Serial anterior segment optical coherence tomography post autologous simple limbal epithelial transplantation

Abstract

A 24-year-old young man presented to us with total limbal stem cell deficiency (LSCD) in the right eye 1 year post ocular chemical burn. The patient subsequently underwent limbal biopsy in the healthy contralateral eye and autologous simple limbal epithelial transplantation in the right eye. The patient was followed up with sequential imaging of the cornea with high-resolution anterior segment optical coherence tomography (HR-ASOCT) for 3 years. The serial HR-ASOCT imaging in the operated eye showed regeneration of the epithelium from the limbal transplant over the human amniotic membrane (hAM) with integration of the transplant within the cornea with subepithelial retention of the hAM. Over the long-term follow-up, thinning of the hAM and thickening of the epithelium was noted. At 3 years, the cornea maintained an intact epithelium with no signs of recurrence of LSCD.

Background

Simple limbal epithelial transplantation (SLET) has revolutionised the management of limbal stem cell deficiency (LSCD) due to chemical injuries.¹ The rationale of this approach is to transfer healthy limbal cells from the fellow normal eye to the diseased eye in order to replace the conjunctivalised cornea with healthy corneal epithelium.¹ The procedure consists of harvesting healthy limbal stem cells from the unaffected eye from a one-clock-hour limbal biopsy, dividing and anchoring it to the diseased cornea with fibrin glue over human amniotic membrane (hAM), which acts as a scaffold for the epithelium regenerating and emerging out of the limbal tissue transplants.² While the behaviour of the individual transplants after SLET in the postoperative period within the first 2 weeks has been a subject of interest,^{3 4} there is no literature regarding the long-term follow-up post SLET with serial imaging of the transplanted ocular surface. Here, we report a patient who underwent SLET following an ocular chemical burn, and thereafter was followed up long term with serial imaging using high-resolution anterior segment optical coherence tomography (HR-ASOCT). The behaviour of the transplants and the changes in the hAM as well as cornea–hAM interaction were studied.

Case presentation

Our patient, a 24-year-old man, presented with complaints of diminution of vision and pain in the right eye following an ocular chemical burn 6 months prior to presentation. Though the patient had received supportive treatment in another hospital, there was no history of any surgical intervention following the injury. At presentation, the vision in the affected eye was counting fingers close to face. Slit-lamp examination revealed mild conjunctival congestion with conjunctivalisation of the cornea, findings clinically suggestive of total LSCD (figure 1A). Digital tension was normal and fundus could not be visualised. The left eye which was unaffected was within normal limits, with a healthy limbal architecture.

Figure 1.

(A) Intraoperative image of the operated eye before simple limbal epithelial transplantation (SLET) depicting total limbal stem cell deficiency; (B) preoperative high-resolution anterior segment optical coherence tomography (HR-ASOCT) before SLET showing a hyper-reflective membrane (pannus) over the cornea with absence of hyporeflective corneal epithelium; (C) preoperative HR-ASOCT of the donor eye showing normal hyporeflective corneal epithelium.

Investigations

At presentation, the patient underwent thorough clinical examination with relevant ocular investigations. In addition, B scan ultrasound of the affected eye was advised, which confirmed an attached retina. Slit-lamp photographs were taken preoperatively and postoperatively at every visit. HR-ASOCT (Optovue, Fremont, CA) was performed preoperatively and postoperatively at every outpatient visit. Preoperatively, HR-ASOCT showed a hyper-reflective membrane over the corneal stroma (figure 1B). HR-ASOCT of the left eye, that is, the donor eye shows the hyporeflective corneal epithelium (figure 1C), which is not seen in the HR-ASOCT of the right eye preoperatively (figure 1B).

Treatment

A diagnosis of right-eye total LSCD was made; however, there is no role of medical therapy in such cases. Hence, we decided to perform autologous SLET for the diseased eye. The other modes of autologous limbal stem cell transplantation are conjunctival limbal autograft (CLAu) and cultivated limbal epithelial transplantation (CLET).⁵ However, since CLET requires cultivating the epithelial cells in the laboratory and CLAu requires harvesting a larger limbal biopsy from the unaffected eye of two-three clock hours, we performed SLET. Also, SLET is an effective and safe surgical modality to treat unilateral total LSCD.⁵

The patient underwent autologous SLET in the right eye after harvesting of one-clock hour of limbus from the left eye. The surgery was performed under peribulbar anaesthesia in the right eye and under topical anaesthesia in the left eye. The technique of SLET was performed as has been described before—harvesting one-clock hour from the limbus of the left eye, dissection of the pannus from the corneal surface in the right eye, glueing of hAM (fresh) with the epithelial side of the hAM facing upwards and division of the limbal tissue from the left eye into 8–12 transplants which were then fixed with fibrin glue (Tisseel Kit, Baxter AG, Vienna, Austria) over the hAM. At the end of the procedure, a bandage contact lens was placed on the corneal surface.⁶

Outcome and follow-up

Postoperatively, the patient was prescribed prednisolone acetate 1% eye drops six times a day, moxifloxacin 0.5% eyedrops four times a day and lubricants in both eyes. Following surgery, the patient was followed up daily for a period of 12 days, and subsequently, at days 21, 42 and 90. Thereafter, the patient was followed up at intervals of 3 months up to a year, and then yearly. The total duration of postoperative follow-up was 3 years. At each visit, besides a full clinical evaluation, slit-lamp photographs were taken and HR-ASOCT was performed on the operated eye. Postoperatively, the visual acuity improved from counting fingers close to face to 20/80 on the fifth postoperative day, which improved to 20/50 on the seventh postoperative day. At the last follow-up visit, 3 years postoperatively, the patient had an unaided visual acuity of 20/20 in the affected eye.

The changes on the ocular surface in the operated eye on HR-ASOCT within the first 2 weeks are depicted in figure 2. Four stages of healing were noted in the recipient eye: (a) adherence of the hAM and disappearance of fibrin glue (starting at day 2 and ending at day 5); (b) epithelial outgrowth from transplants (starting at day 2 and ending at day 7); (c) confluence and stratification of regenerated epithelium (starting at day 7 and ending at day 23) and (d) retention of and progressive remodelling of the transplants and hAM (starting at day 24 and continuous remodelling noted). Thus, the re-epithelisation of the corneal surface was completed by 2 weeks and stabilisation of the ocular surface was achieved by the third week, as confirmed by HR-ASOCT.

Figure 2.

Corneal epithelial regeneration post simple limbal epithelial transplantation in the immediate postoperative period.¹ High-resolution anterior segment optical coherence tomography (HR-ASOCT) images (A, B, C, D). (A) On postoperative day 1, HR-ASOCT shows a single limbal piece labelled as transplant with the other components labelled—bandage contact lens, fibrin glue overlying the transplant and underneath the human amniotic membrane (hAM), hyper-reflective hAM and corneal stroma; (B) on post-operative day 5, HR-ASOCT shows corneal epithelium growing from the single transplant, with disappearance of fibrin glue and thinning of hAM; (C) on post-operative day 8, HR-ASOCT shows corneal epithelium growing from two different transplants merging to form a single layer with a thinner underlying hAM and (D) on post-operative day 11, HR-ASOCT shows thinning and integration of the transplants with epithelium covering the entire cornea with a retained underlying hAM.

On long-term follow-up over 3 years, progressive thickening of the epithelium and thinning of the hAM was noted. However, the hAM could still be discerned as a thin hyper-reflective membrane in the subepithelial region on the HR-ASOCT until the last follow-up (figure 3).

Figure 3.

Corneal regeneration post successful simple limbal epithelial transplantation at long-term follow-up over 3 years. Slit-lamp Photographs (A, C, E, G, I) on the right panel with corresponding high-resolution anterior segment optical coherence tomography (HR-ASOCT) images (B, D, F, H, J) on the left panel. (A) At postoperative 6 weeks, diffuse illumination shows a transparent cornea with intact epithelium with some visible transplants with no vascularisation of the surface; (B) HR-ASOCT shows thinned transplants with thin corneal epithelium with underlying human amniotic membrane (hAM); (C) postoperative 6 months, cornea with intact epithelium with no visible transplants; (D) HR-ASOCT shows thinner transplants than previously shown; (E, G, I) postoperative 1 year, 2 years and 3 years shows a transparent cornea with intact corneal epithelium maintained over follow-up; (F, H, J) HR-ASOCT at postoperative 1 year, 2 year, and 3 years shows disappearance of the transplants, a prominent and thicker corneal epithelium, and a thin hAM which is still visible as a retained membrane at last follow-up.

Discussion

With most studies on SLET focusing on clinical outcomes, there is a dearth of information on the structural changes that occur on the ocular surface after SLET. While a few studies have attempted to fill these lacunae, there is no long-term follow-up of a case of SLET using HR-ASOCT imaging. Mittal et al studied the pattern of epithelisation of the ocular surface following SLET in vivo with detailed photographic documentation and concluded that the pattern of epithelisation is the same as in in-vitro epithelisation after CLET.^{3 7} Also, each transplant showed a centrifugal growth of epithelium from each transplant in all directions.³ They reported that corneal epithelisation begins at 2 days postoperatively and is completed within 14 days.³ In contrast, Chaudhuri et al did not find any evidence of epithelial proliferation even on the eighth day following SLET.⁴ In our case, epithelial proliferation commenced on the third day and was completed by day 12. Also, the corneal epithelium, which appeared thin in the first few days after SLET, continued to stratify and thicken over the next 3–4 weeks. Chaudhuri et al, in their report, did not find any change in the thickness and consistency of the epithelium, although this is not surprising as the follow-up duration was only 14 days postoperatively.⁴

We also noted that the transplants slowly thinned out and could not be clearly discerned on slit-lamp examination or HR-ASOCT after 2 years of follow-up. However, we do concede here that it could be difficult to always image the corneal transplant at the same position. With regard to the behaviour of the hAM in the initial postoperative period, the hAM was thicker, thereafter, settling and adhering to the cornea. The hAM remained intact throughout the postoperative course, until the last follow-up visit; though, it appeared to thin out with time. Our report confirms our previous hypothesis that the presence of a periodic acid Schiff -positive basement membrane in the histopathological sections in post-SLET corneal buttons suggest retention of hAM on a long-term basis.² Amescua et al also showed a normalised corneal epithelium, and a hyper-reflective subepithelial layer, which they postulated to be the residual hAM 4 months post SLET on HR-ASOCT imaging.⁸

Thus, this case shows us the process of epithelial regeneration from the transplants in a case with a successful anatomical and functional outcome post autologous SLET in unilateral total LSCD. HR-ASOCT clearly showed the thinning and the persistence of the hAM under the regenerated corneal epithelium over 3 years of follow-up post SLET. Retention of the hAM under the corneal epithelium was a unique finding we noted, which has not been reported before via imaging, to the best of our knowledge. In vivo imaging using HR-ASOCT is extremely helpful in understanding the process of epithelial regeneration after SLET.

Learning points.

• The serial recording of the high-resolution anterior segment optical coherence tomography images post limbal epithelial stem cell transplantation helps in depicting the exact sequence of events during corneal epithelial regeneration over a long follow-up period.

• Over the immediate postoperative period post simple limbal epithelial transplantation (SLET), corneal epithelium grows from each transplant on the human amniotic membrane (hAM), and thus, an epithelial sheet coalesces from multiple transplants to form intact corneal epithelium over the hAM which acts as a scaffold.

• Over the long-term follow-up period post SLET, the hAM is retained under the regenerated corneal epithelium.