Results

A total of 17 eyes of 15 patients underwent AMT for partial LSCD and each was followed up for more than 1 year with a mean period of 25.8 (SD 2.5) months. Nine patients were male and six female with a mean age of 42.3 (4.6) years (Table 1). The extent of the LSCD involved from 90° to nearly 360° of the limbus. In two cases (Nos 14 and 15) of nearly 360° of limbal involvement the central corneal epithelium appeared intact and clear. Surgery was uneventful in all cases; in particular there were no episodes of postoperative graft infection or rejection. All eyes showed less inflammation and vascularisation immediately postoperatively, and this effect was maintained or improved over the entire follow up period (for examples see Fig 2B, D, F, H) even in those whose stromal vascularisation recurred with time (patient Nos 2, 4, 7, and 10). In the 10 eyes in which it was recorded, the mean time to complete corneal and conjunctival epithelialisation was 22.8 (5.0) days. All eyes maintained a smooth and stable corneal epithelial surface at the last follow up visit without recurrent erosion or persistent epithelial defect. As a result, visual acuity was improved in the large majority of cases. Excluding those whose visual potential was limited by pre-existing or concomitant disease (case No 2 by radiation induced optic neuropathy, case No 9 by total retinal detachment, and case No 12 by established age related macular degeneration), visual acuity improved in 13 eyes (92.9%) and decreased in one eye (7.1%) of the remaining 14 eyes. Of these cases, five eyes (38.5%) gained at least six lines, two eyes (15.4%) gained four to five lines, and six eyes (46.2%) gained one to three lines of Snellen acuity. The one eye of case No 3 lost three lines; this eye suffered from a bilateral alkali burn and had previously undergone a rotational autopenetrating keratoplasty followed by a conjunctival flap. The cause was due to progression of central corneal fibrosis and thinning after AMT. Of the 14 cases with the complaint of pain or photophobia, these symptoms completely resolved in 12 cases (85.7%) and significantly reduced in two cases (14.3%). One patient (case No 9) underwent evisceration for phthisis bulbi 14 months following AMT. Owing to trauma induced dislocation, the AM had to be replaced in the child (case No 2). Postoperative complications were minimal and included the removal of loose sutures and in one case temporary treatment with systemic cyclosporin A for an idiopathic inflammatory reaction between the AM edge and the host conjunctiva. In particular no cases of inadvertent perforation or elevated intraocular pressure occurred.

• Download figure
• Open in new tab
• Download powerpoint

Figure 2

Comparison of preoperative (left panels) and postoperative (right panels) corneal appearance following AMT. Patient No 8 complained of severe photophobia and decreased vision. Bilateral contact lens induced LSCD involved 300° of the right limbus (A) and 270° of the left limbus (C). Following AMT, a clear, smooth, and avascular epithelial surface was noted in the right cornea 23.5 months (B) and in the left cornea 21 months postoperatively (D). Patient No 4 with 270° of LSCD due to a chemical burn (E) resulting in prominent vascularisation (delineated by white stars) and an irregular epithelium (white arrows) affecting the visual axis. 23 months after AMT, the cornea became less inflamed and vascularised with residual stromal haze (white arrows) (F). Patient No 12 with CIN (white star) involving 330° of the limbus (G). Following excision of the lesion, removal of fibrovascular pannus, and AMT, the cornea became avascular, smooth, and stable 6 months postoperatively (H). Black arrow indicates the limbal running 10/0 nylon suture.

REPRESENTATIVE CASES

Case 13

A 28 year old woman sustained a chemical burn to her left eye resulting in photophobia, decreased vision and sustained hyperaemia, the right eye was unaffected. At presentation the best corrected visual acuity (BCVA) was 20/25 left eye with a mild ptosis. Slit lamp examination revealed loss of the palisades of Vogt and fibrovascular pannus invading the peripheral cornea for 300°; the central cornea was intact and clear (Fig 3A). The diagnosis of partial LSCD was made clinically and the patient underwent AMT. At surgery, a peritomy was performed in the area of the LSCD and subconjunctival fibrous tissue that appeared to be the worst, followed by a conjunctival recession of between 5–7 mm from the limbus. Using this plane, fibrovascular pannus was excised from the cornea by blunt dissection and an AMT graft placed over the defect only using interrupted 10/0 Vicryl sutures over the resected sclera and completed with a 10/0 nylon running suture over the limbal region. The area from 7 to 10 o' clock was left uncovered with no dissection at the limbus. Nine days postoperatively the membrane was noted to be securely in place (Fig 3B) and partially epithelialised (Fig 3C). Three months postoperatively the AM dissolved over the cornea (Fig 3D) resulting in a transparent and stable ocular surface with a BCVA of 20/60. Eight months postoperatively, however, the area that was left unoperated—that is, not covered by AM, showed progressive vascularisation and conjunctivalisation (Fig 3E). This region was characterised by an irregular epithelial surface which displayed delayed fluorescein uptake caused by poor epithelial integrity (Fig3F). At final follow up 21 months following surgery the operated area remained stable and non-inflamed and the patient was minimally symptomatic with a BCVA of 20/20.

• Download figure
• Open in new tab
• Download powerpoint

Figure 3

Comparison of preoperative and postoperative appearances of case No 13 with LSCD arising from a chemical burn. Prominent fibrovascular pannus of 300° noted at presentation extending from 11 to 9 o' clock (A). Nine days following AMT and superficial keratectomy (B), the eye was non-inflamed and AM was held in place by a running 10/0 nylon suture. The AM was partially epithelialised with a residual central defect apparent on fluorescein staining (arrows) (C). Four months postoperatively, the cornea was transparent with an intact and smooth epithelial surface (D). However, 8 months postoperatively progressive vascularisation and conjunctivalisation (arrows) arose from the limbal area that was originally thought only to be mildly affected and not excised or covered with AM (E). Delayed fluorescein staining was noted in the area of corresponding epithelial irregularity (F).

Case 14

A 52 year old woman initially presented to the referring ophthalmologist with a 5 year history of photophobia, ocular irritation, and decreasing vision worse on the right eye. No previous history of atopy or significant systemic illness was obtained. Bilateral corneal peripheral fibrovascular pannus was observed with conjunctival foreshortening worse on the right, and a BCVA of 20/60 right eye and 20/40 left eye. Immunofluorescence studies of a conjunctival biopsy were inconsistent with ocular cicatricial pemphigoid and no eosinophils were observed in the specimen. Cataract surgery of the right eye was followed by deterioration of the condition and no improvement was obtained with topical steroids or topical and systemic antibiotics. Corneal topography revealed increasing astigmatism and at the time of referral she complained of severe photophobia, foreign body sensation, ocular irritation, and progressive loss of vision. Examination revealed a BCVA of 20/100 right eye and 20/60 left eye with fine pannus predominantly of the superior and inferior limbus affecting the right eye (Fig 4A) more than the left and associated with poor epithelial integrity (Fig 4B). Lipid tear deficiency was diagnosed on the basis of meibomium orifice metaplasia, short tear break up time (3 seconds), and facial rosacea. Floppy eyelid syndrome was also present but corneal, conjunctival, and lid margin sensitivity measured with the Bonnet-Cochet aesthesiometer was normal. The diagnosis of idiopathic, bilateral LSCD affecting almost 360° of the right limbal zone was made on the basis of the clinical examination and impression cytology findings. Excision of the fibrovascular pannus to the intact corneal epithelium followed by AMT of the right eye (Fig1). Despite topical treatment with preservative-free methylprednisolone (BPEI pharmacy), the procedure was followed by marked inflammation of the host conjunctiva and systemic immunosuppression with cyclosporin A was commenced. The membrane was re-epithelialised 30 days postoperatively and the cyclosporin A discontinued 2 months later. Two months following the procedure the patient reported that the right eye was comfortable and non-photophobic, examination revealed the eye to be non-inflamed with a clear, smooth, intact corneal epithelial surface (Fig 4C and D). The BCVA was improved at 20/30 right eye and unchanged on the left at 20/60 at final follow up 13 months following surgery. This contrasted with the left eye which had been maintained on medical treatment only and displayed no change in the corneal findings of vascularisation (Fig 4E) and poor epithelial integrity (Fig 4F).

• Download figure
• Open in new tab
• Download powerpoint

Figure 4

Preoperative and postoperative appearances of case 14 with idiopathic LSCD. At presentation, prominent fibrovascular pannus was noted (white stars) to involve nearly 360° of the limbus (A). This was associated with an irregular epithelium (white arrows) characterised by delayed fluorescein uptake (B). Two months following AMT the corneal surface showed no recurrence of the fibrovascular pannus (C) with a restored epithelial integrity demonstrated by fluorescein staining (D) although a prominent stromal vessel was still apparent at 12 o' clock. This appearance contrasted with the fellow eye (E), which acted as a control as no surgery was performed. In this eye the epithelial irregularity is still evidenced by delayed fluorescein uptake (F).

Discussion

In this report, we have shown that AMT ameliorated annoying photophobia or pain, facilitated rapid epithelialisation, restored a normal corneal epithelial surface, and improved the final visual acuity in the majority of these 17 consecutive eyes with partial LSCD. Photophobia and ocular discomfort were abolished in 85.7% and substantially reduced in the remaining 14.3% of the 14 patients who presented with these complaints. Epithelialisation of the entire membrane covered surface took an average of 22.8 days. Excluding the three eyes with limited visual potential due to radiation optic neuropathy, macular degeneration, or total retinal detachment, 92.9% of eyes gained visual acuity with 42.9% of eyes gaining one to three lines, 14.3% gaining four to five lines, and 35.7% gaining six or more lines. These findings substantiate our earlier report,32 and more importantly confirmed that this favourable outcome could be maintained in all of the above cases for more than 1 year of follow up (with a mean of 25.8 months). Collectively, these data support the hypothesis that AMT can help preserve and expand the remaining limbal epithelial stem cell population in vivo that is left in partial LSCD. This effect was noted even in eyes with more than 300° of LSCD. In two cases of nearly total LSCD (cases 14 and 15), we found AMT was still workable if a central island of epithelium remained intact. This observation suggested that surviving islands of transient amplifying cells were worth preserving and may have contributed to the regeneration of the corneal epithelium. In these two cases, the defect healed in 15 and 30 days, respectively, despite the nearly total loss of limbus. Taken together these observations have prompted us to propose the use of AMT as a first line procedure to treat patients suffering from partial LSCD. Because no transplantation of autologous or allogeneic limbal epithelial stem cells is needed, the potential complications of systemic immunosuppression and risk to the donor eye can be avoided. In one of our reported cases (case 14) we did employ systemic immunosuppression for a 2 month period. In this case the idiopathic inflammation at the junction between the conjunctiva and AM was noted to resolve fairly rapidly without the features of hypopyon uveitis recently reported following repeated AMT for deep trophic ulcer.37

The beneficial effect of AMT may be due in part to the restoration of an intact basement membrane that is invariably damaged in LSCD. Basement membrane is known to support epithelial cell adhesion, differentiation, and migration (see review by Tseng and Tsubota38), and to suppress epithelial cell apoptosis.39 Compositionally, the basement membrane component of the amniotic membrane resembles that of the conjunctiva.40 Amniotic membrane basement membrane is an ideal substrate for supporting the growth of epithelial progenitor cells by prolonging their life span and maintaining their clonogenicity.41 This action explains why AMT facilitates epithelialisation for persistent corneal epithelial defects with stromal ulceration.3542-45 In tissue culture, amniotic membrane supports epithelial cells grown from explant cultures444647 and other cultures,48 and maintains their normal morphology and differentiation. The resultant graft with an epithelial cell layer and amniotic membrane can be successfully transplanted back to reconstruct the damaged corneal surface in rabbits4748 and in humans.4849

The stromal side of the membrane may also provide additional benefit in treating LSCD. We have reported that this side contains a unique matrix component that suppresses TGF-β signalling, and the proliferation and myofibroblast differentiation of normal human corneal and limbal fibroblasts50 and normal conjunctival and pterygium body fibroblasts.51 This action explains why AMT reduces fibrosis during conjunctival surface reconstruction,3652 prevents recurrent scarring after pterygium removal,53-55 and reduces corneal haze following phototherapeutic keratectomy and photorefractive keratectomy.55-58 The stromal matrix of the membrane can also exclude inflammatory cells,5859 and contains growth factors60 and several forms of protease inhibitors.61 These actions explain why stromal inflammation is reduced after AMT3536 and corneal neovascularisation is mitigated.62 Collectively, these actions are useful to prepare a stromal bed that may subsequently support limbal epithelial stem cells (for review see Tseng3). In this manner, AMT has been used either simultaneously or following the transplantation of allogeneic limbal epithelial stem cells to treat patients with total LSCD.31325463-66 Taken together, these findings and the results of the present study suggest that AMT may be superior to repeated debridement of the conjunctivalised corneal surface which has been reported to be effective in six cases of partial LSCD with a follow up less than 8 months6768 although a randomised clinical trial would be required to answer this question.

In summary, this report demonstrates that AMT is a safe and effective procedure to restore the corneal epithelial surface in patients with partial LSCD. By avoiding the potential complications of autograft or allograft transplantation of limbal tissue, AMT may offer a superior alternative. Future studies directed to the molecular mechanism by which AM may help preserve and expand limbal epithelial stem cells may clarify our understanding of this unique biological matrix and unravel other potential clinical applications.