Dalia G. Said

Human corneal anatomy redefined: a novel pre-Descemet's layer (Dua's layer).

PURPOSE To define and characterize a novel pre-Descemets layer in the human cornea. DESIGN Clinical and experimental study. PARTICIPANTS We included 31 human donor sclerocorneal discs, including 6 controls (mean age, 77.7 years). METHODS Air was injected into the stroma of donor whole globes (n = 4) and sclerocorneal discs (n = 21) as in the clinical deep anterior lamellar keratoplasty procedure with the big bubble (BB) technique. The following experiments were performed: (1) creation of BB followed by peeling of the Descemets membrane (DM); (2) peeling off of the DM followed by creation of the BB, and (3) creation of the BB and continued inflation until the bubble popped to measure the popping pressure. Tissue obtained from these experiments was subjected to histologic examination. MAIN OUTCOME MEASURES Demonstration of a novel pre-Descemets layer (Duas layer) in the human cornea. RESULTS Three types of BB were obtained. Type-1, is a well-circumscribed, central dome-shaped elevation up to 8.5 mm in diameter (n = 14). Type-2, is a thin-walled, large BB of maximum 10.5 mm diameter, which always started at the periphery, enlarging centrally to form a large BB (n = 5), and a mixed type (n = 3). With type-1 BB, unlike type-2 BB, it was possible to peel off DM completely without deflating the BB, indicating the presence of an additional layer of tissue. A type-1 BB could be created after first peeling off the DM (n = 5), confirming that DM was not essential to create a type-1 BB. The popping pressure was 1.45 bar and 0.6 bar for type-1 BB and type-2 BB, respectively. Histology confirmed that the cleavage occurred beyond the last row of keratocytes. This layer was acellular, measured 10.15 ± 3.6 microns composed of 5 to 8 lamellae of predominantly type-1 collagen bundles arranged in transverse, longitudinal, and oblique directions. CONCLUSIONS There exists a novel, well-defined, acellular, strong layer in the pre-Descemets cornea. This separates along the last row of keratocytes in most cases performed with the BB technique. Its recognition will have considerable impact on posterior corneal surgery and the understanding of corneal biomechanics and posterior corneal pathology such as acute hydrops, Descematocele and pre-Descemets dystrophies. FINANCIAL DISCLOSURE(S) The authors have no proprietary or commercial interest in any materials discussed in this article.

Amniotic membrane in ophthalmology: indications and limitations

The amniotic membrane remains a useful tool in the treatment of several ophthalmic conditions, especially those related to the ocular surface. However, the ‘success’ reported in individual case reports and case series is not substantiated in the few published randomised controlled trials. More often than not, it is not as good as existing alternative options and, at best, is as good but with probably an increased cost. The variable outcomes could be related to inter and intra donor variations in the membrane and the depletion or alterations in its constituents subsequent to processing and storage. The membrane thus is a fairly ‘non-standardised product’ making comparisons of different applications and indications difficult. The potential for ‘epidemic’ infections, such as HIV, hepatitis B and C, is a serious issue as, in many parts of the world, fresh unquarantined membrane, at times with no tests for the above infectious agents, is being used. The exact mechanism of action of the membrane is not known but the consensus is that it acts as a substrate or scaffold for host cells to populate and thus facilitate healing and repair. The development of a standard ‘synthetic membrane’ using collagen or polymer matrices impregnated with putative beneficial ingredients, such as growth factors and antimicrobials, is being considered and may prove to be a step in the right direction.

Collagen Cross-Linking with Photoactivated Riboflavin (PACK-CXL) for the Treatment of Advanced Infectious Keratitis with Corneal Melting

PURPOSE To investigate the efficacy and safety of corneal collagen cross-linking (CXL) with photoactivated riboflavin (photoactivated chromophore for infectious keratitis [PACK]-CXL) in the management of infectious keratitis with corneal melting. DESIGN Prospective clinical trial. PARTICIPANTS Forty eyes from 40 patients with advanced infectious keratitis and coexisting corneal melting. METHODS Twenty-one patients (21 eyes) underwent PACK-CXL treatment in addition to antimicrobial therapy. The control group consisted of 19 patients (19 eyes) who received only antimicrobial therapy. MAIN OUTCOME MEASURES The slit-lamp characteristics of the corneal ulceration, corrected distance visual acuity, duration until healing, and complications were documented in each group. The Mann-Whitney U test was used for statistical analysis. P values less than 0.05 were considered statistically significant. RESULTS The average time until healing was 39.76 ± 18.22 days in the PACK-CXL group and 46.05 ± 27.44 days in the control group (P = 0.68). After treatment and healing, corrected distance visual acuity was 1.64 ± 0.62 in the PACK-CXL group and 1.67 ± 0.48 in the control group (P = 0.68). The corneal ulcerations width and length was significantly bigger in the PACK-CXL group (P = 0.004 and P = 0.007). Three patients in the control group demonstrated corneal perforation; infection recurred in 1 of them. No serious complications occurred in the PACK-CXL group. CONCLUSIONS Corneal CXL with photoactivated riboflavin did not shorten the time to corneal healing; however, the complication rate was 21% in the control group, whereas there was no incidence of corneal perforation or recurrence of the infection in the PACK-CXL group. These results indicate that PACK-CXL may be an effective adjuvant therapy in the management of severe infectious keratitis associated with corneal melting.

Contemporary limbal stem cell transplantation – a review

Conjunctivalization of the cornea is the hallmark of limbal stem cell deficiency (LSCD). This is often associated with persistent corneal epithelial defects and a fibrovascular pannus. LSCD can be unilateral or bilateral and partial or total. In partial LSCD involving the visual axis sequential sector conjunctival epitheliectomy (SSCE) is a useful option. In total LSCD, transplantation of limbal tissue or of ex vivo expanded sheets is the mainstay. In unilateral cases autolimbal transplant is the procedure of choice. In bilateral cases living (related) and cadaver donors are considered. The former has the advantage of being fresh and can be human leucocyte antigen matched. Procedures for harvesting limbal tissue from living donors are identical. Different strategies are required for harvesting tissue from cadaver whole globes or sclero‐corneal rims. Recipient eye preparation requires removal of the fibrovascular tissue. Donor explants are generally sutured directly on the denuded recipient surface without the preparation of a ‘bed’ to fit the explant. It is imperative that inflammation is meticulously controlled before limbal transplantation especially if tissue from living donors is used. Limbal transplantation, with the exception of a corneal graft, should be the last surgical intervention planned. Meticulous postoperative care and treatment with antibiotics, steroids, artificial tears and autologous serum are required. With allografts long‐term immunosuppression is necessary. Limbal transplantation is contraindicated in the presence of severe dry eye. Despite its complexities limbal transplantation does significantly improve vision related quality of life. Autografts give the best results and living related donor grafts are next best. Majority of cadaver grafts fail in 5 years.

Outcomes of deep anterior lamellar keratoplasty following successful and failed ‘big bubble’

Aim The most popular technique for deep anterior lamellar keratoplasty (DALK) is the ‘big bubble’ (BB) technique wherein air is injected in the cornea to create a bubble that separates Descemets membrane (DM) from the stroma. An attempt to create a BB often results in the cornea being filled with numerous small bubbles without the formation of a BB. Manual dissection is then required to complete the procedure. The aim of the study is to compare these two groups, successful BB versus failed bubble (FB) dissection to determine whether the clinical outcomes were different. Methods In this retrospective comparative study, 46 patients out of 52 who underwent DALK for various corneal stromal diseases such as keratoconus, stromal dystrophy or corneal scarring (caused by different conditions) were included in the analysis. BB was achieved in 25 patients and in the remaining 21 patients a BB separation of the DM was not possible necessitating manual lamellar dissection of stroma to get as close to the DM as possible. Results The authors compared best-corrected visual acuity, contrast sensitivity, astigmatism, interface densitometry and Scheimpflug pachymetry in the two groups. Postoperative corneal thickness was higher in the ‘small bubbles’ group (mean 628.9 vs 564.1 μm; p<0.0005), but there was no significant difference in best-corrected visual acuity, astigmatism, contrast sensitivity and densitometry between the groups. Conclusions In DALK, manual lamellar dissection is a reasonable alternative when BB separation of the DM is not achieved.

In vivo confocal microscopic findings in patients with limbal stem cell deficiency

Aim To describe in vivo confocal microscopy (IVCM) findings in patients with limbal stem cell deficiency (LSCD). Methods 23 eyes of 17 consecutive patients suffering from LSCD were included in this study. A detailed examination by IVCM was performed in addition to a routine slit-lamp biomicroscopy. Size and density of corneal epithelial and conjunctival epithelial cells on cornea were measured and statistically analysed using SPSS version 8.0 software. Results were compared with histology in select cases. Results Anatomical and morphological differences were observed between normal corneal cells and conjunctival epithelial cells on cornea. Size and density differences reached statistically significant levels between the normal corneal cells and the conjunctival epithelial cells on cornea (p<0.01). Goblet cells were visible throughout the conjunctivalised corneal epithelium in eight eyes. Several IVCM features could be correlated with histology in six of our patients. Conclusions A number of features were demonstrated by laser IVCM in patients presenting clinically with LSCD. Some of these features were corroborated with features observed on histological examination of tissue samples.

In vivo confocal microscopic features of normal limbus

Aim To describe in vivo confocal microscopy (IVCM) features of the limbus in normal eyes as related to the palisades of Vogts. Methods 46 eyes of 29 consecutive volunteers were recruited in this observational study. A detailed examination by IVCM was performed in addition to a routine slit-lamp biomicroscopy. Size and density of corneal and limbal epithelial cells were measured and statistically analysed using SPSS version 8.0 software. Results Anatomical and morphological features were noted between corneal and limbal cells. Size and density differences reached to significant levels (p<0.05). Different shapes of palisades of Vogt have been described clearly by confocal microscope. Cell-like structures were observed in the peripheral end of the palisades which might represent limbal stem cell crypts. Conclusions Laser IVCM can be used to establish the features of the normal limbus. The identified features demonstrate quantitative changes in the basal epithelium between the limbus and the central cornea and morphological differences between pigmented or non-pigmented studied subjects. Further studies should be performed to correlate with histology the possible crypts which were observed in this study.

In Vivo Analysis of Stromal Integration of Multilayer Amniotic Membrane Transplantation in Corneal Ulcers

PURPOSE To evaluate integration of amniotic membrane into the corneal stroma using laser scanning in vivo confocal microscopy and anterior segment optical coherence tomography (AS-OCT). DESIGN Prospective noncomparative interventional case series. METHODS Twenty-two eyes of 22 consecutive patients (mean age 53.9 ± 9.2 years) presenting with noninfectious corneal ulcers and stromal thinning unresponsive to medical treatment were enrolled. Multiple layers of amniotic membrane were applied over the ulcer bed to fill the ulcer crater and held in place with an overlying amniotic membrane patch, which was anchored to the surrounding cornea with 10-0 nylon interrupted sutures. Outcome measures were healing of the corneal ulcers, corneal morphology and stromal thickness changes at the ulcer site as measured by AS-OCT and surface epithelialization, stromal repopulation, and structural modifications of the amniotic membrane grafts as evaluated by confocal microscopy. RESULTS Follow-up extended to 12 months. Successful result was observed in 20 of 22 eyes (90.9%). AS-OCT showed that the mean residual stromal thickness at the ulcer bed was 222 ± 70 μm before surgery. The mean thickness of amniotic membrane layers at the same site was 394 ± 80 μm while the mean total corneal thickness was 623 ± 51 μm at day 1 post surgery. Thereafter a progressive reduction in thickness to 420 ± 61 μm at 6 months occurred, after which the thickness stabilized. Confocal microscopy showed that integration of the amniotic membrane tissues with corneal stroma was preceded by epithelialization over the amniotic membrane covering the ulcer. This occurred 15 ± 5 days post surgery in the successful cases. Confocal microscopy also showed that the amniotic membrane patch was degraded during the first few weeks after surgery, while the integrated amniotic tissues underwent progressive modifications characterized by early loss of amniotic epithelial cells, changes in fibrillar structure, and migration into the amniotic stroma by corneal stroma-derived cells. CONCLUSIONS Multiple layers of amniotic membrane can integrate into the corneal stroma with resulting increase in corneal thickness. This appears to be related to re-epithelialization of the transplanted membrane. Integrated amnion within the stromal defect undergoes progressive changes including contraction of tissue and repopulation by corneal stroma-derived cells.

Variations in amniotic membrane: relevance for clinical applications

The amniotic membrane (AM) has found several clinical applications for ophthalmic indications, in particular, those related to ocular surface (OS) diseases. Successful results have been reported after use in treatment of persistent corneal epithelial defects, bullous keratopathy, acute and late stages of chemical burns and OS inflammatory diseases such as Stevens Johnson syndrome and after excision of conjunctival lesions, besides others.1–3 The AM has a complex structure, and several layers have been described. Essentially, it is composed of a metabolically active epithelium, which rests on a basement membrane and an avascular stroma. The epithelium and the stroma contain several growth factors, cytokines and other metabolically active substances. The transforming growth factor (TGFb) and the epidermal growth factor (EGF) are major and important growth factors. Proinflammatory and anti-inflammatory cytokines, such as interleukin 6 (IL-6), IL-8, IL-10 and IL-1ra, metalloproteases and tissue inhibitors of metalloproteases, and others have also been described.3 4 The mechanism of action of the membrane is not precisely known. Much of its beneficial effect can be attributed to its role as a substrate or scaffold supporting cell growth, migration and adhesion.5 The actions …

The collagen matrix of the human trabecular meshwork is an extension of the novel pre-Descemet's layer (Dua's layer)

Background The trabecular meshwork (TM) located at the angle of the anterior chamber of the eye contributes to aqueous drainage. A novel layer in the posterior part of the human cornea has recently been reported (the pre-Descemets layer (Duas layer (PDL)). We examined the peripheral part of this layer in relation to the origin of the TM. Methods The PDL and TM of 19 human donor eyes and one exenterated sample were studied. Samples were examined by light and electron microscopy (EM) for tissue architecture and by immunohistology for four matricellular proteins, five collagen types and CD34. Results EM revealed that beams of collagen emerged from the periphery of PDL on the anterior surface of the Descemets membrane and divided and subdivided to continue as the beams of the TM. Long-spacing collagen was seen in the PDL and TM. Trabecular cells (CD34-ve) associated with basement membrane were seen in the peripheral part of the PDL and corresponded to the start of the separation of the collagen lamellae of PDL. Collagen VI was present continuously in PDL and extended into the TM. Matricellular proteins were seen predominantly in the TM with only laminin extending into the periphery of PDL. Conclusions This study provides an insight into the origins of the collagen core of the TM as an extension of the PDL of the cornea. This finding adds to the knowledge base of the TM and cornea and has the potential to impact future research into the TM and glaucoma.