I. Introduction

The cornea is lined by non-keratinized, stratified squamous epithelium. At the limbus, there is a gradual transition of these cells to non-keratinized, stratified columnar epithelial cells, along with mucin-secreting goblet cells, forming the conjunctival epithelium. The corneoscleral limbus lies between these two epithelia in a healthy eye. The limbus contains palisades of Vogt, the basal layer of which function as a repository for the corneal epithelial stem cells.(1, 2)

These limbal epithelial stem cells (LESC’s) divide whenever the corneal epithelium is required to be replenished. As the stem cells divide, one of the daughter cells retains the primitive nature of the parent cell and functions to maintain the stem cell pool. The other daughter stem cell differentiates to acquire the physiological nature of a specific tissue, like the corneal epithelium. These cells are known as ‘transient amplifying cells’, which then divide and migrate centripetally, circumferentially, and vertically from the basal layer to replace the surface epithelial cells that are desquamated. This theory for corneal homeostasis was given by Thoft and is known as the XYZ hypothesis of corneal epithelial maintenance.(2)

The LESC’s act as a mechanical barrier preventing the conjunctival epithelium from growing over the corneal surface. Any insult to this barrier disrupts it, allowing the conjunctival epithelium to grow over the cornea resulting in limbal stem cell deficiency (LSCD).(3, 4)

II. Definition and pathogenesis of LSCD

LSCD is an ocular surface disease caused secondary to a decrease in the number of, or abnormal physiological functions of the LESC's. This interferes with the function of the LESC’s to regenerate and maintain a healthy corneal surface.(3) LSCD is characterized by progressive conjunctivalization (the corneal epithelium is replaced by the conjunctival epithelium) along with signs of poor corneal epithelial healing such as chronic inflammation, persistent epithelial defects, corneal neovascularization, and corneal scarring.(5)

The complete loss of LESC’s and their function, results in Total LSCD, where the corneal epithelium is completely replaced by the conjunctival epithelium, thus resulting in corneal blindness. (Figure 1) Partial LSCD may occur in the presence of few functional LESCS’s, characterized by incomplete conjunctivalization of the corneal surface.(3)

Our understanding of the physiology of LESC’s, their role in maintaining the ocular surface, and that LSCD is a manifestation of their dysfunction, has fueled many surgical techniques involving transplantation of these stem cells, over the past few decades. LSCD that was once considered as an irreversible blinding disease can now be treated with various limbal stem cell transplantation (LSCT) techniques, the most recent of which is simple limbal epithelial transplantation (SLET).[3],[4],[5]

Simple limbal epithelial transplant (SLET) is a type of LSCT where one clock hour of the limbal tissue containing the LESC’s is harvested from the healthy donor eye and transferred to the eye with LSCD. This innovative surgical technique was first described by Sangwan et al in 2012.(6) Depending on the source of the donor tissue, LSCT can either be autologous (from the unaffected fellow eye of the same person) or allogeneic (from a cadaveric donor or from a live-related donor).

III. What are the other types of autologous LSCT for unilateral LSCD, other than SLET?

A. Conjunctival-limbal autograft (CLAU) described by Kenyon and Tseng, 1989(7)

Technique: Limbal tissue attached to a conjunctival carrier (of around 3 clock hours) is transplanted from the healthy donor eye to the contralateral recipient eye with LSCD, at 12 and 6’o clock positions. The transplanted tissue is hence located at the physiological position, that is, the limbus.

Indications: In cases of unilateral total LSCD with severe symblepharon which require a complex procedure to address the LSCD with symblepharon simultaneously, with or without a corneal transplant

Limitations: Iatrogenic LSCD in the donor eye.(8)

B. Cultivated limbal epithelial transplantation (CLET) described by Pellegrini et al 1997.(9)

Technique: Limbal tissue (2 × 2 mm) harvested from the contralateral healthy eye is expanded in vivo (in the laboratory), to a multilayer sheet of corneal epithelial cells on an amniotic membrane over a period of 10-14 days, this is then transferred to the recipient cornea with LSCD

Advantages: The amount of limbus harvested is small, thus no risk of iatrogenic LSCD, hence the procedure is repeatable

Limitations:

1. Considering this technique requires a laboratory for in vivo cultivation of the epithelial cells, difficult in a resource-limited setting.

2. The cultivated corneal epithelial cells may get contaminated while transporting them, strict protocols are required to be followed.

3. The cultivated cells are xenogeneic material; thus, potential safety issues arise.

4. Difficult logistically for both the patient and the surgeon as a gap of 10-14 days exists between the harvesting of limbal tissue and the transplant of the multilayer sheet of epithelial cells.

5. Expansion of the epithelial cells over the amniotic membrane might fail.

IV. Clinical presentation of LSCD (3, 4, 10, 11)

A) Symptoms:

• Conjunctival hyperemia (chronic)
• Decreased vision
• Photophobia
• Tearing
• Blepharospasm
• Foreign body sensation
• Recurrent episodes of pain (due to repeated epithelial breakdown)

B) Signs:

*These features are better appreciated under cobalt blue filter on slit-lamp examination, after staining the corneal surface with fluorescein dye.

V. Indications of SLET:

A) Autologous SLET for unilateral LSCD (source of the donor tissue is the unaffected fellow eye of the same person)

1. Ocular chemical injury (12-14)
2. Iatrogenic LSCD (post topical Mitomycin C use, post excision of OSSN, post multiple surgical interventions)(15-18)
3. Primary/recurrent pterygium (19-21)
4. Previously failed LSCT (22)

B) Allogeneic SLET for bilateral LSCD (source of the donor tissue is the cadaveric or live-related limbus) (23)

Inflammatory causes such as

• Stevens-Johnson Syndrome (SJS),
• Mucous membrane pemphigoid (MMP)
• Chronic ocular allergy – Vernal Keratoconjunctivitis (VKC)
• Graft versus host disease

V. Pre-operative Considerations (23)

A. Evaluation of the donor cornea

1. Most common site for harvesting the donor tissue is the superior limbus as the limbal palisades of Vogt are maximum in this area.

2. The donor limbus should be healthy. Ensure there is no previous history of trauma (mechanical/chemical), inflammation, or surgery performed at the site from where the tissue is to be harvested.

3. If cadaveric limbal tissue is used (cadaveric allogeneic SLET) (24)

• It should be utilized within 48 hours of harvesting the tissue
• Have clinically visible limbal palisades of Vogt
• No associated epithelial sloughing
• Donor age less than 60 years

These criteria ensure that the proliferative potential of the cadaveric tissue is as good as tissue from a live-related donor.(24)

B. Evaluation of the Recipient Cornea (23)

1.Ensure that the recipient corneal surface is wet

• Repeated Schirmer’s score value of more than 10 mm after instilling topical anesthetic drops
• There should be no evidence of conjunctival/corneal surface keratinization

2. No existing adnexal pathologies (lagophthalmos, entropion/ectropion, trichiasis/distichiasis)

• If adnexal pathologies are present, they need to be corrected first before performing SLET

3. Look for location and extent of symblepharon

• A symblepharon is a pathological condition where the palpebral conjunctiva partially or completely adheres to the bulbar conjunctiva
• Cases that need symblepharon release with conjunctival autograft along with SLET are cases where:

1. The symblepharon extends till the limbus (Figure 2)- this may interfere with placement of eye speculum intra-operatively and placement of bandage contact lens (BCL) or scleral lenses in the post-operative period
2. The symblepharon limits the extraocular movement of the eye causing diplopia.

4. Presence of underlying stromal scarring and corneal thickness

• An ideal case would be an eye with clear to translucent underlying stroma of the cornea (Figure 3) – this can be delineated on an anterior segment optical coherence tomography (AS-OCT)

• In cases that have dense stromal opacification (Figure 4A), an AS-OCT can provide additional information like:

a. Extent of underlying stromal scarring (Figure 4B) - These cases may need additional surgery like a full-thickness penetrating keratoplasty or anterior lamellar keratoplasty in addition to SLET to address the scarring, either as a single surgery or a staged procedure later.

b. Extent of corneal thickness (linear scan) - Areas that are thin need careful dissection of pannus over them, as there is a risk of perforation in these areas

Absolute contraindications for SLET (23)

1. Dry ocular surface (defined as repeated Schirmer's I score with anaesthesia of less than 10 mm or presence of corneal or bulbar conjunctival keratinization) (Figure 5)
2. blind eye with no visual potential
3. disorganized anterior segment (adherent leukoma, anterior staphyloma, or extensive peripheral anterior synechiae)
4. presence of uncorrected adnexal pathologies like lagophthalmos, ectropion, entropion, trichiasis, and dacryocystitis

C. Pre-operative counseling:

• Since harvesting the limbal graft involves operating on the unaffected eye, it is important to explain to the patient and relatives/guardians clearly about the procedure and address all their queries.
• The goal of surgery should be explained:

1. The cosmesis of the affected eye is likely to improve after surgery, however SLET being an epithelial regenerative procedure has limited impact on corneal stromal opacification. The patient should be counselled that additional surgeries may be required later to improve vision.
2. In long-standing cases, especially when initial insult has occurred in childhood, certain coexisting pathologies like ptosis, squint, or poor vision due to amblyopia may not improve after surgery. For better cosmesis, conditions like ptosis and squint may need additional surgical procedures.

• Consent should be taken for surgical intervention in both the eyes, after explaining the procedure and associated risks in a language the patient/relative/guardian best understands.

VI. Surgical technique(23)

The surgical steps are sequentially depicted in the following videos:

A. Anesthesia

Surgery in children should always be performed under general anesthesia. For adults, harvesting the limbal graft can be done under topical/subtenon’s/peribulbar anesthesia. Surgery on the affected eye should be performed under peribulbar anesthesia.

B. Harvesting the limbal graft from the donor's eye

Marking the biopsy site is not mandatory, but it is advisable to do so. In live donors (autologous/allogeneic), the marking is done using a skin marking pen, slightly beyond the limbus on the conjunctival surface. The limbus should not be marked directly as the alcohol in the marking ink may damage the LESC’s.

The limbal graft of around one clock hour/3.5-4mm limbal tissue is sufficient. Conjunctival bleb is created using fluid, slightly behind the marked area for biopsy. A limbus-based conjunctival flap is lifted till the insertion of Tenon’s capsule at the limbus. This area is marked by blood vessels and defines the posterior limit of the limbus. Dissection is carried out using a 15 number blade on a Bard-Parker handle.

The flap is reposited, and the conjunctival part is separated from the limbus by excising the grey demarcation line (anterior to this line is the limbus and posterior to the line is the white conjunctival tissue)

The conjunctival part is preserved or discarded depending upon whether the conjunctival graft needs to be performed with SLET. For example, in cases with extensive symblepharon, the limbal tissue is excised separately using Vannas or Wescott scissors and preserved in a balanced salt solution (BSS)

In cadaveric donors, it is not possible to make a conjunctival flap, as the corneoscleral rim may not have any conjunctival tissue. ‘Snip biopsy’ technique has been described to harvest the limbal tissue from cadaveric donors. In this technique, the limbal tissue is pinched using a Lim’s forceps and one clock hour is cut using a Vannas or Wescott’s scissors. This tissue is bulky as it contains more stromal tissue as compared to a graft harvested from a live donor and requires stromal trimming before transplantation.

C. Dissection in the recipient eye

Any symblepharon interfering with insertion of eye speculum should be excised first. Conjunctival peritomy is performed 360 degrees around the presumed limbus using Vannas scissor. This provides a good amount of tissue to hold on to, while dissecting the pannus over the cornea. Careful dissection of the pannus is performed over the corneal surface using an appropriate techniquelike peeling, and blunt and sharp dissection. Dissection over the central cornea is performed last, as the cornea is thinnest in this area and there is a higher risk of inadvertent corneal perforation. Excessive manipulation of the corneal stroma to achieve a clear corneal surface is best avoided.

The hAM is placed epithelium side up and secured over the recipient corneal surface with fibrin glue (TISSEEL kit from Baxter AG, Austria). The hAM is ironed out with a blunt spatula and tucked all around the recessed conjunctival margins. Folds in the hAM over the cornea should be avoided.

The limbal tissue is then removed from the BSS and divided into 6-10 pieces using a Vannas scissor. These grafts are then placed in a concentric fashion in the midperiphery of the cornea, outside the pupillary area, over the hAM.

Ensure that the grafts are placed in the correct orientation with the epithelial side up.

A drop of fibrin glue is placed over each piece to ensure that they adhere to the underlying hAM. Allow sufficient time for the glue to polymerize (seen as white lines on the surface) over the transplants, before placing the BCL on the corneal surface.

The speculum should be removed carefully by simultaneously cutting the fibrin glue strands attached to the speculum with Vannas scissor. If adequate care is not taken while removing the speculum, the sheet of fibrin glue holding the transplants may be pulled and dislodged, and hAM may also be dislodged simultaneously.

In children, temporary lateral suture tarsorrhaphy is recommended postoperatively, to avoid accidental loss of BCL/hAM with transplants as children often tend to rub their eyes.

VII. Post-operative regimen(23)

VIII. Mechanism of action of SLET

The epithelial cells grow from all directions, usually from each transplant, until they form a uniform sheet of confluent epithelial cells covering the surface of the cornea. This has been demonstrated by Mittal et al in study to serially demonstrate this process using fluorescein staining.(25) Other conclusions drawn from the study were:

1. It takes around 14 days for complete epithelization of the ocular surface.
2. All the transplants usually do not assume activity at the same time or at the same rate.
3. Inactive transplants were overrun by epithelial cells growing from the adjoining transplant.
4. The hAM provides a substrate for these cells to proliferate, migrate and adhere better to the ocular surface.

In another study, sequential corneal imaging (High-resolution anterior segment optical coherence tomography, HR-ASOCT) was performed post SLET, for total LSCD after ocular chemical burn.(26) The observations made during the 3 year follow up were:

1. The corneal epithelization started on the 3^rd day post-operatively and was completed on the 14^th day.
2. The thickness of the corneal transplants gradually reduced as they integrated in the corneal epithelium and could not be clinically appreciated on slit-lamp examination after 2 years.
3. The hAM was seen as a hyper-reflective layer below the corneal epithelium that could be recognized for the entire follow-up duration of 3 years, however, the thickness gradually reduced over the period of time.

The epithelial cells grown from each transplant not only have cells of corneal phenotype (CK3⁺, CK12⁺, CK19^-, MUC5AC^-) but also there is focal retention of stem cells (ABCG2⁺, ΔP63α⁺) in the basal epithelial layer of this newly regenerated epithelium. This was confirmed by the histopathology, immunohistochemistry, and cytokeratin studies of the excised corneal buttons undergoing PK after SLET.(12)

IX. Outcomes of SLET

• Promising results post SLET have been published in large studies with a success rate of 70-84%, over a mean follow-up period of 1-3 years.(12-14) In all these three studies, in a majority of cases SLET was performed for the primary indication of ocular chemical burns. Successful outcome was labelled in eyes that maintained a well epithelized and avascular corneal surface without progressive conjunctivalization.(Figure 6)

• A few risk factors contributing to the failure of SLET were the presence of symblepharon and cases where simultaneous keratoplasty was performed. (12-14) Better outcomes were seen where keratoplasty was performed (DALK or PK) as a sequential surgery in cases where the ocular surface inflammation was well-controlled and stable after autologous SLET, as compared to combined surgery (SLET with PK).(27, 28)
• Outcomes of SLET in children has not been extensively published. However, successful results were seen in all 4 eyes that underwent SLET six months after AMT was done for acute ocular burns, either as a primary procedure or repeat SLET with CLAG for a failed SLET.(29)
• Exceptional outcomes were also seen where simultaneous SLET was performed with OSSN excision,(15) and recurrent pterygium excision. (19)

X. Complications (12, 23)

XI. Limitations and advantages of SLET (6, 23)

1. Advantages:

• Single staged procedure
• Requires a small amount of healthy limbus to regenerate the entire corneal surface in the affected eye – hence it is a repeatable procedure and minimizes chances of iatrogenic LSCD in the donor eye
• Can be performed in a resource-limited setting - Does not require a lab for the cultivation of the epithelial cells, hence is also cost-effective

2. Limitations:

• Cases with bilateral LSCD, need allogeneic SLET which needs long-term systemic immunosuppression
• Cases with bilateral total LSCD with extensive symblepharon, CAG cannot be performed. Hence in these cases an allogeneic SLET needs to be combined with oral mucous membrane graft taken from the labial mucosa, or a CLAU is an alternative procedure that may be performed, although CLAU may not be able to address eyes with multiple symblephara

XII. Conclusions

SLET has shown promising results in eyes with unilateral LSCD. This surgery has been commonly performed in eyes with LSCD secondary to ocular surface burns. This procedure helps in epithelial regeneration and maintaining a healthy stable ocular surface. The success rate of this technique is high, especially when the case selection is appropriate. This technique does not have a steep learning curve and has been adopted by corneal surgeons worldwide. This surgical procedure can also be performed easily in a resource-limited setting without the need for an expensive laboratory set-up.

References