominal adhesions, where they prevent contact between damaged tissues by serving as a barrier. In a study comparing anti-adhesion products in a cecum to abdominal wall adhesion model, seprafilm was found to be superior in preventing adhesions, followed by polylactic acid, hyaluronic acid, chitosan, and PEG⁴⁷. Product limitations include difficulty in handling and inability to prevent all adhesion types. To overcome this, Stapleton and coworkers⁷⁶ developed a dynamic cross-linked supramolecular polymer-nanoparticle hydrogel to prevent pericardial adhesions. They found marked adhesion reduction using hydrogels compared to seprafilm. This hydrogel has complex viscoelastic properties and sustained local retention making it a good candidate for testing in ocular adhesion formation models.

4.3. Cell lineage tracing

While dendritic cells and fibrocytes may cause ocular inflammation and scarring, there may be other symblephara etiologies that have not been explored. Tsai and coworkers⁸³ found by cell lineage tracing and clonal analysis that the molecular events leading to peritoneal adhesions originate in the injured surface mesothelial cells after surgery. They found that mesothelial cells upregulated Hif1a that preceded adhesion development. Mesothelial cells are also found in the human cornea, limbus and conjunctiva, as shown by Jirsova and coworkers.³² Future studies are needed to determine if mesothelial cells are potentially involved in symblephara formation. In addition, it is likely that lymphocytes also infiltrate into symblephara. If that is the case, lifitegrast (Xiidra®, Novartis) an ICAM-1 and LFA-1 antagonist used for dry eye disease^57,27, could also be a potential therapeutic agent to prevent symblephara.

5. Conclusion

Symblephara have long been recognized as an ocular complication of chemical injuries and autoimmune diseases; however, the molecular mechanisms involved in symblephara formation are still poorly understood. While there are various treatment options to prevent symblephara formation, including AMT and contact lenses, most methods are not effective in treating recurrent symblephara. We have summarized current symblephara treatments and the molecular pathways that may be involved in symblephara formation. TGF-β appears to play a major role in symblephara formation along with the BMP and HDAC signaling pathways. In conjunction, myofibroblasts, fibrocytes, CAMs and dendritic cells all may contribute to symblephara formation. Future studies are needed to determine if mesothelial cells are involved in symblephara formation and to test potential antiadhesive therapies to prevent recurrent symblephara.